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Sample records for messenger rna profiling

  1. The microRNA and messengerRNA profile of the RNA-induced silencing complex in human primary astrocyte and astrocytoma cells.

    Moser, Joanna J; Fritzler, Marvin J

    2010-10-18

    GW/P bodies are cytoplasmic ribonucleoprotein-rich foci involved in microRNA (miRNA)-mediated messenger RNA (mRNA) silencing and degradation. The mRNA regulatory functions within GW/P bodies are mediated by GW182 and its binding partner hAgo2 that bind miRNA in the RNA-induced silencing complex (RISC). To date there are no published reports of the profile of miRNA and mRNA targeted to the RISC or a comparison of the RISC-specific miRNA/mRNA profile differences in malignant and non-malignant cells. RISC mRNA and miRNA components were profiled by microarray analysis of malignant human U-87 astrocytoma cells and its non-malignant counterpart, primary human astrocytes. Total cell RNA as well as RNA from immunoprecipitated RISC was analyzed. The novel findings were fourfold: (1) miRNAs were highly enriched in astrocyte RISC compared to U-87 astrocytoma RISC, (2) astrocytoma and primary astrocyte cells each contained unique RISC miRNA profiles as compared to their respective cellular miRNA profiles, (3) miR-195, 10b, 29b, 19b, 34a and 455-3p levels were increased and the miR-181b level was decreased in U-87 astrocytoma RISC as compared to astrocyte RISC, and (4) the RISC contained decreased levels of mRNAs in primary astrocyte and U-87 astrocytoma cells. The observation that miR-34a and miR-195 levels were increased in the RISC of U-87 astrocytoma cells suggests an oncogenic role for these miRNAs. Differential regulation of mRNAs by specific miRNAs is evidenced by the observation that three miR34a-targeted mRNAs and two miR-195-targeted mRNAs were downregulated while one miR-195-targeted mRNA was upregulated. Biological pathway analysis of RISC mRNA components suggests that the RISC plays a pivotal role in malignancy and other conditions. This study points to the importance of the RISC and ultimately GW/P body composition and function in miRNA and mRNA deregulation in astrocytoma cells and possibly in other malignancies.

  2. The microRNA and messengerRNA profile of the RNA-induced silencing complex in human primary astrocyte and astrocytoma cells.

    Joanna J Moser

    2010-10-01

    Full Text Available GW/P bodies are cytoplasmic ribonucleoprotein-rich foci involved in microRNA (miRNA-mediated messenger RNA (mRNA silencing and degradation. The mRNA regulatory functions within GW/P bodies are mediated by GW182 and its binding partner hAgo2 that bind miRNA in the RNA-induced silencing complex (RISC. To date there are no published reports of the profile of miRNA and mRNA targeted to the RISC or a comparison of the RISC-specific miRNA/mRNA profile differences in malignant and non-malignant cells.RISC mRNA and miRNA components were profiled by microarray analysis of malignant human U-87 astrocytoma cells and its non-malignant counterpart, primary human astrocytes. Total cell RNA as well as RNA from immunoprecipitated RISC was analyzed. The novel findings were fourfold: (1 miRNAs were highly enriched in astrocyte RISC compared to U-87 astrocytoma RISC, (2 astrocytoma and primary astrocyte cells each contained unique RISC miRNA profiles as compared to their respective cellular miRNA profiles, (3 miR-195, 10b, 29b, 19b, 34a and 455-3p levels were increased and the miR-181b level was decreased in U-87 astrocytoma RISC as compared to astrocyte RISC, and (4 the RISC contained decreased levels of mRNAs in primary astrocyte and U-87 astrocytoma cells.The observation that miR-34a and miR-195 levels were increased in the RISC of U-87 astrocytoma cells suggests an oncogenic role for these miRNAs. Differential regulation of mRNAs by specific miRNAs is evidenced by the observation that three miR34a-targeted mRNAs and two miR-195-targeted mRNAs were downregulated while one miR-195-targeted mRNA was upregulated. Biological pathway analysis of RISC mRNA components suggests that the RISC plays a pivotal role in malignancy and other conditions. This study points to the importance of the RISC and ultimately GW/P body composition and function in miRNA and mRNA deregulation in astrocytoma cells and possibly in other malignancies.

  3. Nuclear Export of Messenger RNA

    Jun Katahira

    2015-03-01

    Full Text Available Transport of messenger RNA (mRNA from the nucleus to the cytoplasm is an essential step of eukaryotic gene expression. In the cell nucleus, a precursor mRNA undergoes a series of processing steps, including capping at the 5' ends, splicing and cleavage/polyadenylation at the 3' ends. During this process, the mRNA associates with a wide variety of proteins, forming a messenger ribonucleoprotein (mRNP particle. Association with factors involved in nuclear export also occurs during transcription and processing, and thus nuclear export is fully integrated into mRNA maturation. The coupling between mRNA maturation and nuclear export is an important mechanism for providing only fully functional and competent mRNA to the cytoplasmic translational machinery, thereby ensuring accuracy and swiftness of gene expression. This review describes the molecular mechanism of nuclear mRNA export mediated by the principal transport factors, including Tap-p15 and the TREX complex.

  4. Nuclear Export of Messenger RNA

    Katahira, Jun

    2015-01-01

    Transport of messenger RNA (mRNA) from the nucleus to the cytoplasm is an essential step of eukaryotic gene expression. In the cell nucleus, a precursor mRNA undergoes a series of processing steps, including capping at the 5' ends, splicing and cleavage/polyadenylation at the 3' ends. During this process, the mRNA associates with a wide variety of proteins, forming a messenger ribonucleoprotein (mRNP) particle. Association with factors involved in nuclear export also occurs during transcription and processing, and thus nuclear export is fully integrated into mRNA maturation. The coupling between mRNA maturation and nuclear export is an important mechanism for providing only fully functional and competent mRNA to the cytoplasmic translational machinery, thereby ensuring accuracy and swiftness of gene expression. This review describes the molecular mechanism of nuclear mRNA export mediated by the principal transport factors, including Tap-p15 and the TREX complex. PMID:25836925

  5. Radiation sensitivity of messenger RNA

    Ponta, H.; Pfennig-Yeh, M.L.; Herrlich, P.; Karlsruhe Univ.; Wagner, E.F.; Schweiger, M.

    1979-01-01

    Messenger RNA function is inactivated by irradiation with ultraviolet light. A unit length mRNA (in bases) is 2-3 times more sensitive than a unit length of DNA (in base pairs) with respect to the inactivation of template function. These data stem from four experimental systems all of which do not repair DNA: the translation of E. coli mRNA in rifampicin-treated cells, of T7 mRNA in infected E.coli, of f2 phage RNA in vivo, and of stable mRNA in chromosomeless minicells. The comparison of relative sensitivities to UV is relevant to the technique of UV mapping of transcription units which enjoys increasing popularity in pro- and eukaryotic genetic research. (orig.) [de

  6. Radiation sensitivity of messenger RNA

    Ponta, H; Pfennig-Yeh, M L; Herrlich, P [Kernforschungszentrum Karlsruhe G.m.b.H. (Germany, F.R.). Inst. fuer Genetik und Toxikologie von Spaltstoffen; Karlsruhe Univ. (TH) (Germany, F.R.). Inst. fuer Genetik); Wagner, E F; Schweiger, M [Innsbruck Univ. (Austria). Inst. fuer Biochemie

    1979-08-01

    Messenger RNA function is inactivated by irradiation with ultraviolet light. A unit length mRNA (in bases) is 2-3 times more sensitive than a unit length of DNA (in base pairs) with respect to the inactivation of template function. These data stem from four experimental systems all of which do not repair DNA: the translation of E. coli mRNA in rifampicin-treated cells, of T7 mRNA in infected E.coli, of f2 phage RNA in vivo, and of stable mRNA in chromosomeless minicells. The comparison of relative sensitivities to UV is relevant to the technique of UV mapping of transcription units which enjoys increasing popularity in pro- and eukaryotic genetic research.

  7. MCT-1 protein interacts with the cap complex and modulates messenger RNA translational profiles

    Reinert, Line; Shi, B; Nandi, S

    2006-01-01

    MCT-1 is an oncogene that was initially identified in a human T cell lymphoma and has been shown to induce cell proliferation as well as activate survival-related pathways. MCT-1 contains the PUA domain, a recently described RNA-binding domain that is found in several tRNA and rRNA modification...... enzymes. Here, we established that MCT-1 protein interacts with the cap complex through its PUA domain and recruits the density-regulated protein (DENR/DRP), containing the SUI1 translation initiation domain. Through the use of microarray analysis on polysome-associated mRNAs, we showed that up......-regulation of MCT-1 was able to modulate the translation profiles of BCL2L2, TFDP1, MRE11A, cyclin D1, and E2F1 mRNAs, despite equivalent levels of mRNAs in the cytoplasm. Our data establish a role for MCT-1 in translational regulation, and support a linkage between translational control and oncogenesis....

  8. Messenger RNA 3' end formation in plants.

    Hunt, A G

    2008-01-01

    Messenger RNA 3' end formation is an integral step in the process that gives rise to mature, translated messenger RNAs in eukaryotes. With this step, a pre-messenger RNA is processed and polyadenylated, giving rise to a mature mRNA bearing the characteristic poly(A) tract. The poly(A) tract is a fundamental feature of mRNAs, participating in the process of translation initiation and being the focus of control mechanisms that define the lifetime of mRNAs. Thus messenger RNA 3' end formation impacts two steps in mRNA biogenesis and function. Moreover, mRNA 3' end formation is something of a bridge that integrates numerous other steps in mRNA biogenesis and function. While the process is essential for the expression of most genes, it is also one that is subject to various forms of regulation, such that both quantitative and qualitative aspects of gene expression may be modulated via the polyadenylation complex. In this review, the current status of understanding of mRNA 3' end formation in plants is discussed. In particular, the nature of mRNA 3' ends in plants is reviewed, as are recent studies that are beginning to yield insight into the functioning and regulation of plant polyadenylation factor subunits.

  9. Messenger RNA surveillance: neutralizing natural nonsense

    Weischelfeldt, Joachim Lütken; Lykke-Andersen, Jens; Porse, Bo

    2005-01-01

    Messenger RNA transcripts that contain premature stop codons are degraded by a process termed nonsense-mediated mRNA decay (NMD). Although previously thought of as a pathway that rids the cell of non-functional mRNAs arising from mutations and processing errors, new research suggests a more general...

  10. Skeletal muscle microRNA and messenger RNA profiling in cofilin-2 deficient mice reveals cell cycle dysregulation hindering muscle regeneration.

    Sarah U Morton

    Full Text Available Congenital myopathies are rare skeletal muscle diseases presenting in early age with hypotonia and weakness often linked to a genetic defect. Mutations in the gene for cofilin-2 (CFL2 have been identified in several families as a cause of congenital myopathy with nemaline bodies and cores. Here we explore the global messenger and microRNA expression patterns in quadriceps muscle samples from cofillin-2-null mice and compare them with sibling-matched wild-type mice to determine the molecular pathways and mechanisms involved. Cell cycle processes are markedly dysregulated, with altered expression of genes involved in mitotic spindle formation, and evidence of loss of cell cycle checkpoint regulation. Importantly, alterations in cell cycle, apoptosis and proliferation pathways are present in both mRNA and miRNA expression patterns. Specifically, p21 transcript levels were increased, and the expression of p21 targets, such as cyclin D and cyclin E, was decreased. We therefore hypothesize that deficiency of cofilin-2 is associated with interruption of the cell cycle at several checkpoints, hindering muscle regeneration. Identification of these pathways is an important step towards developing appropriate therapies against various congenital myopathies.

  11. RNA decay by messenger RNA interferases

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

    2008-01-01

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

  12. Processivity and coupling in messenger RNA transcription.

    Stuart Aitken

    2010-01-01

    Full Text Available The complexity of messenger RNA processing is now being uncovered by experimental techniques that are capable of detecting individual copies of mRNA in cells, and by quantitative real-time observations that reveal the kinetics. This processing is commonly modelled by permitting mRNA to be transcribed only when the promoter is in the on state. In this simple on/off model, the many processes involved in active transcription are represented by a single reaction. These processes include elongation, which has a minimum time for completion and processing that is not captured in the model.In this paper, we explore the impact on the mRNA distribution of representing the elongation process in more detail. Consideration of the mechanisms of elongation leads to two alternative models of the coupling between the elongating polymerase and the state of the promoter: Processivity allows polymerases to complete elongation irrespective of the promoter state, whereas coupling requires the promoter to be active to produce a full-length transcript. We demonstrate that these alternatives have a significant impact on the predicted distributions. Models are simulated by the Gillespie algorithm, and the third and fourth moments of the resulting distribution are computed in order to characterise the length of the tail, and sharpness of the peak. By this methodology, we show that the moments provide a concise summary of the distribution, showing statistically-significant differences across much of the feasible parameter range.We conclude that processivity is not fully consistent with the on/off model unless the probability of successfully completing elongation is low--as has been observed. The results also suggest that some form of coupling between the promoter and a rate-limiting step in transcription may explain the cell's inability to maintain high mRNA levels at low noise--a prediction of the on/off model that has no supporting evidence.

  13. Effects of ionizing radiation and partial hepatectomy on messenger RNA synthesis

    Abdel-Halim, M.N.

    1979-01-01

    Newly synthesized messenger RNA, as measured by a 40 min uptake of the radioactive precursor (6- 14 C) orotic acid, was studied in the regenerating livers of non-irradiated and gamma-irradiated (1800 rad) adrenal-intact and adrenalectomized rats 24 and 48 hours after partial hepatectomy. Two groups of rats, one with and one without adrenal glands were each divided into four subgroups: (1) control rats, (2) irradiated rats, (3) partially hepatectomized rats and (4) irradiated, partially hepatectomized rats. The radioactive profile of polyribosome formation and distribution was determined by sucrose density gradient centrifugation (10 to 40 per cent). The result of this study indicates that ionizing radiation decreases the synthesis of newly formed messenger RNA in regenerating livers of adrenal-intact rats. However, adrenalectomy largely abolished that inhibition. These data suggest that the decrease in messenger RNA synthesis may be explained by the disturbance of adrenal hormones induced by partial hepatectomy and ionizing radiation. (author)

  14. Bifurcations in the interplay of messenger RNA, protein and nonprotein coding RNA

    Zhdanov, Vladimir P

    2008-01-01

    The interplay of messenger RNA (mRNA), protein, produced via translation of this RNA, and nonprotein coding RNA (ncRNA) may include regulation of the ncRNA production by protein and (i) ncRNA-protein association resulting in suppression of the protein regulatory activity or (ii) ncRNA-mRNA association resulting in degradation of the miRNA-mRNA complex. The kinetic models describing these two scenarios are found to predict bistability provided that protein suppresses the ncRNA formation

  15. Tissue distribution of human acetylcholinesterase and butyrylcholinesterase messenger RNA

    Jbilo, O.; Barteles, C.F.; Chatonnet, A.; Toutant, J.P.; Lockridge, O.

    1994-12-31

    Tissue distribution of human acetyicholinesterase and butyryicholinesterase messenger RNA. 1 Cholinesterase inhibitors occur naturally in the calabar bean (eserine), green potatoes (solanine), insect-resistant crab apples, the coca plant (cocaine) and snake venom (fasciculin). There are also synthetic cholinesterase inhibitors, for example man-made insecticides. These inhibitors inactivate acetyicholinesterase and butyrylcholinesterase as well as other targets. From a study of the tissue distribution of acetylcholinesterase and butyrylcholinesterase mRNA by Northern blot analysis, we have found the highest levels of butyrylcholinesterase mRNA in the liver and lungs, tissues known as the principal detoxication sites of the human body. These results indicate that butyrylcholinesterase may be a first line of defense against poisons that are eaten or inhaled.

  16. Characterization of long noncoding RNA and messenger RNA signatures in melanoma tumorigenesis and metastasis.

    Siqi Wang

    Full Text Available The incidence of melanoma, the most aggressive and life-threatening form of skin cancer, has significantly risen over recent decades. Therefore, it is essential to identify the mechanisms that underlie melanoma tumorigenesis and metastasis and to explore novel and effective melanoma treatment strategies. Accumulating evidence s uggests that aberrantly expressed long noncoding RNAs (lncRNAs have vital functions in multiple cancers. However, lncRNA functions in melanoma tumorigenesis and metastasis remain unclear. In this study, we investigated lncRNA and messenger RNA (mRNA expression profiles in primary melanomas, metastatic melanomas and normal skin samples from the Gene Expression Omnibus database. We used GSE15605 as the training set (n = 74 and GSE7553 as the validation set (n = 58. In three comparisons (primary melanoma versus normal skin, metastatic melanoma versus normal skin, and metastatic melanoma versus primary melanoma, 178, 295 and 48 lncRNAs and 847, 1758, and 295 mRNAs were aberrantly expressed, respectively. We performed Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses to examine the differentially expressed mRNAs, and potential core lncRNAs were predicted by lncRNA-mRNA co-expression networks. Based on our results, 15 lncRNAs and 144 mRNAs were significantly associated with melanoma tumorigenesis and metastasis. A subsequent analysis suggested a critical role for a five-lncRNA signature during melanoma tumorigenesis and metastasis. Low expression of U47924.27 was significantly associated with decreased survival of patients with melanoma. To the best of our knowledge, this study is the first to explore the expression patterns of lncRNAs and mRNAs during melanoma tumorigenesis and metastasis by re-annotating microarray data from the Gene Expression Omnibus (GEO microarray dataset. These findings reveal potential roles for lncRNAs during melanoma tumorigenesis and metastasis and provide a rich candidate

  17. Shielding the messenger (RNA): microRNA-based anticancer therapies

    Sotillo, Elena; Thomas-Tikhonenko, Andrei

    2011-01-01

    It has been a decade since scientists realized that microRNAs (miRNAs) are not an oddity invented by worms to regulate gene expression at post-transcriptional levels. Rather, many of these 21–22-nucleotide-short RNAs exist in invertebrates and vertebrates alike and some of them are in fact highly conserved. miRNAs are now recognized as an important class of non-coding small RNAs that inhibit gene expression by targeting mRNA stability and translation. In the last ten years, our knowledge of the miRNAs world was expanding at vertiginous speed, propelled by the development of computational engines for miRNA identification and target prediction, biochemical tools and techniques to modulate miRNA activity, and last but not least, the emergence of miRNA-centric animal models. One important conclusion that has emerged from this effort is that many microRNAs and their cognate targets are strongly implicated in cancer, either as oncogenes or tumor and metastasis suppressors. In this review we will discuss the diverse role that miRNAs play in cancer initiation and progression and also the tools with which miRNA expression could be corrected in vivo. While the idea of targeting microRNAs towards therapeutic ends is getting considerable traction, basic, translational, and clinical research done in the next few years will tell whether this promise is well-founded. PMID:21514318

  18. Alterations in messenger RNA and small nuclear RNA metabolism resulting from fluorouracil incorporation

    Armstrong, R.D.; Cadman, E.C.

    1985-01-01

    Studies were completed to examine the effect of 5-fluorouracil (FUra) incorporation on messenger RNA (mRNA) and small molecular weight nuclear RNA (SnRNA) metabolism. Studies of mRNA were completed using cDNA-mRNA hybridization methods to specifically examine dihydrofolate reductase (DHFR) mRNA. C 3 -L5178Y murine leukemia cells which are gene-amplified for DHFR, were exposed to FUra for 6, 12 or 24 hr, and the nuclear and cytoplasmic levels of DHFR-mRNA determined by hybridization with 32 P-DHFR-cDNA. FUra produced a dose-dependent increase in nuclear DHFR-mRNA levels, while total cytoplasmic DHFR-mRNA levels appeared to be unchanged. To examine only mRNA synthesized during FUra exposure, cells were also treated concurrently with [ 3 H] cytidine, and the [ 3 H]mRNA-cDNA hybrids measured following S 1 -nuclease treatment. FUra produced a concentration-dependent increase in nascent nuclear DHFR-mRNA levels, and a decrease in nascent cytoplasmic DHFR-mRNAs levels. These results suggest that FUra produces either an inhibition of mRNA processing, or an inhibition of nuclear-cytoplasmic transport. Preliminary experiments to examine ATP-dependent mRNA transport were completed with isolated nuclei from cells treated with FUra for 1 or 24 hr and then pulse-labeled for 1 hr with [ 3 H] cytidine. The results demonstrate a FUra-concentration and time-dependent inhibition of ATP-mediated mRNA efflux

  19. ESTRADIOL-INDUCED SYNTHESIS OF VITELLOGENIN .3. ISOLATION AND CHARACTERIZATION OF VITELLOGENIN MESSENGER-RNA FROM AVIAN LIVER

    AB, G.; Roskam, W. G.; Dijkstra, J.; Mulder, J.; Willems, M.; van der Ende, A.; Gruber, M.

    1976-01-01

    The messenger RNA of the hormone-induced protein vitellogenin was isolated from the liver of estrogen-treated roosters. Starting from total polysomal RNA, the vitellogenin messenger was purified 67-fold by oligo (dT)-cellulose chromatography and sizing on a sucrose gradient. The messenger was

  20. A Contemporary, Laboratory-Intensive Course on Messenger RNA Transcription and Processing

    Carson, Sue; Miller, Heather

    2012-01-01

    Messenger ribonucleic acid (mRNA) plays a pivotal role in the central dogma of molecular biology. Importantly, molecular events occurring during and after mRNA synthesis have the potential to create multiple proteins from one gene, leading to some of the remarkable protein diversity that genomes hold. The North Carolina State University…

  1. Guardian of Genetic Messenger-RNA-Binding Proteins

    Antje Anji

    2016-01-01

    Full Text Available RNA in cells is always associated with RNA-binding proteins that regulate all aspects of RNA metabolism including RNA splicing, export from the nucleus, RNA localization, mRNA turn-over as well as translation. Given their diverse functions, cells express a variety of RNA-binding proteins, which play important roles in the pathologies of a number of diseases. In this review we focus on the effect of alcohol on different RNA-binding proteins and their possible contribution to alcohol-related disorders, and discuss the role of these proteins in the development of neurological diseases and cancer. We further discuss the conventional methods and newer techniques that are employed to identify RNA-binding proteins.

  2. Effects of insulin on messenger RNA activities in rat liver

    Hill, R.E.; Lee, K.L.; Kenney, F.T.

    1981-01-01

    Liver poly(A) RNA, isolated from adrenalectomized rats after insulin treatment, was translated in a nuclease-treated lysate of rabbit reticulocytes and quantitated for both total activity and the capacity to synthesize the insulin-inducible enzyme tyrosine amino-transferase. Analysis of the translated products from poly(A) RNA isolated 1 h after insulin treatment showed a 2.7-fold increase in activity of tyrosine aminotransferase mRNA. During the same interval, the capacity of poly(A) RNA to direct the synthesis of total protein in lysates also changed, showing a 30 to 40% increase in translational activity/unit of RNA. Increased translatability was apparent in all fractions of poly(A) RNA separated by centrifugation on sucrose gradients. Insulin thus appears to mediated a generalized changed in mRNAs leading to increased capacity for translation; induction of tyrosine aminotransferase may reflect unusual sensitivity to this effect of the hormone

  3. Localization of calcium-binding proteins and GABA transporter (GAT-1) messenger RNA in the human subthalamic nucleus

    Augood, S.J.; Waldvogel, H.J.; Muenkle, M.C.; Faull, R.L.M.; Emson, P.C.

    1999-01-01

    The distribution of messenger RNA encoding the human GAT-1 (a high-affinity GABA transporter) was investigated in the subthalamic nucleus of 10 neurologically normal human post mortem cases. Further, the distribution of messenger RNA and protein encoding the three neuronally expressed calcium-binding proteins (calbindin D28k, parvalbumin and calretinin) was similarly investigated using in situ hybridization and immunohistochemical techniques. Cellular sites of calbindin D28k, parvalbumin, calretinin and GAT-1 messenger RNA expression were localized using human-specific oligonucleotide probes radiolabelled with [ 35 S]dATP. Sites of protein localization were visualized using specific anti-calbindin D28k, anti-parvalbumin and anti-calretinin antisera. Examination of emulsion-coated tissue sections processed for in situ hybridization revealed an intense signal for GAT-1 messenger RNA within the human subthalamic nucleus, indeed the majority of Methylene Blue-counterstained cells were enriched in this transcript. Further, a marked heterogeneity was noted with regard to the expression of the messenger RNA's encoding the three calcium-binding proteins; this elliptical nucleus was highly enriched in parvalbumin messenger RNA-positive neurons and calretinin mRNA-positive cells but not calbindin messenger RNA-positive cells. Indeed, only an occasional calbindin messenger RNA-positive cell was detected within the mediolateral extent of the nucleus. In marked contrast, numerous parvalbumin messenger RNA-positive cells and calretinin messenger RNA-positive cells were detected and they were topographically distributed; parvalbumin messenger RNA-positive cells were highly enriched in the dorsal subthalamic nucleus extending mediolaterally; calretinin messenger RNA-positive cells were more enriched ventrally although some degree of overlap was apparent. Computer-assisted analysis of the average cross-sectional somatic area of parvalbumin, calretinin and GAT-1 messenger RNA

  4. The synthesis of polyadenylated messenger RNA in herpes simplex type I virus infected BHK cells.

    Harris, T J; Wildy, P

    1975-09-01

    The pattern of polyadenylated messenger RNA (mRNA) synthesis in BHK cell monolayers, infected under defined conditions with herpes simplex type I virus has been investigated by polyacrylamide gel electrophoresis or pulse-labelled RNA isolated by oligo dT-cellulose chromatography. Two classes of mRNA molecules were synthesized in infected cells; these were not detected in uninfected cells. The rate of synthesis of the larger, 18 to 30S RNA class reached a maximum soon after injection and then declined, whereas the rate of synthesis of the 7 to 11 S RNA class did not reach a maximum until much later and did not decline. In the presence of cytosine arabinoside, the rate of mRNA synthesis in infected cells was reduced but the electrophoretic pattern remained the same.

  5. A thermostable messenger RNA based vaccine against rabies.

    Stitz, Lothar; Vogel, Annette; Schnee, Margit; Voss, Daniel; Rauch, Susanne; Mutzke, Thorsten; Ketterer, Thomas; Kramps, Thomas; Petsch, Benjamin

    2017-12-01

    Although effective rabies virus vaccines have been existing for decades, each year, rabies virus infections still cause around 50.000 fatalities worldwide. Most of these cases occur in developing countries, where these vaccines are not available. The reasons for this are the prohibitive high costs of cell culture or egg grown rabies virus vaccines and the lack of a functional cold chain in many regions in which rabies virus is endemic. Here, we describe the excellent temperature resistance of a non-replicating mRNA based rabies virus vaccine encoding the rabies virus glycoprotein (RABV-G). Prolonged storage of the vaccine from -80°C to up to +70°C for several months did not impact the protective capacity of the mRNA vaccine. Efficacy after storage was demonstrated by the induction of rabies specific virus neutralizing antibodies and protection in mice against lethal rabies infection. Moreover, storing the vaccine at oscillating temperatures between +4° and +56°C for 20 cycles in order to simulate interruptions of the cold chain during vaccine transport, did not affect the vaccine's immunogenicity and protective characteristics, indicating that maintenance of a cold chain is not essential for this vaccine.

  6. Postage for the messenger: Designating routes for Nuclear mRNA Export

    Natalizio, Barbara J.; Wente, Susan R.

    2013-01-01

    Transcription of messenger(m) RNA occurs in the nucleus, making the translocation of mRNA across the nuclear envelope (NE) boundary a critical determinant of proper gene expression and cell survival. A major mRNA export route occurs via the NXF1-dependent pathway through the nuclear pore complexes (NPCs) embedded in the NE. However, recent findings have discovered new evidence supporting the existence of multiple mechanisms for crossing the NE, including both NPC-mediated and NE budding-mediated pathways. An analysis of the trans-acting factors and cis components that define these pathways reveals shared elements as well as mechanistic differences. We review here the current understanding of the mechanisms that characterize each pathway and highlight the determinants that influence mRNA transport fate. PMID:23583578

  7. DNA Methylation of MMP9 Is Associated with High Levels of MMP-9 Messenger RNA in Periapical Inflammatory Lesions.

    Campos, Kelma; Gomes, Carolina Cavalieri; Farias, Lucyana Conceição; Silva, Renato Menezes; Letra, Ariadne; Gomez, Ricardo Santiago

    2016-01-01

    Matrix metalloproteinases (MMPs) are the major class of enzymes responsible for degradation of extracellular matrix components and participate in the pathogenesis of periapical inflammatory lesions. MMP expression may be regulated by DNA methylation. The purpose of the present investigation was to analyze the expression of MMP2 and MMP9 in periapical granulomas and radicular cysts and to test the hypothesis that, in these lesions, their transcription may be modulated by DNA methylation. Methylation-specific polymerase chain reaction was used to evaluate the DNA methylation pattern of the MMP2 gene in 13 fresh periapical granuloma samples and 10 fresh radicular cyst samples. Restriction enzyme digestion was used to assess methylation of the MMP9 gene in 12 fresh periapical granuloma samples and 10 fresh radicular cyst samples. MMP2 and MMP9 messenger RNA transcript levels were measured by quantitative real-time polymerase chain reaction. All periapical lesions and healthy mucosa samples showed partial methylation of the MMP2 gene; however, periapical granulomas showed higher MMP2 mRNA expression levels than healthy mucosa (P = .014). A higher unmethylated profile of the MMP9 gene was found in periapical granulomas and radicular cysts compared with healthy mucosa. In addition, higher MMP9 mRNA expression was observed in the periapical lesions compared with healthy tissues. The present study suggests that the unmethylated status of the MMP9 gene in periapical lesions may explain the observed up-regulation of messenger RNA transcription in these lesions. Copyright © 2016 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

  8. Messenger RNA biomarker signatures for forensic body fluid identification revealed by targeted RNA sequencing.

    Hanson, E; Ingold, S; Haas, C; Ballantyne, J

    2018-05-01

    The recovery of a DNA profile from the perpetrator or victim in criminal investigations can provide valuable 'source level' information for investigators. However, a DNA profile does not reveal the circumstances by which biological material was transferred. Some contextual information can be obtained by a determination of the tissue or fluid source of origin of the biological material as it is potentially indicative of some behavioral activity on behalf of the individual that resulted in its transfer from the body. Here, we sought to improve upon established RNA based methods for body fluid identification by developing a targeted multiplexed next generation mRNA sequencing assay comprising a panel of approximately equal sized gene amplicons. The multiplexed biomarker panel includes several highly specific gene targets with the necessary specificity to definitively identify most forensically relevant biological fluids and tissues (blood, semen, saliva, vaginal secretions, menstrual blood and skin). In developing the biomarker panel we evaluated 66 gene targets, with a progressive iteration of testing target combinations that exhibited optimal sensitivity and specificity using a training set of forensically relevant body fluid samples. The current assay comprises 33 targets: 6 blood, 6 semen, 6 saliva, 4 vaginal secretions, 5 menstrual blood and 6 skin markers. We demonstrate the sensitivity and specificity of the assay and the ability to identify body fluids in single source and admixed stains. A 16 sample blind test was carried out by one lab with samples provided by the other participating lab. The blinded lab correctly identified the body fluids present in 15 of the samples with the major component identified in the 16th. Various classification methods are being investigated to permit inference of the body fluid/tissue in dried physiological stains. These include the percentage of reads in a sample that are due to each of the 6 tissues/body fluids tested and

  9. Genetic recombination in plant-infecting messenger-sense RNA viruses: overview and research perspectives

    Jozef Julian Bujarski

    2013-03-01

    Full Text Available RNA recombination is one of the driving forces of genetic variability in (+-strand RNA viruses. Various types of RNA-RNA crossovers were described including crosses between the same or different viral RNAs or between viral and cellular RNAs. Likewise, a variety of molecular mechanisms are known to support RNA recombination, such as replicative events (based on internal or end-to-end replicase switchings along with nonreplicative joining among RNA fragments of viral and/or cellular origin. Such mechanisms as RNA decay or RNA interference are responsible for RNA fragmentation and trans-esterification reactions which are likely accountable for ligation of RNA fragments. Numerous host factors were found to affect the profiles of viral RNA recombinants and significant differences in recombination frequency were observed among various RNA viruses. Comparative analyses of viral sequences allowed for the development of evolutionary models in order to explain adaptive phenotypic changes and co-evolving sites. Many questions remain to be answered by forthcoming RNA recombination research. (i How various factors modulate the ability of viral replicase to switch templates, (ii What is the intracellular location of RNA-RNA template switchings, (iii Mechanisms and factors responsible for non-replicative RNA recombination, (iv Mechanisms of integration of RNA viral sequences with cellular genomic DNA, and (v What is the role of RNA splicing and ribozyme activity. From an evolutionary stand point, it is not known how RNA viruses parasitize new host species via recombination, nor is it obvious what the contribution of RNA recombination is among other RNA modification pathways. We do not understand why the frequency of RNA recombination varies so much among RNA viruses and the status of RNA recombination as a form of sex is not well documented.

  10. Genetic recombination in plant-infecting messenger-sense RNA viruses: overview and research perspectives.

    Bujarski, Jozef J

    2013-01-01

    RNA recombination is one of the driving forces of genetic variability in (+)-strand RNA viruses. Various types of RNA-RNA crossovers were described including crosses between the same or different viral RNAs or between viral and cellular RNAs. Likewise, a variety of molecular mechanisms are known to support RNA recombination, such as replicative events (based on internal or end-to-end replicase switchings) along with non-replicative joining among RNA fragments of viral and/or cellular origin. Such mechanisms as RNA decay or RNA interference are responsible for RNA fragmentation and trans-esterification reactions which are likely accountable for ligation of RNA fragments. Numerous host factors were found to affect the profiles of viral RNA recombinants and significant differences in recombination frequency were observed among various RNA viruses. Comparative analyses of viral sequences allowed for the development of evolutionary models in order to explain adaptive phenotypic changes and co-evolving sites. Many questions remain to be answered by forthcoming RNA recombination research. (1) How various factors modulate the ability of viral replicase to switch templates, (2) What is the intracellular location of RNA-RNA template switchings, (3) Mechanisms and factors responsible for non-replicative RNA recombination, (4) Mechanisms of integration of RNA viral sequences with cellular genomic DNA, and (5) What is the role of RNA splicing and ribozyme activity. From an evolutionary stand point, it is not known how RNA viruses parasitize new host species via recombination, nor is it obvious what the contribution of RNA recombination is among other RNA modification pathways. We do not understand why the frequency of RNA recombination varies so much among RNA viruses and the status of RNA recombination as a form of sex is not well documented.

  11. Antigen-presenting cells transfected with Hsp65 messenger RNA fail to treat experimental tuberculosis

    Rocha, C.D.; Trombone, A.P.F.; Lorenzi, J.C.C.; Almeida, L.P.; Gembre, A.F.; Padilha, E. [Departamento de Bioquímica e Imunologia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP (Brazil); Ramos, S.G. [Departamento de Patologia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP (Brazil); Silva, C.L.; Coelho-Castelo, A.A.M. [Departamento de Bioquímica e Imunologia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP (Brazil)

    2012-09-21

    In the last several years, the use of dendritic cells has been studied as a therapeutic strategy against tumors. Dendritic cells can be pulsed with peptides or full-length protein, or they can be transfected with DNA or RNA. However, comparative studies suggest that transfecting dendritic cells with messenger RNA (mRNA) is superior to other antigen-loading techniques in generating immunocompetent dendritic cells. In the present study, we evaluated a new therapeutic strategy to fight tuberculosis using dendritic cells and macrophages transfected with Hsp65 mRNA. First, we demonstrated that antigen-presenting cells transfected with Hsp65 mRNA exhibit a higher level of expression of co-stimulatory molecules, suggesting that Hsp65 mRNA has immunostimulatory properties. We also demonstrated that spleen cells obtained from animals immunized with mock and Hsp65 mRNA-transfected dendritic cells were able to generate a mixed Th1/Th2 response with production not only of IFN-γ but also of IL-5 and IL-10. In contrast, cells recovered from mice immunized with Hsp65 mRNA-transfected macrophages were able to produce only IL-5. When mice were infected with Mycobacterium tuberculosis and treated with antigen-presenting cells transfected with Hsp65 mRNA (therapeutic immunization), we did not detect any decrease in the lung bacterial load or any preservation of the lung parenchyma, indicating the inability of transfected cells to confer curative effects against tuberculosis. In spite of the lack of therapeutic efficacy, this study reports for the first time the use of antigen-presenting cells transfected with mRNA in experimental tuberculosis.

  12. The ribosome uses two active mechanisms to unwind messenger RNA during translation.

    Qu, Xiaohui; Wen, Jin-Der; Lancaster, Laura; Noller, Harry F; Bustamante, Carlos; Tinoco, Ignacio

    2011-07-06

    The ribosome translates the genetic information encoded in messenger RNA into protein. Folded structures in the coding region of an mRNA represent a kinetic barrier that lowers the peptide elongation rate, as the ribosome must disrupt structures it encounters in the mRNA at its entry site to allow translocation to the next codon. Such structures are exploited by the cell to create diverse strategies for translation regulation, such as programmed frameshifting, the modulation of protein expression levels, ribosome localization and co-translational protein folding. Although strand separation activity is inherent to the ribosome, requiring no exogenous helicases, its mechanism is still unknown. Here, using a single-molecule optical tweezers assay on mRNA hairpins, we find that the translation rate of identical codons at the decoding centre is greatly influenced by the GC content of folded structures at the mRNA entry site. Furthermore, force applied to the ends of the hairpin to favour its unfolding significantly speeds translation. Quantitative analysis of the force dependence of its helicase activity reveals that the ribosome, unlike previously studied helicases, uses two distinct active mechanisms to unwind mRNA structure: it destabilizes the helical junction at the mRNA entry site by biasing its thermal fluctuations towards the open state, increasing the probability of the ribosome translocating unhindered; and it mechanically pulls apart the mRNA single strands of the closed junction during the conformational changes that accompany ribosome translocation. The second of these mechanisms ensures a minimal basal rate of translation in the cell; specialized, mechanically stable structures are required to stall the ribosome temporarily. Our results establish a quantitative mechanical basis for understanding the mechanism of regulation of the elongation rate of translation by structured mRNAs. ©2011 Macmillan Publishers Limited. All rights reserved

  13. Phenotypic characterization of neurotensin messenger RNA-expressing cells in the neuroleptic-treated rat striatum: a detailed cellular co-expression study

    Emson, P.C.; Westmore, K.; Augood, S.J.

    1996-01-01

    The chemical phenotype of proneurotensin messenger RNA-expressing cells was determined in the acute haloperidol-treated rat striatum using a combination of [ 35 S]-labelled and alkaline phosphatase-labelled oligonucleotides. Cellular sites of proneurotensin messenger RNA expression were visualized simultaneously on tissue sections processed to reveal cellular sites of preproenkephalin A messenger RNA or the dopamine and adenylate cyclase phosphoprotein-32, messenger RNA. The cellular co-expression of preproenkepahlin A and preprotachykinin messenger RNA was also examined within forebrain structures. Cellular sites of preproenkephalin A and dopamine and adenylate cyclase phosphoprotein-32 messenger RNAs were visualized using alkaline phosphatase-labelled oligonucleotides whilst sites of preprotachykinin and proneurotensin messenger RNA expression were detected using [ 35 S]-labelled oligos. Cellular sites of enkephalin and dopamine and adenylate cyclase phosphoprotein-32 gene expression were identified microscopically by the concentration of purple alkaline phosphatase reaction product within the cell cytoplasm, whereas sites of substance P and proneurotensin gene expression were identified by the dense clustering of silver grains overlying cells.An intense hybridization signal was detected for all three neuropeptide messenger RNAs in the striatum, the nucleus accumbens and septum. Dopamine and adenylate cyclase phosphoprotein-32 messenger RNA was detected within the neostriatum but not within the septum. In all forebrain regions examined, with the exception of the islands of Cajella, the cellular expression of enkephalin messenger RNA and substance P messenger RNA was discordant; the two neuropeptide messenger RNAs were detected essentially in different cells, although in the striatum and nucleus accumbens occasional isolated cells were detected which contained both hybridization signals; dense clusters of silver grains overlay alkaline phosphatase-positive cells

  14. Phenotypic characterization of neurotensin messenger RNA-expressing cells in the neuroleptic-treated rat striatum: a detailed cellular co-expression study

    Emson, P C; Westmore, K; Augood, S J [MRC Molecular Neuroscience Group, The Department of Neurobiology, The Babraham Institute, Babraham, Cambridge (United Kingdom)

    1996-12-11

    The chemical phenotype of proneurotensin messenger RNA-expressing cells was determined in the acute haloperidol-treated rat striatum using a combination of [{sup 35}S]-labelled and alkaline phosphatase-labelled oligonucleotides. Cellular sites of proneurotensin messenger RNA expression were visualized simultaneously on tissue sections processed to reveal cellular sites of preproenkephalin A messenger RNA or the dopamine and adenylate cyclase phosphoprotein-32, messenger RNA. The cellular co-expression of preproenkepahlin A and preprotachykinin messenger RNA was also examined within forebrain structures. Cellular sites of preproenkephalin A and dopamine and adenylate cyclase phosphoprotein-32 messenger RNAs were visualized using alkaline phosphatase-labelled oligonucleotides whilst sites of preprotachykinin and proneurotensin messenger RNA expression were detected using [{sup 35}S]-labelled oligos. Cellular sites of enkephalin and dopamine and adenylate cyclase phosphoprotein-32 gene expression were identified microscopically by the concentration of purple alkaline phosphatase reaction product within the cell cytoplasm, whereas sites of substance P and proneurotensin gene expression were identified by the dense clustering of silver grains overlying cells.An intense hybridization signal was detected for all three neuropeptide messenger RNAs in the striatum, the nucleus accumbens and septum. Dopamine and adenylate cyclase phosphoprotein-32 messenger RNA was detected within the neostriatum but not within the septum. In all forebrain regions examined, with the exception of the islands of Cajella, the cellular expression of enkephalin messenger RNA and substance P messenger RNA was discordant; the two neuropeptide messenger RNAs were detected essentially in different cells, although in the striatum and nucleus accumbens occasional isolated cells were detected which contained both hybridization signals; dense clusters of silver grains overlay alkaline phosphatase

  15. Early changes of placenta-derived messenger RNA in maternal plasma – potential value for preeclampsia prediction?

    Surugiu Sebastian

    2015-12-01

    Full Text Available Objective: the pourpose of the study was to determine if there are any differences between placenta derived plasmatic levels of messenger RNA in normal and future preeclamptic pregnancies and if these placental transcripts can predict preeclampsia long before clinical onset

  16. Fox-2 protein regulates the alternative splicing of scleroderma-associated lysyl hydroxylase 2 messenger RNA.

    Seth, Puneet; Yeowell, Heather N

    2010-04-01

    Scleroderma (systemic sclerosis [SSc]) is a complex connective tissue disorder characterized by hardening and thickening of the skin. One hallmark of scleroderma is excessive accumulation of collagen accompanied by increased levels of pyridinoline collagen crosslinks derived from hydroxylysine residues in the collagen telopeptide domains. Lysyl hydroxylase 2 (LH2), an important alternatively spliced enzyme in collagen biosynthesis, acts as a collagen telopeptide hydroxylase. Changes in the pattern of LH2 alternative splicing, favoring increased inclusion of the alternatively spliced LH2 exon 13A, thereby increasing the levels of the long transcript of LH2 (LH2[long]), are linked to scleroderma disease. This study was undertaken to examine the role played by RNA binding protein Fox-2 in regulating exon 13A inclusion, which leads to the generation of scleroderma-associated LH2(long) messenger RNA (mRNA). Phylogenetic sequence analysis of introns flanking exon 13A was performed. A tetracycline-inducible system in T-Rex 293 cells was used to induce Fox-2 protein, and endogenous LH2(long) mRNA was determined by reverse transcriptase-polymerase chain reaction. An LH2 minigene was designed, validated, and used in Fox-2 overexpression and mutagenesis experiments. Knockdown of Fox-2 was performed in mouse embryonic fibroblasts and in fibroblasts from SSc patients. Overexpression of Fox-2 enhanced the inclusion of exon 13A and increased the generation of LH2(long) mRNA, whereas knockdown of Fox-2 decreased LH2(long) transcripts. Mutational analysis of an LH2 minigene demonstrated that 2 of the 4 Fox binding motifs flanking LH2 exon 13A are required for inclusion of exon 13A. In early passage fibroblasts derived from patients with scleroderma, the knockdown of Fox-2 protein significantly decreased the endogenous levels of LH2(long) mRNA. Our findings indicate that Fox-2 plays an integral role in the regulation of LH2 splicing. Knockdown of Fox-2 and other methods to decrease

  17. Combining miRNA and mRNA Expression Profiles in Wilms Tumor Subtypes

    Nicole Ludwig

    2016-03-01

    Full Text Available Wilms tumor (WT is the most common childhood renal cancer. Recent findings of mutations in microRNA (miRNA processing proteins suggest a pivotal role of miRNAs in WT genesis. We performed miRNA expression profiling of 36 WTs of different subtypes and four normal kidney tissues using microarrays. Additionally, we determined the gene expression profile of 28 of these tumors to identify potentially correlated target genes and affected pathways. We identified 85 miRNAs and 2107 messenger RNAs (mRNA differentially expressed in blastemal WT, and 266 miRNAs and 1267 mRNAs differentially expressed in regressive subtype. The hierarchical clustering of the samples, using either the miRNA or mRNA profile, showed the clear separation of WT from normal kidney samples, but the miRNA pattern yielded better separation of WT subtypes. A correlation analysis of the deregulated miRNA and mRNAs identified 13,026 miRNA/mRNA pairs with inversely correlated expression, of which 2844 are potential interactions of miRNA and their predicted mRNA targets. We found significant upregulation of miRNAs-183, -301a/b and -335 for the blastemal subtype, and miRNAs-181b, -223 and -630 for the regressive subtype. We found marked deregulation of miRNAs regulating epithelial to mesenchymal transition, especially in the blastemal subtype, and miRNAs influencing chemosensitivity, especially in regressive subtypes. Further research is needed to assess the influence of preoperative chemotherapy and tumor infiltrating lymphocytes on the miRNA and mRNA patterns in WT.

  18. Identification of messenger RNA of fetoplacental source in maternal plasma of women with normal pregnancies and pregnancies with intrauterine growth restriction.

    Ayala Ramírez, Paola; García Robles, Reggie; Rojas, Juan Diego; Bermúdez, Martha; Bernal, Jaime

    2012-07-01

    to quantify placenta-specific RNA in plasma of women carrying foetuses with intrauterine growth restriction and pregnant women with normal pregnancies. 8 pregnant women with foetuses with intrauterine growth restriction were studied as well as 18 women with uncomplicated pregnancies in the third pregnancy trimester. Total free RNA was quantified in maternal plasma by spectrophotometry and the gene expression of hPL (Human Placental Lactogen) at the messenger RNA level through technical Real Time-Chain Reaction Polymerase. plasma RNA of fetoplacental origin was successfully detected in 100% of pregnant women. There were no statistically significant differences between the values of total RNA extracted from plasma (p= 0.5975) nor in the messenger RNA expression of hPL gene (p= 0.5785) between cases and controls. messenger RNA of fetoplacental origin can be detected in maternal plasma during pregnancy.

  19. In vivo expression of ß-galactosidase by rat oviduct exposed to naked DNA or messenger RNA

    MARIANA RIOS

    2002-01-01

    Full Text Available Intra-oviductal administration of RNA obtained from oviducts of estradiol-treated rats resulted in accelerated egg transport (Ríos et al., 1997. It is probable that estradiol-induced messenger RNA (mRNA entered oviductal cells and was translated into the proteins involved in accelerated egg transport. In order to test this interpretation we deposited in vivo 50 µg of pure ß-galactosidase (ß-gal mRNA, 50 µg of pure DNA from the reporter gene ß-gal under SV40 promoter or the vehicle (control oviducts into the oviductal lumen of rats. Twenty four hours later the ß-gal activity was assayed in oviductal tissue homogenates using o-nitrophenyl-ß-D-galactopyranoside as a substrate. The administration of ß-gal mRNA and pSVBgal plasmid increased ß-gal activity by 71% and 142%, respectively, over the control oviducts. These results indicate that naked DNA and mRNA coding for ß-gal can enter oviductal cells and be translated into an active enzyme. They are consistent with the interpretation that embryo transport acceleration caused by the injection of estradiol-induced RNA in the oviduct involves translation of the injected mRNA

  20. Vanillin inhibits translation and induces messenger ribonucleoprotein (mRNP) granule formation in saccharomyces cerevisiae: application and validation of high-content, image-based profiling.

    Iwaki, Aya; Ohnuki, Shinsuke; Suga, Yohei; Izawa, Shingo; Ohya, Yoshikazu

    2013-01-01

    Vanillin, generated by acid hydrolysis of lignocellulose, acts as a potent inhibitor of the growth of the yeast Saccharomyces cerevisiae. Here, we investigated the cellular processes affected by vanillin using high-content, image-based profiling. Among 4,718 non-essential yeast deletion mutants, the morphology of those defective in the large ribosomal subunit showed significant similarity to that of vanillin-treated cells. The defects in these mutants were clustered in three domains of the ribosome: the mRNA tunnel entrance, exit and backbone required for small subunit attachment. To confirm that vanillin inhibited ribosomal function, we assessed polysome and messenger ribonucleoprotein granule formation after treatment with vanillin. Analysis of polysome profiles showed disassembly of the polysomes in the presence of vanillin. Processing bodies and stress granules, which are composed of non-translating mRNAs and various proteins, were formed after treatment with vanillin. These results suggest that vanillin represses translation in yeast cells.

  1. Translation affects YoeB and MazF messenger RNA interferase activities by different mechanisms

    Christensen-Dalsgaard, Mikkel; Gerdes, Kenn

    2008-01-01

    Prokaryotic toxin-antitoxin loci encode mRNA cleaving enzymes that inhibit translation. Two types are known: those that cleave mRNA codons at the ribosomal A site and those that cleave any RNA site specifically. RelE of Escherichia coli cleaves mRNA at the ribosomal A site in vivo and in vitro bu...

  2. Blockade of OX40/OX40 ligand to decrease cytokine messenger RNA expression in acute renal allograft rejection in vitro.

    Wang, Y-L; Li, G; Fu, Y-X; Wang, H; Shen, Z-Y

    2013-01-01

    The aim of this study was to investigate cytokine messenger RNA (mRNA) expression by peripheral blood mononuclear cells (PBMCs) from renal recipients experiencing acute rejection by blocking OX40-OX40L interactions with recombinant human OX40-Fc fusion protein (rhOX40Fc) in vitro. PBMCs were isolated from 20 recipients experiencing acute rejection episodes (rejection group) and 20 recipients with stable graft function (stable group). Levels of Th1 (interferon [IFN]-γ) and Th2 (interleukin [IL]-4) mRNA expressions by PBMCs were measured using real-time reverse transcriptase-polymerase chain reactions. IFN-γ mRNA expression levels were significantly higher in the rejection than the stable group (P rejection group, rhOX40Fc reduced significantly the expression of IFN-γ and IL-4 mRNA by anti-CD3-monoclonal antibody stimulated PBMCs (P type cytokines. Copyright © 2013 Elsevier Inc. All rights reserved.

  3. Maternal provision of non-sex-specific transformer messenger RNA in sex determination of the wasp Asobara tabida.

    Geuverink, E; Verhulst, E C; van Leussen, M; van de Zande, L; Beukeboom, L W

    2018-02-01

    In many insect species maternal provision of sex-specifically spliced messenger RNA (mRNA) of sex determination genes is an essential component of the sex determination mechanism. In haplodiploid Hymenoptera, maternal provision in combination with genomic imprinting has been shown for the parasitoid Nasonia vitripennis, known as maternal effect genomic imprinting sex determination (MEGISD). Here, we characterize the sex determination cascade of Asobara tabida, another hymenopteran parasitoid. We show the presence of the conserved sex determination genes doublesex (dsx), transformer (tra) and transformer-2 (tra2) orthologues in As. tabida. Of these, At-dsx and At-tra are sex-specifically spliced, indicating a conserved function in sex determination. At-tra and At-tra2 mRNA is maternally provided to embryos but, in contrast to most studied insects, As. tabida females transmit a non-sex-specific splice form of At-tra mRNA to the eggs. In this respect, As. tabida sex determination differs from the MEGISD mechanism. How the paternal genome can induce female development in the absence of maternal provision of sex-specifically spliced mRNA remains an open question. Our study reports a hitherto unknown variant of maternal effect sex determination and accentuates the diversity of insect sex determination mechanisms. © 2017 The Authors. Insect Molecular Biology published by John Wiley & Sons Ltd on behalf of Royal Entomological Society.

  4. Electrochemical Branched-DNA Assay for Polymerase Chain Reaction-Free Detection and Quantification of Oncogenes in Messenger RNA

    Lee, Ai Cheng; Dai, Ziyu; Chen, Baowei; Wu, Hong; Wang, Jun; Zhang, Aiguo; Zhang, Lurong; Lim, Tit-Meng; Lin, Yuehe

    2008-12-01

    We describe a novel electrochemical branched-DNA (bDNA) assay for polymerase chain reaction (PCR)-free detection and quantification of p185 BCR-ABL leukemia fusion transcript in the population of messenger RNA (mRNA) extracted from cell lines. The bDNA amplifier carrying high loading of alkaline phosphatase (ALP) tracers was used to amplify targets signal. The targets were captured on microplate well surfaces through cooperative sandwich hybridization prior to the labeling of bDNA. The activity of captured ALP was monitored by square-wave voltammetric (SWV) analysis of the electroactive enzymatic product in the presence of 1-napthyl-phosphate. The specificity and sensitivity of assay enabled direct detection of target transcript in as little as 4.6 ng mRNA without PCR amplification. In combination with the use of a well-quantified standard, the electrochemical bDNA assay was capable of direct use for a PCR-free quantitative analysis of target transcript in total mRNA population. The approach thus provides a simple, sensitive, accurate and quantitative tool alternate to the RQ-PCR for early disease diagnosis.

  5. Efficient ex vivo delivery of chemically modified messenger RNA using lipofection and magnetofection.

    Badieyan, Zohreh Sadat; Pasewald, Tamara; Mykhaylyk, Olga; Rudolph, Carsten; Plank, Christian

    2017-01-22

    Recently, chemically modified mRNA (cmRNA) therapeutics have been the subject of extensive application-oriented research in both academia and industry as a safer alternative for gene and recombinant protein therapies. However, the lack of an efficient delivery system hinders widespread application. Here we used ∼100-nm lipoplexes and magnetic lipoplexes that can protect cmRNA from RNases and efficiently deliver it into muscle and fat tissues as well as to the endothelium of the carotid artery. Establishing magnetofection for ex vivo cmRNA delivery for the first time, we suggest this method for potential enhanced and targeted delivery of cmRNA. This study introduces optimal cmRNA complexes with high ex vivo efficiency as good candidates for further in vivo cmRNA delivery. Copyright © 2016 Elsevier Inc. All rights reserved.

  6. Physical change in cytoplasmic messenger ribonucleoproteins in cells treated with inhibitors of mRNA transcription

    Dreyfuss, G.; Adam, S.A.; Choi, Y.D.

    1984-01-01

    Exposure of intact cells to UV light brings about cross-linking of polyadenylated mRNA to a set of cytoplasmic proteins which are in direct contact with the mRNA in vivo. Substantial amounts of an additional protein of molecular weight 38,000 become cross-linked to the mRNA when cells are treated with inhibitors of mRNA synthesis (actinomycin D, camptothecin, and 5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole) or after infection with vesicular stomatitis virus. Cordycepin, which inhibits polyadenylation but not mRNA synthesis, has no such effect. Inhibitors of protein synthesis and of rRNA synthesis are also without effect on 38K cross-linking to mRNA. The onset of the effect of inhibitors of mRNA synthesis on the UV cross-linkable interaction between mRNA and 38K is rapid and reaches a maximal level in less than 60 min, and it is completely and rapidly reversible. In cells treated with actinomycin D, the amount of 38K which becomes cross-linked to mRNA is proportional to the extent of inhibition of mRNA synthesis. The association of 38K with mRNA during transcriptional arrest does not require protein synthesis because simultaneous treatment with the protein synthesis inhibitor emetine does not interfere with it. The effectors which promote the interaction of 38K with mRNA do not affect the proteins which are in contact with polyadenylated heterogeneous nuclear RNA and do not markedly affect protein synthesis in the cell. The 38K protein can be isolated with the polyribosomal polyadenylated fraction from which it was purified, and monoclonal antibodies against it were prepared

  7. Transfer-messenger RNA controls the translation of cell-cycle and stress proteins in Streptomyces

    Barends, Sharief; Zehl, Martin; Bialek, Sylwia

    2010-01-01

    coelicolor, trans-translation has a specialized role in stress management. Analysis of proteins that were carboxy-terminally His(8)-tagged by a recombinant tmRNA identified only 10 targets, including the stress proteins: DnaK heat-shock protein 70, thiostrepton-induced protein A, universal stress protein A...... functionality for tmRNA, promoting the translation of the same mRNA it targets, at the expense of sacrificing the first nascent protein. In streptomycetes, tmRNA has evolved into a dedicated task force that ensures the instantaneous response to the exposure to stress....

  8. Covalent Strategies for Targeting Messenger and Non-Coding RNAs: An Updated Review on siRNA, miRNA and antimiR Conjugates

    Santiago Grijalvo

    2018-02-01

    Full Text Available Oligonucleotide-based therapy has become an alternative to classical approaches in the search of novel therapeutics involving gene-related diseases. Several mechanisms have been described in which demonstrate the pivotal role of oligonucleotide for modulating gene expression. Antisense oligonucleotides (ASOs and more recently siRNAs and miRNAs have made important contributions either in reducing aberrant protein levels by sequence-specific targeting messenger RNAs (mRNAs or restoring the anomalous levels of non-coding RNAs (ncRNAs that are involved in a good number of diseases including cancer. In addition to formulation approaches which have contributed to accelerate the presence of ASOs, siRNAs and miRNAs in clinical trials; the covalent linkage between non-viral vectors and nucleic acids has also added value and opened new perspectives to the development of promising nucleic acid-based therapeutics. This review article is mainly focused on the strategies carried out for covalently modifying siRNA and miRNA molecules. Examples involving cell-penetrating peptides (CPPs, carbohydrates, polymers, lipids and aptamers are discussed for the synthesis of siRNA conjugates whereas in the case of miRNA-based drugs, this review article makes special emphasis in using antagomiRs, locked nucleic acids (LNAs, peptide nucleic acids (PNAs as well as nanoparticles. The biomedical applications of siRNA and miRNA conjugates are also discussed.

  9. Appendix: a solution hybridization assay to detect radioactive globin messenger RNA nucleotide sequences

    Ross, J

    1976-09-15

    In view of the sensitivity and specificity of the solution hybridization assay for unlabeled globin mRNA a similar technique has been devised to detect radioactive globin mRNA sequences with unlabeled globin cDNA. Several properties of the hybridization reaction are presented since RNA kinetic experiments reported recently depend on the validity of this assay. Data on hybridization analysis of (/sup 3/H)RNA from mouse fetal liver or erythroleukemia cell cytoplasm are presented. These data indicate that the excess cDNA solution assay for radioactive globin mRNA detection is specific for globin mRNA sequences. It can be performed rapidly and is highly reproducible from experiment. It is at least 500-fold less sensitive than the assay for unlabeled globin mRNA, due to the RNAase backgrounds of 0.05 to 0.15 %. However, this limitation has not affected kinetic experiments with non-dividing fetal liver erythroid cells, which synthesize relatively large quantities of globin mRNA.

  10. Illuminating Messengers: An Update and Outlook on RNA Visualization in Bacteria

    Lieke A. van Gijtenbeek

    2017-06-01

    Full Text Available To be able to visualize the abundance and spatiotemporal features of RNAs in bacterial cells would permit obtaining a pivotal understanding of many mechanisms underlying bacterial cell biology. The first methods that allowed observing single mRNA molecules in individual cells were introduced by Bertrand et al. (1998 and Femino et al. (1998. Since then, a plethora of techniques to image RNA molecules with the aid of fluorescence microscopy has emerged. Many of these approaches are useful for the large eukaryotic cells but their adaptation to study RNA, specifically mRNA molecules, in bacterial cells progressed relatively slow. Here, an overview will be given of fluorescent techniques that can be used to reveal specific RNA molecules inside fixed and living single bacterial cells. It includes a critical evaluation of their caveats as well as potential solutions.

  11. Increased efficiency of exogenous messenger RNA translation in a Krebs ascites cell lysate.

    Metafora, S; Terada, M; Dow, L W; Marks, P A; Bank, A

    1972-05-01

    Addition of a 0.5 M KCl wash fraction from rabbit reticulocyte ribosomes causes a 3- to 10-fold increase in the extent of translation of natural mRNAs by Krebs-cell lysates. In the presence of the wash fraction, 1 pmol of rabbit or mouse 10S RNA directs the incorporation of 80 pmol of leucine into rabbit globin. The addition of human 10S RNA results in the synthesis of equal amounts of human alpha and beta chains, identified by column chromatography. The stimulation by the wash fraction is almost completely dependent on added mammalian tRNA. In contrast to the wash fraction from rabbit reticulocytes, the wash fraction isolated from Krebs-cell ribosomes is inhibitory to both endogenous and exogenous mRNA translation. The stimulation by the wash fraction from rabbit ribosomes is not specific for globin mRNAs, but also increases endogenous, phage Qbeta, and viral RNA-directed protein synthesis.

  12. Expression of μ, κ, and δ opioid receptor messenger RNA in the human CNS: a 33P in situ hybridization study

    Peckys, D.; Landwehrmeyer, G.B.

    1999-01-01

    The existence of at least three opioid receptor types, referred to as μ, κ, and δ, is well established. Complementary DNAs corresponding to the pharmacologically defined μ, κ, and δ opioid receptors have been isolated in various species including man. The expression patterns of opioid receptor transcripts in human brain has not been established with a cellular resolution, in part because of the low apparent abundance of opioid receptor messenger RNAs in human brain. To visualize opioid receptor messenger RNAs we developed a sensitive in situ hybridization histochemistry method using 33 P-labelled RNA probes. In the present study we report the regional and cellular expression of μ, κ, and δ opioid receptor messenger RNAs in selected areas of the human brain. Hybridization of the different opioid receptor probes resulted in distinct labelling patterns. For the μ and κ opioid receptor probes, the most intense regional signals were observed in striatum, thalamus, hypothalamus, cerebral cortex, cerebellum and certain brainstem areas as well as the spinal cord. The most intense signals for the δ opioid receptor probe were found in cerebral cortex. Expression of opioid receptor transcripts was restricted to subpopulations of neurons within most regions studied demonstrating differences in the cellular expression patterns of μ, κ, and δ opioid receptor messenger RNAs in numerous brain regions. The messenger RNA distribution patterns for each opioid receptor corresponded in general to the distribution of opioid receptor binding sites as visualized by receptor autoradiography. However, some mismatches, for instance between μ opioid receptor receptor binding and μ opioid receptor messenger RNA expression in the anterior striatum, were observed. A comparison of the distribution patterns of opioid receptor messenger RNAs in the human brain and that reported for the rat suggests a homologous expression pattern in many regions. However, in the human brain, κ

  13. Photoreversible UV-inactivation of messenger RNA in an insect embryo (Smittia spec., chironomidae, diptera)

    Jaeckle, H.; Kalthoff, K.

    1980-01-01

    Smittia embryos were UV-irradiated during intravitelline cleavage while nuclei are heavily shielded by yolk-rich cytoplasm and do not synthesize detectable amounts of RNA. Irradiation at 265, 285 and 295 nm wavelength caused biological inactivation, and pyrimidine dimer formation in maternal RNA. Marked effects on protein synthesis were also observed: (1) the overall rate of 35 S-methionine incorporation in vivo was reduced to less than half of the normal rate, (2) two dimensional gel electrophoresis revealed quantitative variations in the synthetic rate of some polypeptides and the appearance of new ones in UV-irradiated embryos, (3) translation of polyadenylated RNA from Smittia embryos in a cell-free system was inhibited by UV-irradiation in vivo, (4) the apparent degradation during early embryogenesis, of maternal polyadenylated RNA was retarded in UV-irradiated embryos. Exposure to light (400 nm) after UV caused partial photoreversal of all UV effects observed. This is the first data showing that animal mRNA, after UV-irradiation, can be photoreactivated in vivo. The results also strongly suggest that the photorepairable lesions consist of pyrimidine dimers generated in a photosensitized reaction. (author)

  14. Transforming growth factor-beta messenger RNA and protein in murine colitis

    Whiting, C V; Williams, A M; Claesson, Mogens Helweg

    2001-01-01

    Using a CD4+ T-cell-transplanted SCID mouse model of colitis, we have analyzed TGF-beta transcription and translation in advanced disease. By in situ hybridization, the epithelium of both control and inflamed tissues transcribed TGF-beta1 and TGF-beta3 mRNAs, but both were expressed significantly...... farther along the crypt axis in disease. Control lamina propria cells transcribed little TGF-beta1 or TGF-beta3 mRNA, but in inflamed tissues many cells expressed mRNA for both isoforms. No TGF-beta2 message was detected in either control or inflamed tissues. Immunohistochemistry for latent and active TGF...

  15. INDUCTION OF INTERLEUKIN-1-BETA MESSENGER-RNA AFTER FOCAL CEREBRAL-ISCHEMIA IN THE RAT

    BUTTINI, M; SAUTER, A; BODDEKE, HWGM

    The expression of interleukin-1beta (IL-1beta) mRNA in the brain in response to cerebral ischaemia in rats was examined using in situ hybridization histochemistry. Focal cerebral ischaemia was induced in spontaneously hypertensive rats by permanent occlusion of the left middle cerebral artery

  16. Rational design of avian metapneumovirus live attenuated vaccines by inhibiting viral messenger RNA cap methyltransferase

    Avian metapneumovirus (aMPV), also known as avian pneumovirus or turkey rhinotracheitis, is a non-segmented negative-sense RNA virus belonging to the family of Paramyxoviridae, the subfamily Pneumovirinae, and the genus Metapneumovirus. aMPV is the causative agent of respiratory tract infection and ...

  17. Fetuin and fetuin messenger RNA in granulosa cells of the rat ovary

    Høyer, Poul Erik; Terkelsen, O B; Grete Byskov, A

    2001-01-01

    during maturation of the oocyte. We demonstrated fetuin mRNA in the rat ovary by reverse transcriptase-polymerase chain reaction and localized it by in situ hybridization. Fetuin mRNA was present in all granulosa cells of growing and large follicles. Immunohistochemical analysis revealed that the fetuin...... protein was only present in some of the small, growing follicles. In large, healthy follicles, fetuin protein was confined to cumulus cells and granulosa cells bordering the antrum. Fetuin was present in atretic follicles, but the staining pattern differed from that of healthy follicles. The follicular...... antrum contained a substantial amount of fetuin, but whether granulosa cells secreted it or it originated in the ovarian blood supply could not be confirmed. We concluded that at least a portion of the fetuin is produced by granulosa cells of growing and large follicles, suggesting that fetuin may...

  18. Tissue-specific splicing pattern of fibronectin messenger RNA precursor during development and aging in rat

    1991-01-01

    Fibronectin isoforms are generated by the alternative splicing of a primary transcript derived from a single gene. In rat at least three regions of the molecule are involved: EIIIA, EIIIB, and V. This study investigated the splicing patterns of these regions during development and aging, by means of ribonuclease protection analysis. Between fetal and adult rat, the extent of inclusion of the EIIIA and/or EIIIB region in fibronectin mRNA varied according to the type of tissue analyzed; but the...

  19. Expression of preprotachykinin-A and neuropeptide-Y messenger RNA in the thymus.

    Ericsson, A; Geenen, V; Robert, F; Legros, J J; Vrindts-Gevaert, Y; Franchimont, P; Brene, S; Persson, H

    1990-08-01

    The preprotachykinin-A gene, the common gene of mRNAs encoding both substance-P (SP) and neurokinin-A (NKA), was shown to be expressed in Sprague-Dawley rat thymus by detection of specific mRNA in gel-blot analyses. In situ hybridization revealed dispersed PPT-A-labeled cells in sections from rat thymus, with a concentration of grains over a subpopulation of cells in the thymic medulla. Also, neuropeptide-Y mRNA-expressing cells were found in the rat thymus, primarily in the thymic medulla. Rat thymic extracts contained SP-like immunoreactivity (SP-LI), and the major part of the immunoreactivity coeluted with authentic SP and SP sulfoxide standards. SP-LI was also detected in human thymus, which contained between 0.09-0.88 ng SP-LI/g wet wt. Evidence for translation of preprotachykinin-A mRNA in the rat thymus was obtained from the demonstration of NKA-LI in thymic cells with an epithelial-like cell morphology. Combined with previous observations on the immunoregulatory roles of tachykinin peptides and the existence of specific receptors on immunocompetent cells, the demonstration of intrathymic synthesis of NKA suggests a role for NKA-LI peptides in T-cell differentiation in the thymus.

  20. Quantitative correlation between promoter methylation and messenger RNA levels of the reduced folate carrier

    Kheradpour Albert

    2008-05-01

    Full Text Available Abstract Background Methotrexate (MTX uptake is mediated by the reduced folate carrier (RFC. Defective drug uptake in association with decreased RFC expression is a common mechanism of MTX resistance in many tumor types. Heavy promoter methylation was previously identified as a basis for the complete silencing of RFC in MDA-MB-231 breast cancer cells, its role and prevalence in RFC transcription regulation are, however, not widely studied. Methods In the current study, RFC promoter methylation was assessed using methylation specific PCR in a panel of malignant cell lines (n = 8, including MDA-MB-231, and M805, a MTX resistant cell line directly established from the specimen of a patient with malignant fibrohistocytoma, whom received multiple doses of MTX. A quantitative approach of real-time PCR for measuring the extent of RFC promoter methylation was developed, and was validated by direct bisulfite genomic sequencing. RFC mRNA levels were determined by quantitative real-time RT-PCR and were related to the extent of promoter methylation in these cell lines. Results A partial promoter methylation and RFC mRNA down-regulation were observed in M805. Using the quantitative approach, a reverse correlation (correlation coefficient = -0.59, p Conclusion This study further suggests that promoter methylation is a potential basis for MTX resistance. The quantitative correlation identified in this study implies that promoter methylation is possibly a mechanism involved in the fine regulation of RFC transcription.

  1. Insulin-like growth factor II messenger RNA-binding protein-3 is an independent prognostic factor in uterine leiomyosarcoma.

    Yasutake, Nobuko; Ohishi, Yoshihiro; Taguchi, Kenichi; Hiraki, Yuka; Oya, Masafumi; Oshiro, Yumi; Mine, Mari; Iwasaki, Takeshi; Yamamoto, Hidetaka; Kohashi, Kenichi; Sonoda, Kenzo; Kato, Kiyoko; Oda, Yoshinao

    2018-04-01

    The aim of this study was to identify the prognostic factors of uterine leiomyosarcoma (ULMS). We reviewed 60 cases of surgically resected ULMSs and investigated conventional clinicopathological factors, together with the expression of insulin-like growth factor II messenger RNA-binding protein-3 (IMP3), hormone receptors and cell cycle regulatory markers by immunohistochemistry. Mediator complex subunit 12 (MED12) mutation analysis was also performed. Univariate analyses revealed that advanced stage (P < 0.0001), older age (P = 0.0244) and IMP3 expression (P = 0.0011) were significant predictors of a poor outcome. Multivariate analysis revealed advanced stage (P < 0.0001) and IMP3 (P = 0.0373) as independent predictors of a poor prognosis. Expressions of cell cycle markers and hormone receptors, and MED12 mutations (12% in ULMSs) were not identified as prognostic markers in this study. IMP3 expression in ULMS could be a marker of a poor prognosis. © 2017 John Wiley & Sons Ltd.

  2. RNA amplification for successful gene profiling analysis

    Wang Ena

    2005-07-01

    Full Text Available Abstract The study of clinical samples is often limited by the amount of material available to study. While proteins cannot be multiplied in their natural form, DNA and RNA can be amplified from small specimens and used for high-throughput analyses. Therefore, genetic studies offer the best opportunity to screen for novel insights of human pathology when little material is available. Precise estimates of DNA copy numbers in a given specimen are necessary. However, most studies investigate static variables such as the genetic background of patients or mutations within pathological specimens without a need to assess proportionality of expression among different genes throughout the genome. Comparative genomic hybridization of DNA samples represents a crude exception to this rule since genomic amplification or deletion is compared among different specimens directly. For gene expression analysis, however, it is critical to accurately estimate the proportional expression of distinct RNA transcripts since such proportions directly govern cell function by modulating protein expression. Furthermore, comparative estimates of relative RNA expression at different time points portray the response of cells to environmental stimuli, indirectly informing about broader biological events affecting a particular tissue in physiological or pathological conditions. This cognitive reaction of cells is similar to the detection of electroencephalographic patterns which inform about the status of the brain in response to external stimuli. As our need to understand human pathophysiology at the global level increases, the development and refinement of technologies for high fidelity messenger RNA amplification have become the focus of increasing interest during the past decade. The need to increase the abundance of RNA has been met not only for gene specific amplification, but, most importantly for global transcriptome wide, unbiased amplification. Now gene

  3. Culture temperature affects human chondrocyte messenger RNA expression in monolayer and pellet culture systems.

    Akira Ito

    Full Text Available Cell-based therapy has been explored for articular cartilage regeneration. Autologous chondrocyte implantation is a promising cell-based technique for repairing articular cartilage defects. However, there are several issues such as chondrocyte de-differentiation. While numerous studies have been designed to overcome some of these issues, only a few have focused on the thermal environment that can affect chondrocyte metabolism and phenotype. In this study, the effects of different culture temperatures on human chondrocyte metabolism- and phenotype-related gene expression were investigated in 2D and 3D environments. Human chondrocytes were cultured in a monolayer or in a pellet culture system at three different culture temperatures (32°C, 37°C, and 41°C for 3 days. The results showed that the total RNA level, normalized to the threshold cycle value of internal reference genes, was higher at lower temperatures in both culture systems. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH and citrate synthase (CS, which are involved in glycolysis and the citric acid cycle, respectively, were expressed at similar levels at 32°C and 37°C in pellet cultures, but the levels were significantly lower at 41°C. Expression of the chondrogenic markers, collagen type IIA1 (COL2A1 and aggrecan (ACAN, was higher at 37°C than at 32°C and 41°C in both culture systems. However, this phenomenon did not coincide with SRY (sex-determining region Y-box 9 (SOX9, which is a fundamental transcription factor for chondrogenesis, indicating that a SOX9-independent pathway might be involved in this phenomenon. In conclusion, the expression of chondrocyte metabolism-related genes at 32°C was maintained or enhanced compared to that at 37°C. However, chondrogenesis-related genes were further induced at 37°C in both culture systems. Therefore, manipulating the culture temperature may be an advantageous approach for regulating human chondrocyte metabolic activity and

  4. Culture temperature affects human chondrocyte messenger RNA expression in monolayer and pellet culture systems.

    Ito, Akira; Nagai, Momoko; Tajino, Junichi; Yamaguchi, Shoki; Iijima, Hirotaka; Zhang, Xiangkai; Aoyama, Tomoki; Kuroki, Hiroshi

    2015-01-01

    Cell-based therapy has been explored for articular cartilage regeneration. Autologous chondrocyte implantation is a promising cell-based technique for repairing articular cartilage defects. However, there are several issues such as chondrocyte de-differentiation. While numerous studies have been designed to overcome some of these issues, only a few have focused on the thermal environment that can affect chondrocyte metabolism and phenotype. In this study, the effects of different culture temperatures on human chondrocyte metabolism- and phenotype-related gene expression were investigated in 2D and 3D environments. Human chondrocytes were cultured in a monolayer or in a pellet culture system at three different culture temperatures (32°C, 37°C, and 41°C) for 3 days. The results showed that the total RNA level, normalized to the threshold cycle value of internal reference genes, was higher at lower temperatures in both culture systems. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and citrate synthase (CS), which are involved in glycolysis and the citric acid cycle, respectively, were expressed at similar levels at 32°C and 37°C in pellet cultures, but the levels were significantly lower at 41°C. Expression of the chondrogenic markers, collagen type IIA1 (COL2A1) and aggrecan (ACAN), was higher at 37°C than at 32°C and 41°C in both culture systems. However, this phenomenon did not coincide with SRY (sex-determining region Y)-box 9 (SOX9), which is a fundamental transcription factor for chondrogenesis, indicating that a SOX9-independent pathway might be involved in this phenomenon. In conclusion, the expression of chondrocyte metabolism-related genes at 32°C was maintained or enhanced compared to that at 37°C. However, chondrogenesis-related genes were further induced at 37°C in both culture systems. Therefore, manipulating the culture temperature may be an advantageous approach for regulating human chondrocyte metabolic activity and chondrogenesis.

  5. Primary structure of the α-subunit of Na+, K+-ATPase. II. Isolation, reverse transcription, and cloning of messenger RNA

    Petrukhin, K.E.; Broude, N.E.; Arsenyan, S.G.; Grishin, A.V.; Dzhandzhugazyan, K.N.; Modyanov, N.N.

    1986-01-01

    The messenger RNA coding the α-subunit of Na + ,K + -ATPase has been isolated from the outer medullary layer of porcine kidneys. The mRNA gives a specific hybridization band in the 25S-26S region with three oligonucleotide probes synthesized on the basis of information on the structure of three peptides isolated from a tryptic hydrolyzate of the α-subunit of Na + ,K + -ATPase. The translation of the mRNA in Xenopus laevis oocytes followed by immunochemical identification of the products of synthesis confirmed the presence of the mRNA of the α-subunit of Na + ,K + -ATPase in an enriched fraction of poly(A + )-RNA. This preparation has been used for the synthesis of cloning of double-stranded cDNA

  6. Primary structure of the. cap alpha. -subunit of Na/sup +/, K/sup +/-ATPase. II. Isolation, reverse transcription, and cloning of messenger RNA

    Petrukhin, K.E.; Broude, N.E.; Arsenyan, S.G.; Grishin, A.V.; Dzhandzhugazyan, K.N.; Modyanov, N.N.

    1986-10-01

    The messenger RNA coding the ..cap alpha..-subunit of Na/sup +/,K/sup +/-ATPase has been isolated from the outer medullary layer of porcine kidneys. The mRNA gives a specific hybridization band in the 25S-26S region with three oligonucleotide probes synthesized on the basis of information on the structure of three peptides isolated from a tryptic hydrolyzate of the ..cap alpha..-subunit of Na/sup +/,K/sup +/-ATPase. The translation of the mRNA in Xenopus laevis oocytes followed by immunochemical identification of the products of synthesis confirmed the presence of the mRNA of the ..cap alpha..-subunit of Na/sup +/,K/sup +/-ATPase in an enriched fraction of poly(A/sup +/)-RNA. This preparation has been used for the synthesis of cloning of double-stranded cDNA.

  7. MicroRNA profiling in intraocular medulloepitheliomas.

    Deepak P Edward

    Full Text Available To study the differential expression of microRNA (miRNA profiles between intraocular medulloepithelioma (ME and normal control tissue (CT.Total RNA was extracted from formalin fixed paraffin embedded (FFPE intraocular ME (n=7 and from age matched ciliary body controls (n=8. The clinical history and phenotype was recorded. MiRNA profiles were determined using the Affymetrix GeneChip miRNA Arrays analyzed using expression console 1.3 software. Validation of significantly dysregulated miRNA was confirmed by quantitative real-time PCR. The web-based DNA Intelligent Analysis (DIANA-miRPath v2.0 was used to perform enrichment analysis of differentially expressed (DE miRNA gene targets in Kyoto Encyclopedia of Genes and Genomes (KEGG pathway.The pathologic evaluation revealed one benign (benign non-teratoid, n=1 and six malignant tumors (malignant teratoid, n=2; malignant non-teratoid, n = 4. A total of 88 miRNAs were upregulated and 43 miRNAs were downregulated significantly (P<0.05 in the tumor specimens. Many of these significantly dysregulated miRNAs were known to play various roles in carcinogenesis and tumor behavior. RT-PCR validated three significantly upregulated miRNAs and three significantly downregulated miRNAs namely miR-217, miR-216a, miR-216b, miR-146a, miR-509-3p and miR-211. Many DE miRNAs that were significant in ME tumors showed dysregulation in retinoblastoma, glioblastoma, and precursor, normal and reactive human cartilage. Enriched pathway analysis suggested a significant association of upregulated miRNAs with 15 pathways involved in prion disease and several types of cancer. The pathways involving significantly downregulated miRNAs included the toll-like receptor (TLR (p<4.36E-16 and Nuclear Factor kappa B (NF-κB signaling pathways (p<9.00E-06.We report significantly dysregulated miRNAs in intraocular ME tumors, which exhibited abnormal profiles in other cancers as well such as retinoblastoma and glioblastoma. Pathway analysis

  8. Diagnostic accuracy of circulating thyrotropin receptor messenger RNA combined with neck ultrasonography in patients with Bethesda III-V thyroid cytology.

    Aliyev, Altay; Patel, Jinesh; Brainard, Jennifer; Gupta, Manjula; Nasr, Christian; Hatipoglu, Betul; Siperstein, Allan; Berber, Eren

    2016-01-01

    The aim of this study was to analyze the usefulness of thyrotropin receptor messenger RNA (TSHR-mRNA) combined with neck ultrasonography (US) in the management of thyroid nodules with Bethesda III-V cytology. Cytology slides of patients with a preoperative fine needle aspiration (FNA) and TSHR-mRNA who underwent thyroidectomy between 2002 and 2011 were recategorized based on the Bethesda classification. Results of thyroid FNA, TSHR-mRNA, and US were compared with the final pathology. Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated. There were 12 patients with Bethesda III, 112 with Bethesda IV, and 58 with Bethesda V cytology. The sensitivity of TSHR-mRNA in predicting cancer was 33%, 65%, and 79 %, and specificity was 67%, 66%, and 71%, for Bethesda III, IV, and V categories, respectively. For the same categories, the PPV of TSHR-mRNA was 25%, 33%, and 79%, respectively; whereas the NPV was 75%, 88%, and 71%, respectively. The addition of neck US to TSHR-mRNA increased the NPV to 100% for Bethesda III, and 86%, for Bethesda IV, and 82% for Bethesda V disease. This study documents the potential usefulness of TSHR-mRNA for thyroid nodules with Bethesda III-V FNA categories. TSHR-mRNA may be used to exclude Bethesda IV disease. A large sample analysis is needed to determine its accuracy for Bethesda category III nodules. Copyright © 2016 Elsevier Inc. All rights reserved.

  9. Ultra-sensitive DNA assay based on single-molecule detection coupled with fluorescent quantum dot-labeling and its application to determination of messenger RNA

    Li Li; Li Xincang; Li Lu; Wang Jinxing; Jin Wenrui

    2011-01-01

    An ultra-sensitive single-molecule detection (SMD) method for quantification of DNA using total internal reflection fluorescence microscopy (TIRFM) coupled with fluorescent quantum dot (QD)-labeling was developed. In this method, the target DNA (tDNA) was captured by the capture DNA immobilized on the silanized coverslip blocked with ethanolamine and bovine serum albumin. Then, the QD-labeled probe DNA was hybridized to the tDNA. Ten fluorescent images of the QD-labeled sandwich DNA hybrids on the coverslip were taken by a high-sensitive CCD. The tDNA was quantified by counting the bright spots on the images using a calibration curve. The LOD of the method was 1 x 10 -14 mol L -1 . Several key factors, including image acquirement, fluorescence probe, substrate preparation, noise elimination from solutions and glass coverslips, and nonspecific adsorption and binding of solution-phase detection probes were discussed in detail. The method could be applied to quantify messenger RNA (mRNA) in cells. In order to determine mRNA, double-stranded RNA-DNA hybrids consisting of mRNA and corresponding cDNA were synthesized from the cellular mRNA template using reverse transcription in the presence of reverse transcriptase. After removing the mRNA in the double-stranded hybrids using ribonuclease, cDNA was quantified using the SMD-based TIRFM. Osteopontin mRNA in decidual stromal cells was chosen as the model analyte.

  10. Ultra-sensitive DNA assay based on single-molecule detection coupled with fluorescent quantum dot-labeling and its application to determination of messenger RNA

    Li Li [School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100 (China); Li Xincang [School of Life Sciences, Shandong University, Jinan 250100 (China); Li Lu [School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100 (China); Wang Jinxing [School of Life Sciences, Shandong University, Jinan 250100 (China); Jin Wenrui, E-mail: jwr@sdu.edu.cn [School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100 (China)

    2011-01-24

    An ultra-sensitive single-molecule detection (SMD) method for quantification of DNA using total internal reflection fluorescence microscopy (TIRFM) coupled with fluorescent quantum dot (QD)-labeling was developed. In this method, the target DNA (tDNA) was captured by the capture DNA immobilized on the silanized coverslip blocked with ethanolamine and bovine serum albumin. Then, the QD-labeled probe DNA was hybridized to the tDNA. Ten fluorescent images of the QD-labeled sandwich DNA hybrids on the coverslip were taken by a high-sensitive CCD. The tDNA was quantified by counting the bright spots on the images using a calibration curve. The LOD of the method was 1 x 10{sup -14} mol L{sup -1}. Several key factors, including image acquirement, fluorescence probe, substrate preparation, noise elimination from solutions and glass coverslips, and nonspecific adsorption and binding of solution-phase detection probes were discussed in detail. The method could be applied to quantify messenger RNA (mRNA) in cells. In order to determine mRNA, double-stranded RNA-DNA hybrids consisting of mRNA and corresponding cDNA were synthesized from the cellular mRNA template using reverse transcription in the presence of reverse transcriptase. After removing the mRNA in the double-stranded hybrids using ribonuclease, cDNA was quantified using the SMD-based TIRFM. Osteopontin mRNA in decidual stromal cells was chosen as the model analyte.

  11. Reprogramming human umbilical cord mesenchymal stromal cells to islet-like cells with the use of in vitro-synthesized pancreatic-duodenal homebox 1 messenger RNA.

    Wang, Xiao Li; Hu, Pei; Guo, Xing Rong; Yan, Ding; Yuan, Yahong; Yan, Shi Rong; Li, Dong Sheng

    2014-11-01

    Human umbilical cord mesenchymal stromal cells (hUC-MSCs) hold great potential as a therapeutic candidate to treat diabetes, owing to their unlimited source and ready availability. In this study, we differentiated hUC-MSCs with in vitro-synthesized pancreatic-duodenal homebox 1 (PDX1) messenger (m)RNA into islet-like cell clusters. hUC-MSCs were confirmed by both biomarker detection and functional differentiation. In vitro-synthesized PDX1 messenger RNA can be transfected into hUC-MSCs efficiently. The upregulated expression of PDX1 protein can be detected 4 h after transfection and remains detectable for 36 h. The induction of islet-like structures was confirmed by means of morphology and dithizone staining. Reverse transcriptase-polymerase chain reaction results revealed the expression of some key pancreatic transcription factors, such as PDX1, NeuroD, NKX6.1, Glut-2 and insulin in islet-like cell clusters. Immunofluorescence analysis showed that differentiated cells express both insulin and C-peptide. Enzyme-linked immunosorbent assay analysis validated the insulin secretion of islet-like cell clusters in response to the glucose stimulation. Our results demonstrate the use of in vitro-synthesized PDX1 messenger RNA to differentiate hUC-MSCs into islet-like cells and pave the way toward the development of reprogramming and directed-differentiation methods for the expression of encoded proteins. Copyright © 2014 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  12. Mercury's Messenger

    Chapman, Clark R.

    2004-01-01

    Forty years after Mariner 2, planetary exploration has still only just begun, and many more missions are on drawing boards, nearing the launch pad, or even en route across interplanetary space to their targets. One of the most challenging missions that will be conducted this decade is sending the MESSENGER spacecraft to orbit the planet Mercury.…

  13. Growth differentiation factor 3 is induced by bone morphogenetic protein 6 (BMP-6) and BMP-7 and increases luteinizing hormone receptor messenger RNA expression in human granulosa cells.

    Shi, Jia; Yoshino, Osamu; Osuga, Yutaka; Akiyama, Ikumi; Harada, Miyuki; Koga, Kaori; Fujimoto, Akihisa; Yano, Tetsu; Taketani, Yuji

    2012-04-01

    To examine the relevance of growth differentiation factor 3 (GDF-3) and bone morphogenetic protein (BMP) cytokines in human ovary. Molecular studies. Research laboratory. Eight women undergoing salpingo-oophorectomy and 30 women undergoing ovarian stimulation for in vitro fertilization. Localizing GDF-3 protein in human ovaries; granulosa cells (GC) cultured with GDF-3, BMP-6, or BMP-7 followed by RNA extraction. The localization of GDF-3 protein in normal human ovaries via immunohistochemical analysis, GDF-3 messenger RNA (mRNA) expression evaluation via quantitative real-time reverse transcription and polymerase chain reaction (RT-PCR), and evaluation of the effect of GDF-3 on leuteinizing hormone (LH) receptor mRNA expression via quantitative real-time RT-PCR. In the ovary, BMP cytokines, of the transforming growth factor beta (TGF-β) superfamily, are known as a luteinization inhibitor by suppressing LH receptor expression in GC. Growth differentiation factor 3, a TGF-β superfamily cytokine, is recognized as an inhibitor of BMP cytokines in other cells. Immunohistochemical analysis showed that GDF-3 was strongly detected in the GC of antral follicles. An in vitro assay revealed that BMP-6 or BMP-7 induced GDF-3 mRNA in GC. Also, GDF-3 increased LH receptor mRNA expression and inhibited the effect of BMP-7, which suppressed the LH receptor mRNA expression in GC. GDF-3, induced with BMP-6 and BMP-7, might play a role in folliculogenesis by inhibiting the effect of BMP cytokines. Copyright © 2012 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  14. Lower glutamic acid decarboxylase 65-kDa isoform messenger RNA and protein levels in the prefrontal cortex in schizoaffective disorder but not schizophrenia.

    Glausier, Jill R; Kimoto, Sohei; Fish, Kenneth N; Lewis, David A

    2015-01-15

    Altered gamma-aminobutyric acid (GABA) signaling in the prefrontal cortex (PFC) has been associated with cognitive dysfunction in patients with schizophrenia and schizoaffective disorder. Levels of the GABA-synthesizing enzyme glutamic acid decarboxylase 67-kDa isoform (GAD67) in the PFC have been consistently reported to be lower in patients with these disorders, but the status of the second GABA-synthesizing enzyme, glutamic acid decarboxylase 65-kDa isoform (GAD65), remains unclear. GAD65 messenger RNA (mRNA) levels were quantified in PFC area 9 by quantitative polymerase chain reaction from 62 subjects with schizophrenia or schizoaffective disorder and 62 matched healthy comparison subjects. In a subset of subject pairs, GAD65 relative protein levels were quantified by confocal immunofluorescence microscopy. Mean GAD65 mRNA levels were 13.6% lower in subjects with schizoaffective disorder but did not differ in subjects with schizophrenia relative to their matched healthy comparison subjects. In the subjects with schizoaffective disorder, mean GAD65 protein levels were 19.4% lower and were correlated with GAD65 mRNA levels. Lower GAD65 mRNA and protein levels within subjects with schizoaffective disorder were not attributable to factors commonly comorbid with the diagnosis. In concert with previous studies, these findings suggest that schizoaffective disorder is associated with lower levels of both GAD65 and GAD67 mRNA and protein in the PFC, whereas subjects with schizophrenia have lower mean levels of only GAD67 mRNA and protein. Because cognitive function is generally better preserved in patients with schizoaffective disorder relative to patients with schizophrenia, these findings may support an interpretation that GAD65 downregulation provides a homeostatic response complementary to GAD67 downregulation that serves to reduce inhibition in the face of lower PFC network activity. Copyright © 2015 Society of Biological Psychiatry. Published by Elsevier Inc

  15. Identification and Characterization of 293T Cell-Derived Exosomes by Profiling the Protein, mRNA and MicroRNA Components.

    Jing Li

    Full Text Available Cell-derived exosomes are leading candidates for in vivo drug delivery carriers. In particular, exosomes derived from 293T cells are used most frequently, although exosome dosing has varied greatly among studies. Considering their biological origin, it is crucial to characterize the molecular composition of exosomes if large doses are to be administered in clinical settings. In this study, we present the first comprehensive analysis of the protein, messenger RNA and microRNA profiles of 293T cell-derived exosomes; then, we characterized these data using Gene Ontology annotation and Kyoto Encyclopedia for Genes and Genomes pathway analysis. Our study will provide the basis for the selection of 293T cell-derived exosome drug delivery systems. Profiling the exosomal signatures of 293T cells will lead to a better understanding of 293T exosome biology and will aid in the identification of any harmful factors in exosomes that could cause adverse clinical effects.

  16. Labeling of eukaryotic messenger RNA 5' terminus with phosphorus -32: use of tobacco acid pyrophosphatase for removal of cap structures

    Lockard, R.E.; Rieser, L.; Vournakis, J.N.

    1981-01-01

    In recent years, there has been a growing appreciation of the potential applications of 5'- 32 P-end-labeled mRNA, not only for screening recombinant clones and mapping gene structure, but also for revealing possible nucleotide sequence and structural signals within mRNA molecules themselves, which may be important for eukaryotic mRNA processing and turnover and for controlling differential rates of translational initiation. Three major problems, however, have retarded progress in this area, lack of methods for efficient and reproducible removal of m7G5ppp5'-cap structures, which maintain the integrity of an RNA molecule; inability to generate a sufficient amount of labeled mRNA, owing to the limited availability of most pure mRNA species; and the frequent problem of RNA degradation during in vitro end-labeling owing to RNAse contamination. The procedures presented here permit one to decap and label minute quantities of mRNA, effectively. Tobacco acid pyrophosphatase is relatively efficient in removing cap structures from even nanogram quantities of available mRNA, and enough radioactivity can be easily generated from minute amounts ofintact mRNA with very high-specific-activity [gamma- 32 P]ATP and the inhibition of ribonuclease contamination with diethylpyrocarbonate. These procedures can be modified and applied to almost any other type of RNA molecule as well. In Section III of this volume, we explore in detail how effectively 5'-end-labeled mRNA can be used not only for nucleotide sequence analysis, but also for mapping mRNA secondary structure

  17. Complex degradation processes lead to non-exponential decay patterns and age-dependent decay rates of messenger RNA.

    Carlus Deneke

    Full Text Available Experimental studies on mRNA stability have established several, qualitatively distinct decay patterns for the amount of mRNA within the living cell. Furthermore, a variety of different and complex biochemical pathways for mRNA degradation have been identified. The central aim of this paper is to bring together both the experimental evidence about the decay patterns and the biochemical knowledge about the multi-step nature of mRNA degradation in a coherent mathematical theory. We first introduce a mathematical relationship between the mRNA decay pattern and the lifetime distribution of individual mRNA molecules. This relationship reveals that the mRNA decay patterns at steady state expression level must obey a general convexity condition, which applies to any degradation mechanism. Next, we develop a theory, formulated as a Markov chain model, that recapitulates some aspects of the multi-step nature of mRNA degradation. We apply our theory to experimental data for yeast and explicitly derive the lifetime distribution of the corresponding mRNAs. Thereby, we show how to extract single-molecule properties of an mRNA, such as the age-dependent decay rate and the residual lifetime. Finally, we analyze the decay patterns of the whole translatome of yeast cells and show that yeast mRNAs can be grouped into three broad classes that exhibit three distinct decay patterns. This paper provides both a method to accurately analyze non-exponential mRNA decay patterns and a tool to validate different models of degradation using decay data.

  18. Association of Cocaine- and Amphetamine-Regulated Transcript (CART) Messenger RNA Level, Food Intake, and Growth in Channel Catfish

    Cocaine-and Amphetamine-Regulated Transcript (CART) is a potent hypothalamic anorectic peptide in mammals and fish. We hypothesized that increased food intake is associated with changes in expression of CART mRNA within the brain of channel catfish. Objectives were to clone the CART gene, examine ...

  19. P-body loss is concomitant with formation of a messenger RNA storage domain in mouse oocytes

    Flemr, Matyáš; Ma, J.; Schultz, R. M.; Svoboda, Petr

    2010-01-01

    Roč. 82, č. 5 (2010), s. 1008-1017 ISSN 0006-3363 R&D Project s: GA MŠk ME09039 Grant - others:EMBO SDIG(DE) project 1483 Institutional research plan: CEZ:AV0Z50520514 Keywords : oocyte * mRNA * cortex Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.870, year: 2010

  20. SIMULTANEOUS EXPRESSION AND REGULATION OF G-CSF AND IL-6 MESSENGER-RNA IN ADHERENT HUMAN MONOCYTES AND FIBROBLASTS

    VELLENGA, E; VANDERVINNE, B; DEWOLF, JTM; HALIE, MR

    The regulation of granulocyte-colony stimulating factor (G-CSF) and interleukin-6 (IL-6) mRNA was studied in human adherent monocytes in response to the protein kinase C activator, oleolyl-acetylglycerol (OAG), the calcium-ionophore A23187 and the cyclic AMP elevating agents, dibutyryl c-AMP

  1. Detailed mapping of serotonin 5-HT1B and 5-HT1D receptor messenger RNA and ligand binding sites in guinea-pig brain and trigeminal ganglion: clues for function

    Leysen, J.E.; Schotte, A.; Jurzak, M.; Luyten, W.H.M.L.; Voorn, P.; Bonaventure, P.

    1997-01-01

    The similar pharmacology of the 5-HT 1B and 5-HT 1D receptors, and the lack of selective compounds sufficiently distinguishing between the two receptor subtypes, have hampered functional studies on these receptors. In order to provide clues for differential functional roles of the two subtypes, we performed a parallel localization study throughout the guinea-pig brain and the trigeminal ganglia by means of quantitative in situ hybridization histochemistry (using [ 35 S]-labelled riboprobes probes for receptor messenger RNA) and receptor autoradiography (using a new radioligand [ 3 H]alniditan).The anatomical patterns of 5-HT 1B and 5-HT 1D receptor messenger RNA were quite different. While 5-HT 1B receptor messenger RNA was abundant throughout the brain (with highest levels in the striatum, nucleus accumbens, olfactory tubercle, cortex, hypothalamus, hippocampal formation, amygdala, thalamus, dorsal raphe and cerebellum), 5-HT 1D receptor messenger RNA exhibited a more restricted pattern; it was found mainly in the olfactory tubercle, entorhinal cortex, dorsal raphe, cerebellum, mesencephalic trigeminal nucleus and in the trigeminal ganglion. The density of 5-HT 1B/1D binding sites (combined) obtained with [ 3 H]alniditan autoradiography was high in the substantia nigra, superior colliculus and globus pallidus, whereas lower levels were detected in the caudate-putamen, hypothalamus, hippocampal formation, amygdala, thalamus and central gray. This distribution pattern was indistinguishable from specific 5-HT 1B receptor labelling in the presence of ketanserin under conditions to occlude 5-HT 1D receptor labelling; hence the latter were below detection level. Relationships between the regional distributions of the receptor messenger RNAs and binding sites and particular neuroanatomical pathways are discussed with respect to possible functional roles of the 5-HT 1B and 5-HT 1D receptors. (Copyright (c) 1997 Elsevier Science B.V., Amsterdam. All rights reserved.)

  2. Distribution of precursor amyloid-β-protein messenger RNA in human cerebral cortex: relationship to neurofibrillary tangles and neuritic plaques

    Lewis, D.A.; Higgins, G.A.; Young, W.G.; Goldgaber, D.; Gajdusek, D.C.; Wilson, M.C.; Morrison, J.H.

    1988-01-01

    Neurofibrillary tangles (NFT) and neuritic plaques (NP), two neuropathological markers of Alzheimer disease, may both contain peptide fragments derived from the human amyloid β protein. However, the nature of the relationship between NFT and NP and the source of the amyloid β proteins found in each have remained unclear. The authors used in situ hybridization techniques to map the anatomical distribution of precursor amyloid-β-protein mRNA in the neocortex of brains from three subjects with no known neurologic disease and from five patients with Alzheimer disease. In brains from control subjects, positively hybridizing neurons were present in cortical regions and layers that contain a high density of neuropathological markers in Alzheimer disease, as well as in those loci that contain NP but few NFT. Quantitative analyses of in situ hybridization patterns within layers III and V of the superior frontal cortex revealed that the presence of high numbers of NFT in Alzheimer-diseased brains was associated with a decrease in the number of positively hybridizing neurons compared to controls and Alzheimer-diseased brains with few NFT. These findings suggest that the expression of precursor amyloid-β-protein mRNA may be a necessary but is clearly not a sufficient prerequisite for NFT formation. In addition, these results may indicate that the amyloid β protein, present in NP in a given region or layer of cortex, is not derived from the resident neuronal cell bodies that express the mRNA for the precursor protein

  3. Translational recognition of the 5'-terminal 7-methylguanosine of globin messenger RNA as a function of ionic strength.

    Chu, L Y; Rhoads, R E

    1978-06-13

    The translation of rabbit globin mRNA in cell-free systems derived from either wheat germ or rabbit reticulocyte was studied in the presence of various analogues of the methylated 5' terminus (cap) as a function of ionic strength. Inhibition by these analogues was strongly enhanced by increasing concentrations of KCl, K(OAc), Na(OAc), or NH4(OAc). At appropriate concentrations of K(OAc), both cell-free systems were equally sensitive to inhibition by m7GTP. At 50 mM K(OAc), the reticulocyte system was not sensitive to m7GMP or m7GTP, but at higher concentrations up to 200 mM K(OAc), both nucleotides caused strong inhibition. The compound in m7G5'ppp5'Am was inhibitory at all concentrations of K(OAc) ranging from 50 to 200 mM, although more strongly so at the higher concentrations. Over the same range of nucleotide concentrations, the compounds GMP, GTP, and G5'ppp5'Am were not inhibitors. The mobility on sodium dodecyl sulfate-polyacrylamide electrophoresis of the translation product was that of globin at all K(OAc) concentrations in the presence of m7GTP. Globin mRNA from which the terminal m7GTP group had been removed by chemical treatment (periodate-cyclohexylamine-alkaline phosphatase) or enzymatic treatment (tobacco acid pyrophosphatase-alkaline phosphatase) was translated less efficiently than untreated globin mRNA at higher K(OAc) concentrations, but retained appreciable activity at low K(OAc) concentrations.

  4. Development of Anti-Human Mesothelin-Targeted Chimeric Antigen Receptor Messenger RNA-transfected Peripheral Blood Lymphocytes for Ovarian Cancer Therapy.

    Hung, Chien-Fu; Xu, Xuequn; Li, Linhong; Ma, Ying; Jin, Qiu; Viley, Angelia; Allen, Cornell; Natarajan, Pachai; Shivakumar, Rama; Peshwa, Madhusudan V; Emens, Leisha A

    2018-04-02

    CD19-targeted chimeric antigen receptor (CAR) engineered T/natural killer (NK)-cell therapies can result in durable clinical responses in B-cell malignancies. However, CAR-based immunotherapies have been much less successful in solid cancers, in part due to "on-target off-tumor" toxicity related to expression of target tumor antigens on normal tissue. Based on preliminary observations of safety and clinical activity in proof-of-concept clinical trials, tumor antigen-specific messenger RNA (mRNA) CAR transfection into selected, activated, and expanded T/NK cells may permit prospective control of "on-target off-tumor" toxicity. To develop a commercial product for solid tumors, mesothelin was selected as an antigen target based on its association with poor prognosis and overexpression in multiple solid cancers. It was hypothesized that selecting, activating, and expanding cells ex vivo prior to mRNA CAR transfection would not be necessary, thus simplifying the complexity and cost of manufacturing. Now, the development of anti-human mesothelin mRNA CAR transfected peripheral blood lymphocytes (CARMA-hMeso) is reported, demonstrating the manufacture and cryopreservation of multiple cell aliquots for repeat administrations from a single human leukapheresis. A rapid, automated, closed system for cGMP-compliant transfection of mRNA CAR in up to 20 × 10 9 peripheral blood lymphocytes was developed. Here we show that CARMA-hMeso cells recognize and lyse tumor cells in a mesothelin-specific manner. Expression of CAR was detectable over approximately 7 days in vitro, with a progressive decline of CAR expression that appears to correlate with in vitro cell expansion. In a murine ovarian cancer model, a single intraperitoneal injection of CARMA-hMeso resulted in the dose-dependent inhibition of tumor growth and improved survival of mice. Furthermore, repeat weekly intraperitoneal administrations of the optimal CARMA-hMeso dose further prolonged disease control and survival

  5. Immunohistochemical identification of messenger RNA-related proteins in basophilic inclusions of adult-onset atypical motor neuron disease.

    Fujita, Kengo; Ito, Hidefumi; Nakano, Satoshi; Kinoshita, Yoshimi; Wate, Reika; Kusaka, Hirofumi

    2008-10-01

    This report concerns an immunohistochemical investigation on RNA-related proteins in the basophilic inclusions (BIs) from patients with adult-onset atypical motor neuron disease. Formalin-fixed, paraffin-embedded sections of the motor cortex and the lumbar spinal cord were examined. The BIs appeared blue in color with H&E and Nissl stain, and pink with methylgreen-pyronin stain. Ribonuclease pretreatment abolished the methylgreen-pyronin staining, suggesting that the BIs contained RNA. Immunohistochemically, the BIs were distinctly labeled with the antibodies against poly(A)-binding protein 1, T cell intracellular antigen 1, and ribosomal protein S6. These proteins are essential constituents of stress granules. In contrast, the BIs were not immunoreactive for ribosomal protein L28 and decapping enzyme 1, which are core components of transport ribonucleoprotein particles and processing bodies, respectively. Moreover, the BIs were not immunopositive for TDP-43. Our results imply that translation attenuation could be involved in the processes of BI formation in this disorder.

  6. Common acute lymphoblastic leukemia antigen: partial characterization by in vivo labeling and isolation of its messenger RNA

    Heinsohn, S.; Kabisch, H.

    1987-01-01

    Common acute lymphoblastic leukemia (ALL) antigen (CALLA)-like proteins were detected by in vivo labeling experiments carried out with human lymphoblastoid cell line KM3 and also in cell-free translation, directed by CALLA-specific mRNA prepared from immunoadsorbed KM3 polysomes. The CALLA-like structure found in both systems shows an Mr of 95kDa. Additional CALLA-like proteins could be identified in the in vivo experiments with calculated Mrs of 40kDa in the cells and 85 and 38kDa in the culture medium. In the cell-free translation system, an additional product of Mr 80kDa could be detected

  7. Isolation and characterization of the messenger RNA and the gene coding for a proline-rich zein from corn endosperm

    Wang, S.Z.

    1985-01-01

    Gamma-zein, a proline-rich protein from corn endosperm, was investigated at the molecular level. Immunological and electrophoretic data indicated that gamma-zein was deposited into protein bodies in corn endosperm. Both isolated polysomes and poly(A) + mRNA were found to direct into vitro synthesis of gamma-zein in a wheat germ system. In vitro synthesized gamma-zein was immunoprecipitated from the total in vitro translation products. A cDNA expression library was constructed by reverse transcription of total poly(A) + mRNA using pUC8 plasmid as vector and E. coli strain DH1 as host. The library was screened for the expression of gamma-zein and alpha-zein by specific antibodies. The library was also screened with 32 P-labeled gamma-zein and alpha-zein cDNA probes. The results indicated that gamma-zein and its fragments were readily expressed in E. coli while alpha-zein was not. Seven independently selected clones, six of which were selected by antibody and one by a cDNA probe, were sequenced. A comparison of sequence information from seven clones revealed that their overlapping regions were identical. This suggests that gamma-zein is encoded by a single gene. This finding is in conflict with what was expected on the basis of extensive charge heterogeneity of gamma-zein in isoelectric focusing. Individual bands cut from an IEF gel were rerun and shown to give several bands suggesting that the charge heterogeneity of gamma-zein may be an artifact. Sequence information of gamma-zein indicated that the gene encodes a mature protein whose primary structure includes 204 amino acids and has a molecular weight of 21,824 daltons

  8. Stability of Reference Genes for Messenger RNA Quantification by Real-Time PCR in Mouse Dextran Sodium Sulfate Experimental Colitis.

    Nour Eissa

    Full Text Available Many animal models have been developed to characterize the complexity of colonic inflammation. In dextran sodium sulfate (DSS experimental colitis in mice the choice of reference genes is critical for accurate quantification of target genes using quantitative real time PCR (RT-qPCR. No studies have addressed the performance of reference genes in mice DSS-experimental colitis. This study aimed to determine the stability of reference genes expression (RGE in DSS-experimental murine colitis.Colitis was induced in male C57BL/6 mice using DSS5% for 5 days, control group received water. RNA was extracted from inflamed and non-inflamed colon. Using RT-qPCR, comparative analysis of 13 RGE was performed according to predefined criteria and relative colonic TNF-α and IL-1β gene expression was determined by calculating the difference in the threshold cycle.Colitis significantly altered the stability of mucosal RGE. Commonly used glyceraldehyde-3-phosphate dehydrogenase (Gapdh, β-actin (Actb, or β2-microglobulin (β2m showed the highest variability within the inflamed and control groups. Conversely, TATA-box-binding protein (Tbp and eukaryotic translation elongation factor 2 (Eef2 were not affected by inflammation and were the most stable genes. Normalization of colonic TNF-α and IL-1β mRNA levels was dependent on the reference gene used. Depending on the genes used to normalize the data, statistical significance varied from significant when TBP / Eef2 were used to non-significant when Gapdh, Actb or β2m were used.This study highlights the appropriate choice of RGE to ensure adequate normalization of RT-qPCR data when using this model. Suboptimal RGE may explain controversial results from published studies. We recommend using Tbp and Eef2 instead of Gapdh, Actb or β2m as reference genes.

  9. Host apolipoprotein B messenger RNA-editing enzyme catalytic polypeptide-like 3G is an innate defensive factor and drug target against hepatitis C virus.

    Peng, Zong-Gen; Zhao, Zhi-Yun; Li, Yan-Ping; Wang, Yu-Ping; Hao, Lan-Hu; Fan, Bo; Li, Yu-Huan; Wang, Yue-Ming; Shan, Yong-Qiang; Han, Yan-Xing; Zhu, Yan-Ping; Li, Jian-Rui; You, Xue-Fu; Li, Zhuo-Rong; Jiang, Jian-Dong

    2011-04-01

    Host cellular factor apolipoprotein B messenger RNA (mRNA)-editing enzyme catalytic polypeptide-like 3G (hA3G) is a cytidine deaminase that inhibits a group of viruses including human immunodeficiency virus-1 (HIV-1). In the continuation of our research on hA3G, we found that hA3G stabilizing compounds significantly inhibited hepatitis C virus (HCV) replication. Therefore, this study investigated the role of hA3G in HCV replication. Introduction of external hA3G into HCV-infected Huh7.5 human hepatocytes inhibited HCV replication; knockdown of endogenous hA3G enhanced HCV replication. Exogenous HIV-1 virion infectivity factor (Vif) decreased intracellular hA3G and therefore enhanced HCV proliferation, suggesting that the presence of Vif might be an explanation for the HIV-1/HCV coinfection often observed in HIV-1(+) individuals. Treatment of the HCV-infected Huh7.5 cells with RN-5 or IMB-26, two known hA3G stabilizing compounds, increased intracellular hA3G and accordingly inhibited HCV replication. The compounds inhibit HCV through increasing the level of hA3G incorporated into HCV particles, but not through inhibiting HCV enzymes. However, G/A hypermutation in the HCV genome were not detected, suggesting a new antiviral mechanism of hA3G in HCV, different from that in HIV-1. Stabilization of hA3G by RN-5 was safe in vivo. hA3G appears to be a cellular restrict factor against HCV and could be a potential target for drug discovery. 2011 American Association for the Study of Liver Diseases.

  10. Changes in growth hormone (GH) messenger RNA (GH mRNA) expression in the rat anterior pituitary after single interferon (IFN) alpha administration

    Romanowski, W.; Braczkowski, R.; Nowakowska-Zajdel, E.; Muc-Wierzgon, M.; Zubelewicz-Szkodzinska, B.; Kosiewicz, J.; Korzonek, I.

    2006-01-01

    Introduction: Interferon a (IFN-a) is a cytokine with pleiotropic effects which, via different pathways, influences the secretion of certain cytokines and hormones. Growth hormone (GH) secreted from the pituitary has physiological effects on various target tissues. The question is how IFN-a administered in various types of disease influences GH secretion. This study investigated the acute effect of IFN-a on GH mRNA expression in the rat anterior pituitary. Objective: The aim of the study was to measure the cellular expression of GH mRNA by in situ hybridisation in the anterior pituitary after a single administration of IFN-a. Material and methods: Rats were administered an intraperitoneal injection of IFN-a or saline. The rat pituitaries were taken 2 and 4 hours after IFN/saline administration and kept frozen until in situ hybridisation histochemistry. A 31 - base 35S -labelled oligonucleotide probe complementary to part of the exonic mRNA sequence coding for GH mRNA was used. All control and experimental sections were hybridised in the same hybridisation reaction. Results: Acute administration of interferon a increased GH mRNA expression in the anterior pituitary in the 4-hour group in comparison with the control group, and there was no difference between the control group and the 2-hour rats. Conclusion: A single IFN-a administration was found to exert an influence on anterior pituitary GH mRNA expression. These observations may pave the way for presenting a possible new action of IFN-a. (author) GH mRNA, anterior pituitary, interferon

  11. Messenger RNA for membrane-type 2 matrix metalloproteinase, MT2-MMP, is expressed in human placenta of first trimester.

    Bjørn, S F; Hastrup, N; Larsen, J F; Lund, L R; Pyke, C

    2000-01-01

    An intimately regulated cell surface activation of matrix metalloproteinases (MMPs) is believed to be of critical importance for the control of trophoblast invasion. A histological investigation of the expression and localization of three different MMPs, the membrane-type matrix metalloproteinases 1 and 2 (MT1-MMP, MT2-MMP) and matrix metalloproteinase 2 (MMP-2/gelatinase A) was performed by in situ hybridization on consecutive sections from human placentae of first trimester pregnancies. Cytokeratin immunostaining identified trophoblast cells. Both normal and tubal implantation sites were studied. We observed a high degree of coexpression of MT2-MMP, MT1-MMP and MMP-2 mRNAs in single extravillous cytotrophoblasts that had invaded the endometrium and tubal wall. Furthermore, mRNAs for all three genes were also seen in cytotrophoblasts of cell islands. In contrast to this coexpression pattern, MT2-MMP expression was absent from cell columns and decidual cells, in which signals for MT1-MMP and MMP-2 mRNAs were seen. The present data on the cellular expression of MT2-MMP mRNA in placenta extend our knowledge of the proteolytic events that take place during early pregnancy. The data suggest that MT2-MMP, capable of activating MMP-2 in vitro, is involved in the invasion of extravillous cytotrophoblast, possibly related to the physiological activation of MMP-2. Copyright 2000 Harcourt Publishers Ltd.

  12. A probabilistic approach for the interpretation of RNA profiles as cell type evidence.

    de Zoete, Jacob; Curran, James; Sjerps, Marjan

    2016-01-01

    DNA profiles can be used as evidence to distinguish between possible donors of a crime stain. In some cases, both the prosecution and the defence claim that the cell material was left by the suspect but they dispute which cell type was left behind. For example, in sexual offense cases the prosecution could claim that the sample contains semen cells where the defence argues that the sample contains skin cells. In these cases, traditional methods (e.g. a phosphatase test) can be used to examine the cell type contained in the sample. However, there are some drawbacks when using these methods. For instance, many of these techniques need to be carried out separately for each cell type and each of them requires part of the available sample, which reduces the amount that can be used for DNA analysis. Another option is messenger RNA (mRNA) evidence. mRNA expression levels vary among cell types and can be used to make (probability) statements about the cell type(s) present in a sample. Existing methods for the interpretation of RNA profiles as evidence for the presence of certain cell types aim at making categorical statements. Such statements limit the possibility to report the associated uncertainty. Some of these existing methods will be discussed. Most notably, a method based on a 'n/2' scoring rule (Lindenbergh et al.) and a method using marker values and cell type scoring thresholds (Roeder et al.). From a statistical point of view, a probabilistic approach is the most obvious choice. Two approaches (multinomial logistic regression and naïve Bayes') are suggested. All methods are compared, using two different datasets and several criteria regarding their ability to assess the evidential value of RNA profiles. We conclude that both the naïve Bayes' method and a method based on multinomial logistic regression, that produces a probabilistic statement as measure of the evidential value, are an important improvement of the existing methods. Besides a better performance

  13. Differential effects of common variants in SCN2A on general cognitive ability, brain physiology, and messenger RNA expression in schizophrenia cases and control individuals.

    Dickinson, Dwight; Straub, Richard E; Trampush, Joey W; Gao, Yuan; Feng, Ningping; Xie, Bin; Shin, Joo Heon; Lim, Hun Ki; Ursini, Gianluca; Bigos, Kristin L; Kolachana, Bhaskar; Hashimoto, Ryota; Takeda, Masatoshi; Baum, Graham L; Rujescu, Dan; Callicott, Joseph H; Hyde, Thomas M; Berman, Karen F; Kleinman, Joel E; Weinberger, Daniel R

    2014-06-01

    One approach to understanding the genetic complexity of schizophrenia is to study associated behavioral and biological phenotypes that may be more directly linked to genetic variation. To identify single-nucleotide polymorphisms associated with general cognitive ability (g) in people with schizophrenia and control individuals. Genomewide association study, followed by analyses in unaffected siblings and independent schizophrenia samples, functional magnetic resonance imaging studies of brain physiology in vivo, and RNA sequencing in postmortem brain samples. The discovery cohort and unaffected siblings were participants in the National Institute of Mental Health Clinical Brain Disorders Branch schizophrenia genetics studies. Additional schizophrenia cohorts were from psychiatric treatment settings in the United States, Japan, and Germany. The discovery cohort comprised 339 with schizophrenia and 363 community control participants. Follow-up analyses studied 147 unaffected siblings of the schizophrenia cases and independent schizophrenia samples including a total of an additional 668 participants. Imaging analyses included 87 schizophrenia cases and 397 control individuals. Brain tissue samples were available for 64 cases and 61 control individuals. We studied genomewide association with g, by group, in the discovery cohort. We used selected genotypes to test specific associations in unaffected siblings and independent schizophrenia samples. Imaging analyses focused on activation in the prefrontal cortex during working memory. Brain tissue studies yielded messenger RNA expression levels for RefSeq transcripts. The schizophrenia discovery cohort showed genomewide-significant association of g with polymorphisms in sodium channel gene SCN2A, accounting for 10.4% of g variance (rs10174400, P = 9.27 × 10(-10)). Control individuals showed a trend for g/genotype association with reversed allelic directionality. The genotype-by-group interaction was also genomewide

  14. Circulating microRNA Profile throughout the menstrual cycle.

    Kadri Rekker

    Full Text Available Normal physiological variables, such as age and gender, contribute to alterations in circulating microRNA (miRNA expression levels. The changes in the female body during the menstrual cycle can also be reflected in plasma miRNA expression levels. Therefore, this study aimed to determine the plasma miRNA profile of healthy women during the menstrual cycle and to assess which circulating miRNAs are derived from blood cells. The plasma miRNA expression profiles in nine healthy women were determined by quantitative real time PCR using Exiqon Human Panel I assays from four time-points of the menstrual cycle. This platform was also used for studying miRNAs from pooled whole blood RNA samples at the same four time-points. Our results indicated that circulating miRNA expression levels in healthy women were not significantly altered by the processes occurring during the menstrual cycle. No significant differences in plasma miRNA expression levels were observed between the menstrual cycle time-points, but the number of detected miRNAs showed considerable variation among the studied individuals. miRNA analysis from whole blood samples revealed that majority of miRNAs in plasma are derived from blood cells. The most abundant miRNA in plasma and blood was hsa-miR-451a, but a number of miRNAs were only detected in one or the other sample type. In conclusion, our data suggest that the changes in the female body during the menstrual cycle do not affect the expression of circulating miRNAs at measurable levels.

  15. Effect of escitalopram versus placebo on GRα messenger RNA expression in peripheral blood cells of healthy individuals with a family history of depression - a secondary outcome analysis from the randomized AGENDA trial

    Knorr, Ulla; Koefoed, Pernille; Gluud, Christian

    2016-01-01

    Background Selective serotonin reuptake inhibitors (SSRIs) are widely prescribed as first-line drugs for the treatment of depression. However, the mechanisms of action for SSRIs are unclear and besides neurotransmitter modulation may depend on modulation of the hypothalamic-pituitary-adrenal (HPA......) system. The glucocorticoid receptor (GR) isoform α plays an important role in the negative feedback regulation of the HPA axis and reduced GRα messenger RNA (mRNA) expression has been shown in mood disorder patients and first-degree relatives compared to healthy individuals with no family history...

  16. Preeclampsia: novel insights from global RNA profiling of trophoblast subpopulations.

    Gormley, Matthew; Ona, Katherine; Kapidzic, Mirhan; Garrido-Gomez, Tamara; Zdravkovic, Tamara; Fisher, Susan J

    2017-08-01

    The maternal signs of preeclampsia, which include the new onset of high blood pressure, can occur because of faulty placentation. We theorized that transcriptomic analyses of trophoblast subpopulations in situ would lend new insights into the role of these cells in preeclampsia pathogenesis. Our goal was to enrich syncytiotrophoblasts, invasive cytotrophoblasts, or endovascular cytotrophoblasts from the placentas of severe preeclampsia cases. Total RNA was subjected to global transcriptional profiling to identify RNAs that were misexpressed compared with controls. This was a cross-sectional analysis of placentas from women who had been diagnosed with severe preeclampsia. Gestational age-matched controls were placentas from women who had a preterm birth with no signs of infection. Laser microdissection enabled enrichment of syncytiotrophoblasts, invasive cytotrophoblasts, or endovascular cytotrophoblasts. After RNA isolation, a microarray approach was used for global transcriptional profiling. Immunolocalization identified changes in messenger RNA expression that carried over to the protein level. Differential expression of non-protein-coding RNAs was confirmed by in situ hybridization. A 2-way analysis of variance of non-coding RNA expression identified particular classes that distinguished trophoblasts in cases vs controls. Cajal body foci were visualized by coilin immunolocalization. Comparison of the trophoblast subtype data within each group (severe preeclampsia or noninfected preterm birth) identified many highly differentially expressed genes. They included molecules that are known to be expressed by each subpopulation, which is evidence that the method worked. Genes that were expressed differentially between the 2 groups, in a cell-type-specific manner, encoded a combination of molecules that previous studies associated with severe preeclampsia and those that were not known to be dysregulated in this pregnancy complication. Gene ontology analysis of the

  17. Detailed mapping of serotonin 5-HT{sub 1B} and 5-HT{sub 1D} receptor messenger RNA and ligand binding sites in guinea-pig brain and trigeminal ganglion: clues for function

    Leysen, J.E. [Graduate School Neurosciences, Amsterdam (Netherlands); Schotte, A.; Jurzak, M.; Luyten, W.H.M.L. [Department of Biochemical Pharmacology, Janssen Research Foundation, Beerse (Belgium); Voorn, P.; Bonaventure, P. [Graduate School Neurosciences, Amsterdam (Netherlands)

    1997-10-17

    The similar pharmacology of the 5-HT{sub 1B} and 5-HT{sub 1D} receptors, and the lack of selective compounds sufficiently distinguishing between the two receptor subtypes, have hampered functional studies on these receptors. In order to provide clues for differential functional roles of the two subtypes, we performed a parallel localization study throughout the guinea-pig brain and the trigeminal ganglia by means of quantitative in situ hybridization histochemistry (using [{sup 35}S]-labelled riboprobes probes for receptor messenger RNA) and receptor autoradiography (using a new radioligand [{sup 3}H]alniditan).The anatomical patterns of 5-HT{sub 1B} and 5-HT{sub 1D} receptor messenger RNA were quite different. While 5-HT{sub 1B} receptor messenger RNA was abundant throughout the brain (with highest levels in the striatum, nucleus accumbens, olfactory tubercle, cortex, hypothalamus, hippocampal formation, amygdala, thalamus, dorsal raphe and cerebellum), 5-HT{sub 1D} receptor messenger RNA exhibited a more restricted pattern; it was found mainly in the olfactory tubercle, entorhinal cortex, dorsal raphe, cerebellum, mesencephalic trigeminal nucleus and in the trigeminal ganglion. The density of 5-HT{sub 1B/1D} binding sites (combined) obtained with [{sup 3}H]alniditan autoradiography was high in the substantia nigra, superior colliculus and globus pallidus, whereas lower levels were detected in the caudate-putamen, hypothalamus, hippocampal formation, amygdala, thalamus and central gray. This distribution pattern was indistinguishable from specific 5-HT{sub 1B} receptor labelling in the presence of ketanserin under conditions to occlude 5-HT{sub 1D} receptor labelling; hence the latter were below detection level. Relationships between the regional distributions of the receptor messenger RNAs and binding sites and particular neuroanatomical pathways are discussed with respect to possible functional roles of the 5-HT{sub 1B} and 5-HT{sub 1D} receptors. (Copyright (c

  18. MicroRNA expression profiling of the porcine developing brain

    Podolska, Agnieszka; Kaczkowski, Bogumil; Busk, Peter Kamp

    2011-01-01

    MicroRNAs are small, non-coding RNA molecules that regulate gene expression at the post-transcriptional level and play an important role in the control of developmental and physiological processes. In particular, the developing brain contains an impressive diversity of microRNAs. Most micro...... and the growth curve when compared to humans. Considering these similarities, studies examining microRNA expression during porcine brain development could potentially be used to predict the expression profile and role of microRNAs in the human brain....

  19. Schizosaccharomyces pombe Polysome Profile Analysis and RNA Purification.

    Wolf, Dieter A; Bähler, Jürg; Wise, Jo Ann

    2017-04-03

    Polysome profile analysis is widely used by investigators studying the mechanism and regulation of translation. The method described here uses high-velocity centrifugation of whole cell extracts on linear sucrose gradients to separate 40S and 60S ribosomal subunits from 80S monosomes and polysomes. Cycloheximide is included in the lysis buffer to "freeze" polysomes by blocking translation. After centrifugation, the gradient is fractionated and RNA (and/or protein) is prepared from each fraction for subsequent analysis of individual species using northern or western blots. The entire RNA population in each fraction can be analyzed by hybridization to microarrays or by high-throughput RNA sequencing, and the proteins present can be identified by mass spectrometry analysis. © 2017 Cold Spring Harbor Laboratory Press.

  20. Cytochrome c oxidase subunit 1-based human RNA quantification to enhance mRNA profiling in forensic biology

    Dong Zhao

    2017-01-01

    Full Text Available RNA analysis offers many potential applications in forensic science, and molecular identification of body fluids by analysis of cell-specific RNA markers represents a new technique for use in forensic cases. However, due to the nature of forensic materials that often admixed with nonhuman cellular components, human-specific RNA quantification is required for the forensic RNA assays. Quantification assay for human RNA has been developed in the present study with respect to body fluid samples in forensic biology. The quantitative assay is based on real-time reverse transcription-polymerase chain reaction of mitochondrial RNA cytochrome c oxidase subunit I and capable of RNA quantification with high reproducibility and a wide dynamic range. The human RNA quantification improves the quality of mRNA profiling in the identification of body fluids of saliva and semen because the quantification assay can exclude the influence of nonhuman components and reduce the adverse affection from degraded RNA fragments.

  1. Profiling microRNA expression in bovine alveolar macrophages using RNA-seq.

    Vegh, Peter; Foroushani, Amir B K; Magee, David A; McCabe, Matthew S; Browne, John A; Nalpas, Nicolas C; Conlon, Kevin M; Gordon, Stephen V; Bradley, Daniel G; MacHugh, David E; Lynn, David J

    2013-10-01

    MicroRNAs (miRNAs) are important regulators of gene expression and are known to play a key role in regulating both adaptive and innate immunity. Bovine alveolar macrophages (BAMs) help maintain lung homeostasis and constitute the front line of host defense against several infectious respiratory diseases, such as bovine tuberculosis. Little is known, however, about the role miRNAs play in these cells. In this study, we used a high-throughput sequencing approach, RNA-seq, to determine the expression levels of known and novel miRNAs in unchallenged BAMs isolated from lung lavages of eight different healthy Holstein-Friesian male calves. Approximately 80 million sequence reads were generated from eight BAM miRNA Illumina sequencing libraries, and 80 miRNAs were identified as being expressed in BAMs at a threshold of at least 100 reads per million (RPM). The expression levels of miRNAs varied over a large dynamic range, with a few miRNAs expressed at very high levels (up to 800,000RPM), and the majority lowly expressed. Notably, many of the most highly expressed miRNAs in BAMs have known roles in regulating immunity in other species (e.g. bta-let-7i, bta-miR-21, bta-miR-27, bta-miR-99b, bta-miR-146, bta-miR-147, bta-miR-155 and bta-miR-223). The most highly expressed miRNA in BAMs was miR-21, which has been shown to regulate the expression of antimicrobial peptides in Mycobacterium leprae-infected human monocytes. Furthermore, the predicted target genes of BAM-expressed miRNAs were found to be statistically enriched for roles in innate immunity. In addition to profiling the expression of known miRNAs, the RNA-seq data was also analysed to identify potentially novel bovine miRNAs. One putatively novel bovine miRNA was identified. To the best of our knowledge, this is the first RNA-seq study to profile miRNA expression in BAMs and provides an important reference dataset for investigating the regulatory roles miRNAs play in this important immune cell type. Copyright

  2. Rapid and inexpensive body fluid identification by RNA profiling-based multiplex High Resolution Melt (HRM) analysis.

    Hanson, Erin K; Ballantyne, Jack

    2013-01-01

    Positive identification of the nature of biological material present on evidentiary items can be crucial for understanding the circumstances surrounding a crime. However, traditional protein-based methods do not permit the identification of all body fluids and tissues, and thus molecular based strategies for the conclusive identification of all forensically relevant biological fluids and tissues need to be developed. Messenger RNA (mRNA) profiling is an example of such a molecular-based approach. Current mRNA body fluid identification assays involve capillary electrophoresis (CE) or quantitative RT-PCR (qRT-PCR) platforms, each with its own limitations. Both platforms require the use of expensive fluorescently labeled primers or probes. CE-based assays require separate amplification and detection steps thus increasing the analysis time. For qRT-PCR assays, only 3-4 markers can be included in a single reaction since each requires a different fluorescent dye. To simplify mRNA profiling assays, and reduce the time and cost of analysis, we have developed single- and multiplex body fluid High Resolution Melt (HRM) assays for the identification of common forensically relevant biological fluids and tissues. The incorporated biomarkers include IL19 (vaginal secretions), IL1F7 (skin), ALAS2 (blood), MMP10 (menstrual blood), HTN3 (saliva) and TGM4 (semen).  The HRM assays require only unlabeled PCR primers and a single saturating intercalating fluorescent dye (Eva Green). Each body-fluid-specific marker can easily be identified by the presence of a distinct melt peak. Usually, HRM assays are used to detect variants or isoforms for a single gene target. However, we have uniquely developed duplex and triplex HRM assays to permit the simultaneous detection of multiple targets per reaction. Here we describe the development and initial performance evaluation of the developed HRM assays. The results demonstrate the potential use of HRM assays for rapid, and relatively inexpensive

  3. tRNA modification profiles of the fast-proliferating cancer cells

    Dong, Chao; Niu, Leilei; Song, Wei [State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Department of Obstetrics and Gynecology, Peking University Third Hospital, Peking University, Beijing 100191 (China); Xiong, Xin; Zhang, Xianhua [Departmentof Pharmacy, Peking University Third Hospital, Peking University, Beijing 100191 (China); Zhang, Zhenxi; Yang, Yi; Yi, Fan [State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Department of Obstetrics and Gynecology, Peking University Third Hospital, Peking University, Beijing 100191 (China); Zhan, Jun; Zhang, Hongquan [Department of Anatomy, Histology and Embryology, Laboratory of Molecular Cell Biology and Tumor Biology, Peking University, Beijing 100191 (China); Yang, Zhenjun; Zhang, Li-He [State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Department of Obstetrics and Gynecology, Peking University Third Hospital, Peking University, Beijing 100191 (China); Zhai, Suodi [Departmentof Pharmacy, Peking University Third Hospital, Peking University, Beijing 100191 (China); Li, Hua, E-mail: huali88@sina.com [State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Department of Obstetrics and Gynecology, Peking University Third Hospital, Peking University, Beijing 100191 (China); Ye, Min, E-mail: yemin@bjmu.edu.cn [State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Department of Obstetrics and Gynecology, Peking University Third Hospital, Peking University, Beijing 100191 (China); Du, Quan, E-mail: quan.du@pku.edu.cn [State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Department of Obstetrics and Gynecology, Peking University Third Hospital, Peking University, Beijing 100191 (China)

    2016-08-05

    Despite the recent progress in RNA modification study, a comprehensive modification profile is still lacking for mammalian cells. Using a quantitative HPLC/MS/MS assay, we present here a study where RNA modifications are examined in term of the major RNA species. With paired slow- and fast-proliferating cell lines, distinct RNA modification profiles are first revealed for diverse RNA species. Compared to mRNAs, increased ribose and nucleobase modifications are shown for the highly-structured tRNAs and rRNAs, lending support to their contribution to the formation of high-order structures. This study also reveals a dynamic tRNA modification profile in the fast-proliferating cells. In addition to cultured cells, this unique tRNA profile has been further confirmed with endometrial cancers and their adjacent normal tissues. Taken together, the results indicate that tRNA is a actively regulated RNA species in the fast-proliferating cancer cells, and suggest that they may play a more active role in biological process than expected. -- Highlights: •RNA modifications were first examined in term of the major RNA species. •A dynamic tRNA modifications was characterized for the fast-proliferating cells. •The unique tRNA profile was confirmed with endometrial cancers and their adjacent normal tissues. •tRNA was predicted as an actively regulated RNA species in the fast-proliferating cancer cells.

  4. tRNA modification profiles of the fast-proliferating cancer cells

    Dong, Chao; Niu, Leilei; Song, Wei; Xiong, Xin; Zhang, Xianhua; Zhang, Zhenxi; Yang, Yi; Yi, Fan; Zhan, Jun; Zhang, Hongquan; Yang, Zhenjun; Zhang, Li-He; Zhai, Suodi; Li, Hua; Ye, Min; Du, Quan

    2016-01-01

    Despite the recent progress in RNA modification study, a comprehensive modification profile is still lacking for mammalian cells. Using a quantitative HPLC/MS/MS assay, we present here a study where RNA modifications are examined in term of the major RNA species. With paired slow- and fast-proliferating cell lines, distinct RNA modification profiles are first revealed for diverse RNA species. Compared to mRNAs, increased ribose and nucleobase modifications are shown for the highly-structured tRNAs and rRNAs, lending support to their contribution to the formation of high-order structures. This study also reveals a dynamic tRNA modification profile in the fast-proliferating cells. In addition to cultured cells, this unique tRNA profile has been further confirmed with endometrial cancers and their adjacent normal tissues. Taken together, the results indicate that tRNA is a actively regulated RNA species in the fast-proliferating cancer cells, and suggest that they may play a more active role in biological process than expected. -- Highlights: •RNA modifications were first examined in term of the major RNA species. •A dynamic tRNA modifications was characterized for the fast-proliferating cells. •The unique tRNA profile was confirmed with endometrial cancers and their adjacent normal tissues. •tRNA was predicted as an actively regulated RNA species in the fast-proliferating cancer cells.

  5. rRNA:mRNA pairing alters the length and the symmetry of mRNA-protected fragments in ribosome profiling experiments

    O?Connor, Patrick B. F.; Li, Gene-Wei; Weissman, Jonathan S.; Atkins, John F.; Baranov, Pavel V.

    2013-01-01

    Motivation: Ribosome profiling is a new technique that allows monitoring locations of translating ribosomes on mRNA at a whole transcriptome level. A recent ribosome profiling study demonstrated that internal Shine?Dalgarno (SD) sequences have a major global effect on translation rates in bacteria: ribosomes pause at SD sites in mRNA. Therefore, it is important to understand how SD sites effect mRNA movement through the ribosome and generation of ribosome footprints. Results: Here, we provide...

  6. microRNA expression profiles in human peripheral blood lymphocytes cultured in modeled microgravity

    National Aeronautics and Space Administration — In the present study we analyzed miRNA and mRNA expression profiles in human peripheral blood lymphocytes (PBLs) incubated in microgravity condition simulated by a...

  7. Changes of microRNA profile and microRNA-mRNA regulatory network in bones of ovariectomized mice.

    An, Jee Hyun; Ohn, Jung Hun; Song, Jung Ah; Yang, Jae-Yeon; Park, Hyojung; Choi, Hyung Jin; Kim, Sang Wan; Kim, Seong Yeon; Park, Woog-Yang; Shin, Chan Soo

    2014-03-01

    Growing evidence shows the possibility of a role of microRNAs (miRNA) in regulating bone mass. We investigated the change of miRNAs and mRNA expression profiles in bone tissue in an ovariectomized mice model and evaluated the regulatory mechanism of bone mass mediated by miRNAs in an estrogen-deficiency state. Eight-week-old female C3H/HeJ mice underwent ovariectomy (OVX) or sham operation (Sham-op), and their femur and tibia were harvested to extract total bone RNAs after 4 weeks for microarray analysis. Eight miRNAs (miR-127, -133a, -133a*, -133b, -136, -206, -378, -378*) were identified to be upregulated after OVX, whereas one miRNA (miR-204) was downregulated. Concomitant analysis of mRNA microarray revealed that 658 genes were differentially expressed between OVX and Sham-op mice. Target prediction of differentially expressed miRNAs identified potential targets, and integrative analysis using the mRNA microarray results showed that PPARγ and CREB pathways are activated in skeletal tissues after ovariectomy. Among the potential candidates of miRNA, we further studied the role of miR-127 in vitro, which exhibited the greatest changes after OVX. We also studied the effects of miR-136, which has not been studied in the context of bone mass regulation. Transfection of miR-127 inhibitor has enhanced osteoblastic differentiation in UAMS-32 cells as measured by alkaline phosphatase activities and mRNA expression of osteoblast-specific genes, whereas miR-136 precursor has inhibited osteoblastic differentiation. Furthermore, transfection of both miR-127 and miR-136 inhibitors enhanced the osteocyte-like morphological changes and survival in MLO-Y4 cells, whereas precursors of miR-127 and -136 have aggravated dexamethasone-induced cell death. Both of the precursors enhanced osteoclastic differentiation in bone marrow macrophages, indicating that both miR-127 and -136 are negatively regulating bone mass. Taken together, these results suggest a novel insight into the

  8. Fragile X Mental Retardation Protein and Dendritic Local Translation of the Alpha Subunit of the Calcium/Calmodulin-Dependent Kinase II Messenger RNA Are Required for the Structural Plasticity Underlying Olfactory Learning.

    Daroles, Laura; Gribaudo, Simona; Doulazmi, Mohamed; Scotto-Lomassese, Sophie; Dubacq, Caroline; Mandairon, Nathalie; Greer, Charles August; Didier, Anne; Trembleau, Alain; Caillé, Isabelle

    2016-07-15

    In the adult brain, structural plasticity allowing gain or loss of synapses remodels circuits to support learning. In fragile X syndrome, the absence of fragile X mental retardation protein (FMRP) leads to defects in plasticity and learning deficits. FMRP is a master regulator of local translation but its implication in learning-induced structural plasticity is unknown. Using an olfactory learning task requiring adult-born olfactory bulb neurons and cell-specific ablation of FMRP, we investigated whether learning shapes adult-born neuron morphology during their synaptic integration and its dependence on FMRP. We used alpha subunit of the calcium/calmodulin-dependent kinase II (αCaMKII) mutant mice with altered dendritic localization of αCaMKII messenger RNA, as well as a reporter of αCaMKII local translation to investigate the role of this FMRP messenger RNA target in learning-dependent structural plasticity. Learning induces profound changes in dendritic architecture and spine morphology of adult-born neurons that are prevented by ablation of FMRP in adult-born neurons and rescued by an metabotropic glutamate receptor 5 antagonist. Moreover, dendritically translated αCaMKII is necessary for learning and associated structural modifications and learning triggers an FMRP-dependent increase of αCaMKII dendritic translation in adult-born neurons. Our results strongly suggest that FMRP mediates structural plasticity of olfactory bulb adult-born neurons to support olfactory learning through αCaMKII local translation. This reveals a new role for FMRP-regulated dendritic local translation in learning-induced structural plasticity. This might be of clinical relevance for the understanding of critical periods disruption in autism spectrum disorder patients, among which fragile X syndrome is the primary monogenic cause. Copyright © 2016 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.

  9. Intracellular microRNA profiles form in the Xenopus laevis oocyte that may contribute to asymmetric cell division

    Šídová, Monika; Šindelka, Radek; Castoldi, M.; Benes, V.; Kubista, Mikael

    2015-01-01

    Roč. 5, č. 11157 (2015) ISSN 2045-2322 R&D Projects: GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:86652036 Keywords : VG1 MESSENGER-RNA * VEGETAL CORTEX * FROG OOCYTE Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 5.228, year: 2015

  10. MicroRNA transcriptome profiles during swine skeletal muscle development

    Sonstegard Tad S

    2009-02-01

    Full Text Available Abstract Background MicroRNA (miR are a class of small RNAs that regulate gene expression by inhibiting translation of protein encoding transcripts. To evaluate the role of miR in skeletal muscle of swine, global microRNA abundance was measured at specific developmental stages including proliferating satellite cells, three stages of fetal growth, day-old neonate, and the adult. Results Twelve potential novel miR were detected that did not match previously reported sequences. In addition, a number of miR previously reported to be expressed in mammalian muscle were detected, having a variety of abundance patterns through muscle development. Muscle-specific miR-206 was nearly absent in proliferating satellite cells in culture, but was the highest abundant miR at other time points evaluated. In addition, miR-1 was moderately abundant throughout developmental stages with highest abundance in the adult. In contrast, miR-133 was moderately abundant in adult muscle and either not detectable or lowly abundant throughout fetal and neonate development. Changes in abundance of ubiquitously expressed miR were also observed. MiR-432 abundance was highest at the earliest stage of fetal development tested (60 day-old fetus and decreased throughout development to the adult. Conversely, miR-24 and miR-27 exhibited greatest abundance in proliferating satellite cells and the adult, while abundance of miR-368, miR-376, and miR-423-5p was greatest in the neonate. Conclusion These data present a complete set of transcriptome profiles to evaluate miR abundance at specific stages of skeletal muscle growth in swine. Identification of these miR provides an initial group of miR that may play a vital role in muscle development and growth.

  11. Embryonic miRNA profiles of normal and ectopic pregnancies.

    Francisco Dominguez

    Full Text Available Our objective was to investigate the miRNA profile of embryonic tissues in ectopic pregnancies (EPs and controlled abortions (voluntary termination of pregnancy; VTOP. Twenty-three patients suffering from tubal EP and twenty-nine patients with a normal ongoing pregnancy scheduled for a VTOP were recruited. Embryonic tissue samples were analyzed by miRNA microarray and further validated by real time PCR. Microarray studies showed that four miRNAs were differentially downregulated (hsa-mir-196b, hsa-mir-30a, hsa-mir-873, and hsa-mir-337-3p and three upregulated (hsa-mir-1288, hsa-mir-451, and hsa-mir-223 in EP compared to control tissue samples. Hsa-miR-196, hsa-miR-223, and hsa-miR-451 were further validated by real time PCR in a wider population of EP and control samples. We also performed a computational analysis to identify the gene targets and pathways which might be modulated by these three differentially expressed miRNAs. The most significant pathways found were the mucin type O-glycan biosynthesis and the ECM-receptor-interaction pathways. We also checked that the dysregulation of these three miRNAs was able to alter the expression of the gene targets in the embryonic tissues included in these pathways such as GALNT13 and ITGA2 genes. In conclusion, analysis of miRNAs in ectopic and eutopic embryonic tissues shows different expression patterns that could modify pathways which are critical for correct implantation, providing new insights into the understanding of ectopic implantation in humans.

  12. Regulation of human histone gene expression: transcriptional and posttranscriptional control in the coupling of histone messenger RNA stability with DNA replication

    Baumbach, L.L.; Stein, G.S.; Stein, J.L.

    1987-01-01

    The extent to which transcriptional and posttranscriptional regulation contributes to the coupling of histone gene expression and DNA replication was examined during the cell cycle in synchronized HeLa S3 cells. Rates of transcription were determined in vitro in isolated nuclei. A 3-5-fold increase in cell cycle dependent histone gene transcription was observed in early S phase, prior to the peak of DNA synthesis. This result is consistent with a previous determination of histone mRNA synthesis in intact cells. The transcription of these genes did not change appreciably after inhibition of DNA replication by hydroxyurea treatment, although Northern blot analysis indicated that cellular levels of histone mRNA decreased rapidly in the presence of the drug. Total cellular levels of histone mRNA closely parallel the rate of DNA synthesis as a function of cell cycle progression, reaching a maximal 20-fold increase as compared with non S phase levels. This DNA synthesis dependent accumulation of histone mRNA occurs predominantly in the cytoplasm and appears to be mediated primarily by control of histone mRNA stability. Changes in nuclear histone mRNA levels were less pronounced. These combined observations suggest that both transcriptional regulation and posttranscriptional regulation contribute toward control of the cell cycle dependent accumulation of histone mRNA during S phase, while the stability of histone mRNA throughout S phase and the selective turnover of histone mRNAs, either at the natural termination of S phase or following inhibition of DNA synthesis, are posttranscriptionally regulated

  13. Quantitation of O6-methylguanine-DNA methyltransferase gene messenger RNA in gliomas by means of real-time RT-PCR and clinical response to nitrosoureas.

    Tanaka, Satoshi; Oka, Hidehiro; Fujii, Kiyotaka; Watanabe, Kaoru; Nagao, Kumi; Kakimoto, Atsushi

    2005-09-01

    1. O6-methylguanine-DNA methyltransferase (MGMT) mRNA was measured in 50 malignant gliomas that had received 1-(4-amino-2-methyl-5-pyrimidynyl) methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride (ACNU) after the resection of the tumor by real-time reverse transcription-polymerase chain reaction (RT-PCR) using TaqMan probe. 2. The mean absolute value of MGMTmRNA normalized to the level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) for 50 tumors was 1.29 x 10(4)+/- 1.28 x 10(4) copy/microg RNA (mean +/- SD). The amount of MGMTmRNA less than 6 x 10(3) copy/microg RNA was the most significant factor in predicting the initial effect of treatment with ACNU by multi-variant regression analysis (p = 0.0157). 3. These results suggest that quantitation of MGMTmRNA is the excellent method for predicting for the effect of ACNU in glioma therapy.

  14. Integration of extracellular RNA profiling data using metadata, biomedical ontologies and Linked Data technologies

    Sai Lakshmi Subramanian

    2015-08-01

    Full Text Available The large diversity and volume of extracellular RNA (exRNA data that will form the basis of the exRNA Atlas generated by the Extracellular RNA Communication Consortium pose a substantial data integration challenge. We here present the strategy that is being implemented by the exRNA Data Management and Resource Repository, which employs metadata, biomedical ontologies and Linked Data technologies, such as Resource Description Framework to integrate a diverse set of exRNA profiles into an exRNA Atlas and enable integrative exRNA analysis. We focus on the following three specific data integration tasks: (a selection of samples from a virtual biorepository for exRNA profiling and for inclusion in the exRNA Atlas; (b retrieval of a data slice from the exRNA Atlas for integrative analysis and (c interpretation of exRNA analysis results in the context of pathways and networks. As exRNA profiling gains wide adoption in the research community, we anticipate that the strategies discussed here will increasingly be required to enable data reuse and to facilitate integrative analysis of exRNA data.

  15. Integration of extracellular RNA profiling data using metadata, biomedical ontologies and Linked Data technologies.

    Subramanian, Sai Lakshmi; Kitchen, Robert R; Alexander, Roger; Carter, Bob S; Cheung, Kei-Hoi; Laurent, Louise C; Pico, Alexander; Roberts, Lewis R; Roth, Matthew E; Rozowsky, Joel S; Su, Andrew I; Gerstein, Mark B; Milosavljevic, Aleksandar

    2015-01-01

    The large diversity and volume of extracellular RNA (exRNA) data that will form the basis of the exRNA Atlas generated by the Extracellular RNA Communication Consortium pose a substantial data integration challenge. We here present the strategy that is being implemented by the exRNA Data Management and Resource Repository, which employs metadata, biomedical ontologies and Linked Data technologies, such as Resource Description Framework to integrate a diverse set of exRNA profiles into an exRNA Atlas and enable integrative exRNA analysis. We focus on the following three specific data integration tasks: (a) selection of samples from a virtual biorepository for exRNA profiling and for inclusion in the exRNA Atlas; (b) retrieval of a data slice from the exRNA Atlas for integrative analysis and (c) interpretation of exRNA analysis results in the context of pathways and networks. As exRNA profiling gains wide adoption in the research community, we anticipate that the strategies discussed here will increasingly be required to enable data reuse and to facilitate integrative analysis of exRNA data.

  16. Messengers of the universe

    Becker, J.K.; Spurio, M.

    2011-01-01

    The observation of the solar neutrinos and of a neutrino burst from the supernova explosion 1987A opened a new observation field which in the next years could be complemented with the detection of astrophysical highenergy neutrinos. Neutrino astronomy is a young discipline derived from the fundamental necessity of extending conventional astronomy beyond the usual electro-magnetic messengers. This is a summary of recent results on those new 'messengers of the universe', based on the presentations in Branch IV of the Neutrino Oscillation Workshop 2010 (NOW2010).

  17. Interleukin 18 messenger RNA and proIL-18 protein expression in chorioamniotic membranes from pregnant women with preterm prelabor rupture of membranes.

    Polettini, Jossimara; Vieira, Eliane Passarelli; Santos, Mariana Perlati dos; Peraçoli, José Carlos; Witkin, Steven S; da Silva, Márcia Guimarães

    2012-04-01

    To quantify the expression of IL-18 mRNA and protein in the chorioamniotic membranes of pregnant women with PPROM and correlate expression with histological chorioamnionitis. A case control study that included 42 pregnant women not in labor in the following groups: PPROM (n=28) and controls with intact membranes submitted to selective cesarean section at term (n=14). Expression of IL-18 mRNA in chorioamniotic membranes was determined by real-time polymerase chain reaction, and IL-18 protein expression was measured by western blot. Histopathological analyses and immunolocalization of IL-18 by immunohistochemistry were also performed. Analyses were performed using the Mann-Whitney or Fisher's exact tests and the group effect was considered significant if the adjusted p-values were <0.05 and the magnitude of change was greater than 2-fold for mRNA expression. IL-18 mRNA was present in 100% of samples and no difference in expression was observed between term vs. PPROM membranes (fold-change 0.12; p=0.88). In the PPROM group, no difference was observed in IL-18 mRNA regarding gestational age (fold-change 0.11; p=0.42) or the presence of histological chorioamnionitis (fold-change 0.26; p=0.15). ProIL-18 was present in all samples. IL-18 was immunolocalized to amnion, chorion and decidua cells, with intense immunohistochemical staining at the choriodecidual junction. Chorioamniotic membranes are sources of IL-18 mRNA and proIL-18, and their expression is unrelated to PPROM or histological chorioamnionitis. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  18. Integrated microRNA and gene expression profiling reveals the crucial miRNAs in curcumin anti-lung cancer cell invasion.

    Zhan, Jian-Wei; Jiao, De-Min; Wang, Yi; Song, Jia; Wu, Jin-Hong; Wu, Li-Jun; Chen, Qing-Yong; Ma, Sheng-Lin

    2017-09-01

    Curcumin (diferuloylmethane) has chemopreventive and therapeutic properties against many types of tumors, both in vitro and in vivo. Previous reports have shown that curcumin exhibits anti-invasive activities, but the mechanisms remain largely unclear. In this study, both microRNA (miRNA) and messenger RNA (mRNA) expression profiles were used to characterize the anti-metastasis mechanisms of curcumin in human non-small cell lung cancer A549 cell line. Microarray analysis revealed that 36 miRNAs were differentially expressed between the curcumin-treated and control groups. miR-330-5p exhibited maximum upregulation, while miR-25-5p exhibited maximum downregulation in the curcumin treatment group. mRNA expression profiles and functional analysis indicated that 226 differentially expressed mRNAs belonged to different functional categories. Significant pathway analysis showed that mitogen-activated protein kinase, transforming growth factor-β, and Wnt signaling pathways were significantly downregulated. At the same time, axon guidance, glioma, and ErbB tyrosine kinase receptor signaling pathways were significantly upregulated. We constructed a miRNA gene network that contributed to the curcumin inhibition of metastasis in lung cancer cells. let-7a-3p, miR-1262, miR-499a-5p, miR-1276, miR-331-5p, and miR-330-5p were identified as key microRNA regulators in the network. Finally, using miR-330-5p as an example, we confirmed the role of miR-330-5p in mediating the anti-migration effect of curcumin, suggesting the importance of miRNAs in the regulation of curcumin biological activity. Our findings provide new insights into the anti-metastasis mechanism of curcumin in lung cancer. © 2017 The Authors. Thoracic Cancer published by China Lung Oncology Group and John Wiley & Sons Australia, Ltd.

  19. IL-2 induction of IL-1 beta mRNA expression in monocytes. Regulation by agents that block second messenger pathways

    Kovacs, E J; Brock, B; Varesio, L

    1989-01-01

    We have previously shown that in mixed cultures of PBL incubation with human rIL-2 induces the rapid expression of IL-1 alpha and IL-1 beta mRNA. Because studies have demonstrated that IL-2R can be expressed on the surface of human peripheral blood monocytes, we chose to investigate whether IL-1 ...

  20. Effects of dietary nitrogen concentration on messenger RNA expression and protein abundance of urea transporter-B and aquaporins in ruminal papillae from lactating Holstein cows

    Røjen, Betina Amdisen; Poulsen, Søren Brandt; Theil, Peter Kappel

    2011-01-01

    To test the hypothesis that dietary N concentrations affect gut epithelial urea transport by modifying the expression of urea transporter B (UT-B) and aquaporins (AQP), the mRNA expression and protein abundance of UT-B and AQP3, AQP7, AQP8, and AQP10 were investigated in ruminal papillae from 9...... lactating dairy cows. Ruminal papillae were harvested from cows fed low N (12.9% crude protein) and high N (17.1% crude protein) diets in a crossover design with 21-d periods. The mRNA expression was determined by real-time reverse transcription-PCR and protein abundance by immunoblotting. The m......RNA expression of UT-B was not affected by dietary treatment, whereas mRNA expression of AQP3, 7, and 10 were greater in the high N compared with the low N fed cows. Using peptide-derived rabbit antibodies to cow AQP3, 7, and 8, immunoblotting revealed bands of approximately 27, 27, and 24 kDa in ruminal...

  1. miRNA and mRNA Expression Profiles Reveal Insight into Chitosan-Mediated Regulation of Plant Growth.

    Zhang, Xiaoqian; Li, Kecheng; Xing, Ronge; Liu, Song; Chen, Xiaolin; Yang, Haoyue; Li, Pengcheng

    2018-04-18

    Chitosan has been numerously studied as a plant growth regulator and stress tolerance inducer. To investigate the roles of chitosan as bioregulator on plant and unravel its possible metabolic responses mechanisms, we simultaneously investigated mRNAs and microRNAs (miRNAs) expression profiles of wheat seedlings in response to chitosan heptamer. We found 400 chitosan-responsive differentially expressed genes, including 268 up-regulated and 132 down-regulated mRNAs, many of which were related to photosynthesis, primary carbon and nitrogen metabolism, defense responses, and transcription factors. Moreover, miRNAs also participate in chitosan-mediated regulation on plant growth. We identified 87 known and 21 novel miRNAs, among which 56 miRNAs were induced or repressed by chitosan heptamer, such as miRNA156, miRNA159a, miRNA164, miRNA171a, miRNA319, and miRNA1127. The integrative analysis of miRNA and mRNA expression profiles in this case provides fundamental information for further investigation of regulation mechanisms of chitosan on plant growth and will facilitate its application in agriculture.

  2. MicroRNA Expression Profile in Penile Cancer Revealed by Next-Generation Small RNA Sequencing.

    Li Zhang

    Full Text Available Penile cancer (PeCa is a relatively rare tumor entity but possesses higher morbidity and mortality rates especially in developing countries. To date, the concrete pathogenic signaling pathways and core machineries involved in tumorigenesis and progression of PeCa remain to be elucidated. Several studies suggested miRNAs, which modulate gene expression at posttranscriptional level, were frequently mis-regulated and aberrantly expressed in human cancers. However, the miRNA profile in human PeCa has not been reported before. In this present study, the miRNA profile was obtained from 10 fresh penile cancerous tissues and matched adjacent non-cancerous tissues via next-generation sequencing. As a result, a total of 751 and 806 annotated miRNAs were identified in normal and cancerous penile tissues, respectively. Among which, 56 miRNAs with significantly different expression levels between paired tissues were identified. Subsequently, several annotated miRNAs were selected randomly and validated using quantitative real-time PCR. Compared with the previous publications regarding to the altered miRNAs expression in various cancers and especially genitourinary (prostate, bladder, kidney, testis cancers, the most majority of deregulated miRNAs showed the similar expression pattern in penile cancer. Moreover, the bioinformatics analyses suggested that the putative target genes of differentially expressed miRNAs between cancerous and matched normal penile tissues were tightly associated with cell junction, proliferation, growth as well as genomic instability and so on, by modulating Wnt, MAPK, p53, PI3K-Akt, Notch and TGF-β signaling pathways, which were all well-established to participate in cancer initiation and progression. Our work presents a global view of the differentially expressed miRNAs and potentially regulatory networks of their target genes for clarifying the pathogenic transformation of normal penis to PeCa, which research resource also

  3. A high ratio of insulin-like growth factor II/insulin-like growth factor binding protein 2 messenger RNA as a marker for anaplasia in meningiomas.

    Nordqvist, A C; Peyrard, M; Pettersson, H; Mathiesen, T; Collins, V P; Dumanski, J P; Schalling, M

    1997-07-01

    Insulin-like growth factors (IGFs) I and II have been implicated as autocrine or paracrine growth promoters. These growth factors bind to specific receptors, and the response is modulated by interaction with IGF-binding proteins (IGFBPs). We observed a strong correlation between anaplastic/atypical histopathology and a high IGF-II/IGFBP-2 mRNA ratio in a set of 68 sporadic meningiomas. A strong correlation was also found between clinical outcome and IGF-II/IGFBP-2 ratio, whereas previously used histochemical markers were less correlated to outcome. We suggest that a high IGF-II/IGFBP-2 mRNA ratio may be a sign of biologically aggressive behavior in meningiomas that can influence treatment strategies. We propose that low IGFBP-2 levels in combination with increased levels of IGF-II would result in more free IGF-II and consequently greater stimulation of proliferation.

  4. Correlations between RNA and protein expression profiles in 23 human cell lines

    Pontén Fredrik

    2009-08-01

    Full Text Available Abstract Background The Central Dogma of biology holds, in famously simplified terms, that DNA makes RNA makes proteins, but there is considerable uncertainty regarding the general, genome-wide correlation between levels of RNA and corresponding proteins. Therefore, to assess degrees of this correlation we compared the RNA profiles (determined using both cDNA- and oligo-based microarrays and protein profiles (determined immunohistochemically in tissue microarrays of 1066 gene products in 23 human cell lines. Results A high mean correlation coefficient (0.52 was obtained from the pairwise comparison of RNA levels determined by the two platforms. Significant correlations, with correlation coefficients exceeding 0.445, between protein and RNA levels were also obtained for a third of the specific gene products. However, the correlation coefficients between levels of RNA and protein products of specific genes varied widely, and the mean correlations between the protein and corresponding RNA levels determined using the cDNA- and oligo-based microarrays were 0.25 and 0.20, respectively. Conclusion Significant correlations were found in one third of the examined RNA species and corresponding proteins. These results suggest that RNA profiling might provide indirect support to antibodies' specificity, since whenever a evident correlation between the RNA and protein profiles exists, this can sustain that the antibodies used in the immunoassay recognized their cognate antigens.

  5. mRNA profiling for the identification of blood-Results of a collaborative EDNAP exercise

    Haas, Cordula; Hanson, E; Bär, W

    2010-01-01

    of the laboratories had no prior experience with RNA. Despite some expected variation in sensitivity between laboratories, the method proved to be reproducible and sensitive using different analysis strategies. The results of this collaborative exercise support the potential use of mRNA profiling as an alternative......A collaborative exercise on mRNA profiling for the identification of blood was organized by the European DNA Profiling Group (EDNAP). Seven blood samples and one blood dilution series were analyzed by the participating laboratories for the reportedly blood-specific markers HBB, SPTB and PBGD, using...

  6. mRNA profiling for the identification of blood--Results of a collaborative EDNAP exercise

    Haas, C.; Hanson, E.; Bär, W.

    2011-01-01

    of the laboratories had no prior experience with RNA. Despite some expected variation in sensitivity between laboratories, the method proved to be reproducible and sensitive using different analysis strategies. The results of this collaborative exercise support the potential use of mRNA profiling as an alternative......A collaborative exercise on mRNA profiling for the identification of blood was organized by the European DNA Profiling Group (EDNAP). Seven blood samples and one blood dilution series were analyzed by the participating laboratories for the reportedly blood-specific markers HBB, SPTB and PBGD, using...

  7. Complementary DNA and derived amino acid sequence of the α subunit of human complement protein C8: evidence for the existence of a separate α subunit messenger RNA

    Rao, A.G.; Howard, O.M.Z.; Ng, S.C.; Whitehead, A.S.; Colten, H.R.; Sodetz, J.M.

    1987-01-01

    The entire amino acid sequence of the α subunit (M/sub r/ 64,000) of the eight component of complement (C8) was determined by characterizing cDNA clones isolated from a human liver cDNA library. Two clones with overlapping inserts of net length 2.44 kilobases (kb) were isolated and found to contain the entire α coding region [1659 base pairs (bp)]. The 5' end consists of an untranslated region and a leader sequence of 30 amino acids. This sequence contains an apparent initiation Met, signal peptide, and propeptide which ends with an arginine-rich sequence that is characteristic of proteolytic processing sites found in the pro form of protein precursors. The 3' untranslated region contains two polyadenylation signals and a poly(A)sequence. RNA blot analysis of total cellular RNA from the human hepatoma cell line HepG2 revealed a message size of ∼2.5 kb. Features of the 5' and 3' sequences and the message size suggest that a separate mRNA codes for α and argues against the occurrence of a single-chain precursor form of the disulfide-linked α-λ subunit found in mature C8. Analysis of the derived amino acid sequence revealed several membrane surface seeking domains and a possible transmembrane domain. Analysis of the carbohydrate composition indicates 1 or 2 asparagine-linked but no O-linked oligosaccharide chains, a result consistent with predictions from the amino acid sequence. Most significantly, it exhibits a striking overall homology to human C9, with values of 24% on the basis of identity and 46% when conserved substitutions are allowed. As described in an accompanying report this homology also extends to the β subunit of C8

  8. Transcriptional profiling of cells sorted by RNA abundance

    Klemm, Sandy; Semrau, Stefan; Wiebrands, Kay; Mooijman, Dylan; Faddah, Dina A; Jaenisch, Rudolf; van Oudenaarden, Alexander

    We have developed a quantitative technique for sorting cells on the basis of endogenous RNA abundance, with a molecular resolution of 10-20 transcripts. We demonstrate efficient and unbiased RNA extraction from transcriptionally sorted cells and report a high-fidelity transcriptome measurement of

  9. Integrated Analysis of Dysregulated ncRNA and mRNA Expression Profiles in Humans Exposed to Carbon Nanotubes.

    Anna A Shvedova

    Full Text Available As the application of carbon nanotubes (CNT in consumer products continues to rise, studies have expanded to determine the associated risks of exposure on human and environmental health. In particular, several lines of evidence indicate that exposure to multi-walled carbon nanotubes (MWCNT could pose a carcinogenic risk similar to asbestos fibers. However, to date the potential markers of MWCNT exposure are not yet explored in humans.In the present study, global mRNA and ncRNA expression profiles in the blood of exposed workers, having direct contact with MWCNT aerosol for at least 6 months (n = 8, were compared with expression profiles of non-exposed (n = 7 workers (e.g., professional and/or technical staff from the same manufacturing facility.Significant changes in the ncRNA and mRNA expression profiles were observed between exposed and non-exposed worker groups. An integrative analysis of ncRNA-mRNA correlations was performed to identify target genes, functional relationships, and regulatory networks in MWCNT-exposed workers. The coordinated changes in ncRNA and mRNA expression profiles revealed a set of miRNAs and their target genes with roles in cell cycle regulation/progression/control, apoptosis and proliferation. Further, the identified pathways and signaling networks also revealed MWCNT potential to trigger pulmonary and cardiovascular effects as well as carcinogenic outcomes in humans, similar to those previously described in rodents exposed to MWCNTs.This study is the first to investigate aberrant changes in mRNA and ncRNA expression profiles in the blood of humans exposed to MWCNT. The significant changes in several miRNAs and mRNAs expression as well as their regulatory networks are important for getting molecular insights into the MWCNT-induced toxicity and pathogenesis in humans. Further large-scale prospective studies are necessary to validate the potential applicability of such changes in mRNAs and miRNAs as prognostic markers

  10. Integrating microRNA and mRNA expression profiles in response to radiation-induced injury in rat lung

    Xie, Ling; Zhou, Jundong; Zhang, Shuyu; Chen, Qing; Lai, Rensheng; Ding, Weiqun; Song, ChuanJun; Meng, XingJun; Wu, Jinchang

    2014-01-01

    Exposure to radiation provokes cellular responses, which are likely regulated by gene expression networks. MicroRNAs are small non-coding RNAs, which regulate gene expression by promoting mRNA degradation or inhibiting protein translation. The expression patterns of both mRNA and miRNA during the radiation-induced lung injury (RILI) remain less characterized and the role of miRNAs in the regulation of this process has not been studied. The present study sought to evaluate miRNA and mRNA expression profiles in the rat lung after irradiation. Male Wistar rats were subjected to single dose irradiation with 20 Gy using 6 MV x-rays to the right lung. (A dose rate of 5 Gy/min was applied). Rats were sacrificed at 3, 12 and 26 weeks after irradiation, and morphological changes in the lung were examined by haematoxylin and eosin. The miRNA and mRNA expression profiles were evaluated by microarrays and followed by quantitative RT-PCR analysis. A cDNA microarray analysis found 2183 transcripts being up-regulated and 2917 transcripts down-regulated (P ≤ 0.05, ≥2.0 fold change) in the lung tissues after irradiation. Likewise, a miRNAs microarray analysis indicated 15 miRNA species being up-regulated and 8 down-regulated (P ≤ 0.05). Subsequent bioinformatics anal -yses of the differentially expressed mRNA and miRNAs revealed that alterations in mRNA expression following irradiation were negatively correlated with miRNAs expression. Our results provide evidence indicating that irradiation induces alterations of mRNA and miRNA expression in rat lung and that there is a negative correlation of mRNA and miRNA expression levels after irradiation. These findings significantly advance our understanding of the regulatory mechanisms underlying the pathophysiology of radiation-induced lung injury. In summary, RILI does not develop gradually in a linear process. In fact, different cell types interact via cytokines in a very complex network. Furthermore, this study suggests that

  11. Evaluation of a new high-dimensional miRNA profiling platform

    Lamblin Anne-Francoise

    2009-08-01

    Full Text Available Abstract Background MicroRNAs (miRNAs are a class of approximately 22 nucleotide long, widely expressed RNA molecules that play important regulatory roles in eukaryotes. To investigate miRNA function, it is essential that methods to quantify their expression levels be available. Methods We evaluated a new miRNA profiling platform that utilizes Illumina's existing robust DASL chemistry as the basis for the assay. Using total RNA from five colon cancer patients and four cell lines, we evaluated the reproducibility of miRNA expression levels across replicates and with varying amounts of input RNA. The beta test version was comprised of 735 miRNA targets of Illumina's miRNA profiling application. Results Reproducibility between sample replicates within a plate was good (Spearman's correlation 0.91 to 0.98 as was the plate-to-plate reproducibility replicates run on different days (Spearman's correlation 0.84 to 0.98. To determine whether quality data could be obtained from a broad range of input RNA, data obtained from amounts ranging from 25 ng to 800 ng were compared to those obtained at 200 ng. No effect across the range of RNA input was observed. Conclusion These results indicate that very small amounts of starting material are sufficient to allow sensitive miRNA profiling using the Illumina miRNA high-dimensional platform. Nonlinear biases were observed between replicates, indicating the need for abundance-dependent normalization. Overall, the performance characteristics of the Illumina miRNA profiling system were excellent.

  12. Short communication: Acute but transient increase in serum insulin reduces messenger RNA expression of hepatic enzymes associated with progesterone catabolism in dairy cows.

    Vieira, F V R; Cooke, R F; Aboin, A C; Lima, P; Vasconcelos, J L M

    2013-02-01

    The objective of this experiment was to evaluate the effects of glucose infusion on serum concentrations of glucose, insulin, and progesterone (P4), as well as mRNA expression of hepatic CYP2C19 and CYP3A4 in nonlactating, ovariectomized cows in adequate nutritional status. Eight Gir × Holstein cows were maintained on a low-quality Brachiaria brizantha pasture with reduced forage availability, but they individually received, on average, 3 kg/cow daily (as fed) of a corn-based concentrate from d -28 to 0 of the experiment. All cows had an intravaginal P4-releasing device inserted on d -14, which remained in cows until the end of the experiment (d 1). On d 0, cows were randomly assigned to receive, in a crossover design containing 2 periods of 24h each (d 0 and 1), (1) an intravenous glucose infusion (GLUC; 0.5 g of glucose/kg of BW, over a 3-h period) or (2) an intravenous saline infusion (SAL; 0.9%, over a 3-h period). Cows were fasted for 12h before infusions, and they remained fasted during infusion and sample collections. Blood samples were collected at 0, 3, and 6h relative to the beginning of infusions. Liver biopsies were performed concurrently with blood collections at 0 and 3h. After the last blood collection of period 1, cows received concentrate and returned to pasture. Cows gained BW (16.5 ± 3.6 kg) and BCS (0.08 ± 0.06) from d -28 to 0. Cows receiving GLUC had greater serum glucose and insulin concentrations at 3h compared with SAL cohorts. No treatment effects were detected for serum P4 concentrations, although mRNA expression of CYP2C19 and CYP3A4 after the infusion period was reduced for cows in the GLUC treatment compared with their cohorts in the SAL treatment. In conclusion, hepatic CYP3A4 and CYP2C19 mRNA expression can be promptly modulated by glucose infusion followed by acute increases in circulating insulin, which provides novel insight into the physiological mechanisms associating nutrition and reproductive function in dairy cows

  13. Effect of N-Feruloylserotonin and Methotrexate on Severity of Experimental Arthritis and on Messenger RNA Expression of Key Proinflammatory Markers in Liver

    Ľudmila Pašková

    2016-01-01

    Full Text Available Rheumatoid arthritis (RA is a chronic inflammatory disease, leading to progressive destruction of joints and extra-articular tissues, including organs such as liver and spleen. The purpose of this study was to compare the effects of a potential immunomodulator, natural polyphenol N-feruloylserotonin (N-f-5HT, with methotrexate (MTX, the standard in RA therapy, in the chronic phase of adjuvant-induced arthritis (AA in male Lewis rats. The experiment included healthy controls (CO, arthritic animals (AA, AA given N-f-5HT (AA-N-f-5HT, and AA given MTX (AA-MTX. N-f-5HT did not affect the body weight change and clinical parameters until the 14th experimental day. Its positive effect was rising during the 28-day experiment, indicating a delayed onset of N-f-5HT action. Administration of either N-f-5HT or MTX caused reduction of inflammation measured as the level of CRP in plasma and the activity of LOX in the liver. mRNA transcription of TNF-α and iNOS in the liver was significantly attenuated in both MTX and N-f-5HT treated groups of arthritic rats. Interestingly, in contrast to MTX, N-f-5HT significantly lowered the level of IL-1β in plasma and IL-1β mRNA expression in the liver and spleen of arthritic rats. This speaks for future investigations of N-f-5HT as an agent in the treatment of RA in combination therapy with MTX.

  14. In vivo genome-wide profiling of RNA secondary structure reveals novel regulatory features.

    Ding, Yiliang; Tang, Yin; Kwok, Chun Kit; Zhang, Yu; Bevilacqua, Philip C; Assmann, Sarah M

    2014-01-30

    RNA structure has critical roles in processes ranging from ligand sensing to the regulation of translation, polyadenylation and splicing. However, a lack of genome-wide in vivo RNA structural data has limited our understanding of how RNA structure regulates gene expression in living cells. Here we present a high-throughput, genome-wide in vivo RNA structure probing method, structure-seq, in which dimethyl sulphate methylation of unprotected adenines and cytosines is identified by next-generation sequencing. Application of this method to Arabidopsis thaliana seedlings yielded the first in vivo genome-wide RNA structure map at nucleotide resolution for any organism, with quantitative structural information across more than 10,000 transcripts. Our analysis reveals a three-nucleotide periodic repeat pattern in the structure of coding regions, as well as a less-structured region immediately upstream of the start codon, and shows that these features are strongly correlated with translation efficiency. We also find patterns of strong and weak secondary structure at sites of alternative polyadenylation, as well as strong secondary structure at 5' splice sites that correlates with unspliced events. Notably, in vivo structures of messenger RNAs annotated for stress responses are poorly predicted in silico, whereas mRNA structures of genes related to cell function maintenance are well predicted. Global comparison of several structural features between these two categories shows that the mRNAs associated with stress responses tend to have more single-strandedness, longer maximal loop length and higher free energy per nucleotide, features that may allow these RNAs to undergo conformational changes in response to environmental conditions. Structure-seq allows the RNA structurome and its biological roles to be interrogated on a genome-wide scale and should be applicable to any organism.

  15. Integrated Analysis of Long Noncoding RNA and mRNA Expression Profile in Advanced Laryngeal Squamous Cell Carcinoma.

    Ling Feng

    Full Text Available Long non-coding RNA (lncRNA plays an important role in tumorigenesis. However, the expression pattern and function of lncRNAs in laryngeal squamous cell carcinoma (LSCC are still unclear. To investigate the aberrantly expressed lncRNAs and mRNAs in advanced LSCC, we screened lncRNA and mRNA expression profiles in 9 pairs of primary Stage IVA LSCC tissues and adjacent non-neoplastic tissues by lncRNA and mRNA integrated microarrays. Gene Ontology and pathway analysis were performed to find out the significant function and pathway of the differentially expressed mRNAs, gene-gene functional interaction network and ceRNA network were constructed to select core mRNAs, and lncRNA-mRNA expression correlation network was built to identify the interactions between lncRNA and mRNA. qRT-PCR was performed to further validate the expressions of selected lncRNAs and mRNAs in advanced LSCC. We found 1459 differentially expressed lncRNAs and 2381 differentially expressed mRNAs, including 846 up-regulated lncRNAs and 613 down-regulated lncRNAs, 1542 up-regulated mRNAs and 839 down-regulated mRNAs. The mRNAs ITGB1, HIF1A, and DDIT4 were selected as core mRNAs, which are mainly involved in biological processes, such as matrix organization, cell cycle, adhesion, and metabolic pathway. LncRNA-mRNA expression correlation network showed LncRNA NR_027340, MIR31HG were positively correlated with ITGB1, HIF1A respectively. LncRNA SOX2-OT was negatively correlated with DDIT4. qRT-PCR further validated the expression of these lncRNAs and mRNAs. The work provides convincing evidence that the identified lncRNAs and mRNAs are potential biomarkers in advanced LSCC for further future studies.

  16. Methods for small RNA preparation for digital gene expression profiling by next-generation sequencing

    Linsen, S.E.V.; Cuppen, E.

    2012-01-01

    Digital gene expression (DGE) profiling techniques are playing an eminent role in the detection, localization, and differential expression quantification of many small RNA species, including microRNAs (1-3). Procedures in small RNA library preparation techniques typically include adapter ligation by

  17. Profile of HIV-1 RNA viral load among HIV-TB co-infected patients in ...

    Profile of HIV-1 RNA viral load among HIV-TB co-infected patients in a tertiary health facility in Maiduguri, Northeastern Nigeria. ... This study aims to estimate the HIV-1 RNA viral load and impact of anti TB therapy (ATT) ... HOW TO USE AJOL.

  18. Performance comparison of digital microRNA profiling technologies applied on human breast cancer cell lines.

    Erik Knutsen

    Full Text Available MicroRNA profiling represents an important first-step in deducting individual RNA-based regulatory function in a cell, tissue, or at a specific developmental stage. Currently there are several different platforms to choose from in order to make the initial miRNA profiles. In this study we investigate recently developed digital microRNA high-throughput technologies. Four different platforms were compared including next generation SOLiD ligation sequencing and Illumina HiSeq sequencing, hybridization-based NanoString nCounter, and miRCURY locked nucleic acid RT-qPCR. For all four technologies, full microRNA profiles were generated from human cell lines that represent noninvasive and invasive tumorigenic breast cancer. This study reports the correlation between platforms, as well as a more extensive analysis of the accuracy and sensitivity of data generated when using different platforms and important consideration when verifying results by the use of additional technologies. We found all the platforms to be highly capable for microRNA analysis. Furthermore, the two NGS platforms and RT-qPCR all have equally high sensitivity, and the fold change accuracy is independent of individual miRNA concentration for NGS and RT-qPCR. Based on these findings we propose new guidelines and considerations when performing microRNA profiling.

  19. Reduction in PSA messenger-RNA expression and clinical recurrence in patients with prostatic cancer undergoing neoadjuvant therapy before radical prostatectomy

    Baruffi Marco

    2004-04-01

    Full Text Available Abstract Background We assessed the incidence of micro-metastases at surgical margins (SM and pelvic lymph nodes (LN in patients submitted to radical retropubic prostatectomy (RP after neoadjuvant therapy (NT or to RP alone. We compared traditional staging to molecular detection of PSA using Taqman-based quantitative real-time PCR (qrt-PCR never used before for this purpose. Methods 29 patients were assigned to NT plus RP (arm A or RP alone (arm B. Pelvic LN were dissected for qrt-PCR analysis, together with right and left lateral SM. Results 64,3% patients of arm B and 26.6% of arm A had evidence of PSA mRNA expression in LN and/or SM. 17,2% patients, all of arm B, had biochemical recurrence. Conclusions Qrt-PCR may be more sensitive, compared to conventional histology, in identifying presence of viable prostate carcinoma cells in SM and LN. Gene expression of PSA in surgical periprostatic samples might be considered as a novel and reliable indicator of minimal residual disease after NT.

  20. Reduction in PSA messenger-RNA expression and clinical recurrence in patients with prostatic cancer undergoing neoadjuvant therapy before radical prostatectomy

    Grasso, Marco; Lania, Caterina; Blanco, Salvatore; Baruffi, Marco; Mocellin, Simone

    2004-01-01

    Background We assessed the incidence of micro-metastases at surgical margins (SM) and pelvic lymph nodes (LN) in patients submitted to radical retropubic prostatectomy (RP) after neoadjuvant therapy (NT) or to RP alone. We compared traditional staging to molecular detection of PSA using Taqman-based quantitative real-time PCR (qrt-PCR) never used before for this purpose. Methods 29 patients were assigned to NT plus RP (arm A) or RP alone (arm B). Pelvic LN were dissected for qrt-PCR analysis, together with right and left lateral SM. Results 64,3% patients of arm B and 26.6% of arm A had evidence of PSA mRNA expression in LN and/or SM. 17,2% patients, all of arm B, had biochemical recurrence. Conclusions Qrt-PCR may be more sensitive, compared to conventional histology, in identifying presence of viable prostate carcinoma cells in SM and LN. Gene expression of PSA in surgical periprostatic samples might be considered as a novel and reliable indicator of minimal residual disease after NT. PMID:15104791

  1. Changes in apoptotic microRNA and mRNA expression profiling in Caenorhabditis elegans during the Shenzhou-8 mission

    Gao Ying; Li Shuai; Xu Dan; Wang Junjun; Sun Yeqing

    2015-01-01

    Radiation and microgravity exposure have been proven to induce abnormal apoptosis in microRNA (miRNA) and mRNA expression, but whether space conditions, including radiation and microgravity, activate miRNAs to regulate the apoptosis is undetermined. For that purpose, we investigated miRNome and mRNA expression in the ced-1 Caenorhabditis elegans mutant vs the wild-type, both of which underwent spaceflight, spaceflight 1g-centrifuge control and ground control conditions during the Shenzhou-8 mission. Results showed that no morphological changes in the worms were detected, but differential miRNA expression increased from 43 (ground control condition) to 57 and 91 in spaceflight and spaceflight control conditions, respectively. Microgravity altered miRNA expression profiling by decreasing the number and significance of differentially expressed miRNA compared with 1 g incubation during spaceflight. Alterations in the miRNAs were involved in alterations in apoptosis, neurogenesis larval development, ATP metabolism and GTPase-mediated signal transduction. Among these, 17 altered miRNAs potentially involved in apoptosis were screened and showed obviously different expression signatures between space conditions. By integrated analysis of miRNA and mRNA, miR-797 and miR-81 may be involved in apoptosis by targeting the genes ced-10 and both drp-1 and hsp-1, respectively. Compared with ground condition, space conditions regulated apoptosis though a different manner on transcription, by altering expression of seven core apoptotic genes in spaceflight condition, and eight in spaceflight control condition. Results indicate that, miRNA of Caenorhabditis elegans probably regulates apoptotic gene expression in response to space environmental stress, and shows different behavior under microgravity condition compared with 1 g condition in the presence of space radiation. (author)

  2. mRNA/microRNA gene expression profile in microsatellite unstable colorectal cancer

    Calin George A

    2007-08-01

    Full Text Available Abstract Background Colorectal cancer develops through two main genetic instability pathways characterized by distinct pathologic features and clinical outcome. Results We investigated colon cancer samples (23 characterized by microsatellite stability, MSS, and 16 by high microsatellite instability, MSI-H for genome-wide expression of microRNA (miRNA and mRNA. Based on combined miRNA and mRNA gene expression, a molecular signature consisting of twenty seven differentially expressed genes, inclusive of 8 miRNAs, could correctly distinguish MSI-H versus MSS colon cancer samples. Among the differentially expressed miRNAs, various members of the oncogenic miR-17-92 family were significantly up-regulated in MSS cancers. The majority of protein coding genes were also up-regulated in MSS cancers. Their functional classification revealed that they were most frequently associated with cell cycle, DNA replication, recombination, repair, gastrointestinal disease and immune response. Conclusion This is the first report that indicates the existence of differences in miRNA expression between MSS versus MSI-H colorectal cancers. In addition, the work suggests that the combination of mRNA/miRNA expression signatures may represent a general approach for improving bio-molecular classification of human cancer.

  3. Dynamical Messengers for Gauge Mediation

    Hook, Anson; Torroba, Gonzalo; /SLAC /Stanford U., Phys. Dept.

    2011-08-17

    We construct models of indirect gauge mediation where the dynamics responsible for breaking supersymmetry simultaneously generates a weakly coupled subsector of messengers. This provides a microscopic realization of messenger gauge mediation where the messenger and hidden sector fields are unified into a single sector. The UV theory is SQCD with massless and massive quarks plus singlets, and at low energies it flows to a weakly coupled quiver gauge theory. One node provides the primary source of supersymmetry breaking, which is then transmitted to the node giving rise to the messenger fields. These models break R-symmetry spontaneously, produce realistic gaugino and sfermion masses, and give a heavy gravitino.

  4. MESSENGER: Exploring Mercury's Magnetosphere

    Slavin, James A.

    2008-01-01

    The MESSENGER mission to Mercury offers our first opportunity to explore this planet's miniature magnetosphere since Mariner 10's brief fly-bys in 1974-5. Mercury's magnetosphere is unique in many respects. The magnetosphere of Mercury is the smallest in the solar system with its magnetic field typically standing off the solar wind only - 1000 to 2000 km above the surface. For this reason there are no closed dri-fi paths for energetic particles and, hence, no radiation belts; the characteristic time scales for wave propagation and convective transport are short possibly coupling kinetic and fluid modes; magnetic reconnection at the dayside magnetopause may erode the subsolar magnetosphere allowing solar wind ions to directly impact the dayside regolith; inductive currents in Mercury's interior should act to modify the solar In addition, Mercury's magnetosphere is the only one with its defining magnetic flux tubes rooted in a planetary regolith as opposed to an atmosphere with a conductive ionosphere. This lack of an ionosphere is thought to be the underlying reason for the brevity of the very intense, but short lived, approx. 1-2 min, substorm-like energetic particle events observed by Mariner 10 in Mercury's magnetic tail. In this seminar, we review what we think we know about Mercury's magnetosphere and describe the MESSENGER science team's strategy for obtaining answers to the outstanding science questions surrounding the interaction of the solar wind with Mercury and its small, but dynamic magnetosphere.

  5. Reproducible pattern of microRNA in normal human skin

    Holst, Line; Kaczkowski, Bogumil; Gniadecki, Robert

    2010-01-01

    RNA expression pattern in normal human skin. Here we investigated miRNA expression profiles from skin biopsies of 8 healthy volunteers taken from sun protected and mildly photo damaged skin using the modified protocol for miRNA extraction. We were able to show a constant pattern of miRNA expression between......MicroRNAs (miRNAs) regulate cell growth, differentiation and apoptosis via specific targeting of messenger RNA (mRNA). Aberrant mRNA expression contributes to pathological processes such as carcinogenesis. To take advantage of miRNA profiling in skin disease it is essential to investigate mi...... different individuals. We did not find any significant differences in miRNA expression between sun protected and mildly photodamaged skin. These results may be valuable for future design of studies on miRNA expression in skin disease....

  6. Reproducible pattern of microRNA in normal human skin

    Holst, Line; Kaczkowski, Bogumil; Gniadecki, Robert

    2010-01-01

    RNA expression pattern in normal human skin. Here we investigated miRNA expression profiles from skin biopsies of 8 healthy volunteers taken from sun protected and mildly photo damaged skin using the modified protocol for miRNA extraction. We were able to show a constant pattern of miRNA expression between...... different individuals. We did not find any significant differences in miRNA expression between sun protected and mildly photodamaged skin. These results may be valuable for future design of studies on miRNA expression in skin disease.......MicroRNAs (miRNAs) regulate cell growth, differentiation and apoptosis via specific targeting of messenger RNA (mRNA). Aberrant mRNA expression contributes to pathological processes such as carcinogenesis. To take advantage of miRNA profiling in skin disease it is essential to investigate mi...

  7. Correlation of mRNA Profiles, miRNA Profiles, and Functional Immune Response in Rainbow Trout (Oncorrhynkus Mykiss) Infected With Viral Hemorrhagic Septicemia Virus (VHSV) and in Fish Vaccinated With a DNA Vaccine Against VHSV

    Bela-Ong, Dennis; Schyth, Brian Dall; Jørgensen, Hanne

    2011-01-01

    and are incorporated into the RNA-Induced Silencing Complex (RISC), which target specific mRNA sequences, causing either mRNA degradation or translation repression. This results in altered mRNA and protein profiles characteristic of a particular cellular phenotype or physiological state. By targeting immune relevant m...

  8. Integration analysis of microRNA and mRNA paired expression profiling identifies deregulated microRNA-transcription factor-gene regulatory networks in ovarian endometriosis.

    Zhao, Luyang; Gu, Chenglei; Ye, Mingxia; Zhang, Zhe; Li, Li'an; Fan, Wensheng; Meng, Yuanguang

    2018-01-22

    The etiology and pathophysiology of endometriosis remain unclear. Accumulating evidence suggests that aberrant microRNA (miRNA) and transcription factor (TF) expression may be involved in the pathogenesis and development of endometriosis. This study therefore aims to survey the key miRNAs, TFs and genes and further understand the mechanism of endometriosis. Paired expression profiling of miRNA and mRNA in ectopic endometria compared with eutopic endometria were determined by high-throughput sequencing techniques in eight patients with ovarian endometriosis. Binary interactions and circuits among the miRNAs, TFs, and corresponding genes were identified by the Pearson correlation coefficients. miRNA-TF-gene regulatory networks were constructed using bioinformatic methods. Eleven selected miRNAs and TFs were validated by quantitative reverse transcription-polymerase chain reaction in 22 patients. Overall, 107 differentially expressed miRNAs and 6112 differentially expressed mRNAs were identified by comparing the sequencing of the ectopic endometrium group and the eutopic endometrium group. The miRNA-TF-gene regulatory network consists of 22 miRNAs, 12 TFs and 430 corresponding genes. Specifically, some key regulators from the miR-449 and miR-34b/c cluster, miR-200 family, miR-106a-363 cluster, miR-182/183, FOX family, GATA family, and E2F family as well as CEBPA, SOX9 and HNF4A were suggested to play vital regulatory roles in the pathogenesis of endometriosis. Integration analysis of the miRNA and mRNA expression profiles presents a unique insight into the regulatory network of this enigmatic disorder and possibly provides clues regarding replacement therapy for endometriosis.

  9. Translational Influence on Messenger Stability

    Eriksen, Mette

    -termination to be a global phenomena in gene regulation. The influence of codon usage in the early coding region on messenger stability was examined, in order to establish how fast or slow the ribosome has to decode the sequence for it to protect the messenger from degradation. The experiments demonstrated that very fast...

  10. Cigarette smoking substantially alters plasma microRNA profiles in healthy subjects

    Takahashi, Kei; Yokota, Shin-ichi; Tatsumi, Naoyuki; Fukami, Tatsuki; Yokoi, Tsuyoshi; Nakajima, Miki, E-mail: nmiki@p.kanazawa-u.ac.jp

    2013-10-01

    Circulating microRNAs (miRNAs) are receiving attention as potential biomarkers of various diseases, including cancers, chronic obstructive pulmonary disease, and cardiovascular disease. However, it is unknown whether the levels of circulating miRNAs in a healthy subject might vary with external factors in daily life. In this study, we investigated whether cigarette smoking, a habit that has spread throughout the world and is a risk factor for various diseases, affects plasma miRNA profiles. We determined the profiles of 11 smokers and 7 non-smokers by TaqMan MicroRNA array analysis. A larger number of miRNAs were detected in smokers than in non-smokers, and the plasma levels of two-thirds of the detected miRNAs (43 miRNAs) were significantly higher in smokers than in non-smokers. A principal component analysis of the plasma miRNA profiles clearly separated smokers and non-smokers. Twenty-four of the miRNAs were previously reported to be potential biomarkers of disease, suggesting the possibility that smoking status might interfere with the diagnosis of disease. Interestingly, we found that quitting smoking altered the plasma miRNA profiles to resemble those of non-smokers. These results suggested that the differences in the plasma miRNA profiles between smokers and non-smokers could be attributed to cigarette smoking. In addition, we found that an acute exposure of ex-smokers to cigarette smoke (smoking one cigarette) did not cause a dramatic change in the plasma miRNA profile. In conclusion, we found that repeated cigarette smoking substantially alters the plasma miRNA profile, interfering with the diagnosis of disease or signaling potential smoking-related diseases. - Highlights: • Plasma miRNA profiles were unambiguously different between smokers and non-smokers. • Smoking status might interfere with the diagnosis of disease using plasma miRNAs. • Changes of plasma miRNA profiles may be a signal of smoking-related diseases.

  11. Cigarette smoking substantially alters plasma microRNA profiles in healthy subjects

    Takahashi, Kei; Yokota, Shin-ichi; Tatsumi, Naoyuki; Fukami, Tatsuki; Yokoi, Tsuyoshi; Nakajima, Miki

    2013-01-01

    Circulating microRNAs (miRNAs) are receiving attention as potential biomarkers of various diseases, including cancers, chronic obstructive pulmonary disease, and cardiovascular disease. However, it is unknown whether the levels of circulating miRNAs in a healthy subject might vary with external factors in daily life. In this study, we investigated whether cigarette smoking, a habit that has spread throughout the world and is a risk factor for various diseases, affects plasma miRNA profiles. We determined the profiles of 11 smokers and 7 non-smokers by TaqMan MicroRNA array analysis. A larger number of miRNAs were detected in smokers than in non-smokers, and the plasma levels of two-thirds of the detected miRNAs (43 miRNAs) were significantly higher in smokers than in non-smokers. A principal component analysis of the plasma miRNA profiles clearly separated smokers and non-smokers. Twenty-four of the miRNAs were previously reported to be potential biomarkers of disease, suggesting the possibility that smoking status might interfere with the diagnosis of disease. Interestingly, we found that quitting smoking altered the plasma miRNA profiles to resemble those of non-smokers. These results suggested that the differences in the plasma miRNA profiles between smokers and non-smokers could be attributed to cigarette smoking. In addition, we found that an acute exposure of ex-smokers to cigarette smoke (smoking one cigarette) did not cause a dramatic change in the plasma miRNA profile. In conclusion, we found that repeated cigarette smoking substantially alters the plasma miRNA profile, interfering with the diagnosis of disease or signaling potential smoking-related diseases. - Highlights: • Plasma miRNA profiles were unambiguously different between smokers and non-smokers. • Smoking status might interfere with the diagnosis of disease using plasma miRNAs. • Changes of plasma miRNA profiles may be a signal of smoking-related diseases

  12. Segundos mensajeros Second messengers

    Diana Patricia Díaz Hernández

    1989-01-01

    Full Text Available

    En esta revisión se describen, de manera esquemática, los mecanismos de acción empleados por los SEGUNDOS MENSAJEROS comenzando por el estimulo del receptor y continuando con las reacciones en cadena que conducen finalmente a una respuesta celular.

    This review schematically describes the different mechanisms of action that Second Messengers employ to stimulate receptors and then Initiate a chain of reactions that finally lead to appropriate cellular responses.

  13. MESSENGER at Mercury: Early Orbital Operations

    McNutt, Ralph L., Jr; Solomon, Sean C.; Bedini, Peter D.; Anderson, Brian J.; Blewett, David T.; Evans, Larry G.; Gold, Robert E.; Krimigis, Stamatios M.; Murchie, Scott L.; Nittler, Larry R.; hide

    2013-01-01

    angles. Targeted areas have been selected for spectral coverage into the ultraviolet with the Ultraviolet and Visible Spectrometer (UVVS). MESSENGER's Mercury Laser Altimeter is acquiring topographic profiles when the slant range to Mercury's surface is less than 1800 km, encompassing latitudes from 20 deg. S to the north pole. Topography over the remainder of the southern hemisphere will be derived from stereo imaging, radio occultations, and limb profiles. MESSENGER's radio science experiment is determining Mercury's gravity field from Doppler signals acquired during frequent downlinks. MESSENGER's Magnetometer is measuring the vector magnetic field both within Mercury's magnetosphere and in Mercury's solar wind environment at an instrument sampling rate of up to 20 samples/s. The UVVS is determining the three-dimensional, time-dependent distribution of Mercury's exospheric neutral and ionic species via their emission lines. During each spacecraft orbit, the Energetic Particle Spectrometer measures energetic electrons and ions, and the Fast Imaging Plasma Spectrometer measures the energies and mass per charge of thermal plasma components, both within Mercury's magnetosphere and in Mercury's solar-wind environment. The primary mission observation sequence will continue for one Earth year, until March 2012. An extended mission, currently under discussion with NASA, would add a second year of orbital observations targeting a set of focused follow-on questions that build on observations to date and take advantage of the more active Sun expected during 2012-2013. MESSENGER's total primary mission cost, projected at $446 M in real-year dollars, is comparable to that of Mariner 10 after adjustment for inflation.

  14. Correlation of mRNA Profiles, miRNA Profiles, and Functional Immune Response in Rainbow Trout (Oncorrhynkus Mykiss) During Infection With Viral Hemorrhagic Septicemia Virus (VHSV) and in Fish Vaccinated With an Anti-VHSV DNA Vaccine

    Bela-Ong, Dennis; Schyth, Brian Dall; Lorenzen, Niels

    fish. Linking mRNA and miRNA profiles with phenotypic, genotypic, and immunological data will provide an integrated view of the mechanisms of resistance and the strong protective immune responses provided by vaccination. This information is important in designing effective strategies to mitigate......-mediated) responses. MRNA and miRNA profiles will be correlated and combined with in vitro work in cell culture to describe target relationships between miRNAs and mRNAs and the effect of this targeting in fish. Vaccinated fish will also be used for mRNA/miRNA profiling and in challenge studies alongside non-vaccinated...

  15. Vitellogenin messenger rna in rooster liver

    Bos, Ebo Sijbren

    1975-01-01

    The investigations described in this thesis were carried out as a part of the studies in our laboratory on the control of gene expression in animal cells. They represent an example of the hormonal regulation of protein synthesis, viz. the induction of vitellogenin synthesis in rooster liver by the

  16. MicroRNA profile changes in human immunodeficiency virus type 1 (HIV-1 seropositive individuals

    Smith Stephen M

    2008-12-01

    Full Text Available Abstract MicroRNAs (miRNAs play diverse roles in regulating cellular and developmental functions. We have profiled the miRNA expression in peripheral blood mononuclear cells from 36 HIV-1 seropositive individuals and 12 normal controls. The HIV-1-positive individuals were categorized operationally into four classes based on their CD4+ T-cell counts and their viral loads. We report that specific miRNA signatures can be observed for each of the four classes.

  17. Brain Gene Expression Signatures From Cerebrospinal Fluid Exosome RNA Profiling

    Zanello, S. B.; Stevens, B.; Calvillo, E.; Tang, R.; Gutierrez Flores, B.; Hu, L.; Skog, J.; Bershad, E.

    2016-01-01

    While the Visual Impairment and Intracranial Pressure (VIIP) syndrome observations have focused on ocular symptoms, spaceflight has been also associated with a number of other performance and neurologic signs, such as headaches, cognitive changes, vertigo, nausea, sleep/circadian disruption and mood alterations, which, albeit likely multifactorial, can also result from elevation of intracranial pressure (ICP). We therefore hypothesize that these various symptoms are caused by disturbances in the neurophysiology of the brain structures and are correlated with molecular markers in the cerebrospinal fluid (CSF) as indicators of neurophysiological changes. Exosomes are 30-200 nm microvesicles shed into all biofluids, including blood, urine, and CSF, carrying a highly rich source of intact protein and RNA cargo. Exosomes have been identified in human CSF, and their proteome and RNA pool is a potential new reservoir for biomarker discovery in neurological disorders. The purpose of this study is to investigate changes in brain gene expression via exosome analysis in patients suffering from ICP elevation of varied severity (idiopathic intracranial hypertension -IIH), a condition which shares some of the neuroophthalmological features of VIIP, as a first step toward obtaining evidence suggesting that cognitive function and ICP levels can be correlated with biomarkers in the CSF. Our preliminary work, reported last year, validated the exosomal technology applicable to CSF analysis and demonstrated that it was possible to obtain gene expression evidence of inflammation processes in traumatic brain injury patients. We are now recruiting patients with suspected IIH requiring lumbar puncture at Baylor College of Medicine. Both CSF (5 ml) and human plasma (10 ml) are being collected in order to compare the pattern of differentially expressed genes observed in CSF and in blood. Since blood is much more accessible than CSF, we would like to determine whether plasma biomarkers for

  18. MESSENGER'S First Flyby of Mercury

    Slavin, James A.

    2008-01-01

    The MESSENGER mission to Mercury offers our first opportunity to explore this planet's miniature magnetosphere since Mariner 10's brief fly-bys in 1974-5. The magnetosphere of Mercury is the smallest in the solar system with its magnetic field typically standing off the solar wind only - 1000 to 2000 km above the surface. An overview of the MESSENGER mission and its January 14th close flyby of Mercury will be provided. Primary science objectives and the science instrumentation will be described. Initial results from MESSENGER'S first flyby on January 14th, 2008 will be discussed with an emphasis on the magnetic field and charged particle measurements.

  19. The Messenger Mission to Mercury

    Domingue, D. L

    2007-01-01

    NASA’s MESSENGER mission, launched on 3 August, 2004 is the seventh mission in the Discovery series. MESSENGER encounters the planet Mercury four times, culminating with an insertion into orbit on 18 March 2011. It carries a comprehensive package of geophysical, geological, geochemical, and space environment experiments to complete the complex investigations of this solar-system end member, which begun with Mariner 10. The articles in this book, written by the experts in each area of the MESSENGER mission, describe the mission, spacecraft, scientific objectives, and payload. The book is of interest to all potential users of the data returned by the MESSENGER mission, to those studying the nature of the planet Mercury, and by all those interested in the design and implementation of planetary exploration missions.

  20. Dysregulation of hepatic microRNA expression profiles with Clonorchis sinensis infection.

    Han, Su; Tang, Qiaoran; Lu, Xi; Chen, Rui; Li, Yihong; Shu, Jing; Zhang, Xiaoli; Cao, Jianping

    2016-11-30

    Clonorchiasis remains an important zoonotic parasitic disease worldwide. The molecular mechanisms of host-parasite interaction are not fully understood. Non-coding microRNAs (miRNAs) are considered to be key regulators in parasitic diseases. The regulation of miRNAs and host micro-environment may be involved in clonorchiasis, and require further investigation. MiRNA microarray technology and bioinformatic analysis were used to investigate the regulatory mechanisms of host miRNA and to compare miRNA expression profiles in the liver tissues of control and Clonorchis sinensis (C. sinensis)-infected rats. A total of eight miRNAs were downregulated and two were upregulated, which showed differentially altered expression profiles in the liver tissue of C. sinensis-infected rats. Further analysis of the differentially expressed miRNAs revealed that many important signal pathways were triggered after infection with C. sinensis, which were related to clonorchiasis pathogenesis, such as cell apoptosis and inflammation, as well as genes involved in signal transduction mechanisms, such as pathways in cancer and the Wnt and Mitogen-activated protein kinases (MAPK) signaling pathways. The present study revealed that the miRNA expression profiles of the host were changed by C. sinensis infection. This dysregulation in miRNA expression may contribute to the etiology and pathophysiology of clonorchiasis. These results also provide new insights into the regulatory mechanisms of miRNAs in clonorchiasis, which may present potential targets for future C. sinensis control strategies.

  1. miRNA Expression Profiles in Cerebrospinal Fluid and Blood of Patients with Acute Ischemic Stroke

    Sørensen, Sofie Sølvsten; Nygaard, Ann-Britt; Nielsen, Ming-Yuan

    2014-01-01

    in the cell-free fractions of CSF and blood were analyzed by a microarray technique (miRCURY LNA™ microRNA Array, Exiqon A/S, Denmark) using a quantitative PCR (qPCR) platform containing 378 miRNA primers. In total, 183 different miRNAs were detected in the CSF, of which two miRNAs (let-7c and miR-221-3p......The aims of the study were (1) to determine whether miRNAs (microRNAs) can be detected in the cerebrospinal fluid (CSF) and blood of patients with ischemic stroke and (2) to compare these miRNA profiles with corresponding profiles from other neurological patients to address whether the mi......RNA profiles of CSF or blood have potential usefulness as diagnostic biomarkers of ischemic stroke. CSF from patients with acute ischemic stroke (n = 10) and patients with other neurological diseases (n = 10) was collected by lumbar puncture. Blood samples were taken immediately after. Expression profiles...

  2. Categorical methods for the interpretation of RNA profiles as cell type evidence and their limitations

    de Zoete, J.; Curran, J.; Sjerps, M.

    2015-01-01

    Existing methods for the interpretation of RNA profiles as evidence for the presence of certain cell types aim for making categorical statements. Such statements limit the possibility to report the associated uncertainty. From a statistical point of view, a probabilistic approach is a preferable

  3. Circulating, Cell-Free Micro-RNA Profiles Reflect Discordant Development of Dementia in Monozygotic Twins

    Mengel-From, Jonas; Rønne, Mette E; Carlsen, Anting L

    2018-01-01

    We aim to examine if circulating micro-RNA and cytokine levels associate with dementia diagnosis and cognitive scores. To test our hypothesis, we use plasma donated from 48 monozygotic twin pairs in 1997 and 46 micro-RNAs and 10 cytokines were quantified using microfluidic RT-qPCR and multiplex...... solid-phase immunoassays, respectively. Micro-RNA and cytokine profiling were examined for associations with dementia diagnoses in a longitudinal registry study or with cognitive scores at baseline. Thirty-six micro-RNAs and all cytokines were detected consistently. Micro-RNA profiles associate...... with diagnoses and cognitive scores at statistically significant levels while cytokine only showed trends pointing at chronic inflammation in twins having or developing dementia. The most notable findings were decreased miR-106a and miR-210, and increased miR-106b expression in twins with a dementia diagnosis...

  4. Gene expression profiles in BCL11B-siRNA treated malignant T cells

    Grabarczyk Piotr

    2011-05-01

    Full Text Available Abstract Background Downregulation of the B-cell chronic lymphocytic leukemia (CLL/lymphoma11B (BCL11B gene by small interfering RNA (siRNA leads to growth inhibition and apoptosis of the human T-cell acute lymphoblastic leukemia (T-ALL cell line Molt-4. To further characterize the molecular mechanism, a global gene expression profile of BCL11B-siRNA -treated Molt-4 cells was established. The expression profiles of several genes were further validated in the BCL11B-siRNA -treated Molt-4 cells and primary T-ALL cells. Results 142 genes were found to be upregulated and 109 genes downregulated in the BCL11B-siRNA -treated Molt-4 cells by microarray analysis. Among apoptosis-related genes, three pro-apoptotic genes, TNFSF10, BIK, BNIP3, were upregulated and one anti-apoptotic gene, BCL2L1 was downregulated. Moreover, the expression of SPP1 and CREBBP genes involved in the transforming growth factor (TGF-β pathway was down 16-fold. Expression levels of TNFSF10, BCL2L1, SPP1, and CREBBP were also examined by real-time PCR. A similar expression pattern of TNFSF10, BCL2L1, and SPP1 was identified. However, CREBBP was not downregulated in the BLC11B-siRNA -treated Molt-4 cells. Conclusion BCL11B-siRNA treatment altered expression profiles of TNFSF10, BCL2L1, and SPP1 in both Molt-4 T cell line and primary T-ALL cells.

  5. A Decade of Global mRNA and miRNA Profiling of HPV-Positive Cell Lines and Clinical Specimens

    Kaczkowski, Bogumil; Morevati, Marya; Rossing, Maria

    2012-01-01

    For more than a decade, global gene expression profiling has been extensively used to elucidate the biology of human papillomaviruses (HPV) and their role in cervical- and head-and-neck cancers. Since 2008, the expression profiling of miRNAs has been reported in multiple HPV studies. Two major...... as the fragmented miRNA-mRNA target correlation evidence. Furthermore, we propose an approach for future research to include more comprehensive miRNA-mRNA target correlation analysis and to apply systems biology/gene networks methodology....... strategies have been employed in the gene and miRNA profiling studies: In the first approach, HPV positive tumors were compared to normal tissues or to HPV negative tumors. The second strategy relied on analysis of cell cultures transfected with single HPV oncogenes or with HPV genomes compared...

  6. Maternal chromium restriction modulates miRNA profiles related to lipid metabolism disorder in mice offspring.

    Zhang, Qian; Xiao, Xinhua; Zheng, Jia; Li, Ming; Yu, Miao; Ping, Fan; Wang, Zhixin; Qi, Cuijuan; Wang, Tong; Wang, Xiaojing

    2017-08-01

    Increasing evidence shows that maternal nutrition status has a vital effect on offspring susceptibility to obesity. MicroRNAs are related to lipid metabolism processes. This study aimed to evaluate whether maternal chromium restriction could affect miRNA expression involved in lipid metabolism in offspring. Weaning C57BL/6J mice born from mothers fed with normal control diet or chromium-restricted diet were fed for 13 weeks. The adipose miRNA expression profile was analyzed by miRNA array analysis. At 16 weeks old, pups from dams fed with chromium-restricted diet exhibit higher body weight, fat weight, and serum TC, TG levels. Six miRNAs were identified as upregulated in the RC group compared with the CC group, whereas eight miRNAs were lower than the threshold level set in the RC group. In the validated target genes of these differentially expressed miRNA, the MAPK signaling pathway serves an important role in the influence of early life chromium-restricted diet on lipid metabolism through miRNA. Long-term programming on various specific miRNA and MAPK signaling pathway may be involved in maternal chromium restriction in the adipose of female offspring. Impact statement For the first time, our study demonstrates important miRNA differences in the effect of maternal chromium restriction in offspring. These miRNAs may serve as "bridges" between the mother and the offspring by affecting the MAPK pathway.

  7. MicroRNA expression in melanocytic nevi: the usefulness of formalin-fixed, paraffin-embedded material for miRNA microarray profiling.

    Glud, Martin; Klausen, Mikkel; Gniadecki, Robert; Rossing, Maria; Hastrup, Nina; Nielsen, Finn C; Drzewiecki, Krzysztof T

    2009-05-01

    MicroRNAs (miRNAs) are small, noncoding RNA molecules that regulate cellular differentiation, proliferation, and apoptosis. MiRNAs are expressed in a developmentally regulated and tissue-specific manner. Aberrant expression may contribute to pathological processes such as cancer, and miRNA may therefore serve as biomarkers that may be useful in a clinical environment for diagnosis of various diseases. Most miRNA profiling studies have used fresh tissue samples. However, in some types of cancer, including malignant melanoma, fresh material is difficult to obtain from primary tumors, and most surgical specimens are formalin fixed and paraffin embedded (FFPE). To explore whether FFPE material would be suitable for miRNA profiling in melanocytic lesions, we compared miRNA expression patterns in FFPE versus fresh frozen samples, obtained from 15 human melanocytic nevi. Out of microarray data, we identified 84 miRNAs that were expressed in both types of samples and represented an miRNA profile of melanocytic nevi. Our results showed a high correlation in miRNA expression (Spearman r-value of 0.80) between paired FFPE and fresh frozen material. The data were further validated by quantitative RT-PCR. In conclusion, FFPE specimens of melanocytic lesions are suitable as a source for miRNA microarray profiling.

  8. MicroRNA expression in melanocytic nevi: the usefulness of formalin-fixed, paraffin-embedded material for miRNA microarray profiling

    Glud, M.; Klausen, M.; Gniadecki, R.

    2009-01-01

    surgical specimens are formalin fixed and paraffin embedded (FFPE). To explore whether FFPE material would be suitable for miRNA profiling in melanocytic lesions, we compared miRNA expression patterns in FFPE versus fresh frozen samples, obtained from 15 human melanocytic nevi. Out of microarray data, we...

  9. MicroRNA and piRNA profiles in normal human testis detected by next generation sequencing.

    Qingling Yang

    Full Text Available BACKGROUND: MicroRNAs (miRNAs are the class of small endogenous RNAs that play an important regulatory role in cells by negatively affecting gene expression at transcriptional and post-transcriptional levels. There have been extensive studies aiming to discover miRNAs and to analyze their functions in the cells from a variety of species. However, there are no published studies of miRNA profiles in human testis using next generation sequencing (NGS technology. RESULTS: We employed Solexa sequencing technology to profile miRNAs in normal human testis. Total 770 known and 5 novel human miRNAs, and 20121 piRNAs were detected, indicating that the human testis has a complex population of small RNAs. The expression of 15 known and 5 novel detected miRNAs was validated by qRT-PCR. We have also predicted the potential target genes of the abundant known and novel miRNAs, and subjected them to GO and pathway analysis, revealing the involvement of miRNAs in many important biological phenomenon including meiosis and p53-related pathways that are implicated in the regulation of spermatogenesis. CONCLUSIONS: This study reports the first genome-wide miRNA profiles in human testis using a NGS approach. The presence of large number of miRNAs and the nature of their target genes suggested that miRNAs play important roles in spermatogenesis. Here we provide a useful resource for further elucidation of the regulatory role of miRNAs and piRNAs in the spermatogenesis. It may also facilitate the development of prophylactic strategies for male infertility.

  10. Circular RNA Profiling and Bioinformatic Modeling Identify Its Regulatory Role in Hepatic Steatosis.

    Guo, Xing-Ya; He, Chong-Xin; Wang, Yu-Qin; Sun, Chao; Li, Guang-Ming; Su, Qing; Pan, Qin; Fan, Jian-Gao

    2017-01-01

    Circular RNAs (circRNAs) exhibit a wide range of physiological and pathological activities. To uncover their role in hepatic steatosis, we investigated the expression profile of circRNAs in HepG2-based hepatic steatosis induced by high-fat stimulation. Differentially expressed circRNAs were subjected to validation using QPCR and functional analyses using principal component analysis, hierarchical clustering, target prediction, gene ontology (GO), and pathway annotation, respectively. Bioinformatic integration established the circRNA-miRNA-mRNA regulatory network so as to identify the mechanisms underlying circRNAs' metabolic effect. Here we reported that hepatic steatosis was associated with a total of 357 circRNAs. Enrichment of transcription-related GOs, especially GO: 0006355, GO: 004589, GO: 0045944, GO: 0045892, and GO: 0000122, demonstrated their specific actions in transcriptional regulation. Lipin 1 (LPIN1) was recognized to mediate the transcriptional regulatory effect of circRNAs on metabolic pathways. circRNA-miRNA-mRNA network further identified the signaling cascade of circRNA_021412/miR-1972/LPIN1, which was characterized by decreased level of circRNA_021412 and miR-1972-based inhibition of LPIN1. LPIN1-induced downregulation of long chain acyl-CoA synthetases (ACSLs) expression finally resulted in the hepatosteatosis. These findings identify circRNAs to be important regulators of hepatic steatosis. Transcription-dependent modulation of metabolic pathways may underlie their effects, partially by the circRNA_021412/miR-1972/LPIN1 signaling.

  11. MiRNA Profiles in Lymphoblastoid Cell Lines of Finnish Prostate Cancer Families.

    Daniel Fischer

    Full Text Available Heritable factors are evidently involved in prostate cancer (PrCa carcinogenesis, but currently, genetic markers are not routinely used in screening or diagnostics of the disease. More precise information is needed for making treatment decisions to distinguish aggressive cases from indolent disease, for which heritable factors could be a useful tool. The genetic makeup of PrCa has only recently begun to be unravelled through large-scale genome-wide association studies (GWAS. The thus far identified Single Nucleotide Polymorphisms (SNPs explain, however, only a fraction of familial clustering. Moreover, the known risk SNPs are not associated with the clinical outcome of the disease, such as aggressive or metastasised disease, and therefore cannot be used to predict the prognosis. Annotating the SNPs with deep clinical data together with miRNA expression profiles can improve the understanding of the underlying mechanisms of different phenotypes of prostate cancer.In this study microRNA (miRNA profiles were studied as potential biomarkers to predict the disease outcome. The study subjects were from Finnish high risk prostate cancer families. To identify potential biomarkers we combined a novel non-parametrical test with an importance measure provided from a Random Forest classifier. This combination delivered a set of nine miRNAs that was able to separate cases from controls. The detected miRNA expression profiles could predict the development of the disease years before the actual PrCa diagnosis or detect the existence of other cancers in the studied individuals. Furthermore, using an expression Quantitative Trait Loci (eQTL analysis, regulatory SNPs for miRNA miR-483-3p that were also directly associated with PrCa were found.Based on our findings, we suggest that blood-based miRNA expression profiling can be used in the diagnosis and maybe even prognosis of the disease. In the future, miRNA profiling could possibly be used in targeted screening

  12. Effect of chronic uremia on the transcriptional profile of the calcified aorta analyzed by RNA sequencing

    Rukov, Jakob Lewin; Gravesen, Eva; Mace, Maria L.

    2016-01-01

    The development of vascular calcification (VC) in chronic uremia (CU) is a tightly regulated process controlled by factors promoting and inhibiting mineralization. Next-generation high-throughput RNA sequencing (RNA-seq) is a powerful and sensitive tool for quantitative gene expression profiling...... with an expression level of >1 reads/kilobase transcript/million mapped reads, 2,663 genes were differentially expressed with 47% upregulated genes and 53% downregulated genes in uremic rats. Significantly deregulated genes were enriched for ontologies related to the extracellular matrix, response to wounding...

  13. MicroRNA profiling of primary cutaneous large B-cell lymphomas.

    Lianne Koens

    Full Text Available Aberrant expression of microRNAs is widely accepted to be pathogenetically involved in nodal diffuse large B-cell lymphomas (DLBCLs. However, the microRNAs profiles of primary cutaneous large B-cell lymphomas (PCLBCLs are not yet described. Its two main subtypes, i.e., primary cutaneous diffuse large B-cell lymphoma, leg type (PCLBCL-LT and primary cutaneous follicle center lymphoma (PCFCL are characterized by an activated B-cell (ABC-genotype and a germinal center B-cell (GCB-genotype, respectively. We performed high-throughput sequencing analysis on frozen tumor biopsies from 19 cases of PCFCL and PCLBCL-LT to establish microRNA profiles. Cluster analysis of the complete microRNome could not distinguish between the two subtypes, but 16 single microRNAs were found to be differentially expressed. Single microRNA RT-qPCR was conducted on formalin-fixed paraffin-embedded tumor biopsies of 20 additional cases, confirming higher expression of miR-9-5p, miR-31-5p, miR-129-2-3p and miR-214-3p in PCFCL as compared to PCLBCL-LT. MicroRNAs previously described to be higher expressed in ABC-type as compared to GCB-type nodal DLBCL were not differentially expressed between PCFCL and PCLBCL-LT. In conclusion, PCFCL and PCLBCL-LT differ in their microRNA profiles. In contrast to their gene expression profile, they only show slight resemblance with the microRNA profiles found in GCB- and ABC-type nodal DLBCL.

  14. Optimization of laser capture microdissection and RNA amplification for gene expression profiling of prostate cancer

    Vasmatzis George

    2007-03-01

    Full Text Available Abstract Background To discover prostate cancer biomarkers, we profiled gene expression in benign and malignant cells laser capture microdissected (LCM from prostate tissues and metastatic prostatic adenocarcinomas. Here we present methods developed, optimized, and validated to obtain high quality gene expression data. Results RNase inhibitor was included in solutions used to stain frozen tissue sections for LCM, which improved RNA quality significantly. Quantitative PCR assays, requiring minimal amounts of LCM RNA, were developed to determine RNA quality and concentration. SuperScript II™ reverse transcriptase was replaced with SuperScript III™, and SpeedVac concentration was eliminated to optimize linear amplification. The GeneChip® IVT labeling kit was used rather than the Enzo BioArray™ HighYield™ RNA transcript labeling kit since side-by-side comparisons indicated high-end signal saturation with the latter. We obtained 72 μg of labeled complementary RNA on average after linear amplification of about 2 ng of total RNA. Conclusion Unsupervised clustering placed 5/5 normal and 2/2 benign prostatic hyperplasia cases in one group, 5/7 Gleason pattern 3 cases in another group, and the remaining 2/7 pattern 3 cases in a third group with 8/8 Gleason pattern 5 cases and 3/3 metastatic prostatic adenocarcinomas. Differential expression of alpha-methylacyl coenzyme A racemase (AMACR and hepsin was confirmed using quantitative PCR.

  15. Gene expression profiling of non-polyadenylated RNA-seq across species

    Xiao-Ou Zhang

    2014-12-01

    Full Text Available Transcriptomes are dynamic and unique, with each cell type/tissue, developmental stage and species expressing a different repertoire of RNA transcripts. Most mRNAs and well-characterized long noncoding RNAs are shaped with a 5′ cap and 3′ poly(A tail, thus conventional transcriptome analyses typically start with the enrichment of poly(A+ RNAs by oligo(dT selection, followed by deep sequencing approaches. However, accumulated lines of evidence suggest that many RNA transcripts are processed by alternative mechanisms without 3′ poly(A tails and, therefore, fail to be enriched by oligo(dT purification and are absent following deep sequencing analyses. We have described an enrichment strategy to purify non-polyadenylated (poly(A−/ribo− RNAs from human total RNAs by removal of both poly(A+ RNA transcripts and ribosomal RNAs, which led to the identification of many novel RNA transcripts with non-canonical 3′ ends in human. Here, we describe the application of non-polyadenylated RNA-sequencing in rhesus monkey and mouse cell lines/tissue, and further profile the transcription of non-polyadenylated RNAs across species, providing new resources for non-polyadenylated RNA identification and comparison across species.

  16. Analysis of miRNA expression profiles in melatonin-exposed GC-1 spg cell line.

    Zhu, Xiaoling; Chen, Shuxiong; Jiang, Yanwen; Xu, Ying; Zhao, Yun; Chen, Lu; Li, Chunjin; Zhou, Xu

    2018-02-05

    Melatonin is an endocrine neurohormone secreted by pinealocytes in the pineal gland. It exerts diverse physiological effects, such as circadian rhythm regulator and antioxidant. However, the functional importance of melatonin in spermatogenesis regulation remains unclear. The objectives of this study are to: (1) detect melatonin affection on miRNA expression profiles in GC-1 spg cells by miRNA deep sequencing (DeepSeq) and (2) define melatonin affected miRNA-mRNA interactions and associated biological processes using bioinformatics analysis. GC-1 spg cells were cultured with melatonin (10 -7 M) for 24h. DeepSeq data were validated using quantitative real-time reverse transcription polymerase chain reaction analysis (qRT-PCR). A total of 176 miRNA expressions were found to be significantly different between two groups (fold change of >2 or melatonin could regulate the expression of miRNA to perform its physiological effects in GC-1 spg cells. These results should be useful to investigate the biological function of miRNAs regulated by melatonin in spermatogenesis and testicular germ cell tumor. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. Comparison of commercial exosome isolation kits for circulating exosomal microRNA profiling.

    Ding, Meng; Wang, Cheng; Lu, Xiaolan; Zhang, Cuiping; Zhou, Zhen; Chen, Xi; Zhang, Chen-Yu; Zen, Ke; Zhang, Chunni

    2018-06-01

    Circulating exosomal microRNAs (miRNAs) are valuable biomarker candidates; however, information on the characterization and mutual agreement of commercial kits for circulating exosomal miRNA profiling is scarce. Here, we analyzed the advantages and weaknesses of four commonly used commercial kits for exosomal miRNA profiling and their application to the sample of serum and/or plasma, respectively. NanoSight and Western blotting were conducted to evaluate the efficiency and purity of the isolated exosomes. In our conditions, the size distribution of the isolated particles was appropriate (40-150 nm), and ExoQuick™ Exosome Precipitation Solution (EXQ) generated a relatively high yield of exosomes. Nevertheless, albumin impurity was ubiquitous for all the four kits, and Total Exosome Isolation for serum or plasma (TEI) yielded a relatively pure isolation. We further performed Illumina sequencing combined with RT-qPCR to determine the ability of these kits for miRNA profiling. There was significant correlation of the exosomal miRNA profile and specific miRNAs between kits, but with differences depending on methods. exoRNeasy Serum/Plasma Midi Kit (EXR) and EXQ performed better in the specific exosomal miRNAs recovery. Intraassay CVs for specific miRNA measurement were 0.88-3.82, 1.19-3.77, 0-2.70, and 1.23-9.11% for EXR, TEI, EXQ, and RIBO™ Exosome Isolation Reagent (REI), respectively. In each kit, serum yielded a higher abundance of exosomes and exosomal miRNAs than plasma, yet with more albumin impurity. In conclusion, our data provide some valuable guidance for the methodology of disease biomarker identification of circulation exosomal miRNAs. Graphical abstract Circulating exosomal microRNAs (miRNAs) are valuable biomarker candidates; however, information on the characterization and mutual agreement of commercial kits for circulating exosomal miRNA profiling is scarce. In this study, we compared four commonly used commercially available kits for exosomal mi

  18. Genomic binding profiles of functionally distinct RNA polymerase III transcription complexes in human cells.

    Moqtaderi, Zarmik; Wang, Jie; Raha, Debasish; White, Robert J; Snyder, Michael; Weng, Zhiping; Struhl, Kevin

    2010-05-01

    Genome-wide occupancy profiles of five components of the RNA polymerase III (Pol III) machinery in human cells identified the expected tRNA and noncoding RNA targets and revealed many additional Pol III-associated loci, mostly near short interspersed elements (SINEs). Several genes are targets of an alternative transcription factor IIIB (TFIIIB) containing Brf2 instead of Brf1 and have extremely low levels of TFIIIC. Strikingly, expressed Pol III genes, unlike nonexpressed Pol III genes, are situated in regions with a pattern of histone modifications associated with functional Pol II promoters. TFIIIC alone associates with numerous ETC loci, via the B box or a novel motif. ETCs are often near CTCF binding sites, suggesting a potential role in chromosome organization. Our results suggest that human Pol III complexes associate preferentially with regions near functional Pol II promoters and that TFIIIC-mediated recruitment of TFIIIB is regulated in a locus-specific manner.

  19. Phenotypic and microRNA transcriptomic profiling of the MDA-MB-231 spheroid-enriched CSCs with comparison of MCF-7 microRNA profiling dataset

    Lily Boo

    2017-07-01

    Full Text Available Breast cancer spheroids have been widely used as in vitro models of cancer stem cells (CSCs, yet little is known about their phenotypic characteristics and microRNAs (miRNAs expression profiles. The objectives of this research were to evaluate the phenotypic characteristics of MDA-MB-231 spheroid-enriched cells for their CSCs properties and also to determine their miRNAs expression profile. Similar to our previously published MCF-7 spheroid, MDA-MB-231 spheroid also showed typical CSCs characteristics namely self-renewability, expression of putative CSCs-related surface markers and enhancement of drug resistance. From the miRNA profile, miR-15b, miR-34a, miR-148a, miR-628 and miR-196b were shown to be involved in CSCs-associated signalling pathways in both models of spheroids, which highlights the involvement of these miRNAs in maintaining the CSCs features. In addition, unique clusters of miRNAs namely miR-205, miR-181a and miR-204 were found in basal-like spheroid whereas miR-125, miR-760, miR-30c and miR-136 were identified in luminal-like spheroid. Our results highlight the roles of miRNAs as well as novel perspectives of the relevant pathways underlying spheroid-enriched CSCs in breast cancer.

  20. microRNA and mRNA profiles in ventral tegmental area relevant to stress-induced depression and resilience.

    Sun, Xiaoyan; Song, Zhenhua; Si, Yawei; Wang, Jin-Hui

    2018-06-01

    Chronic stress with lack of reward presumably may impair brain reward circuit, leading to major depressive disorder (MDD). Most individuals experiencing chronic stress do not suffer from MDD, i.e., resilience, implying the presence of endogenous anti-depression in the brain. Molecular mechanisms underlying stress-induced depression versus resilience were investigated. Mice were treated by chronic unpredictable mild stress (CUMS) for four weeks. Their mood state was assessed by behavioral tasks, such as sucrose preference, Y-maze and forced swimming testes. To reveal comprehensive molecular profiles of major depression versus resilience, mRNA and microRNA profiles were analyzed by high-throughput sequencing in the ventral tegmental area (VTA) harvested from control, CUMS-susceptible and CUMS-resilience mice. In data analyses of control versus CUMS-susceptible mice as well as control versus CUMS-resilience mice, 1.5 fold ratio in reads per kilo-base per million reads was set as the threshold to judge the involvement of mRNAs and microRNAs in the CUMS, depression or resilience. The downregulation of synaptic vesicle cycle, neurotrophin, GABAergic synapse and morphine addiction as well as the upregulation of transmitter release, calcium signal and cAMP-dependent response element binding are associated to CUMS-susceptibility. The downregulation of tyrosine metabolism and protein process in endoplasmic reticulum as well as the upregulation of amino acid biosynthesis, neuroactive ligand-receptor interaction and dopaminergic synapse are associated to CUMS-resilience. Therefore, the impairment of neurons and GABA/dopaminergic synapses in the VTA is associated with major depression. The upregulation of these entities is associated with resilience. Consistent results obtained from analyzing mRNAs and microRNAs as well as using different approaches strengthen our finding and conclusion. Copyright © 2018. Published by Elsevier Inc.

  1. MicroRNA expression profiles of cancer stem cells in head and neck squamous cell carcinoma

    YATA, KAZUYA; BEDER, LEVENT BEKIR; TAMAGAWA, SHUNJI; HOTOMI, MUNEKI; HIROHASHI, YOSHIHIKO; GRENMAN, REIDAR; YAMANAKA, NOBORU

    2015-01-01

    Increasing evidence indicates that cancer stem cells have essential roles in tumor initiation, progression, metastasis and resistance to chemo-radiation. Recent research has pointed out biological importance of microRNAs in cancer stem cell dysregulation. Total number of mature microRNAs in human genome increased to more than 2,500 with the recent up-date of the database. However, currently no information is available regarding microRNA expression profiles of cancer stem cells in head and nec...

  2. MicroRNA Expression Profiling Altered by Variant Dosage of Radiation Exposure

    Kuei-Fang Lee

    2014-01-01

    Full Text Available Various biological effects are associated with radiation exposure. Irradiated cells may elevate the risk for genetic instability, mutation, and cancer under low levels of radiation exposure, in addition to being able to extend the postradiation side effects in normal tissues. Radiation-induced bystander effect (RIBE is the focus of rigorous research as it may promote the development of cancer even at low radiation doses. Alterations in the DNA sequence could not explain these biological effects of radiation and it is thought that epigenetics factors may be involved. Indeed, some microRNAs (or miRNAs have been found to correlate radiation-induced damages and may be potential biomarkers for the various biological effects caused by different levels of radiation exposure. However, the regulatory role that miRNA plays in this aspect remains elusive. In this study, we profiled the expression changes in miRNA under fractionated radiation exposure in human peripheral blood mononuclear cells. By utilizing publicly available microRNA knowledge bases and performing cross validations with our previous gene expression profiling under the same radiation condition, we identified various miRNA-gene interactions specific to different doses of radiation treatment, providing new insights for the molecular underpinnings of radiation injury.

  3. Plasma microRNA profiles distinguish lethal injury in acetaminophen toxicity: A research study

    Jeanine Ward; Shashi Bala; Jan Petrasek; Gyongyi Szabo

    2012-01-01

    AIM:To investigate plasma microRNA (miRNA) profiles indicative of hepatotoxicity in the setting of lethal acetaminophen (APAP) toxicity in mice.METHODS:Using plasma from APAP poisoned mice,either lethally (500 mg/kg) or sublethally (150 mg/kg) dosed,we screened commercially available murine microRNA libraries (SABiosciences,Qiagen Sciences,MD) to evaluate for unique miRNA profiles between these two dosing parameters.RESULTS:We distinguished numerous,unique plasma miRNAs both up- and downregulated in lethally compared to sublethally dosed mice.Of note,many of the greatest up- and downregulated miRNAs,namely 574-5p,466g,466f-3p,375,29c,and 148a,have been shown to be associated with asthma in prior studies.Interestingly,a relationship between APAP and asthma has been previously well described in the literature,with an as yet unknown mechanism of pathology.There was a statistically significant increase in alanine aminotransferase levels in the lethal compared to sublethal APAP dosing groups at the 12 h time point (P <0.001).There was 90% mortality in the lethally compared to sublethally dosed mice at the 48 h time point (P =0.011).CONCLUSION:We identified unique plasma miRNAs both up- and downregulated in APAP poisoning which are correlated to asthma development.

  4. RNA-seq transcriptional profiling of Herbaspirillum seropedicae colonizing wheat (Triticum aestivum) roots.

    Pankievicz, V C S; Camilios-Neto, D; Bonato, P; Balsanelli, E; Tadra-Sfeir, M Z; Faoro, H; Chubatsu, L S; Donatti, L; Wajnberg, G; Passetti, F; Monteiro, R A; Pedrosa, F O; Souza, E M

    2016-04-01

    Herbaspirillum seropedicae is a diazotrophic and endophytic bacterium that associates with economically important grasses promoting plant growth and increasing productivity. To identify genes related to bacterial ability to colonize plants, wheat seedlings growing hydroponically in Hoagland's medium were inoculated with H. seropedicae and incubated for 3 days. Total mRNA from the bacteria present in the root surface and in the plant medium were purified, depleted from rRNA and used for RNA-seq profiling. RT-qPCR analyses were conducted to confirm regulation of selected genes. Comparison of RNA profile of root attached and planktonic bacteria revealed extensive metabolic adaptations to the epiphytic life style. These adaptations include expression of specific adhesins and cell wall re-modeling to attach to the root. Additionally, the metabolism was adapted to the microxic environment and nitrogen-fixation genes were expressed. Polyhydroxybutyrate (PHB) synthesis was activated, and PHB granules were stored as observed by microscopy. Genes related to plant growth promotion, such as auxin production were expressed. Many ABC transporter genes were regulated in the bacteria attached to the roots. The results provide new insights into the adaptation of H. seropedicae to the interaction with the plant.

  5. Quantitative multi-target RNA profiling in Epstein-Barr virus infected tumor cells.

    Greijer, A E; Ramayanti, O; Verkuijlen, S A W M; Novalić, Z; Juwana, H; Middeldorp, J M

    2017-03-01

    Epstein-Barr virus (EBV) is etiologically linked to multiple acute, chronic and malignant diseases. Detection of EBV-RNA transcripts in tissues or biofluids besides EBV-DNA can help in diagnosing EBV related syndromes. Sensitive EBV transcription profiling yields new insights on its pathogenic role and may be useful for monitoring virus targeted therapy. Here we describe a multi-gene quantitative RT-PCR profiling method that simultaneously detects a broad spectrum (n=16) of crucial latent and lytic EBV transcripts. These transcripts include (but are not restricted to), EBNA1, EBNA2, LMP1, LMP2, BARTs, EBER1, BARF1 and ZEBRA, Rta, BGLF4 (PK), BXLF1 (TK) and BFRF3 (VCAp18) all of which have been implicated in EBV-driven oncogenesis and viral replication. With this method we determine the amount of RNA copies per infected (tumor) cell in bulk populations of various origin. While we confirm the expected RNA profiles within classic EBV latency programs, this sensitive quantitative approach revealed the presence of rare cells undergoing lytic replication. Inducing lytic replication in EBV tumor cells supports apoptosis and is considered as therapeutic approach to treat EBV-driven malignancies. This sensitive multi-primed quantitative RT-PCR approach can provide broader understanding of transcriptional activity in latent and lytic EBV infection and is suitable for monitoring virus-specific therapy responses in patients with EBV associated cancers. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  6. Subcellular RNA profiling links splicing and nuclear DICER1 to alternative cleavage and polyadenylation.

    Neve, Jonathan; Burger, Kaspar; Li, Wencheng; Hoque, Mainul; Patel, Radhika; Tian, Bin; Gullerova, Monika; Furger, Andre

    2016-01-01

    Alternative cleavage and polyadenylation (APA) plays a crucial role in the regulation of gene expression across eukaryotes. Although APA is extensively studied, its regulation within cellular compartments and its physiological impact remains largely enigmatic. Here, we used a rigorous subcellular fractionation approach to compare APA profiles of cytoplasmic and nuclear RNA fractions from human cell lines. This approach allowed us to extract APA isoforms that are subjected to differential regulation and provided us with a platform to interrogate the molecular regulatory pathways that shape APA profiles in different subcellular locations. Here, we show that APA isoforms with shorter 3' UTRs tend to be overrepresented in the cytoplasm and appear to be cell-type-specific events. Nuclear retention of longer APA isoforms occurs and is partly a result of incomplete splicing contributing to the observed cytoplasmic bias of transcripts with shorter 3' UTRs. We demonstrate that the endoribonuclease III, DICER1, contributes to the establishment of subcellular APA profiles not only by expected cytoplasmic miRNA-mediated destabilization of APA mRNA isoforms, but also by affecting polyadenylation site choice. © 2016 Neve et al.; Published by Cold Spring Harbor Laboratory Press.

  7. microRNA expression profiling in fetal single ventricle malformation identified by deep sequencing.

    Yu, Zhang-Bin; Han, Shu-Ping; Bai, Yun-Fei; Zhu, Chun; Pan, Ya; Guo, Xi-Rong

    2012-01-01

    microRNAs (miRNAs) have emerged as key regulators in many biological processes, particularly cardiac growth and development, although the specific miRNA expression profile associated with this process remains to be elucidated. This study aimed to characterize the cellular microRNA profile involved in the development of congenital heart malformation, through the investigation of single ventricle (SV) defects. Comprehensive miRNA profiling in human fetal SV cardiac tissue was performed by deep sequencing. Differential expression of 48 miRNAs was revealed by sequencing by oligonucleotide ligation and detection (SOLiD) analysis. Of these, 38 were down-regulated and 10 were up-regulated in differentiated SV cardiac tissue, compared to control cardiac tissue. This was confirmed by real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis. Predicted target genes of the 48 differentially expressed miRNAs were analyzed by gene ontology and categorized according to cellular process, regulation of biological process and metabolic process. Pathway-Express analysis identified the WNT and mTOR signaling pathways as the most significant processes putatively affected by the differential expression of these miRNAs. The candidate genes involved in cardiac development were identified as potential targets for these differentially expressed microRNAs and the collaborative network of microRNAs and cardiac development related-mRNAs was constructed. These data provide the basis for future investigation of the mechanism of the occurrence and development of fetal SV malformations.

  8. Profiling of RNA degradation for estimation of post mortem [corrected] interval.

    Fernanda Sampaio-Silva

    Full Text Available An estimation of the post mortem interval (PMI is frequently touted as the Holy Grail of forensic pathology. During the first hours after death, PMI estimation is dependent on the rate of physical observable modifications including algor, rigor and livor mortis. However, these assessment methods are still largely unreliable and inaccurate. Alternatively, RNA has been put forward as a valuable tool in forensic pathology, namely to identify body fluids, estimate the age of biological stains and to study the mechanism of death. Nevertheless, the attempts to find correlation between RNA degradation and PMI have been unsuccessful. The aim of this study was to characterize the RNA degradation in different post mortem tissues in order to develop a mathematical model that can be used as coadjuvant method for a more accurate PMI determination. For this purpose, we performed an eleven-hour kinetic analysis of total extracted RNA from murine's visceral and muscle tissues. The degradation profile of total RNA and the expression levels of several reference genes were analyzed by quantitative real-time PCR. A quantitative analysis of normalized transcript levels on the former tissues allowed the identification of four quadriceps muscle genes (Actb, Gapdh, Ppia and Srp72 that were found to significantly correlate with PMI. These results allowed us to develop a mathematical model with predictive value for estimation of the PMI (confidence interval of ±51 minutes at 95% that can become an important complementary tool for traditional methods.

  9. Tamarix microRNA Profiling Reveals New Insight into Salt Tolerance

    Jianwen Wang

    2018-04-01

    Full Text Available The halophyte tamarisk (Tamarix is extremely salt tolerant, making it an ideal material for salt tolerance-related studies. Although many salt-responsive genes of Tamarix were identified in previous studies, there are no reports on the role of post-transcriptional regulation in its salt tolerance. We constructed six small RNA libraries of Tamarix chinensis roots with NaCl treatments. High-throughput sequencing of the six libraries was performed and microRNA expression profiles were constructed. We investigated salt-responsive microRNAs to uncover the microRNA-mediated genes regulation. From these analyses, 251 conserved and 18 novel microRNA were identified from all small RNAs. From 191 differentially expressed microRNAs, 74 co-expressed microRNAs were identified as salt-responsive candidate microRNAs. The most enriched GO (gene ontology terms for the 157 genes targeted by differentially expressed microRNAs suggested that transcriptions factors were highly active. Two hub microRNAs (miR414, miR5658, which connected by several target genes into an organic microRNA regulatory network, appeared to be the key regulators of post-transcriptional salt-stress responses. As the first survey on the tamarisk small RNAome, this study improves the understanding of tamarisk salt-tolerance mechanisms and will contribute to the molecular-assisted resistance breeding.

  10. High-Resolution Analysis of Coronavirus Gene Expression by RNA Sequencing and Ribosome Profiling.

    Irigoyen, Nerea; Firth, Andrew E; Jones, Joshua D; Chung, Betty Y-W; Siddell, Stuart G; Brierley, Ian

    2016-02-01

    Members of the family Coronaviridae have the largest genomes of all RNA viruses, typically in the region of 30 kilobases. Several coronaviruses, such as Severe acute respiratory syndrome-related coronavirus (SARS-CoV) and Middle East respiratory syndrome-related coronavirus (MERS-CoV), are of medical importance, with high mortality rates and, in the case of SARS-CoV, significant pandemic potential. Other coronaviruses, such as Porcine epidemic diarrhea virus and Avian coronavirus, are important livestock pathogens. Ribosome profiling is a technique which exploits the capacity of the translating ribosome to protect around 30 nucleotides of mRNA from ribonuclease digestion. Ribosome-protected mRNA fragments are purified, subjected to deep sequencing and mapped back to the transcriptome to give a global "snap-shot" of translation. Parallel RNA sequencing allows normalization by transcript abundance. Here we apply ribosome profiling to cells infected with Murine coronavirus, mouse hepatitis virus, strain A59 (MHV-A59), a model coronavirus in the same genus as SARS-CoV and MERS-CoV. The data obtained allowed us to study the kinetics of virus transcription and translation with exquisite precision. We studied the timecourse of positive and negative-sense genomic and subgenomic viral RNA production and the relative translation efficiencies of the different virus ORFs. Virus mRNAs were not found to be translated more efficiently than host mRNAs; rather, virus translation dominates host translation at later time points due to high levels of virus transcripts. Triplet phasing of the profiling data allowed precise determination of translated reading frames and revealed several translated short open reading frames upstream of, or embedded within, known virus protein-coding regions. Ribosome pause sites were identified in the virus replicase polyprotein pp1a ORF and investigated experimentally. Contrary to expectations, ribosomes were not found to pause at the ribosomal

  11. High-Resolution Analysis of Coronavirus Gene Expression by RNA Sequencing and Ribosome Profiling.

    Nerea Irigoyen

    2016-02-01

    Full Text Available Members of the family Coronaviridae have the largest genomes of all RNA viruses, typically in the region of 30 kilobases. Several coronaviruses, such as Severe acute respiratory syndrome-related coronavirus (SARS-CoV and Middle East respiratory syndrome-related coronavirus (MERS-CoV, are of medical importance, with high mortality rates and, in the case of SARS-CoV, significant pandemic potential. Other coronaviruses, such as Porcine epidemic diarrhea virus and Avian coronavirus, are important livestock pathogens. Ribosome profiling is a technique which exploits the capacity of the translating ribosome to protect around 30 nucleotides of mRNA from ribonuclease digestion. Ribosome-protected mRNA fragments are purified, subjected to deep sequencing and mapped back to the transcriptome to give a global "snap-shot" of translation. Parallel RNA sequencing allows normalization by transcript abundance. Here we apply ribosome profiling to cells infected with Murine coronavirus, mouse hepatitis virus, strain A59 (MHV-A59, a model coronavirus in the same genus as SARS-CoV and MERS-CoV. The data obtained allowed us to study the kinetics of virus transcription and translation with exquisite precision. We studied the timecourse of positive and negative-sense genomic and subgenomic viral RNA production and the relative translation efficiencies of the different virus ORFs. Virus mRNAs were not found to be translated more efficiently than host mRNAs; rather, virus translation dominates host translation at later time points due to high levels of virus transcripts. Triplet phasing of the profiling data allowed precise determination of translated reading frames and revealed several translated short open reading frames upstream of, or embedded within, known virus protein-coding regions. Ribosome pause sites were identified in the virus replicase polyprotein pp1a ORF and investigated experimentally. Contrary to expectations, ribosomes were not found to pause at the

  12. MicroRNA Profiling Reveals Marker of Motor Neuron Disease in ALS Models.

    Hoye, Mariah L; Koval, Erica D; Wegener, Amy J; Hyman, Theodore S; Yang, Chengran; O'Brien, David R; Miller, Rebecca L; Cole, Tracy; Schoch, Kathleen M; Shen, Tao; Kunikata, Tomonori; Richard, Jean-Philippe; Gutmann, David H; Maragakis, Nicholas J; Kordasiewicz, Holly B; Dougherty, Joseph D; Miller, Timothy M

    2017-05-31

    Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder marked by the loss of motor neurons (MNs) in the brain and spinal cord, leading to fatally debilitating weakness. Because this disease predominantly affects MNs, we aimed to characterize the distinct expression profile of that cell type to elucidate underlying disease mechanisms and to identify novel targets that inform on MN health during ALS disease time course. microRNAs (miRNAs) are short, noncoding RNAs that can shape the expression profile of a cell and thus often exhibit cell-type-enriched expression. To determine MN-enriched miRNA expression, we used Cre recombinase-dependent miRNA tagging and affinity purification in mice. By defining the in vivo miRNA expression of MNs, all neurons, astrocytes, and microglia, we then focused on MN-enriched miRNAs via a comparative analysis and found that they may functionally distinguish MNs postnatally from other spinal neurons. Characterizing the levels of the MN-enriched miRNAs in CSF harvested from ALS models of MN disease demonstrated that one miRNA (miR-218) tracked with MN loss and was responsive to an ALS therapy in rodent models. Therefore, we have used cellular expression profiling tools to define the distinct miRNA expression of MNs, which is likely to enrich future studies of MN disease. This approach enabled the development of a novel, drug-responsive marker of MN disease in ALS rodents. SIGNIFICANCE STATEMENT Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease in which motor neurons (MNs) in the brain and spinal cord are selectively lost. To develop tools to aid in our understanding of the distinct expression profiles of MNs and, ultimately, to monitor MN disease progression, we identified small regulatory microRNAs (miRNAs) that were highly enriched or exclusive in MNs. The signal for one of these MN-enriched miRNAs is detectable in spinal tap biofluid from an ALS rat model, where its levels change as disease

  13. Evaluating whole transcriptome amplification for gene profiling experiments using RNA-Seq.

    Faherty, Sheena L; Campbell, C Ryan; Larsen, Peter A; Yoder, Anne D

    2015-07-30

    RNA-Seq has enabled high-throughput gene expression profiling to provide insight into the functional link between genotype and phenotype. Low quantities of starting RNA can be a severe hindrance for studies that aim to utilize RNA-Seq. To mitigate this bottleneck, whole transcriptome amplification (WTA) technologies have been developed to generate sufficient sequencing targets from minute amounts of RNA. Successful WTA requires accurate replication of transcript abundance without the loss or distortion of specific mRNAs. Here, we test the efficacy of NuGEN's Ovation RNA-Seq V2 system, which uses linear isothermal amplification with a unique chimeric primer for amplification, using white adipose tissue from standard laboratory rats (Rattus norvegicus). Our goal was to investigate potential biological artifacts introduced through WTA approaches by establishing comparisons between matched raw and amplified RNA libraries derived from biological replicates. We found that 93% of expressed genes were identical between all unamplified versus matched amplified comparisons, also finding that gene density is similar across all comparisons. Our sequencing experiment and downstream bioinformatic analyses using the Tuxedo analysis pipeline resulted in the assembly of 25,543 high-quality transcripts. Libraries constructed from raw RNA and WTA samples averaged 15,298 and 15,253 expressed genes, respectively. Although significant differentially expressed genes (P < 0.05) were identified in all matched samples, each of these represents less than 0.15% of all shared genes for each comparison. Transcriptome amplification is efficient at maintaining relative transcript frequencies with no significant bias when using this NuGEN linear isothermal amplification kit under ideal laboratory conditions as presented in this study. This methodology has broad applications, from clinical and diagnostic, to field-based studies when sample acquisition, or sample preservation, methods prove

  14. Unravelling the Long Non-Coding RNA Profile of Undifferentiated Large Cell Lung Carcinoma.

    Shukla, Sudhanshu

    2018-02-05

    Undifferentiated large cell lung carcinoma (LCLC) accounts for 2.9-9% of total lung cancers. Recently, RNA-seq based studies have revealed major genomic aberrations in LCLC. In this study, we aim to identify long non-coding RNAs (LncRNAs) expression pattern specific to LCLC. The RNA-seq profile of LCLC and other non-small cell lung carcinoma (NSCLC) was downloaded from Gene Expression Omnibus (GEO) and analyzed. Using 10 LCLC samples, we found that 18% of all the annotated LncRNAs are expressed in LCLC samples. Among 1794 expressed LncRNAs, 11 were overexpressed and 14 were downregulated in LCLC compared to normal samples. Based on receiver operating characteristic (ROC) analysis, we showed that the top five differentially expressed LncRNAs were able to differentiate between LCLC and normal samples with high sensitivity and specificity. Guilt by association analysis using genes correlating with differentially expressed LncRNAs identified several cancer-associated pathways, suggesting the role of these deregulated LncRNA in LCLC biology. We also identified the LncRNA differentially expressed in LCLC compared to lung squamous carcinoma (LUSC) and Lung-adenocarcinoma (LUAD). We found that LCLC sample showed more deregulated LncRNA in LUSC than LUAD. Interestingly, LCLC had more downregulated LncRNA compared to LUAD and LUSC. Our study provides novel insight into LncRNA deregulation in LCLC. This study also finds tools to diagnose LCLC and differentiate LCLC with other Non-Small Cell Lung Cancer.

  15. Mercury's Reference Frames After the MESSENGER Mission

    Stark, A.; Oberst, J.; Preusker, F.; Burmeister, S.; Steinbrügge, G.; Hussmann, H.

    2018-05-01

    We provide an overview of Mercury's reference frames based on MESSENGER observations. We discuss the dynamical, the principal-axes, the ellipsoid, as well as the cartographic frame, which was adopted for MESSENGER data products.

  16. miRNA profiling of naive, effector and memory CD8 T cells.

    Haoquan Wu

    Full Text Available microRNAs have recently emerged as master regulators of gene expression during development and cell differentiation. Although profound changes in gene expression also occur during antigen-induced T cell differentiation, the role of miRNAs in the process is not known. We compared the miRNA expression profiles between antigen-specific naïve, effector and memory CD8+ T cells using 3 different methods--small RNA cloning, miRNA microarray analysis and real-time PCR. Although many miRNAs were expressed in all the T cell subsets, the frequency of 7 miRNAs (miR-16, miR-21, miR-142-3p, miR-142-5p, miR-150, miR-15b and let-7f alone accounted for approximately 60% of all miRNAs, and their expression was several fold higher than the other expressed miRNAs. Global downregulation of miRNAs (including 6/7 dominantly expressed miRNAs was observed in effector T cells compared to naïve cells and the miRNA expression levels tended to come back up in memory T cells. However, a few miRNAs, notably miR-21 were higher in effector and memory T cells compared to naïve T cells. These results suggest that concomitant with profound changes in gene expression, miRNA profile also changes dynamically during T cell differentiation. Sequence analysis of the cloned mature miRNAs revealed an extensive degree of end polymorphism. While 3'end polymorphisms dominated, heterogeneity at both ends, resembling drosha/dicer processing shift was also seen in miR-142, suggesting a possible novel mechanism to generate new miRNA and/or to diversify miRNA target selection. Overall, our results suggest that dynamic changes in the expression of miRNAs may be important for the regulation of gene expression during antigen-induced T cell differentiation. Our study also suggests possible novel mechanisms for miRNA biogenesis and function.

  17. Plasma and White Blood Cells Show Different miRNA Expression Profiles in Parkinson's Disease.

    Schwienbacher, Christine; Foco, Luisa; Picard, Anne; Corradi, Eloina; Serafin, Alice; Panzer, Jörg; Zanigni, Stefano; Blankenburg, Hagen; Facheris, Maurizio F; Giannini, Giulia; Falla, Marika; Cortelli, Pietro; Pramstaller, Peter P; Hicks, Andrew A

    2017-06-01

    Parkinson's disease (PD) diagnosis is based on the assessment of motor symptoms, which manifest when more than 50% of dopaminergic neurons are degenerated. To date, no validated biomarkers are available for the diagnosis of PD. The aims of the present study are to evaluate whether plasma and white blood cells (WBCs) are interchangeable biomarker sources and to identify circulating plasma-based microRNA (miRNA) biomarkers for an early detection of PD. We profiled plasma miRNA levels in 99 L-dopa-treated PD patients from two independent data collections, in ten drug-naïve PD patients, and in unaffected controls matched by sex and age. We evaluated expression levels by reverse transcription and quantitative real-time PCR (RT-qPCR) and combined the results from treated PD patients using a fixed effect inverse-variance weighted meta-analysis. We revealed different expression profiles comparing plasma and WBCs and drug-naïve and L-dopa-treated PD patients. We observed an upregulation trend for miR-30a-5p in L-dopa-treated PD patients and investigated candidate target genes by integrated in silico analyses. We could not analyse miR-29b-3p, normally expressed in WBCs, due to the very low expression in plasma. We observed different expression profiles in WBCs and plasma, suggesting that they are both suitable but not interchangeable peripheral sources for biomarkers. We revealed miR-30a-5p as a potential biomarker for PD in plasma. In silico analyses suggest that miR-30a-5p might have a regulatory role in mitochondrial dynamics and autophagy. Further investigations are needed to confirm miR-30a-5p deregulation and targets and to investigate the influence of L-dopa treatment on miRNA expression levels.

  18. RNA-Seq for gene identification and transcript profiling of three Stevia rebaudiana genotypes.

    Chen, Junwen; Hou, Kai; Qin, Peng; Liu, Hongchang; Yi, Bin; Yang, Wenting; Wu, Wei

    2014-07-07

    Stevia (Stevia rebaudiana) is an important medicinal plant that yields diterpenoid steviol glycosides (SGs). SGs are currently used in the preparation of medicines, food products and neutraceuticals because of its sweetening property (zero calories and about 300 times sweeter than sugar). Recently, some progress has been made in understanding the biosynthesis of SGs in Stevia, but little is known about the molecular mechanisms underlying this process. Additionally, the genomics of Stevia, a non-model species, remains uncharacterized. The recent advent of RNA-Seq, a next generation sequencing technology, provides an opportunity to expand the identification of Stevia genes through in-depth transcript profiling. We present a comprehensive landscape of the transcriptome profiles of three genotypes of Stevia with divergent SG compositions characterized using RNA-seq. 191,590,282 high-quality reads were generated and then assembled into 171,837 transcripts with an average sequence length of 969 base pairs. A total of 80,160 unigenes were annotated, and 14,211 of the unique sequences were assigned to specific metabolic pathways by the Kyoto Encyclopedia of Genes and Genomes. Gene sequences of all enzymes known to be involved in SG synthesis were examined. A total of 143 UDP-glucosyltransferase (UGT) unigenes were identified, some of which might be involved in SG biosynthesis. The expression patterns of eight of these genes were further confirmed by RT-QPCR. RNA-seq analysis identified candidate genes encoding enzymes responsible for the biosynthesis of SGs in Stevia, a non-model plant without a reference genome. The transcriptome data from this study yielded new insights into the process of SG accumulation in Stevia. Our results demonstrate that RNA-Seq can be successfully used for gene identification and transcript profiling in a non-model species.

  19. MicroRNA expression profiles in human cancer cells after ionizing radiation

    Niemoeller, Olivier M; Niyazi, Maximilian; Corradini, Stefanie; Zehentmayr, Franz; Li, Minglun; Lauber, Kirsten; Belka, Claus

    2011-01-01

    MicroRNAs are regulators of central cellular processes and are implicated in the pathogenesis and prognosis of human cancers. MicroRNAs also modulate responses to anti-cancer therapy. In the context of radiation oncology microRNAs were found to modulate cell death and proliferation after irradiation. However, changes in microRNA expression profiles in response to irradiation have not been comprehensively analyzed so far. The present study's intend is to present a broad screen of changes in microRNA expression following irradiation of different malignant cell lines. 1100 microRNAs (Sanger miRBase release version 14.0) were analyzed in six malignant cell lines following irradiation with clinically relevant doses of 2.0 Gy. MicroRNA levels 6 hours after irradiation were compared to microRNA levels in non-irradiated cells using the 'Geniom Biochip MPEA homo sapiens'. Hierarchical clustering analysis revealed a pattern, which significantly (p = 0.014) discerned irradiated from non-irradiated cells. The expression levels of a number of microRNAs known to be involved in the regulation of cellular processes like apoptosis, proliferation, invasion, local immune response and radioresistance (e. g. miR-1285, miR-24-1, miR-151-5p, let-7i) displayed 2 - 3-fold changes after irradiation. Moreover, several microRNAs previously not known to be radiation-responsive were discovered. Ionizing radiation induced significant changes in microRNA expression profiles in 3 glioma and 3 squamous cell carcinoma cell lines. The functional relevance of these changes is not addressed but should by analyzed by future work especially focusing on clinically relevant endpoints like radiation induced cell death, proliferation, migration and metastasis

  20. Plasma microRNA profiles in rat models of hepatocellular injury, cholestasis, and steatosis.

    Yu Yamaura

    Full Text Available MicroRNAs (miRNAs are small RNA molecules that function to modulate the expression of target genes, playing important roles in a wide range of physiological and pathological processes. The miRNAs in body fluids have received considerable attention as potential biomarkers of various diseases. In this study, we compared the changes of the plasma miRNA expressions by acute liver injury (hepatocellular injury or cholestasis and chronic liver injury (steatosis, steatohepatitis and fibrosis using rat models made by the administration of chemicals or special diets. Using miRNA array analysis, we found that the levels of a large number of miRNAs (121-317 miRNAs were increased over 2-fold and the levels of a small number of miRNAs (6-35 miRNAs were decreased below 0.5-fold in all models except in a model of cholestasis caused by bile duct ligation. Interestingly, the expression profiles were different between the models, and the hierarchical clustering analysis discriminated between the acute and chronic liver injuries. In addition, miRNAs whose expressions were typically changed in each type of liver injury could be specified. It is notable that, in acute liver injury models, the plasma level of miR-122, the most abundant miRNA in the liver, was more quickly and dramatically increased than the plasma aminotransferase level, reflecting the extent of hepatocellular injury. This study demonstrated that the plasma miRNA profiles could reflect the types of liver injury (e.g. acute/chronic liver injury or hepatocellular injury/cholestasis/steatosis/steatohepatitis/fibrosis and identified the miRNAs that could be specific and sensitive biomarkers of liver injury.

  1. MicroRNA Profiling in Aqueous Humor of Individual Human Eyes by Next-Generation Sequencing.

    Wecker, Thomas; Hoffmeier, Klaus; Plötner, Anne; Grüning, Björn Andreas; Horres, Ralf; Backofen, Rolf; Reinhard, Thomas; Schlunck, Günther

    2016-04-01

    Extracellular microRNAs (miRNAs) in aqueous humor were suggested to have a role in transcellular signaling and may serve as disease biomarkers. The authors adopted next-generation sequencing (NGS) techniques to further characterize the miRNA profile in single samples of 60 to 80 μL human aqueous humor. Samples were obtained at the outset of cataract surgery in nine independent, otherwise healthy eyes. Four samples were used to extract RNA and generate sequencing libraries, followed by an adapter-driven amplification step, electrophoretic size selection, sequencing, and data analysis. Five samples were used for quantitative PCR (qPCR) validation of NGS results. Published NGS data on circulating miRNAs in blood were analyzed in comparison. One hundred fifty-eight miRNAs were consistently detected by NGS in all four samples; an additional 59 miRNAs were present in at least three samples. The aqueous humor miRNA profile shows some overlap with published NGS-derived inventories of circulating miRNAs in blood plasma with high prevalence of human miR-451a, -21, and -16. In contrast to blood, miR-184, -4448, -30a, -29a, -29c, -19a, -30d, -205, -24, -22, and -3074 were detected among the 20 most prevalent miRNAs in aqueous humor. Relative expression patterns of miR-451a, -202, and -144 suggested by NGS were confirmed by qPCR. Our data illustrate the feasibility of miRNA analysis by NGS in small individual aqueous humor samples. Intraocular cells as well as blood plasma contribute to the extracellular aqueous humor miRNome. The data suggest possible roles of miRNA in intraocular cell adhesion and signaling by TGF-β and Wnt, which are important in intraocular pressure regulation and glaucoma.

  2. mRNA expression profile in DLD-1 and MOLT-4 cancer cell lines cultured under Microgravity

    National Aeronautics and Space Administration — DLD-1 and MOLT-4 cell lines were cultured in a Rotating cell culture system to simulate microgravity and mRNA expression profile was observed in comparison to Static...

  3. RNA.

    Darnell, James E., Jr.

    1985-01-01

    Ribonucleic acid (RNA) converts genetic information into protein and usually must be processed to serve its function. RNA types, chemical structure, protein synthesis, translation, manufacture, and processing are discussed. Concludes that the first genes might have been spliced RNA and that humans might be closer than bacteria to primitive…

  4. OP17MICRORNA PROFILING USING SMALL RNA-SEQ IN PAEDIATRIC LOW GRADE GLIOMAS

    Jeyapalan, Jennie N.; Jones, Tania A.; Tatevossian, Ruth G.; Qaddoumi, Ibrahim; Ellison, David W.; Sheer, Denise

    2014-01-01

    INTRODUCTION: MicroRNAs regulate gene expression by targeting mRNAs for translational repression or degradation at the post-transcriptional level. In paediatric low-grade gliomas a few key genetic mutations have been identified, including BRAF fusions, FGFR1 duplications and MYB rearrangements. Our aim in the current study is to profile aberrant microRNA expression in paediatric low-grade gliomas and determine the role of epigenetic changes in the aetiology and behaviour of these tumours. METHOD: MicroRNA profiling of tumour samples (6 pilocytic, 2 diffuse, 2 pilomyxoid astrocytomas) and normal brain controls (4 adult normal brain samples and a primary glial progenitor cell-line) was performed using small RNA sequencing. Bioinformatic analysis included sequence alignment, analysis of the number of reads (CPM, counts per million) and differential expression. RESULTS: Sequence alignment identified 695 microRNAs, whose expression was compared in tumours v. normal brain. PCA and hierarchical clustering showed separate groups for tumours and normal brain. Computational analysis identified approximately 400 differentially expressed microRNAs in the tumours compared to matched location controls. Our findings will then be validated and integrated with extensive genetic and epigenetic information we have previously obtained for the full tumour cohort. CONCLUSION: We have identified microRNAs that are differentially expressed in paediatric low-grade gliomas. As microRNAs are known to target genes involved in the initiation and progression of cancer, they provide critical information on tumour pathogenesis and are an important class of biomarkers.

  5. MicroRNA Expression Profiling of Human Respiratory Epithelium Affected by Invasive Candida Infection.

    Syed Aun Muhammad

    Full Text Available Invasive candidiasis is potentially life-threatening systemic fungal infection caused by Candida albicans (C. albicans. Candida enters the blood stream and disseminate throughout the body and it is often observed in hospitalized patients, immunocompromised individuals or those with chronic diseases. This infection is opportunistic and risk starts with the colonization of C. albicans on mucocutaneous surfaces and respiratory epithelium. MicroRNAs (miRNAs are small non-coding RNAs which are involved in the regulation of virtually every cellular process. They regulate and control the levels of mRNA stability and post-transcriptional gene expression. Aberrant expression of miRNAs has been associated in many disease states, and miRNA-based therapies are in progress. In this study, we investigated possible variations of miRNA expression profiles of respiratory epithelial cells infected by invasive Candida species. For this purpose, respiratory epithelial tissues of infected individuals from hospital laboratory were accessed before their treatment. Invasive Candida infection was confirmed by isolation of Candia albicans from the blood cultures of the same infected individuals. The purity of epithelial tissues was assessed by flow cytometry (FACSCalibur cytometer; BD Biosciences, Heidelberg, Germany using statin antibody (S-44. TaqMan quantitative real-time PCR (in a TaqMan Low Density Array format was used for miRNA expression profiling. MiRNAs investigated, the levels of expression of 55 miRNA were significantly altered in infected tissues. Some miRNAs showed dramatic increase (miR-16-1 or decrease of expression (miR-17-3p as compared to control. Gene ontology enrichment analysis of these miRNA-targeted genes suggests that Candidal infection affect many important biological pathways. In summary, disturbance in miRNA expression levels indicated the change in cascade of pathological processes and the regulation of respiratory epithelial functions

  6. Transient Gene and miRNA Expression Profile Changes of Confluent Human Fibroblast Cells in Space

    Zhang, Ye; Lu, Tao; Wong, Michael; Feiveson, Alan; Stodieck, Louis; Karouia, Fathi; Wang, Xiaoyu; Wu, Honglu

    2015-01-01

    Microgravity or an altered gravity environment from the static 1 gravitational constant has been shown to influence global gene expression patterns and protein levels in cultured cells. However, most of the reported studies conducted in space or using simulated microgravity on the ground have focused on the growth or differentiation of the cells. Whether non-dividing cultured cells will sense the presence of microgravity in space has not been specifically addressed. In an experiment conducted on the International Space Station, confluent human fibroblast cells were fixed after being cultured in space for 3 and 14 days for investigations of gene and miRNA (microRNA) expression profile changes in these cells. A fibroblast is a type of cell that synthesizes the extracellular matrix and collagen, the structural framework for tissues, and plays a critical role in wound healing and other functions. Results of the experiment showed that on Day 3, both the flown and ground cells were still proliferating slowly even though they were confluent, as measured by the expression of the protein Ki-67 positive cells, and the cells in space grew slightly faster. Gene and miRNA expression data indicated activation of NF(sub kappa)B (nuclear factor kappa-light-chain-enhancer of activated B cells) and other growth related pathways involving HGF and VEGF in the flown cells. On Day 14 when the cells were mostly non-dividing, the gene and miRNA expression profiles between the flight and ground samples were indistinguishable. Comparison of gene and miRNA expressions in the Day 3 samples in respect to Day 14 revealed that most of the changes observed on Day 3 were related to cell growth for both the flown and ground cells. Analysis of cytoskeleton changes by immunohistochemistry staining of the cells with antibodies for alpha-tubulin showed no difference between the flight and ground samples. Results of our study suggest that in true non-dividing human fibroblast cells, microgravity in

  7. Integrating microRNA and mRNA expression profiling in Symbiodinium microadriaticum, a dinoflagellate symbiont of reef-building corals.

    Baumgarten, Sebastian; Bayer, Till; Aranda, Manuel; Liew, Yi Jin; Carr, Adrian; Micklem, Gos; Voolstra, Christian R.

    2013-01-01

    Animal and plant genomes produce numerous small RNAs (smRNAs) that regulate gene expression post-transcriptionally affecting metabolism, development, and epigenetic inheritance. In order to characterize the repertoire of endogenous smRNAs and potential gene targets in dinoflagellates, we conducted smRNA and mRNA expression profiling over 9 experimental treatments of cultures from Symbiodinium microadriaticum, a photosynthetic symbiont of scleractinian corals.

  8. Integrating microRNA and mRNA expression profiling in Symbiodinium microadriaticum, a dinoflagellate symbiont of reef-building corals.

    Baumgarten, Sebastian

    2013-10-12

    Animal and plant genomes produce numerous small RNAs (smRNAs) that regulate gene expression post-transcriptionally affecting metabolism, development, and epigenetic inheritance. In order to characterize the repertoire of endogenous smRNAs and potential gene targets in dinoflagellates, we conducted smRNA and mRNA expression profiling over 9 experimental treatments of cultures from Symbiodinium microadriaticum, a photosynthetic symbiont of scleractinian corals.

  9. Dose-Response Analysis of RNA-Seq Profiles in Archival Formalin-Fixed Paraffin-Embedded (FFPE) Samples.

    Use of archival resources has been limited to date by inconsistent methods for genomic profiling of degraded RNA from formalin-fixed paraffin-embedded (FFPE) samples. RNA-sequencing offers a promising way to address this problem. Here we evaluated transcriptomic dose responses us...

  10. Paper-Based MicroRNA Expression Profiling from Plasma and Circulating Tumor Cells.

    Leong, Sai Mun; Tan, Karen Mei-Ling; Chua, Hui Wen; Huang, Mo-Chao; Cheong, Wai Chye; Li, Mo-Huang; Tucker, Steven; Koay, Evelyn Siew-Chuan

    2017-03-01

    Molecular characterization of circulating tumor cells (CTCs) holds great promise for monitoring metastatic progression and characterizing metastatic disease. However, leukocyte and red blood cell contamination of routinely isolated CTCs makes CTC-specific molecular characterization extremely challenging. Here we report the use of a paper-based medium for efficient extraction of microRNAs (miRNAs) from limited amounts of biological samples such as rare CTCs harvested from cancer patient blood. Specifically, we devised a workflow involving the use of Flinders Technology Associates (FTA) ® Elute Card with a digital PCR-inspired "partitioning" method to extract and purify miRNAs from plasma and CTCs. We demonstrated the sensitivity of this method to detect miRNA expression from as few as 3 cancer cells spiked into human blood. Using this method, background miRNA expression was excluded from contaminating blood cells, and CTC-specific miRNA expression profiles were derived from breast and colorectal cancer patients. Plasma separated out during purification of CTCs could likewise be processed using the same paper-based method for miRNA detection, thereby maximizing the amount of patient-specific information that can be derived from a single blood draw. Overall, this paper-based extraction method enables an efficient, cost-effective workflow for maximized recovery of small RNAs from limited biological samples for downstream molecular analyses. © 2016 American Association for Clinical Chemistry.

  11. Altered retinal microRNA expression profiles in early diabetic retinopathy: an in silico analysis.

    Xiong, Fen; Du, Xinhua; Hu, Jianyan; Li, Tingting; Du, Shanshan; Wu, Qiang

    2014-07-01

    MicroRNAs (miRNAs) - as negative regulators of target genes - are associated with various human diseases, but their precise role(s) in diabetic retinopathy (DR) remains to be elucidated. The aim of this study was to elucidate the involvement of miRNAs in early DR using in silico analysis to explore their gene expression patterns. We used the streptozotocin (STZ)-induced diabetic rat to investigate the roles of miRNAs in early DR. Retinal miRNA expression profiles from diabetic versus healthy control rats were examined by miRNA array analysis. Based on several bioinformatic systems, specifically, gene ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, we identified signatures of the potential pathological processes, gene functions, and signaling pathways that are influenced by dysregulated miRNAs. We used quantitative real-time polymerase chain reaction (qRT-PCR) to validate six (i.e. those with significant changes in expression levels) of the 17 miRNAs that were detected in the miRNA array. We also describe the significant role of the miRNA-gene network, which is based on the interactions between miRNAs and target genes. GO analysis of the 17 miRNAs detected in the miRNA array analysis revealed the most prevalent miRNAs to be those related to biological processes, olfactory bulb development and axonogenesis. These miRNAs also exert significant influence on additional pathways, including the mitogen-activated protein and calcium signaling pathways. Six of the seventeen miRNAs were chosen for qRT-PCR validation. With the exception of a slight difference in miRNA-350, our results are in close agreement with the differential expressions detected by array analysis. This study, which describes miRNA expression during the early developmental phases of DR, revealed extensive miRNA interactions. Based on both their target genes and signaling pathways, we suggest that miRNAs perform critical regulatory functions during the early stages of DR

  12. Phenomenologies of Higgs messenger models

    Zheng Sibo; Yu Yao; Wu Xinggang [Department of Physics, Chongqing University, Chongqing 401331 (China)

    2011-08-11

    In this Letter, we investigate the phenomenologies of models where the Higgs sector plays the role of messengers in gauge mediation. The minimal Higgs sector and its extension are considered respectively. We find that there exist viable models when an appropriate parity is imposed. Phenomenological features in these kind of models include three sum rules for scalar masses, light gluino as well as one-loop {mu} and two-loop B{mu} terms.

  13. MicroRNA expression profiling during the life cycle of the silkworm (Bombyx mori)

    Liu, Shiping; Zhang, Liang; Li, Qibin; Zhao, Ping; Duan, Jun; Cheng, Daojun; Xiang, Zhonghuai; Xia, Qingyou

    2009-01-01

    Abstract Background MicroRNAs (miRNAs) are expressed by a wide range of eukaryotic organisms, and function in diverse biological processes. Numerous miRNAs have been identified in Bombyx mori, but the temporal expression profiles of miRNAs corresponding to each stage transition over the entire life cycle of the silkworm remain to be established. To obtain a comprehensive overview of the correlation between miRNA expression and stage transitions, we performed a whole-life test and subsequent s...

  14. Tissue-specific mRNA expression profiling in grape berry tissues

    Grimplet, Jerome; Deluc, Laurent G; Tillett, Richard L; Wheatley, Matthew D; Schlauch, Karen A; Cramer, Grant R; Cushman, John C

    2007-01-01

    Background Berries of grape (Vitis vinifera) contain three major tissue types (skin, pulp and seed) all of which contribute to the aroma, color, and flavor characters of wine. The pericarp, which is composed of the exocarp (skin) and mesocarp (pulp), not only functions to protect and feed the developing seed, but also to assist in the dispersal of the mature seed by avian and mammalian vectors. The skin provides volatile and nonvolatile aroma and color compounds, the pulp contributes organic acids and sugars, and the seeds provide condensed tannins, all of which are important to the formation of organoleptic characteristics of wine. In order to understand the transcriptional network responsible for controlling tissue-specific mRNA expression patterns, mRNA expression profiling was conducted on each tissue of mature berries of V. vinifera Cabernet Sauvignon using the Affymetrix GeneChip® Vitis oligonucleotide microarray ver. 1.0. In order to monitor the influence of water-deficit stress on tissue-specific expression patterns, mRNA expression profiles were also compared from mature berries harvested from vines subjected to well-watered or water-deficit conditions. Results Overall, berry tissues were found to express approximately 76% of genes represented on the Vitis microarray. Approximately 60% of these genes exhibited significant differential expression in one or more of the three major tissue types with more than 28% of genes showing pronounced (2-fold or greater) differences in mRNA expression. The largest difference in tissue-specific expression was observed between the seed and pulp/skin. Exocarp tissue, which is involved in pathogen defense and pigment production, showed higher mRNA abundance relative to other berry tissues for genes involved with flavonoid biosynthesis, pathogen resistance, and cell wall modification. Mesocarp tissue, which is considered a nutritive tissue, exhibited a higher mRNA abundance of genes involved in cell wall function and

  15. Tissue-specific mRNA expression profiling in grape berry tissues

    Cramer Grant R

    2007-06-01

    Full Text Available Abstract Background Berries of grape (Vitis vinifera contain three major tissue types (skin, pulp and seed all of which contribute to the aroma, color, and flavor characters of wine. The pericarp, which is composed of the exocarp (skin and mesocarp (pulp, not only functions to protect and feed the developing seed, but also to assist in the dispersal of the mature seed by avian and mammalian vectors. The skin provides volatile and nonvolatile aroma and color compounds, the pulp contributes organic acids and sugars, and the seeds provide condensed tannins, all of which are important to the formation of organoleptic characteristics of wine. In order to understand the transcriptional network responsible for controlling tissue-specific mRNA expression patterns, mRNA expression profiling was conducted on each tissue of mature berries of V. vinifera Cabernet Sauvignon using the Affymetrix GeneChip® Vitis oligonucleotide microarray ver. 1.0. In order to monitor the influence of water-deficit stress on tissue-specific expression patterns, mRNA expression profiles were also compared from mature berries harvested from vines subjected to well-watered or water-deficit conditions. Results Overall, berry tissues were found to express approximately 76% of genes represented on the Vitis microarray. Approximately 60% of these genes exhibited significant differential expression in one or more of the three major tissue types with more than 28% of genes showing pronounced (2-fold or greater differences in mRNA expression. The largest difference in tissue-specific expression was observed between the seed and pulp/skin. Exocarp tissue, which is involved in pathogen defense and pigment production, showed higher mRNA abundance relative to other berry tissues for genes involved with flavonoid biosynthesis, pathogen resistance, and cell wall modification. Mesocarp tissue, which is considered a nutritive tissue, exhibited a higher mRNA abundance of genes involved in cell

  16. Distinctive serum miRNA profile in mouse models of striated muscular pathologies.

    Nicolas Vignier

    Full Text Available Biomarkers are critically important for disease diagnosis and monitoring. In particular, close monitoring of disease evolution is eminently required for the evaluation of therapeutic treatments. Classical monitoring methods in muscular dystrophies are largely based on histological and molecular analyses of muscle biopsies. Such biopsies are invasive and therefore difficult to obtain. The serum protein creatine kinase is a useful biomarker, which is however not specific for a given pathology and correlates poorly with the severity or course of the muscular pathology. The aim of the present study was the systematic evaluation of serum microRNAs (miRNAs as biomarkers in striated muscle pathologies. Mouse models for five striated muscle pathologies were investigated: Duchenne muscular dystrophy (DMD, limb-girdle muscular dystrophy type 2D (LGMD2D, limb-girdle muscular dystrophy type 2C (LGMD2C, Emery-Dreifuss muscular dystrophy (EDMD and hypertrophic cardiomyopathy (HCM. Two-step RT-qPCR methodology was elaborated, using two different RT-qPCR miRNA quantification technologies. We identified miRNA modulation in the serum of all the five mouse models. The most highly dysregulated serum miRNAs were found to be commonly upregulated in DMD, LGMD2D and LGMD2C mouse models, which all exhibit massive destruction of striated muscle tissues. Some of these miRNAs were down rather than upregulated in the EDMD mice, a model without massive myofiber destruction. The dysregulated miRNAs identified in the HCM model were different, with the exception of one dysregulated miRNA common to all pathologies. Importantly, a specific and distinctive circulating miRNA profile was identified for each studied pathological mouse model. The differential expression of a few dysregulated miRNAs in the DMD mice was further evaluated in DMD patients, providing new candidates of circulating miRNA biomarkers for DMD.

  17. Microparticle conferred microRNA profiles - implications in the transfer and dominance of cancer traits

    Jaiswal Ritu

    2012-06-01

    Full Text Available Abstract Background Microparticles (MPs are membrane vesicles which are released from normal and malignant cells following a process of budding and detachment from donor cells. MPs contain surface antigens, proteins and genetic material and serve as vectors of intercellular communication. MPs comprise the major source of systemic RNA including microRNA (miRNA, the aberrant expression of which appears to be associated with stage, progression and spread of many cancers. Our previous study showed that MPs carry both transcripts and miRNAs associated with the acquisition of multidrug resistance in cancer. Results Herein, we expand on our previous finding and demonstrate that MPs carry the transcripts of the membrane vesiculation machinery (floppase and scramblase as well as nucleic acids encoding the enzymes essential for microRNA biogenesis (Drosha, Dicer and Argonaute. We also demonstrate using microarray miRNA profiling analysis, the selective packaging of miRNAs (miR-1228*, miR-1246, miR-1308, miR-149*, miR-455-3p, miR-638 and miR-923 within the MP cargo upon release from the donor cells. Conclusions These miRNAs are present in both haematological and non-haematological cancer cells and are involved in pathways implicated in cancer pathogenesis, membrane vesiculation and cascades regulated by ABC transporters. Our recent findings reinforce our earlier reports that MP transfer ‘re-templates’ recipient cells so as to reflect donor cell traits. We now demonstrate that this process is likely to occur via a process of selective packaging of nucleic acid species, including regulatory nucleic acids upon MP vesiculation. These findings have significant implications in understanding the cellular basis governing the intercellular acquisition and dominance of deleterious traits in cancers.

  18. Unique inflammatory RNA profiles of microglia in Creutzfeldt-Jakob disease

    Baker, Christopher A.; Manuelidis, Laura

    2003-01-01

    Previous studies in Creutzfeldt-Jakob disease (CJD) have shown that myeloid cells in the periphery as well as derivative microglial cells in the brain are infectious. Microglia can show an activated phenotype before prion protein (PrP) pathology is detectable in brain, and isolated infectious microglia contain very little PrP. To find whether a set of inflammatory genes are significantly induced or suppressed with infection, we analyzed RNA from isolated microglia with relevant cDNA arrays, and identified 30 transcripts not previously examined in any transmissible spongiform encephalopathy. This CJD expression profile contrasted with that of uninfected microglia exposed to prototypic inflammatory stimuli such as lipopolysaccharide and IFN-, as well as PrP amyloid. These findings underscore inflammatory pathways evoked by the infectious agent in brain. Transcript profiles unique for CJD microglia and other myeloid cells provide opportunities for more sensitive preclinical diagnoses of infectious and noninfectious neurodegenerative diseases.

  19. piRNA Profiling of Dengue Virus Type 2-Infected Asian Tiger Mosquito and Midgut Tissues

    Yanhai Wang

    2018-04-01

    Full Text Available The Asian tiger mosquito, Aedes albopictus, is a competent vector for the majority of arboviruses. The mosquito innate immune response is a primary determinant for arthropod-borne virus transmission, and the midgut is the first barrier to pathogen transmission. Mosquito antiviral immunity is primarily mediated by the small interfering RNA pathway. However, the roles that the P-element induced wimpy testis (PIWI-interacting RNA (piRNA pathway play in antiviral immunity in Ae. albopictus and its midgut still need further exploration. This study aimed to explore the profiles of both viral-derived and host-originated piRNAs in the whole body and midgut infected with Dengue virus 2 (DENV-2 in Ae. albopictus, and to elucidate gene expression profile differences of the PIWI protein family between adult females and their midguts. A deep sequencing-based method was used to identify and analyze small non-coding RNAs, especially the piRNA profiles in DENV-2-infected Ae. albopictus and its midgut. The top-ranked, differentially-expressed piRNAs were further validated using Stem-loop qRT-PCR. Bioinformatics analyses and reverse-transcription PCR (RT-PCR methods were used to detect PIWI protein family members, and their expression profiles. DENV-2 derived piRNAs (vpiRNA, 24–30 nts were observed in both infected Ae. albopictus and its midgut; however, only vpiRNA in the whole-body library had a weak preference for adenine at position 10 (10A in the sense molecules as a feature of secondary piRNA. These vpiRNAs were not equally distributed, instead they were derived from a few specific regions of the genome, especially several hot spots, and displayed an obvious positive strand bias. We refer to the differentially expressed host piRNAs after DENV infection as virus-induced host endogenous piRNAs (vepiRNAs. However, we found that vepiRNAs were abundant in mosquito whole-body tissue, but deficient in the midgut. A total of eleven PIWI family genes were

  20. RBiomirGS: an all-in-one miRNA gene set analysis solution featuring target mRNA mapping and expression profile integration

    Jing Zhang

    2018-01-01

    Full Text Available Background With the continuous discovery of microRNA’s (miRNA association with a wide range of biological and cellular processes, expression profile-based functional characterization of such post-transcriptional regulation is crucial for revealing its significance behind particular phenotypes. Profound advancement in bioinformatics has been made to enable in depth investigation of miRNA’s role in regulating cellular and molecular events, resulting in a huge quantity of software packages covering different aspects of miRNA functional analysis. Therefore, an all-in-one software solution is in demand for a comprehensive yet highly efficient workflow. Here we present RBiomirGS, an R package for a miRNA gene set (GS analysis. Methods The package utilizes multiple databases for target mRNA mapping, estimates miRNA effect on the target mRNAs through miRNA expression profile and conducts a logistic regression-based GS enrichment. Additionally, human ortholog Entrez ID conversion functionality is included for target mRNAs. Results By incorporating all the core steps into one package, RBiomirGS eliminates the need for switching between different software packages. The modular structure of RBiomirGS enables various access points to the analysis, with which users can choose the most relevant functionalities for their workflow. Conclusions With RBiomirGS, users are able to assess the functional significance of the miRNA expression profile under the corresponding experimental condition by minimal input and intervention. Accordingly, RBiomirGS encompasses an all-in-one solution for miRNA GS analysis. RBiomirGS is available on GitHub (http://github.com/jzhangc/RBiomirGS. More information including instruction and examples can be found on website (http://kenstoreylab.com/?page_id=2865.

  1. Geodesy at Mercury with MESSENGER

    Smith, David E.; Zuber, Maria t.; Peale, Stanley J.; Phillips, Roger J.; Solomon, Sean C.

    2006-01-01

    In 2011 the MESSENGER (MErcury Surface, Space ENvironment, GEochemistry, and Ranging) spacecraft will enter Mercury orbit and begin the mapping phase of the mission. As part of its science objectives the MESSENGER mission will determine the shape and gravity field of Mercury. These observations will enable the topography and the crustal thickness to be derived for the planet and will determine the small libration of the planet about its axis, the latter critical to constraining the state of the core. These measurements require very precise positioning of the MESSENGER spacecraft in its eccentric orbit, which has a periapsis altitude as low as 200 km, an apoapsis altitude near 15,000 km, and a closest approach to the surface varying from latitude 60 to about 70 N. The X-band tracking of MESSENGER and the laser altimetry are the primary data that will be used to measure the planetary shape and gravity field. The laser altimeter, which has an expected range of 1000 to 1200 km, is expected to provide significant data only over the northern hemisphere because of MESSENGER's eccentric orbit. For the southern hemisphere, radio occultation measurements obtained as the spacecraft passes behind the planet as seen from Earth and images obtained with the imaging system will be used to provide the long-wavelength shape of the planet. Gravity, derived from the tracking data, will also have greater resolution in the northern hemisphere, but full global models for both topography and gravity will be obtained at low harmonic order and degree. The limiting factor for both gravity and topography is expected to be knowledge of the spacecraft location. Present estimations are that in a combined tracking, altimetry, and occultation solution the spacecraft position uncertainty is likely to be of order 10 m. This accuracy should be adequate for establishing an initial geodetic coordinate system for Mercury that will enable positioning of imaged features on the surface, determination of

  2. MicroRNA and Transcriptomic Profiling Showed miRNA-Dependent Impairment of Systemic Regulation and Synthesis of Biomolecules in Rag2 KO Mice

    Abu Musa Md Talimur Reza

    2018-02-01

    Full Text Available The Rag2 knockout (KO mouse is a well-established immune-compromised animal model for biomedical research. A comparative study identified the deregulated expression of microRNAs (miRNAs and messenger RNAs (mRNAs in Rag2 KO mice. However, the interaction between deregulated genes and miRNAs in the alteration of systemic (cardiac, renal, hepatic, nervous, and hematopoietic regulations and the synthesis of biomolecules (such as l-tryptophan, serotonin, melatonin, dopamine, alcohol, noradrenaline, putrescine, and acetate are unclear. In this study, we analyzed both miRNA and mRNA expression microarray data from Rag2 KO and wild type mice to investigate the possible role of miRNAs in systemic regulation and biomolecule synthesis. A notable finding obtained from this analysis is that the upregulation of several genes which are target molecules of the downregulated miRNAs in Rag2 KO mice, can potentially trigger the degradation of l-tryptophan, thereby leading to the systemic impairment and alteration of biomolecules synthesis as well as changes in behavioral patterns (such as stress and fear responses, and social recognition memory in Rag2 gene-depleted mice. These findings were either not observed or not explicitly described in other published Rag2 KO transcriptome analyses. In conclusion, we have provided an indication of miRNA-dependent regulations of clinical and pathological conditions in cardiac, renal, hepatic, nervous, and hematopoietic systems in Rag2 KO mice. These results may significantly contribute to the prediction of clinical disease caused by Rag2 deficiency.

  3. MicroRNA and Transcriptomic Profiling Showed miRNA-Dependent Impairment of Systemic Regulation and Synthesis of Biomolecules in Rag2 KO Mice.

    Reza, Abu Musa Md Talimur; Choi, Yun-Jung; Kim, Jin-Hoi

    2018-02-27

    The Rag2 knockout (KO) mouse is a well-established immune-compromised animal model for biomedical research. A comparative study identified the deregulated expression of microRNAs (miRNAs) and messenger RNAs (mRNAs) in Rag2 KO mice. However, the interaction between deregulated genes and miRNAs in the alteration of systemic (cardiac, renal, hepatic, nervous, and hematopoietic) regulations and the synthesis of biomolecules (such as l-tryptophan, serotonin, melatonin, dopamine, alcohol, noradrenaline, putrescine, and acetate) are unclear. In this study, we analyzed both miRNA and mRNA expression microarray data from Rag2 KO and wild type mice to investigate the possible role of miRNAs in systemic regulation and biomolecule synthesis. A notable finding obtained from this analysis is that the upregulation of several genes which are target molecules of the downregulated miRNAs in Rag2 KO mice, can potentially trigger the degradation of l-tryptophan, thereby leading to the systemic impairment and alteration of biomolecules synthesis as well as changes in behavioral patterns (such as stress and fear responses, and social recognition memory) in Rag2 gene-depleted mice. These findings were either not observed or not explicitly described in other published Rag2 KO transcriptome analyses. In conclusion, we have provided an indication of miRNA-dependent regulations of clinical and pathological conditions in cardiac, renal, hepatic, nervous, and hematopoietic systems in Rag2 KO mice. These results may significantly contribute to the prediction of clinical disease caused by Rag2 deficiency.

  4. microRNA profiling in the zoonotic parasite Echinococcus canadensis using a high-throughput approach.

    Macchiaroli, Natalia; Cucher, Marcela; Zarowiecki, Magdalena; Maldonado, Lucas; Kamenetzky, Laura; Rosenzvit, Mara Cecilia

    2015-02-06

    microRNAs (miRNAs), a class of small non-coding RNAs, are key regulators of gene expression at post-transcriptional level and play essential roles in fundamental biological processes such as development and metabolism. The particular developmental and metabolic characteristics of cestode parasites highlight the importance of studying miRNA gene regulation in these organisms. Here, we perform a comprehensive analysis of miRNAs in the parasitic cestode Echinococcus canadensis G7, one of the causative agents of the neglected zoonotic disease cystic echinococcosis. Small RNA libraries from protoscoleces and cyst walls of E. canadensis G7 and protoscoleces of E. granulosus sensu stricto G1 were sequenced using Illumina technology. For miRNA prediction, miRDeep2 core algorithm was used. The output list of candidate precursors was manually curated to generate a high confidence set of miRNAs. Differential expression analysis of miRNAs between stages or species was estimated with DESeq. Expression levels of selected miRNAs were validated using poly-A RT-qPCR. In this study we used a high-throughput approach and found transcriptional evidence of 37 miRNAs thus expanding the miRNA repertoire of E. canadensis G7. Differential expression analysis showed highly regulated miRNAs between life cycle stages, suggesting a role in maintaining the features of each developmental stage or in the regulation of developmental timing. In this work we characterize conserved and novel Echinococcus miRNAs which represent 30 unique miRNA families. Here we confirmed the remarkable loss of conserved miRNA families in E. canadensis, reflecting their low morphological complexity and high adaptation to parasitism. We performed the first in-depth study profiling of small RNAs in the zoonotic parasite E. canadensis G7. We found that miRNAs are the preponderant small RNA silencing molecules, suggesting that these small RNAs could be an essential mechanism of gene regulation in this species. We also

  5. Hepatitis C virus (HCV) RNA profiles among chronic HIV/HCV-coinfected individuals in ESPRIT; spontaneous HCV RNA clearance observed in nine individuals.

    Grint, D; Tedaldi, E; Peters, L; Mocroft, A; Edlin, B; Gallien, S; Klinker, H; Boesecke, C; Kokordelis, P; Rockstroh, J K

    2017-07-01

    Studies have shown that hepatitis C virus (HCV) RNA levels remain stable over time in HIV/HCV-coinfected individuals taking combination antiretroviral therapy (cART), while spontaneous clearance of HCV RNA during the persistent infection phase has been documented only rarely among those with the CC interleukin (IL)-28B genotype. This study describes HCV RNA profiles and factors associated with changes over time in HCV RNA levels in the ESPRIT study. HIV/HCV-coinfected individuals positive for HCV RNA were included in the study. Follow-up was counted from the first HCV RNA positive test and censored at the initiation of interferon-based treatment. HCV RNA and IL-28B measurements were performed in the same reference laboratory. Random effects mixed models were used to analyse changes over time in HCV RNA. A total of 312 ESPRIT patients were included in the study (151 in the arm receiving subcutaneous recombinant IL-2 and 161 in the control arm). Most of the patients were white (89%) and male (76%), and they had a median of 5 HCV RNA measurements per person [interquartile range (IQR) 3-6; range 1-9]. Median follow-up was 5 years (IQR: 2-6 years). At baseline, 96% of patients were taking cART and 93% had undetectable HIV RNA. Mean HCV RNA levels decreased by 13% per year over the study period [95% confidence interval (CI) 8-18%; P < 0.0001]. Baseline HCV RNA levels and the change over time in HCV RNA did not differ by randomization arm (P = 0.16 and P = 0.56, respectively). Nine individuals spontaneously cleared HCV RNA during follow-up [IL-28B genotypes: CC, five patients (56%); CT, four patients (44%)]. HCV RNA levels decreased over time in this population with well-controlled HIV infection. Spontaneous clearance of HCV RNA was documented in five individuals with IL-28B genotype CC and four with the CT genotype. © 2016 British HIV Association.

  6. MicroRNA expression profiling identifies activated B cell status in chronic lymphocytic leukemia cells.

    Shuqiang Li

    2011-03-01

    Full Text Available Chronic lymphocytic leukemia (CLL is thought to be a disease of resting lymphocytes. However, recent data suggest that CLL cells may more closely resemble activated B cells. Using microRNA (miRNA expression profiling of highly-enriched CLL cells from 38 patients and 9 untransformed B cells from normal donors before acute CpG activation and 5 matched B cells after acute CpG activation, we demonstrate an activated B cell status for CLL. Gene set enrichment analysis (GSEA identified statistically-significant similarities in miRNA expression between activated B cells and CLL cells including upregulation of miR-34a, miR-155, and miR-342-3p and downregulation of miR-103, miR-181a and miR-181b. Additionally, decreased levels of two CLL signature miRNAs miR-29c and miR-223 are associated with ZAP70(+ and IgV(H unmutated status and with shorter time to first therapy. These data indicate an activated B cell status for CLL cells and suggest that the direction of change of individual miRNAs may predict clinical course in CLL.

  7. MicroRNA profiling reveals distinct signatures in B cell chronic lymphocytic leukemias

    Calin, George Adrian; Liu, Chang-Gong; Sevignani, Cinzia; Ferracin, Manuela; Felli, Nadia; Dumitru, Calin Dan; Shimizu, Masayoshi; Cimmino, Amelia; Zupo, Simona; Dono, Mariella; Dell'Aquila, Marie L.; Alder, Hansjuerg; Rassenti, Laura; Kipps, Thomas J.; Bullrich, Florencia; Negrini, Massimo; Croce, Carlo M.

    2004-01-01

    Little is known about the expression levels or function of micro-RNAs (miRNAs) in normal and neoplastic cells, although it is becoming clear that miRNAs play important roles in the regulation of gene expression during development [Ambros, V. (2003) Cell 113, 673–676; McManus, M. T. (2003) Semin. Cancer Biol. 13, 253–258]. We now report the genomewide expression profiling of miRNAs in human B cell chronic lymphocytic leukemia (CLL) by using a microarray containing hundreds of human precursor and mature miRNA oligonucleotide probes. This approach allowed us to identify significant differences in miRNome expression between CLL samples and normal CD5+ B cells; data were confirmed by Northern blot analyses and real-time RT-PCR. At least two distinct clusters of CLL samples can be identified that were associated with the presence or absence of Zap-70 expression, a predictor of early disease progression. Two miRNA signatures were associated with the presence or absence of mutations in the expressed Ig variableregion genes or with deletions at 13q14, respectively. These data suggest that miRNA expression patterns have relevance to the biological and clinical behavior of this leukemia. PMID:15284443

  8. Analysis of microRNA profile of Anopheles sinensis by deep sequencing and bioinformatic approaches.

    Feng, Xinyu; Zhou, Xiaojian; Zhou, Shuisen; Wang, Jingwen; Hu, Wei

    2018-03-12

    microRNAs (miRNAs) are small non-coding RNAs widely identified in many mosquitoes. They are reported to play important roles in development, differentiation and innate immunity. However, miRNAs in Anopheles sinensis, one of the Chinese malaria mosquitoes, remain largely unknown. We investigated the global miRNA expression profile of An. sinensis using Illumina Hiseq 2000 sequencing. Meanwhile, we applied a bioinformatic approach to identify potential miRNAs in An. sinensis. The identified miRNA profiles were compared and analyzed by two approaches. The selected miRNAs from the sequencing result and the bioinformatic approach were confirmed with qRT-PCR. Moreover, target prediction, GO annotation and pathway analysis were carried out to understand the role of miRNAs in An. sinensis. We identified 49 conserved miRNAs and 12 novel miRNAs by next-generation high-throughput sequencing technology. In contrast, 43 miRNAs were predicted by the bioinformatic approach, of which two were assigned as novel. Comparative analysis of miRNA profiles by two approaches showed that 21 miRNAs were shared between them. Twelve novel miRNAs did not match any known miRNAs of any organism, indicating that they are possibly species-specific. Forty miRNAs were found in many mosquito species, indicating that these miRNAs are evolutionally conserved and may have critical roles in the process of life. Both the selected known and novel miRNAs (asi-miR-281, asi-miR-184, asi-miR-14, asi-miR-nov5, asi-miR-nov4, asi-miR-9383, and asi-miR-2a) could be detected by quantitative real-time PCR (qRT-PCR) in the sequenced sample, and the expression patterns of these miRNAs measured by qRT-PCR were in concordance with the original miRNA sequencing data. The predicted targets for the known and the novel miRNAs covered many important biological roles and pathways indicating the diversity of miRNA functions. We also found 21 conserved miRNAs and eight counterparts of target immune pathway genes in An. sinensis

  9. Changes in Rat Brain MicroRNA Expression Profiles Following Sevoflurane and Propofol Anesthesia

    Yu Lu

    2015-01-01

    Full Text Available Background: Sevoflurane and propofol are widely used anesthetics for surgery. Studies on the mechanisms of general anesthesia have focused on changes in protein expression properties and membrane lipid. MicroRNAs (miRNAs regulate neural function by altering protein expression. We hypothesize that sevoflurane and propofol affect miRNA expression profiles in the brain, expect to understand the mechanism of anesthetic agents. Methods: Rats were randomly assigned to a 2% sevoflurane group, 600 μg·kg − 1·min − 1 propofol group, and a control group without anesthesia (n = 4, respectively. Treatment group was under anesthesia for 6 h, and all rats breathed spontaneously with continuous monitoring of respiration and blood gases. Changes in rat cortex miRNA expression profiles were analyzed by miRNA microarrays and validated by quantitative real-time polymerase chain reaction (qRT-PCR. Differential expression of miRNA using qRT-PCR among the control, sevoflurane, and propofol groups were compared using one-way analysis of variance (ANOVA. Results: Of 677 preloaded rat miRNAs, the microarray detected the expression of 277 miRNAs in rat cortex (40.9%, of which 9 were regulated by propofol and (or sevoflurane. Expression levels of three miRNAs (rno-miR-339-3p, rno-miR-448, rno-miR-466b-1FNx01 were significantly increased following sevoflurane and six (rno-miR-339-3p, rno-miR-347, rno-miR-378FNx01, rno-miR-412FNx01, rno-miR-702-3p, and rno-miR-7a-2FNx01 following propofol. Three miRNAs (rno-miR-466b-1FNx01, rno-miR-3584-5p and rno-miR-702-3p were differentially expressed by the two anesthetic treatment groups. Conclusions: Sevoflurane and propofol anesthesia induced distinct changes in brain miRNA expression patterns, suggesting differential regulation of protein expression. Determining the targets of these differentially expressed miRNAs may help reveal both the common and agent-specific actions of anesthetics on neurological and physiological

  10. Alterations in polyribosome and messenger ribonucleic acid metabolism and messenger ribonucleoprotein utilization in osmotically stressed plant seedlings

    Mason, H.S.

    1986-01-01

    Polyribosome aggregation state in growing tissues of barley and wheat leaf of stems of pea and squash was studied in relation to seedling growth and water status of the growing tissue in plants at various levels of osmotic stress. It was found to be highly correlated with water potential and osmotic potential of the growing tissue and with leaf of stem elongation rate. Stress rapidly reduced polyribosome content and water status in growing tissues of barley leaves; changes were slow and slight in the non-growing leaf blade. Membrane-bound and free polyribosomes were equally sensitive to stress-induced disaggregation. Incorporation of 32 PO 4 3- into ribosomal RNA was rapidly inhibited by stress, but stability of poly(A) + RNA relative to ribosomal RNA was similar in stressed and unstressed tissues, with a half-life of about 12 hours. Stress also caused progressive loss of poly(A) + RNA from these tissues. Quantitation of poly(A) and in vitro messenger template activity in polysome gradient fractions showed a shift of activity from the polysomal region to the region of 20-60 S in stressed plants. Messenger RNA in the 20-60 S region coded for the same peptides as mRNA found in the polysomal fraction. Nonpolysomal and polysome-derived messenger ribonucleoprotein complexes (mRNP) were isolated, and characteristic proteins were found associated with either fraction. Polysomal mRNP from stressed or unstressed plants were translated with similar efficiency in a wheat germ cell-free system. It was concluded that no translational inhibitory activity was associated with nonpolysomal mRNP from barley prepared as described

  11. Rapid and inexpensive body fluid identification by RNA profiling-based multiplex High Resolution Melt (HRM analysis [v1; ref status: indexed, http://f1000r.es/2hj

    Erin K. Hanson

    2013-12-01

    Full Text Available Positive identification of the nature of biological material present on evidentiary items can be crucial for understanding the circumstances surrounding a crime. However, traditional protein-based methods do not permit the identification of all body fluids and tissues, and thus molecular based strategies for the conclusive identification of all forensically relevant biological fluids and tissues need to be developed. Messenger RNA (mRNA profiling is an example of such a molecular-based approach. Current mRNA body fluid identification assays involve capillary electrophoresis (CE or quantitative RT-PCR (qRT-PCR platforms, each with its own limitations. Both platforms require the use of expensive fluorescently labeled primers or probes. CE-based assays require separate amplification and detection steps thus increasing the analysis time. For qRT-PCR assays, only 3-4 markers can be included in a single reaction since each requires a different fluorescent dye. To simplify mRNA profiling assays, and reduce the time and cost of analysis, we have developed single- and multiplex body fluid High Resolution Melt (HRM assays for the identification of common forensically relevant biological fluids and tissues. The incorporated biomarkers include IL19 (vaginal secretions, IL1F7 (skin, ALAS2 (blood, MMP10 (menstrual blood, HTN3 (saliva and TGM4 (semen.  The HRM assays require only unlabeled PCR primers and a single saturating intercalating fluorescent dye (Eva Green. Each body-fluid-specific marker can easily be identified by the presence of a distinct melt peak. Usually, HRM assays are used to detect variants or isoforms for a single gene target. However, we have uniquely developed duplex and triplex HRM assays to permit the simultaneous detection of multiple targets per reaction. Here we describe the development and initial performance evaluation of the developed HRM assays. The results demonstrate the potential use of HRM assays for rapid, and relatively

  12. Splenic marginal zone lymphoma: comprehensive analysis of gene expression and miRNA profiling.

    Arribas, Alberto J; Gómez-Abad, Cristina; Sánchez-Beato, Margarita; Martinez, Nerea; Dilisio, Lorena; Casado, Felipe; Cruz, Miguel A; Algara, Patrocinio; Piris, Miguel A; Mollejo, Manuela

    2013-07-01

    Splenic marginal zone lymphoma is a small B-cell neoplasm whose molecular pathogenesis is still essentially unknown and whose differentiation from other small B-cell lymphomas is hampered by the lack of specific markers. We have analyzed the gene expression and miRNA profiles of 31 splenic marginal zone lymphoma cases. For comparison, 7 spleens with reactive lymphoid hyperplasia, 10 spleens infiltrated by chronic lymphocytic leukemia, 12 spleens with follicular lymphoma, 6 spleens infiltrated by mantle cell lymphoma and 15 lymph nodes infiltrated by nodal marginal zone lymphoma were included. The results were validated by qRT-PCR in an independent series including 77 paraffin-embedded splenic marginal zone lymphomas. The splenic marginal zone lymphoma miRNA signature had deregulated expression of 51 miRNAs. The most highly overexpressed miRNAs were miR-155, miR-21, miR-34a, miR-193b and miR-100, while the most repressed miRNAs were miR-377, miR-27b, miR-145, miR-376a and miR-424. MiRNAs located in 14q32-31 were underexpressed in splenic marginal zone lymphoma compared with reactive lymphoid tissues and other B-cell lymphomas. Finally, the gene expression data were integrated with the miRNA profile to identify functional relationships between genes and deregulated miRNAs. Our study reveals miRNAs that are deregulated in splenic marginal zone lymphoma and identifies new candidate diagnostic molecules for splenic marginal zone lymphoma.

  13. Long Non-Coding RNA Profiling in a Non-Alcoholic Fatty Liver Disease Rodent Model: New Insight into Pathogenesis

    Yi Chen

    2017-01-01

    Full Text Available Non-alcoholic fatty liver disease (NAFLD is one of the most prevalent chronic liver diseases worldwide with an unclear mechanism. Long non-coding RNAs (lncRNAs have recently emerged as important regulatory molecules. To better understand NAFLD pathogenesis, lncRNA and messenger RNA (mRNA microarrays were conducted in an NAFLD rodent model. Potential target genes of significantly changed lncRNA were predicted using cis/trans-regulatory algorithms. Gene Ontology (GO analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG pathway enrichment analysis were then performed to explore their function. In the current analysis, 89 upregulated and 177 downregulated mRNAs were identified, together with 291 deregulated lncRNAs. Bioinformatic analysis of these RNAs has categorized these RNAs into pathways including arachidonic acid metabolism, circadian rhythm, linoleic acid metabolism, peroxisome proliferator-activated receptor (PPAR signaling pathway, sphingolipid metabolism, steroid biosynthesis, tryptophan metabolism and tyrosine metabolism were compromised. Quantitative polymerase chain reaction (qPCR of representative nine mRNAs and eight lncRNAs (named fatty liver-related lncRNA, FLRL was conducted and this verified previous microarray results. Several lncRNAs, such as FLRL1, FLRL6 and FLRL2 demonstrated to be involved in circadian rhythm targeting period circadian clock 3 (Per3, Per2 and aryl hydrocarbon receptor nuclear translocator-like (Arntl, respectively. While FLRL8, FLRL3 and FLRL7 showed a potential role in PPAR signaling pathway through interaction with fatty acid binding protein 5 (Fabp5, lipoprotein lipase (Lpl and fatty acid desaturase 2 (Fads2. Functional experiments showed that interfering of lncRNA FLRL2 expression affected the expression of predicted target, circadian rhythm gene Arntl. Moreover, both FLRL2 and Arntl were downregulated in the NAFLD cellular model. The current study identified lncRNA and corresponding mRNA in NAFLD

  14. Comparative mRNA and microRNA expression profiling of three genitourinary cancers reveals common hallmarks and cancer-specific molecular events.

    Xianxin Li

    Full Text Available Genome-wide gene expression profile using deep sequencing technologies can drive the discovery of cancer biomarkers and therapeutic targets. Such efforts are often limited to profiling the expression signature of either mRNA or microRNA (miRNA in a single type of cancer.Here we provided an integrated analysis of the genome-wide mRNA and miRNA expression profiles of three different genitourinary cancers: carcinomas of the bladder, kidney and testis.Our results highlight the general or cancer-specific roles of several genes and miRNAs that may serve as candidate oncogenes or suppressors of tumor development. Further comparative analyses at the systems level revealed that significant aberrations of the cell adhesion process, p53 signaling, calcium signaling, the ECM-receptor and cell cycle pathways, the DNA repair and replication processes and the immune and inflammatory response processes were the common hallmarks of human cancers. Gene sets showing testicular cancer-specific deregulation patterns were mainly implicated in processes related to male reproductive function, and general disruptions of multiple metabolic pathways and processes related to cell migration were the characteristic molecular events for renal and bladder cancer, respectively. Furthermore, we also demonstrated that tumors with the same histological origins and genes with similar functions tended to group together in a clustering analysis. By assessing the correlation between the expression of each miRNA and its targets, we determined that deregulation of 'key' miRNAs may result in the global aberration of one or more pathways or processes as a whole.This systematic analysis deciphered the molecular phenotypes of three genitourinary cancers and investigated their variations at the miRNA level simultaneously. Our results provided a valuable source for future studies and highlighted some promising genes, miRNAs, pathways and processes that may be useful for diagnostic or

  15. The miRNA Expression Profile in Acute Myocardial Infarct Using Sheep Model with Left Ventricular Assist Device Unloading

    Xiaoqian Yan

    2017-01-01

    Full Text Available This study attempted to establish miRNA expression profiles in acute myocardial infarct (AMI sheep model with left ventricular assist device (LVAD unloading. AMI was established in sheep model and FW-II type axial flow pump was implanted to maintain continuous unloading for 3 days. The cardiomyocyte survival, inflammatory cell infiltration, and myocardial fibrosis were detected by tissue staining, and cardiomyocyte apoptosis was detected by TUNEL assay. High throughput sequencing technique was used to detect miRNA expression in cardiomyocytes and to establish miRNA expression profile. The Gene Ontology (GO and Kyoto Encyclopedia of Genes and Genomes (KEGG analyses were established. miRNA sequencing results identified 152 known mature miRNAs and 1582 new mature miRNAs. The unloading and control groups differentially expressed genes, of which RT-PCR verified oar-miR-19b and oar-miR-26a. The GO and KEGG pathway annotation and enrichment established that the regulating functions and signaling pathways of these miRNAs were closely related to cardiovascular diseases (CVD. In this study, LVAD effectively reduced the cell death degree of cardiomyocyte in MI. The established miRNA expression profiles of AMI and LVAD intervention in this study suggest that the expression profile could be used to explore the unknown miRNA and the regulatory mechanisms involved in AMI.

  16. Expression profiles of mRNA and long noncoding RNA in the ovaries of letrozole-induced polycystic ovary syndrome rat model through deep sequencing.

    Fu, Lu-Lu; Xu, Ying; Li, Dan-Dan; Dai, Xiao-Wei; Xu, Xin; Zhang, Jing-Shun; Ming, Hao; Zhang, Xue-Ying; Zhang, Guo-Qing; Ma, Ya-Lan; Zheng, Lian-Wen

    2018-05-30

    Polycystic ovary syndrome (PCOS) is one of the most common endocrine disorders in reproductive-aged women. However, the exact pathophysiology of PCOS remains largely unclear. We performed deep sequencing to investigate the mRNA and long noncoding RNA (lncRNA) expression profiles in the ovarian tissues of letrozole-induced PCOS rat model and control rats. A total of 2147 mRNAs and 158 lncRNAs were differentially expressed between the PCOS models and control. Gene ontology analysis indicated that differentially expressed mRNAs were associated with biological adhesion, reproduction, and metabolic process. Pathway analysis results indicated that these aberrantly expressed mRNAs were related to several specific signaling pathways, including insulin resistance, steroid hormone biosynthesis, PPAR signaling pathway, cell adhesion molecules, autoimmune thyroid disease, and AMPK signaling pathway. The relative expression levels of mRNAs and lncRNAs were validated through qRT-PCR. LncRNA-miRNA-mRNA network was constructed to explore ceRNAs involved in the PCOS model and were also verified by qRTPCR experiment. These findings may provide insight into the pathogenesis of PCOS and clues to find key diagnostic and therapeutic roles of lncRNA in PCOS. Copyright © 2018 Elsevier B.V. All rights reserved.

  17. Genetic variability in MCF-7 sublines: evidence of rapid genomic and RNA expression profile modifications

    Nugoli, Mélanie; Theillet, Charles; Chuchana, Paul; Vendrell, Julie; Orsetti, Béatrice; Ursule, Lisa; Nguyen, Catherine; Birnbaum, Daniel; Douzery, Emmanuel JP; Cohen, Pascale

    2003-01-01

    Both phenotypic and cytogenetic variability have been reported for clones of breast carcinoma cell lines but have not been comprehensively studied. Despite this, cell lines such as MCF-7 cells are extensively used as model systems. In this work we documented, using CGH and RNA expression profiles, the genetic variability at the genomic and RNA expression levels of MCF-7 cells of different origins. Eight MCF-7 sublines collected from different sources were studied as well as 3 subclones isolated from one of the sublines by limit dilution. MCF-7 sublines showed important differences in copy number alteration (CNA) profiles. Overall numbers of events ranged from 28 to 41. Involved chromosomal regions varied greatly from a subline to another. A total of 62 chromosomal regions were affected by either gains or losses in the 11 sublines studied. We performed a phylogenetic analysis of CGH profiles using maximum parsimony in order to reconstruct the putative filiation of the 11 MCF-7 sublines. The phylogenetic tree obtained showed that the MCF-7 clade was characterized by a restricted set of 8 CNAs and that the most divergent subline occupied the position closest to the common ancestor. Expression profiles of 8 MCF-7 sublines were analyzed along with those of 19 unrelated breast cancer cell lines using home made cDNA arrays comprising 720 genes. Hierarchical clustering analysis of the expression data showed that 7/8 MCF-7 sublines were grouped forming a cluster while the remaining subline clustered with unrelated breast cancer cell lines. These data thus showed that MCF-7 sublines differed at both the genomic and phenotypic levels. The analysis of CGH profiles of the parent subline and its three subclones supported the heteroclonal nature of MCF-7 cells. This strongly suggested that the genetic plasticity of MCF-7 cells was related to their intrinsic capacity to generate clonal heterogeneity. We propose that MCF-7, and possibly the breast tumor it was derived from, evolved

  18. Nitric oxide: a physiologic messenger.

    Lowenstein, C J; Dinerman, J L; Snyder, S H

    1994-02-01

    To review the physiologic role of nitric oxide, an unusual messenger molecule that mediates blood vessel relaxation, neurotransmission, and pathogen suppression. A MEDLINE search of articles published from 1987 to 1993 that addressed nitric oxide and the enzyme that synthesizes it, nitric oxide synthase. Animal and human studies were selected from 3044 articles to analyze the clinical importance of nitric oxide. Descriptions of the structure and function of nitric oxide synthase were selected to show how nitric oxide acts as a biological messenger molecule. Biochemical and physiologic studies were analyzed if the same results were found by three or more independent observers. Two major classes of nitric oxide synthase enzymes produce nitric oxide. The constitutive isoforms found in endothelial cells and neurons release small amounts of nitric oxide for brief periods to signal adjacent cells, whereas the inducible isoform found in macrophages releases large amounts of nitric oxide continuously to eliminate bacteria and parasites. By diffusing into adjacent cells and binding to enzymes that contain iron, nitric oxide plays many important physiologic roles. It regulates blood pressure, transmits signals between neurons, and suppresses pathogens. Excess amounts, however, can damage host cells, causing neurotoxicity during strokes and causing the hypotension associated with sepsis. Nitric oxide is a simple molecule with many physiologic roles in the cardiovascular, neurologic, and immune systems. Although the general principles of nitric oxide synthesis are known, further research is necessary to determine what role it plays in causing disease.

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

    Gwendal Le Martelot

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

  20. Analysis of MicroRNA Expression Profiles in Weaned Pig Skeletal Muscle after Lipopolysaccharide Challenge

    Jing Zhang

    2015-09-01

    Full Text Available MicroRNAs (miRNAs constitute a class of non-coding RNAs that play a crucial regulatory role in skeletal muscle development and disease. Several acute inflammation conditions including sepsis and cancer are characterized by a loss of skeletal muscle due primarily to excessive muscle catabolism. As a well-known inducer of acute inflammation, a lipopolysaccharide (LPS challenge can cause serious skeletal muscle wasting. However, knowledge of the role of miRNAs in the course of inflammatory muscle catabolism is still very limited. In this study, RNA extracted from the skeletal muscle of pigs injected with LPS or saline was subjected to small RNA deep sequencing. We identified 304 conserved and 114 novel candidate miRNAs in the pig. Of these, four were significantly increased in the LPS-challenged samples and five were decreased. The expression of five miRNAs (ssc-miR-146a-5p, ssc-miR-221-5p, ssc-miR-148b-3p, ssc-miR-215 and ssc-miR-192 were selected for validation by quantitative polymerase chain reaction (qPCR, which found that ssc-miR-146a-5p and ssc-miR-221-5p were significantly upregulated in LPS-challenged pig skeletal muscle. Moreover, we treated mouse C2C12 myotubes with 1000 ng/mL LPS as an acute inflammation cell model. Expression of TNF-α, IL-6, muscle atrophy F-box (MAFbx and muscle RING finger 1 (MuRF1 mRNA was strongly induced by LPS. Importantly, miR-146a-5p and miR-221-5p also showed markedly increased expression in LPS-treated C2C12 myotubes, suggesting the two miRNAs may be involved in muscle catabolism systems in response to acute inflammation caused by a LPS challenge. To our knowledge, this study is the first to examine miRNA expression profiles in weaned pig skeletal muscle challenged with LPS, and furthers our understanding of miRNA function in the regulation of inflammatory muscle catabolism.

  1. Altered expression of estrogen receptor-α variant messenger RNAs between adjacent normal breast and breast tumor tissues

    Leygue, Etienne; Dotzlaw, Helmut; Watson, Peter H; Murphy, Leigh C

    2000-01-01

    Using semiquantitative reverse transcription-polymerase chain reaction assays, we investigated the expression of variant messenger RNAs relative to wild-type estrogen receptor (ER)-α messenger RNA in normal breast tissues and their adjacent matched breast tumor tissues. Higher ER variant truncated after sequences encoding exon 2 of the wild-type ER-α (ERC4) messenger RNA and a lower exon 3 deleted ER-α variant (ERD3) messenger RNA relative expression in the tumor compartment were observed in the ER-positive/PR-positive and the ER-positive subsets, respectively. A significantly higher relative expression of exon 5 deleted ER-α varient (ERD5) messenger RNA was observed in tumor components overall. These data demonstrate that changes in the relative expression of ER-α variant messenger RNAs occur between adjacent normal and neoplastic breast tissues. We suggest that these changes might be involved in the mechanisms that underlie breast tumorigenesis. Estrogen receptor (ER)-α and ER-β are believed to mediate the action of estradiol in target tissues. Several ER-α and ER-β variant messenger RNAs have been identified in both normal and neoplastic human tissues. Most of these variants contain a deletion of one or more exons of the wild-type (WT) ER messenger RNAs. The putative proteins that are encoded by these variant messenger RNAs would therefore be missing some functional domains of the WT receptors, and might interfere with WT-ER signaling pathways. The detection of ER-α variants in both normal and neoplastic human breast tissues raised the question of their possible role in breast tumorigenesis. We have previously reported an increased relative expression of exon 5 deleted ER-α variant (ERD5) messenger RNA and of another ER-α variant truncated of all sequences following the exon 2 of the WT ER-α (ERC4) messenger RNA in breast tumor samples versus independent normal breast tissues. In contrast, a decreased relative expression of exon 3 deleted ER

  2. MicroRNA expression profiling during the life cycle of the silkworm (Bombyx mori

    Cheng Daojun

    2009-09-01

    Full Text Available Abstract Background MicroRNAs (miRNAs are expressed by a wide range of eukaryotic organisms, and function in diverse biological processes. Numerous miRNAs have been identified in Bombyx mori, but the temporal expression profiles of miRNAs corresponding to each stage transition over the entire life cycle of the silkworm remain to be established. To obtain a comprehensive overview of the correlation between miRNA expression and stage transitions, we performed a whole-life test and subsequent stage-by-stage examinations on nearly one hundred miRNAs in the silkworm. Results Our results show that miRNAs display a wide variety of expression profiles over the whole life of the silkworm, including continuous expression from embryo to adult (miR-184, up-regulation over the entire life cycle (let-7 and miR-100, down-regulation over the entire life cycle (miR-124, expression associated with embryogenesis (miR-29 and miR-92, up-regulation from early 3rd instar to pupa (miR-275, and complementary pulses in expression between miR-34b and miR-275. Stage-by-stage examinations revealed further expression patterns, such as emergence at specific time-points during embryogenesis and up-regulation of miRNA groups in late embryos (miR-1 and bantam, expression associated with stage transition between instar and molt larval stages (miR-34b, expression associated with silk gland growth and spinning activity (miR-274, continuous high expression from the spinning larval to pupal and adult stages (miR-252 and miR-31a, a coordinate expression trough in day 3 pupae of both sexes (miR-10b and miR-281, up-regulation in pupal metamorphosis of both sexes (miR-29b, and down-regulation in pupal metamorphosis of both sexes (miR-275. Conclusion We present the full-scale expression profiles of miRNAs throughout the life cycle of Bombyx mori. The whole-life expression profile was further investigated via stage-by-stage analysis. Our data provide an important resource for more detailed

  3. MicroRNA expression profiling during the life cycle of the silkworm (Bombyx mori).

    Liu, Shiping; Zhang, Liang; Li, Qibin; Zhao, Ping; Duan, Jun; Cheng, Daojun; Xiang, Zhonghuai; Xia, Qingyou

    2009-09-28

    MicroRNAs (miRNAs) are expressed by a wide range of eukaryotic organisms, and function in diverse biological processes. Numerous miRNAs have been identified in Bombyx mori, but the temporal expression profiles of miRNAs corresponding to each stage transition over the entire life cycle of the silkworm remain to be established. To obtain a comprehensive overview of the correlation between miRNA expression and stage transitions, we performed a whole-life test and subsequent stage-by-stage examinations on nearly one hundred miRNAs in the silkworm. Our results show that miRNAs display a wide variety of expression profiles over the whole life of the silkworm, including continuous expression from embryo to adult (miR-184), up-regulation over the entire life cycle (let-7 and miR-100), down-regulation over the entire life cycle (miR-124), expression associated with embryogenesis (miR-29 and miR-92), up-regulation from early 3rd instar to pupa (miR-275), and complementary pulses in expression between miR-34b and miR-275. Stage-by-stage examinations revealed further expression patterns, such as emergence at specific time-points during embryogenesis and up-regulation of miRNA groups in late embryos (miR-1 and bantam), expression associated with stage transition between instar and molt larval stages (miR-34b), expression associated with silk gland growth and spinning activity (miR-274), continuous high expression from the spinning larval to pupal and adult stages (miR-252 and miR-31a), a coordinate expression trough in day 3 pupae of both sexes (miR-10b and miR-281), up-regulation in pupal metamorphosis of both sexes (miR-29b), and down-regulation in pupal metamorphosis of both sexes (miR-275). We present the full-scale expression profiles of miRNAs throughout the life cycle of Bombyx mori. The whole-life expression profile was further investigated via stage-by-stage analysis. Our data provide an important resource for more detailed functional analysis of miRNAs in this animal.

  4. Long non-coding RNA expression profiles in hereditary haemorrhagic telangiectasia

    Tørring, Pernille M; Larsen, Martin Jakob; Kjeldsen, Anette D

    2014-01-01

    transcriptome, we wanted to assess whether lncRNAs play a role in the molecular pathogenesis of HHT manifestations. By microarray technology, we profiled lncRNA transcripts from HHT nasal telangiectasial and non-telangiectasial tissue using a paired design. The microarray probes were annotated using the GENCODE...... v.16 dataset, identifying 4,810 probes mapping to 2,811 lncRNAs. Comparing HHT telangiectasial tissue with HHT non-telangiectasial tissue, we identified 42 lncRNAs that are differentially expressed (qUsing GREAT, a tool that assumes cis-regulation, we showed that differently expressed lncRNAs...... to the TGF-β signalling pathway. The exact mechanism of how haploinsufficiency of ENG and ACVRL1 leads to HHT manifestations remains to be identified. As long non-coding RNAs (lncRNAs) are increasingly recognized as key regulators of gene expression and constitute a sizable fraction of the human...

  5. HemaExplorer: a database of mRNA expression profiles in normal and malignant haematopoiesis

    Bagger, Frederik Otzen; Rapin, Nicolas; Theilgaard-Mönch, Kim

    2013-01-01

    lead to full integrity of the data in the database. The HemaExplorer has comprehensive visualization interface that can make it useful as a daily tool for biologists and cancer researchers to assess the expression patterns of genes encountered in research or literature. HemaExplorer is relevant for all......The HemaExplorer (http://servers.binf.ku.dk/hemaexplorer) is a curated database of processed mRNA Gene expression profiles (GEPs) that provides an easy display of gene expression in haematopoietic cells. HemaExplorer contains GEPs derived from mouse/human haematopoietic stem and progenitor cells...... as well as from more differentiated cell types. Moreover, data from distinct subtypes of human acute myeloid leukemia is included in the database allowing researchers to directly compare gene expression of leukemic cells with those of their closest normal counterpart. Normalization and batch correction...

  6. Expression profiles of mRNA after exposure yeast and rice to heavy-ion radiation

    Iwahashi, Hitoshi; Mizukami, Satomi; Nojima, Kumie

    2005-01-01

    We have studied expression profiles of mRNA after exposure yeast cells to heavy-ion radiation. Yeast cells was exposed by heavy-ion radiation with the levels of 6, 12, 25, 50, and 100 Gy. We could confirm the reproducibility of physiological state of yeast cells under the experimental conditions by DNA microarray. We could also confirm the reproducibility of viability of yeast cells after exposure to heavy-ion radiation. We thus applied yeast cells exposed with 25 Gy was applied to DNA microarray analysis. The strongly induced genes were HUG1 RAR4 RNR2 for DNA repairing genes and GLC3 GSY1 for energy metabolism genes. (author)

  7. Mercury's Seasonal Sodium Exosphere: MESSENGER Orbital Observations

    Cassidy, Timothy A.; Merkel, Aimee W.; Burger, Matthew H.; Killen, Rosemary M.; McClintock, William E.; Vervack, Ronald J., Jr.; Sarantos, Menelaos

    2014-01-01

    The Mercury Atmospheric and Surface Composition Spectrometer (MASCS) Ultraviolet and Visible Spectrometer (UVVS) on the MErcury Surface, Space ENvironment, GEochemistry, and Ranging (MESSENGER) spacecraft now orbiting Mercury provides the first close-up look at the planet's sodium exosphere. UVVS has observed the exosphere from orbit almost daily for over 10 Mercury years. In this paper we describe and analyze a subset of these data: altitude profiles taken above the low-latitude dayside and south pole. The observations show spatial and temporal variations, but there are no obvious year-to-year variations in most of the observations. We do not see the episodic variability reported by some ground-based observers. We used these altitude profiles to make estimates of sodium density and temperature. The bulk of the exosphere, at about 1200 K, is much warmer than Mercury's surface. This value is consistent with some ground-based measurements and suggests that photon-stimulated desorption is the primary ejection process. We also observe a tenuous energetic component but do not see evidence of the predicted thermalized (or partially thermalized) sodium near Mercury's surface temperature. Overall we do not see the variable mixture of temperatures predicted by most Monte Carlo models of the exosphere.

  8. Prokaryotic community profiling of local algae wastewaters using advanced 16S rRNA gene sequencing.

    Limayem, Alya; Micciche, Andrew; Nayak, Bina; Mohapatra, Shyam

    2018-01-01

    Algae biomass-fed wastewaters are a promising source of lipid and bioenergy manufacture, revealing substantial end-product investment returns. However, wastewaters would contain lytic pathogens carrying drug resistance detrimental to algae yield and environmental safety. This study was conducted to simultaneously decipher through high-throughput advanced Illumina 16S ribosomal RNA (rRNA) gene sequencing, the cultivable and uncultivable bacterial community profile found in a single sample that was directly recovered from the local wastewater systems. Samples were collected from two previously documented sources including anaerobically digested (AD) municipal wastewater and swine wastewater with algae namely Chlorella spp. in addition to control samples, swine wastewater, and municipal wastewater without algae. Results indicated the presence of a significant level of Bacteria in all samples with an average of approximately 95.49% followed by Archaea 2.34%, in local wastewaters designed for algae cultivation. Taxonomic genus identification indicated the presence of Calothrix, Pseudomonas, and Clostridium as the most prevalent strains in both local municipal and swine wastewater samples containing algae with an average of 17.37, 12.19, and 7.84%, respectively. Interestingly, swine wastewater without algae displayed the lowest level of Pseudomonas strains algae indicates potential coexistence between these strains and algae microenvironment, suggesting further investigations. This finding was particularly relevant for the earlier documented adverse effects of some nosocomial Pseudomonas strains on algae growth and their multidrug resistance potential, requiring the development of targeted bioremediation with regard to the beneficial flora.

  9. LncRNA Expression Profile of Human Thoracic Aortic Dissection by High-Throughput Sequencing.

    Sun, Jie; Chen, Guojun; Jing, Yuanwen; He, Xiang; Dong, Jianting; Zheng, Junmeng; Zou, Meisheng; Li, Hairui; Wang, Shifei; Sun, Yili; Liao, Wangjun; Liao, Yulin; Feng, Li; Bin, Jianping

    2018-01-01

    In this study, the long non-coding RNA (lncRNA) expression profile in human thoracic aortic dissection (TAD), a highly lethal cardiovascular disease, was investigated. Human TAD (n=3) and normal aortic tissues (NA) (n=3) were examined by high-throughput sequencing. Bioinformatics analyses were performed to predict the roles of aberrantly expressed lncRNAs. Quantitative real-time polymerase chain reaction (qRT-PCR) was applied to validate the results. A total of 269 lncRNAs (159 up-regulated and 110 down-regulated) and 2, 255 mRNAs (1 294 up-regulated and 961 down-regulated) were aberrantly expressed in human TAD (fold-change> 1.5, PTAD than in NA. The predicted binding motifs of three up-regulated lncRNAs (ENSG00000248508, ENSG00000226530, and EG00000259719) were correlated with up-regulated RUNX1 (R=0.982, PTAD. These findings suggest that lncRNAs are novel potential therapeutic targets for human TAD. © 2018 The Author(s). Published by S. Karger AG, Basel.

  10. Analysis of miRNA and mRNA Expression Profiles Highlights Alterations in Ionizing Radiation Response of Human Lymphocytes under Modeled Microgravity

    Casara, Silvia; Sales, Gabriele; Lanfranchi, Gerolamo; Celotti, Lucia; Mognato, Maddalena

    2012-01-01

    Background Ionizing radiation (IR) can be extremely harmful for human cells since an improper DNA-damage response (DDR) to IR can contribute to carcinogenesis initiation. Perturbations in DDR pathway can originate from alteration in the functionality of the microRNA-mediated gene regulation, being microRNAs (miRNAs) small noncoding RNA that act as post-transcriptional regulators of gene expression. In this study we gained insight into the role of miRNAs in the regulation of DDR to IR under microgravity, a condition of weightlessness experienced by astronauts during space missions, which could have a synergistic action on cells, increasing the risk of radiation exposure. Methodology/Principal Findings We analyzed miRNA expression profile of human peripheral blood lymphocytes (PBL) incubated for 4 and 24 h in normal gravity (1 g) and in modeled microgravity (MMG) during the repair time after irradiation with 0.2 and 2Gy of γ-rays. Our results show that MMG alters miRNA expression signature of irradiated PBL by decreasing the number of radio-responsive miRNAs. Moreover, let-7i*, miR-7, miR-7-1*, miR-27a, miR-144, miR-200a, miR-598, miR-650 are deregulated by the combined action of radiation and MMG. Integrated analyses of miRNA and mRNA expression profiles, carried out on PBL of the same donors, identified significant miRNA-mRNA anti-correlations of DDR pathway. Gene Ontology analysis reports that the biological category of “Response to DNA damage” is enriched when PBL are incubated in 1 g but not in MMG. Moreover, some anti-correlated genes of p53-pathway show a different expression level between 1 g and MMG. Functional validation assays using luciferase reporter constructs confirmed miRNA-mRNA interactions derived from target prediction analyses. Conclusions/Significance On the whole, by integrating the transcriptome and microRNome, we provide evidence that modeled microgravity can affects the DNA-damage response to IR in human PBL. PMID:22347458

  11. Expression of somatotropin receptor messenger ribonucleic acid in bovine tissues

    Lucy, M.C.; Boyd, C.K.; Koenigsfeld, A.T.; Okamura, C.S.

    1998-01-01

    The somatotropin receptor mRNA is controlled by at least two different gene promoters that generate 2 two variants with different exon 1 sequences (1A and 1B). The location of 1A and 1B somatotropin receptor mRNA within cattle tissues and, hence, the tissue specificity of the 1A and 1B promoters are unknown. In addition, the cDNA sequence of the 1B somatotropin receptor has not been determined. Our objective, therefore, was to sequence a cDNA for the 1B somatotropin receptor and to analyze bovine tissues for expression of 1A and 1B somatotropin receptor mRNA. Twenty adult tissues and six fetal tissues were collected at slaughter from each of four cows and two fetuses. Messenger RNA was analyzed using ribonuclease protection assays. The adult liver expressed both 1A and 1B mRNA. All other adult tissues expressed 1B mRNA but not 1A mRNA. The greatest amount of 1B mRNA was detected in liver and adipose (abdominal and subcutaneous) tissues. Other tissues had approximately one-half to one-tenth of the amount of 1B mRNA in the liver or adipose tissue. Fetal tissues (including fetal liver) expressed 1B mRNA and not 1A mRNA. Based on cDNA sequencing, the protein encoded by the 1A and 1B mRNA was nearly identical. We concluded that 1A somatotropin receptor mRNA is specific to adult bovine liver. Other adult and fetal bovine tissues expressed 1B somatotropin receptor mRNA with a predicted protein sequence that was similar to the 1A somatotropin receptor

  12. Gene expression profiling of liver cancer stem cells by RNA-sequencing.

    David W Y Ho

    Full Text Available BACKGROUND: Accumulating evidence supports that tumor growth and cancer relapse are driven by cancer stem cells. Our previous work has demonstrated the existence of CD90(+ liver cancer stem cells (CSCs in hepatocellular carcinoma (HCC. Nevertheless, the characteristics of these cells are still poorly understood. In this study, we employed a more sensitive RNA-sequencing (RNA-Seq to compare the gene expression profiling of CD90(+ cells sorted from tumor (CD90(+CSCs with parallel non-tumorous liver tissues (CD90(+NTSCs and elucidate the roles of putative target genes in hepatocarcinogenesis. METHODOLOGY/PRINCIPAL FINDINGS: CD90(+ cells were sorted respectively from tumor and adjacent non-tumorous human liver tissues using fluorescence-activated cell sorting. The amplified RNAs of CD90(+ cells from 3 HCC patients were subjected to RNA-Seq analysis. A differential gene expression profile was established between CD90(+CSCs and CD90(+NTSCs, and validated by quantitative real-time PCR (qRT-PCR on the same set of amplified RNAs, and further confirmed in an independent cohort of 12 HCC patients. Five hundred genes were differentially expressed (119 up-regulated and 381 down-regulated genes between CD90(+CSCs and CD90(+NTSCs. Gene ontology analysis indicated that the over-expressed genes in CD90(+CSCs were associated with inflammation, drug resistance and lipid metabolism. Among the differentially expressed genes, glypican-3 (GPC3, a member of glypican family, was markedly elevated in CD90(+CSCs compared to CD90(+NTSCs. Immunohistochemistry demonstrated that GPC3 was highly expressed in forty-two human liver tumor tissues but absent in adjacent non-tumorous liver tissues. Flow cytometry indicated that GPC3 was highly expressed in liver CD90(+CSCs and mature cancer cells in liver cancer cell lines and human liver tumor tissues. Furthermore, GPC3 expression was positively correlated with the number of CD90(+CSCs in liver tumor tissues. CONCLUSIONS

  13. Gene Expression Profiling of Liver Cancer Stem Cells by RNA-Sequencing

    Lam, Chi Tat; Ng, Michael N. P.; Yu, Wan Ching; Lau, Joyce; Wan, Timothy; Wang, Xiaoqi; Yan, Zhixiang; Liu, Hang; Fan, Sheung Tat

    2012-01-01

    Background Accumulating evidence supports that tumor growth and cancer relapse are driven by cancer stem cells. Our previous work has demonstrated the existence of CD90+ liver cancer stem cells (CSCs) in hepatocellular carcinoma (HCC). Nevertheless, the characteristics of these cells are still poorly understood. In this study, we employed a more sensitive RNA-sequencing (RNA-Seq) to compare the gene expression profiling of CD90+ cells sorted from tumor (CD90+CSCs) with parallel non-tumorous liver tissues (CD90+NTSCs) and elucidate the roles of putative target genes in hepatocarcinogenesis. Methodology/Principal Findings CD90+ cells were sorted respectively from tumor and adjacent non-tumorous human liver tissues using fluorescence-activated cell sorting. The amplified RNAs of CD90+ cells from 3 HCC patients were subjected to RNA-Seq analysis. A differential gene expression profile was established between CD90+CSCs and CD90+NTSCs, and validated by quantitative real-time PCR (qRT-PCR) on the same set of amplified RNAs, and further confirmed in an independent cohort of 12 HCC patients. Five hundred genes were differentially expressed (119 up-regulated and 381 down-regulated genes) between CD90+CSCs and CD90+NTSCs. Gene ontology analysis indicated that the over-expressed genes in CD90+CSCs were associated with inflammation, drug resistance and lipid metabolism. Among the differentially expressed genes, glypican-3 (GPC3), a member of glypican family, was markedly elevated in CD90+CSCs compared to CD90+NTSCs. Immunohistochemistry demonstrated that GPC3 was highly expressed in forty-two human liver tumor tissues but absent in adjacent non-tumorous liver tissues. Flow cytometry indicated that GPC3 was highly expressed in liver CD90+CSCs and mature cancer cells in liver cancer cell lines and human liver tumor tissues. Furthermore, GPC3 expression was positively correlated with the number of CD90+CSCs in liver tumor tissues. Conclusions/Significance The identified genes

  14. Profiling microRNA expression during multi-staged date palm (Phoenix dactylifera L.) fruit development

    Xin, Chengqi; Liu, Wanfei; Lin, Qiang; Zhang, Xiaowei; Cui, Peng; Li, Fusen; Zhang, Guangyu; Pan, Linlin; Al-Amer, Ali; Mei, Hailiang; Al-Mssallem, Ibrahim S.; Hu, Songnian; Al-Johi, Hasan Awad; Yu, Jun

    2015-01-01

    MicroRNAs (miRNAs) play crucial roles in multiple stages of plant development and regulate gene expression at posttranscriptional and translational levels. In this study, we first identified 238 conserved miRNAs in date palm (Phoenix dactylifera) based on a high-quality genome assembly and defined 78 fruit-development-associated (FDA) miRNAs, whose expression profiles are variable at different fruit development stages. Using experimental data, we subsequently detected 276 novel P. dactylifera-specific FDA miRNAs and predicted their targets. We also revealed that FDA miRNAs function mainly in regulating genes involved in starch/sucrose metabolisms and other carbon metabolic pathways; among them, 221 FDA miRNAs exhibit negative correlation with their corresponding targets, which suggests their direct regulatory roles on mRNA targets. Our data define a comprehensive set of conserved and novel FDA miRNAs along with their expression profiles, which provide a basis for further experimentation in assigning discrete functions of these miRNAs in P. dactylifera fruit development.

  15. Identification of circulating miRNA biomarkers based on global quantitative real-time PCR profiling

    Kang Kang

    2012-02-01

    Full Text Available Abstract MicroRNAs (miRNAs are small noncoding RNAs (18-25 nucleotides that regulate gene expression at the post-transcriptional level. Recent studies have demonstrated the presence of miRNAs in the blood circulation. Deregulation of miRNAs in serum or plasma has been associated with many diseases including cancers and cardiovascular diseases, suggesting the possible use of miRNAs as diagnostic biomarkers. However, the detection of the small amount of miRNAs found in serum or plasma requires a method with high sensitivity and accuracy. Therefore, the current study describes polymerase chain reaction (PCR-based methods for measuring circulating miRNAs. Briefly, the procedure involves four major steps: (1 sample collection and preparation; (2 global miRNAs profiling using quantitative real-time PCR (qRT-PCR; (3 data normalization and analysis; and (4 selection and validation of miRNA biomarkers. In conclusion, qRT-PCR is a promising method for profiling of circulating miRNAs as biomarkers.

  16. Profiling microRNA expression during multi-staged date palm (Phoenix dactylifera L.) fruit development

    Xin, Chengqi

    2015-01-29

    MicroRNAs (miRNAs) play crucial roles in multiple stages of plant development and regulate gene expression at posttranscriptional and translational levels. In this study, we first identified 238 conserved miRNAs in date palm (Phoenix dactylifera) based on a high-quality genome assembly and defined 78 fruit-development-associated (FDA) miRNAs, whose expression profiles are variable at different fruit development stages. Using experimental data, we subsequently detected 276 novel P. dactylifera-specific FDA miRNAs and predicted their targets. We also revealed that FDA miRNAs function mainly in regulating genes involved in starch/sucrose metabolisms and other carbon metabolic pathways; among them, 221 FDA miRNAs exhibit negative correlation with their corresponding targets, which suggests their direct regulatory roles on mRNA targets. Our data define a comprehensive set of conserved and novel FDA miRNAs along with their expression profiles, which provide a basis for further experimentation in assigning discrete functions of these miRNAs in P. dactylifera fruit development.

  17. Optimized high-throughput microRNA expression profiling provides novel biomarker assessment of clinical prostate and breast cancer biopsies

    Fedele Vita

    2006-06-01

    Full Text Available Abstract Background Recent studies indicate that microRNAs (miRNAs are mechanistically involved in the development of various human malignancies, suggesting that they represent a promising new class of cancer biomarkers. However, previously reported methods for measuring miRNA expression consume large amounts of tissue, prohibiting high-throughput miRNA profiling from typically small clinical samples such as excision or core needle biopsies of breast or prostate cancer. Here we describe a novel combination of linear amplification and labeling of miRNA for highly sensitive expression microarray profiling requiring only picogram quantities of purified microRNA. Results Comparison of microarray and qRT-PCR measured miRNA levels from two different prostate cancer cell lines showed concordance between the two platforms (Pearson correlation R2 = 0.81; and extension of the amplification, labeling and microarray platform was successfully demonstrated using clinical core and excision biopsy samples from breast and prostate cancer patients. Unsupervised clustering analysis of the prostate biopsy microarrays separated advanced and metastatic prostate cancers from pooled normal prostatic samples and from a non-malignant precursor lesion. Unsupervised clustering of the breast cancer microarrays significantly distinguished ErbB2-positive/ER-negative, ErbB2-positive/ER-positive, and ErbB2-negative/ER-positive breast cancer phenotypes (Fisher exact test, p = 0.03; as well, supervised analysis of these microarray profiles identified distinct miRNA subsets distinguishing ErbB2-positive from ErbB2-negative and ER-positive from ER-negative breast cancers, independent of other clinically important parameters (patient age; tumor size, node status and proliferation index. Conclusion In sum, these findings demonstrate that optimized high-throughput microRNA expression profiling offers novel biomarker identification from typically small clinical samples such as breast

  18. Laser altimeter observations from MESSENGER's first Mercury flyby.

    Zuber, Maria T; Smith, David E; Solomon, Sean C; Phillips, Roger J; Peale, Stanton J; Head, James W; Hauck, Steven A; McNutt, Ralph L; Oberst, Jürgen; Neumann, Gregory A; Lemoine, Frank G; Sun, Xiaoli; Barnouin-Jha, Olivier; Harmon, John K

    2008-07-04

    A 3200-kilometers-long profile of Mercury by the Mercury Laser Altimeter on the MESSENGER spacecraft spans approximately 20% of the near-equatorial region of the planet. Topography along the profile is characterized by a 5.2-kilometer dynamic range and 930-meter root-mean-square roughness. At long wavelengths, topography slopes eastward by 0.02 degrees , implying a variation of equatorial shape that is at least partially compensated. Sampled craters on Mercury are shallower than their counterparts on the Moon, at least in part the result of Mercury's higher gravity. Crater floors vary in roughness and slope, implying complex modification over a range of length scales.

  19. Early and late effects of prenatal corticosteroid treatment on the microRNA profiles of lung tissue in rats

    YU, HONG-REN; LI, SUNG-CHOU; TSENG, WAN-NING; TAIN, YOU-LIN; CHEN, CHIH-CHENG; SHEEN, JIUNN-MING; TIAO, MAO-MENG; KUO, HO-CHANG; HUANG, CHAO-CHENG; HSIEH, KAI-SHENG; HUANG, LI-TUNG

    2016-01-01

    Glucocorticoids have been administered to mothers at risk of premature delivery to induce maturation of preterm fetal lungs and prevent the development of respiratory distress syndrome. Micro (mi)RNAs serve various crucial functions in cell proliferation, differentiation and organ development; however, few studies have demonstrated an association between miRNAs and lung development. The aim of the present study was to investigate alterations in the miRNA profiles of rat lung tissue following prenatal glucocorticoid therapy for fetal lung development. The differences in miRNA expression profiles were compared between postnatal days 7 (D7) and 120 (D120) rat lung tissues, followed by validation using reverse transcription-quantitative polymerase chain reaction. The miRNA profiles of rat lung tissues following prenatal dexamethasone (DEX) therapy were also investigated. miRNAs with 2-fold changes were selected for further analysis. At D120, 6 upregulated and 6 downregulated miRNAs were detected, compared with D7. Among these differentially expressed miRNAs, miR-101-3p and miR-99b-5p were associated with the lowest and highest expressions of miRNA at D7, respectively. A limited impact on the miRNA profiles of rat lung tissues was observed following prenatal DEX treatment, which may help to further clarify the mechanisms underlying normal lung development. However, the results of the present study cannot entirely elucidate the effects of prenatal DEX treatment on the lung development of premature infants, and further studies investigating the impact of prenatal corticosteroids on fetal lung miRNA profiles are required. PMID:26997989

  20. MicroRNA Expression Profiling to Identify and Validate Reference Genes for the Relative Quantification of microRNA in Rectal Cancer

    Eriksen, Anne Haahr Mellergaard; Andersen, Rikke Fredslund; Pallisgaard, Niels

    2016-01-01

    the miRNA profiling experiment, miR-645, miR-193a-5p, miR-27a and let-7g were identified as stably expressed, both in malignant and stromal tissue. In addition, NormFinder confirmed high expression stability for the four miRNAs. In the RT-qPCR based validation experiments, no significant difference...... management. Real-time quantitative polymerase chain reaction (RT-qPCR) is commonly used, when measuring miRNA expression. Appropriate normalisation of RT-qPCR data is important to ensure reliable results. The aim of the present study was to identify stably expressed miRNAs applicable as normaliser candidates...... in future studies of miRNA expression in rectal cancer.MATERIALS AND METHODS: We performed high-throughput miRNA profiling (OpenArray®) on ten pairs of laser micro-dissected rectal cancer tissue and adjacent stroma. A global mean expression normalisation strategy was applied to identify the most stably...

  1. A Downmodulated MicroRNA Profiling in Patients with Gastric Cancer

    Tao Zhang

    2017-01-01

    Full Text Available Objective. Here, we aim to investigate the microRNA (miR profiling in human gastric cancer (GC. Methods. Tumoral and matched peritumoral gastric specimens were collected from 12 GC patients who underwent routine surgery. A high-throughput miR sequencing method was applied to detect the aberrantly expressed miRs in a subset of 6 paired samples. The stem-loop quantitative real-time polymerase chain reaction (qRT-PCR assay was subsequently performed to confirm the sequencing results in the remaining 6 paired samples. The profiling results were also validated in vitro in three human GC cell lines (BGC-823, MGC-803, and GTL-16 and a normal gastric epithelial cell line (GES-1. Results. The miR sequencing approach detected 5 differentially expressed miRs, hsa-miR-132-3p, hsa-miR-155-5p, hsa-miR-19b-3p, hsa-miR-204-5p, and hsa-miR-30a-3p, which were significantly downmodulated between the tumoral and peritumoral GC tissues. Most of the results were further confirmed by qRT-PCR, while no change was observed for hsa-miR-30a-3p. The in vitro finding also agreed with the results of both miR sequencing and qRT-PCR for hsa-miR-204-5p, hsa-miR-155-5p, and hsa-miR-132-3p. Conclusion. Together, our findings may serve to identify new molecular alterations as well as to enrich the miR profiling in human GC.

  2. iSRAP – a one-touch research tool for rapid profiling of small RNA-seq data

    Quek, Camelia; Jung, Chol-hee; Bellingham, Shayne A.; Lonie, Andrew; Hill, Andrew F.

    2015-01-01

    Small non-coding RNAs have been significantly recognized as the key modulators in many biological processes, and are emerging as promising biomarkers for several diseases. These RNA species are transcribed in cells and can be packaged in extracellular vesicles, which are small vesicles released from many biotypes, and are involved in intercellular communication. Currently, the advent of next-generation sequencing (NGS) technology for high-throughput profiling has further advanced the biological insights of non-coding RNA on a genome-wide scale and has become the preferred approach for the discovery and quantification of non-coding RNA species. Despite the routine practice of NGS, the processing of large data sets poses difficulty for analysis before conducting downstream experiments. Often, the current analysis tools are designed for specific RNA species, such as microRNA, and are limited in flexibility for modifying parameters for optimization. An analysis tool that allows for maximum control of different software is essential for drawing concrete conclusions for differentially expressed transcripts. Here, we developed a one-touch integrated small RNA analysis pipeline (iSRAP) research tool that is composed of widely used tools for rapid profiling of small RNAs. The performance test of iSRAP using publicly and in-house available data sets shows its ability of comprehensive profiling of small RNAs of various classes, and analysis of differentially expressed small RNAs. iSRAP offers comprehensive analysis of small RNA sequencing data that leverage informed decisions on the downstream analyses of small RNA studies, including extracellular vesicles such as exosomes. PMID:26561006

  3. iSRAP - a one-touch research tool for rapid profiling of small RNA-seq data.

    Quek, Camelia; Jung, Chol-Hee; Bellingham, Shayne A; Lonie, Andrew; Hill, Andrew F

    2015-01-01

    Small non-coding RNAs have been significantly recognized as the key modulators in many biological processes, and are emerging as promising biomarkers for several diseases. These RNA species are transcribed in cells and can be packaged in extracellular vesicles, which are small vesicles released from many biotypes, and are involved in intercellular communication. Currently, the advent of next-generation sequencing (NGS) technology for high-throughput profiling has further advanced the biological insights of non-coding RNA on a genome-wide scale and has become the preferred approach for the discovery and quantification of non-coding RNA species. Despite the routine practice of NGS, the processing of large data sets poses difficulty for analysis before conducting downstream experiments. Often, the current analysis tools are designed for specific RNA species, such as microRNA, and are limited in flexibility for modifying parameters for optimization. An analysis tool that allows for maximum control of different software is essential for drawing concrete conclusions for differentially expressed transcripts. Here, we developed a one-touch integrated small RNA analysis pipeline (iSRAP) research tool that is composed of widely used tools for rapid profiling of small RNAs. The performance test of iSRAP using publicly and in-house available data sets shows its ability of comprehensive profiling of small RNAs of various classes, and analysis of differentially expressed small RNAs. iSRAP offers comprehensive analysis of small RNA sequencing data that leverage informed decisions on the downstream analyses of small RNA studies, including extracellular vesicles such as exosomes.

  4. Genome-wide profiling of the PIWI-interacting RNA-mRNA regulatory networks in epithelial ovarian cancers.

    Singh, Garima; Roy, Jyoti; Rout, Pratiti; Mallick, Bibekanand

    2018-01-01

    PIWI-interacting (piRNAs), ~23-36 nucleotide-long small non-coding RNAs (sncRNAs), earlier believed to be germline-specific, have now been identified in somatic cells, including cancer cells. These sncRNAs impact critical biological processes by fine-tuning gene expression at post-transcriptional and epigenetic levels. The expression of piRNAs in ovarian cancer, the most lethal gynecologic cancer is largely uncharted. In this study, we investigated the expression of PIWILs by qRT-PCR and western blotting and then identified piRNA transcriptomes in tissues of normal ovary and two most prevalent epithelial ovarian cancer subtypes, serous and endometrioid by small RNA sequencing. We detected 219, 256 and 234 piRNAs in normal ovary, endometrioid and serous ovarian cancer samples respectively. We observed piRNAs are encoded from various genomic regions, among which introns harbor the majority of them. Surprisingly, piRNAs originated from different genomic contexts showed the varied level of conservations across vertebrates. The functional analysis of predicted targets of differentially expressed piRNAs revealed these could modulate key processes and pathways involved in ovarian oncogenesis. Our study provides the first comprehensive piRNA landscape in these samples and a useful resource for further functional studies to decipher new mechanistic views of piRNA-mediated gene regulatory networks affecting ovarian oncogenesis. The RNA-seq data is submitted to GEO database (GSE83794).

  5. Circular RNA expression profiling of human granulosa cells during maternal aging reveals novel transcripts associated with assisted reproductive technology outcomes.

    Jing Cheng

    Full Text Available Circular RNAs (circRNAs are a unique class of endogenous RNAs which could be used as potential diagnostic and prognostic biomarkers of many diseases. Our study aimed to investigate circRNA profiles in human granulosa cells (GCs during maternal aging and to uncover age-related circRNA variations that potentially reflect decreased oocyte competence. CircRNAs in GCs from in vitro fertilization (IVF patients with young age (YA, ≤ 30 years and advanced age (AA, ≥ 38 years were profiled by microarray, and validated in 20 paired samples. The correlation between circRNAs expression and clinical characteristics was analyzed in additional 80 samples. Chip-based analysis revealed 46 up-regulated and 11 down-regulated circRNAs in AA samples (fold change > 2.0. Specifically, circRNA_103829, circRNA_103827 and circRNA_104816 were validated to be up-regulated, while circRNA_101889 was down-regulated in AA samples. After adjustment for gonadotropin treatment, only circRNA_103827 and circRNA_104816 levels were positively associated with maternal age (partial r = 0.332, P = 0.045; partial r = 0.473, P = 0.003; respectively. Moreover, circRNA_103827 and circRNA_104816 expressions in GCs were negatively correlated with the number of top quality embryos (r = -0.235, P = 0.036; r = -0.221, P = 0.049; respectively. Receiver operating characteristic (ROC curve analysis indicated that the performance of circRNA_103827 for live birth prediction reached 0.698 [0.570-0.825], with 77.2% sensitivity and 60.9% specificity (P = 0.006, and that of circRNA_104816 was 0.645 [0.507-0.783] (P = 0.043. Bioinformatics analysis revealed that both circRNAs were potentially involved in glucose metabolism, mitotic cell cycle, and ovarian steroidogenesis. Therefore, age-related up-regulation of circRNA_103827 and circRNA_104816 might be potential indicators of compromised follicular micro-environment which could be used to predict IVF prognosis, and improve female infertility

  6. MicroRNA Expression Profiling by Bead Array Technology in Human Tumor Cell Lines Treated with Interferon-Alpha-2a

    Siegrist Fredy

    2009-01-01

    Full Text Available Abstract MicroRNAs are positive and negative regulators of eukaryotic gene expression that modulate transcript abundance by specific binding to sequence motifs located prevalently in the 3' untranslated regions of target messenger RNAs (mRNA. Interferon-alpha-2a (IFNα induces a large set of protein coding genes mediating antiproliferative and antiviral responses. Here we use a global microarray-based microRNA detection platform to identify genes that are induced by IFNα in hepatoma- or melanoma-derived human tumor cell lines. Despite the enormous differences in expression levels between these models, we were able to identify microRNAs that are upregulated by IFNα in both lines suggesting the possibility that interferon-regulated microRNAs are involved in the transcriptional repression of mRNA relevant to cytokine responses.

  7. MicroRNA expression profiles in chronic epilepsy rats and neuroprotection from seizures by targeting miR-344a

    Liu XX

    2017-07-01

    Full Text Available Xixia Liu,1,2 Yuhan Liao,1 Xiuxiu Wang,1 Donghua Zou,1 Chun Luo,1 Chongdong Jian,1 Yuan Wu1 1Department of Neurology, First Affiliated Hospital of Guangxi Medical University, 2Department of Rehabilitation, People’s Hospital of Guangxi Zhuang Autonomous Region, Nanning, China Abstract: MicroRNA (miRNA is believed to play a crucial role in the cause and treatment of epilepsy by controlling gene expression. However, it is still unclear how miRNA profiles change after multiple prolonged seizures and aggravation of brain injury in chronic epilepsy (CE. To investigate the role of miRNA in epilepsy, we utilized the CE rat models with pentylenetetrazol (PTZ and miRNA profiles in the hippocampus. miRNA profiles were characterized using miRNA microarray analysis and were compared with the rats in the sham group, which received 0.9% physiological saline treatment at the same dose. Four up-regulated miRNAs (miR-139–3p, -770–5p, -127–5p, -331–3p and 5 down-regulated miRNAs (miR-802–5p, -380–5p, -183–5p, -547–5p, -344a/-344a–5p were found in the CE rats (fold change >1.5, P<0.05. Three of the dysregulated miRNAs were validated by quantitative real-time polymerase chain reaction, which revealed an outcome consistent with the initial results of the miRNA microarray analyses. Then, miR-344a agomir was intracerebroventricularly injected and followed by PTZ induction of CE models to investigate the effect of miR-344a in chronic neocortical epileptogenesis. After miRNA-344a agomir and scramble treatment, results showed a restoration of seizure behavior and a reduction in neuron damage in the cortex in miRNA-334a agomir treated rats. These data suggest that miRNA-344a might have a small modulatory effect on seizure-induced apoptosis signaling pathways in the cortex. Keywords: microRNA, chronic epilepsy, miR-344a, epigenetics, apoptosis

  8. In vivo transcriptional profile analysis reveals RNA splicing and chromatin remodeling as prominent processes for adult neurogenesis.

    Lim, Daniel A; Suárez-Fariñas, Mayte; Naef, Felix; Hacker, Coleen R; Menn, Benedicte; Takebayashi, Hirohide; Magnasco, Marcelo; Patil, Nila; Alvarez-Buylla, Arturo

    2006-01-01

    Neural stem cells and neurogenesis persist in the adult mammalian brain subventricular zone (SVZ). Cells born in the rodent SVZ migrate to the olfactory bulb (Ob) where they differentiate into interneurons. To determine the gene expression and functional profile of SVZ neurogenesis, we performed three complementary sets of transcriptional analysis experiments using Affymetrix GeneChips: (1) comparison of adult mouse SVZ and Ob gene expression profiles with those of the striatum, cerebral cortex, and hippocampus; (2) profiling of SVZ stem cells and ependyma isolated by fluorescent-activated cell sorting (FACS); and (3) analysis of gene expression changes during in vivo SVZ regeneration after anti-mitotic treatment. Gene Ontology (GO) analysis of data from these three separate approaches showed that in adult SVZ neurogenesis, RNA splicing and chromatin remodeling are biological processes as statistically significant as cell proliferation, transcription, and neurogenesis. In non-neurogenic brain regions, RNA splicing and chromatin remodeling were not prominent processes. Fourteen mRNA splicing factors including Sf3b1, Sfrs2, Lsm4, and Khdrbs1/Sam68 were detected along with 9 chromatin remodeling genes including Mll, Bmi1, Smarcad1, Baf53a, and Hat1. We validated the transcriptional profile data with Northern blot analysis and in situ hybridization. The data greatly expand the catalogue of cell cycle components, transcription factors, and migration genes for adult SVZ neurogenesis and reveal RNA splicing and chromatin remodeling as prominent biological processes for these germinal cells.

  9. EFFECTS OF STORAGE, RNA EXTRACTION, GENECHIP TYPE, AND DONOR SEX ON GENE EXPRESSION PROFILING OF HUMAN WHOLE BLOOD

    Background: Gene expression profiling of whole blood may be useful for monitoring toxicological exposure and for diagnosis and monitoring of various diseases. Several methods are available that can be used to transport, store, and extract RNA from whole blood, but it is not clear...

  10. Effect of Thymine Starvation on Messenger Ribonucleic Acid Synthesis in Escherichia coli

    Luzzati, Denise

    1966-01-01

    Luzzati, Denise (Institut de Biologie Physico-Chimique, Paris, France). Effect of thymine starvation on messenger ribonucleic acid synthesis in Escherichia coli. J. Bacteriol. 92:1435–1446. 1966.—During the course of thymine starvation, the rate of synthesis of messenger ribonucleic acid (mRNA, the rapidly labeled fraction of the RNA which decays in the presence of dinitrophenol or which hybridizes with deoxyribonucleic acid) decreases exponentially, in parallel with the viability of the thymine-starved bacteria. The ability of cell-free extracts of starved bacteria to incorporate ribonucleoside triphosphates into RNA was determined; it was found to be inferior to that of extracts from control cells. The analysis of the properties of cell-free extracts of starved cells shows that their decreased RNA polymerase activity is the consequence of a modification of their deoxyribonucleic acid, the ability of which to serve as a template for RNA polymerase decreases during starvation. PMID:5332402

  11. Classifications within molecular subtypes enables identification of BRCA1/BRCA2 mutation carriers by RNA tumor profiling

    Larsen, Martin J; Kruse, Torben A; Tan, Qihua

    2013-01-01

    Pathogenic germline mutations in BRCA1 or BRCA2 are detected in less than one third of families with a strong history of breast cancer. It is therefore expected that mutations still remain undetected by currently used screening methods. In addition, a growing number of BRCA1/2 sequence variants...... of unclear pathogen significance are found in the families, constituting an increasing clinical challenge. New methods are therefore needed to improve the detection rate and aid the interpretation of the clinically uncertain variants. In this study we analyzed a series of 33 BRCA1, 22 BRCA2, and 128 sporadic...... tumors by RNA profiling to investigate the classification potential of RNA profiles to predict BRCA1/2 mutation status. We found that breast tumors from BRCA1 and BRCA2 mutation carriers display characteristic RNA expression patterns, allowing them to be distinguished from sporadic tumors. The majority...

  12. Ionizing Radiation Deregulates the MicroRNA Expression Profile in Differentiated Thyroid Cells.

    Penha, Ricardo Cortez Cardoso; Pellecchia, Simona; Pacelli, Roberto; Pinto, Luis Felipe Ribeiro; Fusco, Alfredo

    2018-03-01

    Ionizing radiation (IR) is a well-known risk factor for papillary thyroid cancer, and it has been reported to deregulate microRNA expression, which is important to thyroid carcinogenesis. Therefore, this study investigated the impact of IR on microRNA expression profile of the normal thyroid cell line (FRTL-5 CL2), as well as its effect on radiosensitivity of thyroid cancer cell lines, especially the human anaplastic thyroid carcinoma cell line (8505c). The global microRNA expression profile of irradiated FRTL-5 CL2 cells (5 Gy X-ray) was characterized, and data were confirmed by quantitative real-time polymerase chain reaction evaluating the expression of rno-miR-10b-5p, rno-miR-33-5p, rno-miR-128-1-5p, rno-miR-199a-3p, rno-miR-296-5p, rno-miR-328a-3p, and rno-miR-541-5p in irradiated cells. The miR-199a-3p and miR-10b-5p targets were validated by quantitative real-time polymerase chain reaction, Western blot, and luciferase target assays. The effects of miR-199a-3p and miR-10b-5p on DNA repair were determined by evaluating the activation of the protein kinases ataxia-telangiectasia mutated, ataxia telangiectasia, and Rad3-related and the serine 39 phosphorylation of variant histone H2AX as an indirect measure of double-strand DNA breaks in irradiated FRTL-5 CL2 cells. The impact of miR-10b-5p on radiosensitivity was analyzed by cell counting and MTT assays in FRTL-5 CL2, Kras-transformed FRTL-5 CL2 (FRTL KiKi), and 8505c cell lines. The results reveal that miR-10b-5p and miR-199a-3p display the most pronounced alterations in expression in irradiated FRTL-5 CL2 cells. Dicer1 and Lin28b were validated as targets of miR-10b-5p and miR-199a-3p, respectively. Functional studies demonstrate that miR-10b-5p increases the growth rate of FRTL-5 CL2 cells, while miR-199a-3p inhibits their proliferation. Moreover, both of these microRNAs negatively affect homologous recombination repair, reducing activated ataxia-telangiectasia mutated and Rad3-related protein levels

  13. MicroRNA and protein profiling of brain metastasis competent cell-derived exosomes.

    Laura Camacho

    Full Text Available Exosomes are small membrane vesicles released by most cell types including tumor cells. The intercellular exchange of proteins and genetic material via exosomes is a potentially effective approach for cell-to-cell communication and it may perform multiple functions aiding to tumor survival and metastasis. We investigated microRNA and protein profiles of brain metastatic (BM versus non-brain metastatic (non-BM cell-derived exosomes. We studied the cargo of exosomes isolated from brain-tropic 70W, MDA-MB-231BR, and circulating tumor cell brain metastasis-selected markers (CTC1BMSM variants, and compared them with parental non-BM MeWo, MDA-MB-231P and CTC1P cells, respectively. By performing microRNA PCR array we identified one up-regulated (miR-210 and two down-regulated miRNAs (miR-19a and miR-29c in BM versus non-BM exosomes. Second, we analyzed the proteomic content of cells and exosomes isolated from these six cell lines, and detected high expression of proteins implicated in cell communication, cell cycle, and in key cancer invasion and metastasis pathways. Third, we show that BM cell-derived exosomes can be internalized by non-BM cells and that they effectively transport their cargo into cells, resulting in increased cell adhesive and invasive potencies. These results provide a strong rationale for additional investigations of exosomal proteins and miRNAs towards more profound understandings of exosome roles in brain metastasis biogenesis, and for the discovery and application of non-invasive biomarkers for new therapies combating brain metastasis.

  14. Long non-coding RNA expression profiling of mouse testis during postnatal development.

    Jin Sun

    Full Text Available Mammalian testis development and spermatogenesis play critical roles in male fertility and continuation of a species. Previous research into the molecular mechanisms of testis development and spermatogenesis has largely focused on the role of protein-coding genes and small non-coding RNAs, such as microRNAs and piRNAs. Recently, it has become apparent that large numbers of long (>200 nt non-coding RNAs (lncRNAs are transcribed from mammalian genomes and that lncRNAs perform important regulatory functions in various developmental processes. However, the expression of lncRNAs and their biological functions in post-natal testis development remain unknown. In this study, we employed microarray technology to examine lncRNA expression profiles of neonatal (6-day-old and adult (8-week-old mouse testes. We found that 8,265 lncRNAs were expressed above background levels during post-natal testis development, of which 3,025 were differentially expressed. Candidate lncRNAs were identified for further characterization by an integrated examination of genomic context, gene ontology (GO enrichment of their associated protein-coding genes, promoter analysis for epigenetic modification, and evolutionary conservation of elements. Many lncRNAs overlapped or were adjacent to key transcription factors and other genes involved in spermatogenesis, such as Ovol1, Ovol2, Lhx1, Sox3, Sox9, Plzf, c-Kit, Wt1, Sycp2, Prm1 and Prm2. Most differentially expressed lncRNAs exhibited epigenetic modification marks similar to protein-coding genes and tend to be expressed in a tissue-specific manner. In addition, the majority of differentially expressed lncRNAs harbored evolutionary conserved elements. Taken together, our findings represent the first systematic investigation of lncRNA expression in the mammalian testis and provide a solid foundation for further research into the molecular mechanisms of lncRNAs function in mammalian testis development and spermatogenesis.

  15. Integrating miRNA and mRNA Expression Profiling Uncovers miRNAs Underlying Fat Deposition in Sheep

    Guangxian Zhou

    2017-01-01

    Full Text Available MicroRNAs (miRNAs are endogenous, noncoding RNAs that regulate various biological processes including adipogenesis and fat metabolism. Here, we adopted a deep sequencing approach to determine the identity and abundance of miRNAs involved in fat deposition in adipose tissues from fat-tailed (Kazakhstan sheep, KS and thin-tailed (Tibetan sheep, TS sheep breeds. By comparing HiSeq data of these two breeds, 539 miRNAs were shared in both breeds, whereas 179 and 97 miRNAs were uniquely expressed in KS and TS, respectively. We also identified 35 miRNAs that are considered to be putative novel miRNAs. The integration of miRNA-mRNA analysis revealed that miRNA-associated targets were mainly involved in the gene ontology (GO biological processes concerning cellular process and metabolic process, and miRNAs play critical roles in fat deposition through their ability to regulate fundamental pathways. These pathways included the MAPK signaling pathway, FoxO and Wnt signaling pathway, and focal adhesion. Taken together, our results define miRNA expression signatures that may contribute to fat deposition and lipid metabolism in sheep.

  16. Research resources: comparative microRNA profiles in human corona radiata cells and cumulus oophorus cells detected by next-generation small RNA sequencing.

    Xian-Hong Tong

    Full Text Available During folliculogenesis, cumulus cells surrounding the oocyte differentiate into corona radiata cells (CRCs and cumulus oophorus cells (COCs, which are involved in gonadal steroidogenesis and the development of germ cells. Several studies suggested that microRNAs (miRNAs play an important regulatory role at the post-transcriptional level in cumulus cells. However, comparative miRNA profiles and associated processes in human CRCs and COCs have not been reported before. In this study, miRNA profiles were obtained from CRCs and COCs using next generation sequencing in women undergoing controlled ovarian stimulation for IVF. A total of 785 and 799 annotated miRNAs were identified in CRCs and COCs, while high expression levels of six novel miRNAs were detected both in CRCs and in COCs. In addition, different expression patterns in CRCs and COCs were detected in 72 annotated miRNAs. To confirm the miRNA profile in COCs and CRCs, quantitative real-time PCR was used to validate the expression of annotated miRNAs, differentially expressed miRNAs, and novel miRNAs. The miRNAs in the let-7 family were found to be involved in the regulation of a broad range of biological processes in both cumulus cell populations, which was accompanied by a large amount of miRNA editing. Bioinformatics analysis showed that amino acid and energy metabolism were targeted significantly by miRNAs that were differentially expressed between CRCs and COCs. Our work extends the current knowledge of the regulatory role of miRNAs and their targeted pathways in folliculogenesis, and provides novel candidates for molecular biomarkers in the research of female infertility.

  17. SHARAKU: an algorithm for aligning and clustering read mapping profiles of deep sequencing in non-coding RNA processing.

    Tsuchiya, Mariko; Amano, Kojiro; Abe, Masaya; Seki, Misato; Hase, Sumitaka; Sato, Kengo; Sakakibara, Yasubumi

    2016-06-15

    Deep sequencing of the transcripts of regulatory non-coding RNA generates footprints of post-transcriptional processes. After obtaining sequence reads, the short reads are mapped to a reference genome, and specific mapping patterns can be detected called read mapping profiles, which are distinct from random non-functional degradation patterns. These patterns reflect the maturation processes that lead to the production of shorter RNA sequences. Recent next-generation sequencing studies have revealed not only the typical maturation process of miRNAs but also the various processing mechanisms of small RNAs derived from tRNAs and snoRNAs. We developed an algorithm termed SHARAKU to align two read mapping profiles of next-generation sequencing outputs for non-coding RNAs. In contrast with previous work, SHARAKU incorporates the primary and secondary sequence structures into an alignment of read mapping profiles to allow for the detection of common processing patterns. Using a benchmark simulated dataset, SHARAKU exhibited superior performance to previous methods for correctly clustering the read mapping profiles with respect to 5'-end processing and 3'-end processing from degradation patterns and in detecting similar processing patterns in deriving the shorter RNAs. Further, using experimental data of small RNA sequencing for the common marmoset brain, SHARAKU succeeded in identifying the significant clusters of read mapping profiles for similar processing patterns of small derived RNA families expressed in the brain. The source code of our program SHARAKU is available at http://www.dna.bio.keio.ac.jp/sharaku/, and the simulated dataset used in this work is available at the same link. Accession code: The sequence data from the whole RNA transcripts in the hippocampus of the left brain used in this work is available from the DNA DataBank of Japan (DDBJ) Sequence Read Archive (DRA) under the accession number DRA004502. yasu@bio.keio.ac.jp Supplementary data are available

  18. Serum microRNA profiles in athyroid patients on and off levothyroxine therapy.

    Massolt, Elske T; Chaker, Layal; Visser, Theo J; Gillis, Ad J M; Dorssers, Lambert C J; Beukhof, Carolien M; Kam, Boen L R; Franssen, Gaston J; Brigante, Giulia; van Ginhoven, Tessa M; Visser, W Edward; Looijenga, Leendert H J; Peeters, Robin P

    2018-01-01

    Levothyroxine replacement treatment in hypothyroidism is unable to restore physiological thyroxine and triiodothyronine concentrations in serum and tissues completely. Normal serum thyroid stimulating hormone (TSH) concentrations reflect only pituitary euthyroidism and, therefore, novel biomarkers representing tissue-specific thyroid state are needed. MicroRNAs (miRNAs), small non-coding regulatory RNAs, exhibit tissue-specific expression patterns and can be detectable in serum. Previous studies have demonstrated differential expression of (precursors of) miRNAs in tissues under the influence of thyroid hormone. To study if serum miRNA profiles are changed in different thyroid states. We studied 13 athyroid patients (6 males) during TSH suppressive therapy and after 4 weeks of thyroid hormone withdrawal. A magnetic bead capture system was used to isolate 384 defined miRNAs from serum. Subsequently, the TaqMan Array Card 3.0 platform was used for profiling after individual target amplification. Mean age of the subjects was 44.0 years (range 20-61 years). Median TSH levels were 88.9 mU/l during levothyroxine withdrawal and 0.006 mU/l during LT4 treatment with a median dosage of 2.1 μg/kg. After normalization to allow inter-sample analysis, a paired analysis did not demonstrate a significant difference in expression of any of the 384 miRNAs analyzed on and off LT4 treatment. Although we previously showed an up-regulation of pri-miRNAs 133b and 206 in hypothyroid state in skeletal muscle, the present study does not supply evidence that thyroid state also affects serum miRNAs in humans.

  19. Systematic gene microarray analysis of the lncRNA expression profiles in human uterine cervix carcinoma.

    Chen, Jie; Fu, Ziyi; Ji, Chenbo; Gu, Pingqing; Xu, Pengfei; Yu, Ningzhu; Kan, Yansheng; Wu, Xiaowei; Shen, Rong; Shen, Yan

    2015-05-01

    The human uterine cervix carcinoma is one of the most well-known malignancy reproductive system cancers, which threatens women health globally. However, the mechanisms of the oncogenesis and development process of cervix carcinoma are not yet fully understood. Long non-coding RNAs (lncRNAs) have been proved to play key roles in various biological processes, especially development of cancer. The function and mechanism of lncRNAs on cervix carcinoma is still rarely reported. We selected 3 cervix cancer and normal cervix tissues separately, then performed lncRNA microarray to detect the differentially expressed lncRNAs. Subsequently, we explored the potential function of these dysregulated lncRNAs through online bioinformatics databases. Finally, quantity real-time PCR was carried out to confirm the expression levels of these dysregulated lncRNAs in cervix cancer and normal tissues. We uncovered the profiles of differentially expressed lncRNAs between normal and cervix carcinoma tissues by using the microarray techniques, and found 1622 upregulated and 3026 downregulated lncRNAs (fold-change>2.0) in cervix carcinoma compared to the normal cervical tissue. Furthermore, we found HOXA11-AS might participate in cervix carcinogenesis by regulating HOXA11, which is involved in regulating biological processes of cervix cancer. This study afforded expression profiles of lncRNAs between cervix carcinoma tissue and normal cervical tissue, which could provide database for further research about the function and mechanism of key-lncRNAs in cervix carcinoma, and might be helpful to explore potential diagnosis factors and therapeutic targets for cervix carcinoma. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

  20. Sweet Spot Supersymmetry and Composite Messengers

    Ibe, Masahiro; Kitano, Ryuichiro

    2007-01-01

    Sweet spot supersymmetry is a phenomenologically and cosmologically perfect framework to realize a supersymmetric world at short distance. We discuss a class of dynamical models of supersymmetry breaking and its mediation whose low-energy effective description falls into this framework. Hadron fields in the dynamical models play a role of the messengers of the supersymmetry breaking. As is always true in the models of the sweet spot supersymmetry, the messenger scale is predicted to be 10 5 GeV ∼ mess ∼ 10 GeV. Various values of the effective number of messenger fields N mess are possible depending on the choice of the gauge group

  1. MESSENGER'S First and Second Flybys of Mercury

    Slavin, James A.

    2009-01-01

    The MESSENGER mission to Mercury offers our first opportunity to explore this planet's miniature magnetosphere since Mariner 10's brief fly-bys in 1974-5. The magnetosphere of Mercury is the smallest in the solar system with its magnetic field typically standing off the solar wind only approximately 1000 km above the surface. An overview of the MESSENGER mission and its January 14th and October 6th, 2008 close flybys of Mercury will be provided. Primary science objectives and the science instrumentation will be described. Initial results from MESSENGER will be discussed with an emphasis on the magnetic field and charged particle measurements.

  2. Inference of RNA decay rate from transcriptional profiling highlights the regulatory programs of Alzheimer's disease.

    Alkallas, Rached; Fish, Lisa; Goodarzi, Hani; Najafabadi, Hamed S

    2017-10-13

    The abundance of mRNA is mainly determined by the rates of RNA transcription and decay. Here, we present a method for unbiased estimation of differential mRNA decay rate from RNA-sequencing data by modeling the kinetics of mRNA metabolism. We show that in all primary human tissues tested, and particularly in the central nervous system, many pathways are regulated at the mRNA stability level. We present a parsimonious regulatory model consisting of two RNA-binding proteins and four microRNAs that modulate the mRNA stability landscape of the brain, which suggests a new link between RBFOX proteins and Alzheimer's disease. We show that downregulation of RBFOX1 leads to destabilization of mRNAs encoding for synaptic transmission proteins, which may contribute to the loss of synaptic function in Alzheimer's disease. RBFOX1 downregulation is more likely to occur in older and female individuals, consistent with the association of Alzheimer's disease with age and gender."mRNA abundance is determined by the rates of transcription and decay. Here, the authors propose a method for estimating the rate of differential mRNA decay from RNA-seq data and model mRNA stability in the brain, suggesting a link between mRNA stability and Alzheimer's disease."

  3. Characterization and Improvement of RNA-Seq Precision in Quantitative Transcript Expression Profiling

    Labaj, Pawel P.; Leparc, German G.; Linggi, Bryan E.; Markillie, Lye Meng; Wiley, H. S.; Kreil, David P.

    2011-07-01

    Measurement precision determines the power of any analysis to reliably identify significant signals, such as in screens for differential expression, independent of whether the experimental design incorporates replicates or not. With the compilation of large scale RNA-Seq data sets with technical replicate samples, however, we can now, for the first time, perform a systematic analysis of the precision of expression level estimates from massively parallel sequencing technology. This then allows considerations for its improvement by computational or experimental means. Results: We report on a comprehensive study of target coverage and measurement precision, including their dependence on transcript expression levels, read depth and other parameters. In particular, an impressive target coverage of 84% of the estimated true transcript population could be achieved with 331 million 50 bp reads, with diminishing returns from longer read lengths and even less gains from increased sequencing depths. Most of the measurement power (75%) is spent on only 7% of the known transcriptome, however, making less strongly expressed transcripts harder to measure. Consequently, less than 30% of all transcripts could be quantified reliably with a relative error < 20%. Based on established tools, we then introduce a new approach for mapping and analyzing sequencing reads that yields substantially improved performance in gene expression profiling, increasing the number of transcripts that can reliably be quantified to over 40%. Extrapolations to higher sequencing depths highlight the need for efficient complementary steps. In discussion we outline possible experimental and computational strategies for further improvements in quantification precision.

  4. MicroRNA expression profiles of drug-resistance breast cancer cells and their exosomes.

    Zhong, Shanliang; Chen, Xiu; Wang, Dandan; Zhang, Xiaohui; Shen, Hongyu; Yang, Sujin; Lv, Mengmeng; Tang, Jinhai; Zhao, Jianhua

    2016-04-12

    Exosomes have been shown to transmit drug resistance through delivering miRNAs. We aimed to explore their roles in breast cancer. Three resistant sublines were established by exposing parental MDA-MB-231 cell line to docetaxel, epirubicin and vinorelbine, respectively. Preneoadjuvant chemotherapy biopsies and paired surgically-resected specimens embedded in paraffin from 23 breast cancer patients were collected. MiRNA expression profiles of the cell lines and their exosomes were evaluated using microarray. The result showed that most miRNAs in exosomes had a lower expression level than that in cells, however, some miRNAs expressed higher in exosomes than in cells, suggesting a number of miRNAs is concentrated in exosomes. Among the dysregulated miRNAs, 22 miRNAs were consistently up-regulated in exosomes and their cells of origin. We further found that 12 of the 22 miRNAs were significantly up-regulated after preneoadjuvant chemotherapy. Further study of the role of these 12 miRNAs in acquisition of drug resistance is needed to clarify their contribution to chemoresistance.

  5. RNA sequencing atopic dermatitis transcriptome profiling provides insights into novel disease mechanisms with potential therapeutic implications

    Suárez-Fariñas, Mayte; Ungar, Benjamin; Correa da Rosa, Joel

    2015-01-01

    . These limitations might be lessened with next-generation RNA sequencing (RNA-seq). Objective: We sought to define the lesional AD transcriptome using RNA-seq and compare it using microarrays performed on the same cohort. Methods: RNA-seq and microarrays were performed to identify differentially expressed genes...... RNA-seq showed somewhat better agreement with RT-PCR (intraclass correlation coefficient, 0.57 and 0.70 for microarrays and RNA-seq vs RT-PCR, respectively), bias was not eliminated. Among genes uniquely identified by using RNA-seq were triggering receptor expressed on myeloid cells 1 (TREM-1......) signaling (eg, CCL2, CCL3, and single immunoglobulin domain IL1R1 related [SIGIRR]) and IL-36 isoform genes. TREM-1 is a surface receptor implicated in innate and adaptive immunity that amplifies infection-related inflammation. Conclusions: This is the first report of a lesional AD phenotype using RNA...

  6. Mercury's Sodium Exosphere: Observations during the MESSENGER Orbital Phase

    Killen, Rosemary M.; Cassidy, Timothy A.; Vervack, Ronald J., Jr.; Burger, Matthew H.; Merkel, Aimee W.; Sarantos, Menelaos; Sprague, Ann L.; McClintock, William E.; Benna, Mehdi; Solomon, Sean C.

    2012-01-01

    The MErcury Surface, Space ENvironment, GEochemistry, and Ranging (MESSENGER) spacecraft entered into orbit about Mercury on March 18,2011. We now have approximately five Mercury years of data from orbit. Prior to the MESSENGER mission, Mercury's surface-bounded exosphere was known to contain H, He, Na. K, and Ca. The Ultraviolet and Visible Spectrometer (UVVS) began routine orbital observations of both the dayside and nightside exosphere on March 29. 2011, measuring altitude profiles for all previously detected neutral species except for He and K. We focus here on what we have learned about the sodium exosphere: its spatial, seasonal, and sporadic variation. Observations to date permit delineation of the relative roles of photon-stimulated desorption (PSD) and impact vaporization (IV) from seasonal and spatial effects, as well as of the roles of ions both as sputtering agents and in their possible role to enhance the efficiency of PSD. Correlations of Mercury's neutral sodium exosphere with measurements from MESSENGER's Magnetometer (MAG) and Energetic Particle and Plasma Spectrometer (EPPS) provide insight into the roles of ions and electrons. Models incorporating MAG observations provide a basis for identifying the location and area of the surface exposed to solar wind plasma, and EPPS observations reveal episodic populations of energetic electrons in the magnetosphere and the presence of planetary He(+), 0(+), and Na(+),

  7. Fecal Microbiota in Healthy Subjects Following Omnivore, Vegetarian and Vegan Diets: Culturable Populations and rRNA DGGE Profiling.

    Ferrocino, Ilario; Di Cagno, Raffaella; De Angelis, Maria; Turroni, Silvia; Vannini, Lucia; Bancalari, Elena; Rantsiou, Kalliopi; Cardinali, Gianluigi; Neviani, Erasmo; Cocolin, Luca

    2015-01-01

    In this study, the fecal microbiota of 153 healthy volunteers, recruited from four different locations in Italy, has been studied by coupling viable counts, on different microbiological media, with ribosomal RNA Denaturing Gradient Gel Electrophoresis (rRNA-DGGE). The volunteers followed three different diets, namely omnivore, ovo-lacto-vegetarian and vegan. The results obtained from culture-dependent and -independent methods have underlined a high level of similarity of the viable fecal microbiota for the three investigated diets. The rRNA DGGE profiles were very complex and comprised a total number of bands that varied from 67 to 64 for the V3 and V9 regions of the 16S rRNA gene, respectively. Only a few bands were specific in/of all three diets, and the presence of common taxa associated with the dietary habits was found. As far as the viable counts are concerned, the high similarity of the fecal microbiota was once again confirmed, with only a few of the investigated groups showing significant differences. Interestingly, the samples grouped differently, according to the recruitment site, thus highlighting a higher impact of the food consumed by the volunteers in the specific geographical locations than that of the type of diet. Lastly, it should be mentioned that the fecal microbiota DGGE profiles obtained from the DNA were clearly separated from those produced using RNA, thus underlining a difference between the total and viable populations in the fecal samples.

  8. Fecal Microbiota in Healthy Subjects Following Omnivore, Vegetarian and Vegan Diets: Culturable Populations and rRNA DGGE Profiling

    Ferrocino, Ilario; Di Cagno, Raffaella; De Angelis, Maria; Turroni, Silvia; Vannini, Lucia; Bancalari, Elena; Rantsiou, Kalliopi; Cardinali, Gianluigi; Neviani, Erasmo; Cocolin, Luca

    2015-01-01

    In this study, the fecal microbiota of 153 healthy volunteers, recruited from four different locations in Italy, has been studied by coupling viable counts, on different microbiological media, with ribosomal RNA Denaturing Gradient Gel Electrophoresis (rRNA-DGGE). The volunteers followed three different diets, namely omnivore, ovo-lacto-vegetarian and vegan. The results obtained from culture-dependent and -independent methods have underlined a high level of similarity of the viable fecal microbiota for the three investigated diets. The rRNA DGGE profiles were very complex and comprised a total number of bands that varied from 67 to 64 for the V3 and V9 regions of the 16S rRNA gene, respectively. Only a few bands were specific in/of all three diets, and the presence of common taxa associated with the dietary habits was found. As far as the viable counts are concerned, the high similarity of the fecal microbiota was once again confirmed, with only a few of the investigated groups showing significant differences. Interestingly, the samples grouped differently, according to the recruitment site, thus highlighting a higher impact of the food consumed by the volunteers in the specific geographical locations than that of the type of diet. Lastly, it should be mentioned that the fecal microbiota DGGE profiles obtained from the DNA were clearly separated from those produced using RNA, thus underlining a difference between the total and viable populations in the fecal samples. PMID:26035837

  9. Fecal Microbiota in Healthy Subjects Following Omnivore, Vegetarian and Vegan Diets: Culturable Populations and rRNA DGGE Profiling.

    Ilario Ferrocino

    Full Text Available In this study, the fecal microbiota of 153 healthy volunteers, recruited from four different locations in Italy, has been studied by coupling viable counts, on different microbiological media, with ribosomal RNA Denaturing Gradient Gel Electrophoresis (rRNA-DGGE. The volunteers followed three different diets, namely omnivore, ovo-lacto-vegetarian and vegan. The results obtained from culture-dependent and -independent methods have underlined a high level of similarity of the viable fecal microbiota for the three investigated diets. The rRNA DGGE profiles were very complex and comprised a total number of bands that varied from 67 to 64 for the V3 and V9 regions of the 16S rRNA gene, respectively. Only a few bands were specific in/of all three diets, and the presence of common taxa associated with the dietary habits was found. As far as the viable counts are concerned, the high similarity of the fecal microbiota was once again confirmed, with only a few of the investigated groups showing significant differences. Interestingly, the samples grouped differently, according to the recruitment site, thus highlighting a higher impact of the food consumed by the volunteers in the specific geographical locations than that of the type of diet. Lastly, it should be mentioned that the fecal microbiota DGGE profiles obtained from the DNA were clearly separated from those produced using RNA, thus underlining a difference between the total and viable populations in the fecal samples.

  10. In Vitro Assays for Mouse Müller Cell Phenotyping Through microRNA Profiling in the Damaged Retina.

    Reyes-Aguirre, Luis I; Quintero, Heberto; Estrada-Leyva, Brenda; Lamas, Mónica

    2018-01-01

    microRNA profiling has identified cell-specific expression patterns that could represent molecular signatures triggering the acquisition of a specific phenotype; in other words, of cellular identity and its associated function. Several groups have hypothesized that retinal cell phenotyping could be achieved through the determination of the global pattern of miRNA expression across specific cell types in the adult retina. This is especially relevant for Müller glia in the context of retinal damage, as these cells undergo dramatic changes of gene expression in response to injury, that render them susceptible to acquire a progenitor-like phenotype and be a source of new neurons.We describe a method that combines an experimental protocol for excitotoxic-induced retinal damage through N-methyl-D-aspartate subretinal injection with magnetic-activated cell sorting (MACS) of Müller cells and RNA isolation for microRNA profiling. Comparison of microRNA patterns of expression should allow Müller cell phenotyping under different experimental conditions.

  11. MicroRNA expression profiling to identify and validate reference genes for relative quantification in colorectal cancer.

    Chang, Kah Hoong

    2010-01-01

    BACKGROUND: Advances in high-throughput technologies and bioinformatics have transformed gene expression profiling methodologies. The results of microarray experiments are often validated using reverse transcription quantitative PCR (RT-qPCR), which is the most sensitive and reproducible method to quantify gene expression. Appropriate normalisation of RT-qPCR data using stably expressed reference genes is critical to ensure accurate and reliable results. Mi(cro)RNA expression profiles have been shown to be more accurate in disease classification than mRNA expression profiles. However, few reports detailed a robust identification and validation strategy for suitable reference genes for normalisation in miRNA RT-qPCR studies. METHODS: We adopt and report a systematic approach to identify the most stable reference genes for miRNA expression studies by RT-qPCR in colorectal cancer (CRC). High-throughput miRNA profiling was performed on ten pairs of CRC and normal tissues. By using the mean expression value of all expressed miRNAs, we identified the most stable candidate reference genes for subsequent validation. As such the stability of a panel of miRNAs was examined on 35 tumour and 39 normal tissues. The effects of normalisers on the relative quantity of established oncogenic (miR-21 and miR-31) and tumour suppressor (miR-143 and miR-145) target miRNAs were assessed. RESULTS: In the array experiment, miR-26a, miR-345, miR-425 and miR-454 were identified as having expression profiles closest to the global mean. From a panel of six miRNAs (let-7a, miR-16, miR-26a, miR-345, miR-425 and miR-454) and two small nucleolar RNA genes (RNU48 and Z30), miR-16 and miR-345 were identified as the most stably expressed reference genes. The combined use of miR-16 and miR-345 to normalise expression data enabled detection of a significant dysregulation of all four target miRNAs between tumour and normal colorectal tissue. CONCLUSIONS: Our study demonstrates that the top six most

  12. MicroRNA expression profiling to identify and validate reference genes for relative quantification in colorectal cancer

    Chang, Kah Hoong

    2010-04-29

    Abstract Background Advances in high-throughput technologies and bioinformatics have transformed gene expression profiling methodologies. The results of microarray experiments are often validated using reverse transcription quantitative PCR (RT-qPCR), which is the most sensitive and reproducible method to quantify gene expression. Appropriate normalisation of RT-qPCR data using stably expressed reference genes is critical to ensure accurate and reliable results. Mi(cro)RNA expression profiles have been shown to be more accurate in disease classification than mRNA expression profiles. However, few reports detailed a robust identification and validation strategy for suitable reference genes for normalisation in miRNA RT-qPCR studies. Methods We adopt and report a systematic approach to identify the most stable reference genes for miRNA expression studies by RT-qPCR in colorectal cancer (CRC). High-throughput miRNA profiling was performed on ten pairs of CRC and normal tissues. By using the mean expression value of all expressed miRNAs, we identified the most stable candidate reference genes for subsequent validation. As such the stability of a panel of miRNAs was examined on 35 tumour and 39 normal tissues. The effects of normalisers on the relative quantity of established oncogenic (miR-21 and miR-31) and tumour suppressor (miR-143 and miR-145) target miRNAs were assessed. Results In the array experiment, miR-26a, miR-345, miR-425 and miR-454 were identified as having expression profiles closest to the global mean. From a panel of six miRNAs (let-7a, miR-16, miR-26a, miR-345, miR-425 and miR-454) and two small nucleolar RNA genes (RNU48 and Z30), miR-16 and miR-345 were identified as the most stably expressed reference genes. The combined use of miR-16 and miR-345 to normalise expression data enabled detection of a significant dysregulation of all four target miRNAs between tumour and normal colorectal tissue. Conclusions Our study demonstrates that the top six most

  13. MicroRNA and mesial temporal lobe epilepsy with hippocampal sclerosis: Whole miRNome profiling of human hippocampus.

    Bencurova, Petra; Baloun, Jiri; Musilova, Katerina; Radova, Lenka; Tichy, Boris; Pail, Martin; Zeman, Martin; Brichtova, Eva; Hermanova, Marketa; Pospisilova, Sarka; Mraz, Marek; Brazdil, Milan

    2017-10-01

    Mesial temporal lobe epilepsy (mTLE) is a severe neurological disorder characterized by recurrent seizures. mTLE is frequently accompanied by neurodegeneration in the hippocampus resulting in hippocampal sclerosis (HS), the most common morphological correlate of drug resistance in mTLE patients. Incomplete knowledge of pathological changes in mTLE+HS complicates its therapy. The pathological mechanism underlying mTLE+HS may involve abnormal gene expression regulation, including posttranscriptional networks involving microRNAs (miRNAs). miRNA expression deregulation has been reported in various disorders, including epilepsy. However, the miRNA profile of mTLE+HS is not completely known and needs to be addressed. Here, we have focused on hippocampal miRNA profiling in 33 mTLE+HS patients and nine postmortem controls to reveal abnormally expressed miRNAs. In this study, we significantly reduced technology-related bias (the most common source of false positivity in miRNA profiling data) by combining two different miRNA profiling methods, namely next generation sequencing and miRNA-specific quantitative real-time polymerase chain reaction. These methods combined have identified and validated 20 miRNAs with altered expression in the human epileptic hippocampus; 19 miRNAs were up-regulated and one down-regulated in mTLE+HS patients. Nine of these miRNAs have not been previously associated with epilepsy, and 19 aberrantly expressed miRNAs potentially regulate the targets and pathways linked with epilepsy (such as potassium channels, γ-aminobutyric acid, neurotrophin signaling, and axon guidance). This study extends current knowledge of miRNA-mediated gene expression regulation in mTLE+HS by identifying miRNAs with altered expression in mTLE+HS, including nine novel abnormally expressed miRNAs and their putative targets. These observations further encourage the potential of microRNA-based biomarkers or therapies. Wiley Periodicals, Inc. © 2017 International League Against

  14. Beyond 16S rRNA Community Profiling: Intra-Species Diversity in the Gut Microbiota

    Ellegaard, Kirsten M.; Engel, Philipp

    2016-01-01

    Interactions with microbes affect many aspects of animal biology, including immune system development, nutrition and health. In vertebrates, the gut microbiota is dominated by a small subset of phyla, but the species composition within these phyla is typically not conserved. Moreover, several recent studies have shown that bacterial species in the gut are composed of a multitude of strains, which frequently co-exist in their host, and may be host-specific. However, since the study of intra-species diversity is challenging, particularly in the setting of complex, host-associated microbial communities, our current understanding of the distribution, evolution and functional relevance of intra-species diversity in the gut is scarce. In order to unravel how genomic diversity translates into phenotypic diversity, community analyses going beyond 16S rRNA profiling, in combination with experimental approaches, are needed. Recently, the honeybee has emerged as a promising model for studying gut bacterial communities, particularly in terms of strain-level diversity. Unlike most other invertebrates, the honeybee gut is colonized by a remarkably consistent and specific core microbiota, which is dominated by only eight bacterial species. As for the vertebrate gut microbiota, these species are composed of highly diverse strains suggesting that similar evolutionary forces shape gut community structures in vertebrates and social insects. In this review, we outline current knowledge on the evolution and functional relevance of strain diversity within the gut microbiota, including recent insights gained from mammals and other animals such as the honeybee. We discuss methodological approaches and propose possible future avenues for studying strain diversity in complex bacterial communities. PMID:27708630

  15. Variation in RNA-Seq transcriptome profiles of peripheral whole blood from healthy individuals with and without globin depletion.

    Heesun Shin

    Full Text Available BACKGROUND: The molecular profile of circulating blood can reflect physiological and pathological events occurring in other tissues and organs of the body and delivers a comprehensive view of the status of the immune system. Blood has been useful in studying the pathobiology of many diseases. It is accessible and easily collected making it ideally suited to the development of diagnostic biomarker tests. The blood transcriptome has a high complement of globin RNA that could potentially saturate next-generation sequencing platforms, masking lower abundance transcripts. Methods to deplete globin mRNA are available, but their effect has not been comprehensively studied in peripheral whole blood RNA-Seq data. In this study we aimed to assess technical variability associated with globin depletion in addition to assessing general technical variability in RNA-Seq from whole blood derived samples. RESULTS: We compared technical and biological replicates having undergone globin depletion or not and found that the experimental globin depletion protocol employed removed approximately 80% of globin transcripts, improved the correlation of technical replicates, allowed for reliable detection of thousands of additional transcripts and generally increased transcript abundance measures. Differential expression analysis revealed thousands of genes significantly up-regulated as a result of globin depletion. In addition, globin depletion resulted in the down-regulation of genes involved in both iron and zinc metal ion bonding. CONCLUSIONS: Globin depletion appears to meaningfully improve the quality of peripheral whole blood RNA-Seq data, and may improve our ability to detect true biological variation. Some concerns remain, however. Key amongst them the significant reduction in RNA yields following globin depletion. More generally, our investigation of technical and biological variation with and without globin depletion finds that high-throughput sequencing by RNA

  16. Integrated analysis of miRNA and mRNA expression profiles in tilapia gonads at an early stage of sex differentiation.

    Tao, Wenjing; Sun, Lina; Shi, Hongjuan; Cheng, Yunying; Jiang, Dongneng; Fu, Beide; Conte, Matthew A; Gammerdinger, William J; Kocher, Thomas D; Wang, Deshou

    2016-05-04

    MicroRNAs (miRNAs) represent a second regulatory network that has important effects on gene expression and protein translation during biological process. However, the possible role of miRNAs in the early stages of fish sex differentiation is not well understood. In this study, we carried an integrated analysis of miRNA and mRNA expression profiles to explore their possibly regulatory patterns at the critical stage of sex differentiation in tilapia. We identified 279 pre-miRNA genes in tilapia genome, which were highly conserved in other fish species. Based on small RNA library sequencing, we identified 635 mature miRNAs in tilapia gonads, in which 62 and 49 miRNAs showed higher expression in XX and XY gonads, respectively. The predicted targets of these sex-biased miRNAs (e.g., miR-9, miR-21, miR-30a, miR-96, miR-200b, miR-212 and miR-7977) included genes encoding key enzymes in steroidogenic pathways (Cyp11a1, Hsd3b, Cyp19a1a, Hsd11b) and key molecules involved in vertebrate sex differentiation (Foxl2, Amh, Star1, Sf1, Dmrt1, and Gsdf). These genes also showed sex-biased expression in tilapia gonads at 5 dah. Some miRNAs (e.g., miR-96 and miR-737) targeted multiple genes involved in steroid synthesis, suggesting a complex miRNA regulatory network during early sex differentiation in this fish. The sequence and expression patterns of most miRNAs in tilapia are conserved in fishes, indicating the basic functions of vertebrate miRNAs might share a common evolutionary origin. This comprehensive analysis of miRNA and mRNA at the early stage of molecular sex differentiation in tilapia XX and XY gonads lead to the discovery of differentially expressed miRNAs and their putative targets, which will facilitate studies of the regulatory network of molecular sex determination and differentiation in fishes.

  17. Genome-wide annotation of porcine microRNA genes and transcriptome profiling during Actinobacillus infection

    Nielsen, Mathilde

    MicroRNAs are small single stranded non-coding RNA molecules which contributes to the regulation of gene expression by primarily binding to the 3´end of protein coding mRNA, hereby inhibiting the translation process or promting degradation of the mRNA. The main focus of this PhD project was to ex......MicroRNAs are small single stranded non-coding RNA molecules which contributes to the regulation of gene expression by primarily binding to the 3´end of protein coding mRNA, hereby inhibiting the translation process or promting degradation of the mRNA. The main focus of this PhD project...

  18. Integrating microRNA and mRNA expression profiles of neuronal progenitors to identify regulatory networks underlying the onset of cortical neurogenesis

    Barker Jeffery L

    2009-08-01

    Full Text Available Abstract Background Cortical development is a complex process that includes sequential generation of neuronal progenitors, which proliferate and migrate to form the stratified layers of the developing cortex. To identify the individual microRNAs (miRNAs and mRNAs that may regulate the genetic network guiding the earliest phase of cortical development, the expression profiles of rat neuronal progenitors obtained at embryonic day 11 (E11, E12 and E13 were analyzed. Results Neuronal progenitors were purified from telencephalic dissociates by a positive-selection strategy featuring surface labeling with tetanus-toxin and cholera-toxin followed by fluorescence-activated cell sorting. Microarray analyses revealed the fractions of miRNAs and mRNAs that were up-regulated or down-regulated in these neuronal progenitors at the beginning of cortical development. Nearly half of the dynamically expressed miRNAs were negatively correlated with the expression of their predicted target mRNAs. Conclusion These data support a regulatory role for miRNAs during the transition from neuronal progenitors into the earliest differentiating cortical neurons. In addition, by supplying a robust data set in which miRNA and mRNA profiles originate from the same purified cell type, this empirical study may facilitate the development of new algorithms to integrate various "-omics" data sets.

  19. microRNA expression profiling on individual breast cancer patients identifies novel panel of circulating microRNA for early detection

    Hamam, Rimi; Ali, Arwa M.; Alsaleh, Khalid A.

    2016-01-01

    Breast cancer (BC) is the most common cancer type and the second cause of cancer-related death among women. Therefore, better understanding of breast cancer tumor biology and the identification of novel biomarkers is essential for the early diagnosis and for better disease stratification and mana......Breast cancer (BC) is the most common cancer type and the second cause of cancer-related death among women. Therefore, better understanding of breast cancer tumor biology and the identification of novel biomarkers is essential for the early diagnosis and for better disease stratification...... and management choices. Herein we developed a novel approach which relies on the isolation of circulating microRNAs through an enrichment step using speed-vacuum concentration which resulted in 5-fold increase in microRNA abundance. Global miRNA microarray expression profiling performed on individual samples...... of 46 BC and 14 controls. The expression of those microRNAs was overall higher in patients with stage I, II, and III, compared to stage IV, with potential utilization for early detection. The expression of this microRNA panel was slightly higher in the HER2 and TN compared to patients with luminal...

  20. Small RNA and mRNA Profiling of Arabidopsis in Response to Phytophthora Infection and PAMP Treatment.

    Hou, Yingnan; Ma, Wenbo

    2017-01-01

    Small non-coding RNAs (smRNAs) regulate gene expression at both transcriptional and post-transcriptional levels. Well known for their roles in development, smRNAs have emerged as important regulators of plant immunity. Upon pathogen perception, accumulation of specific smRNAs are found to be altered, presumably as a host defense response. Therefore, identification of differentially accumulated smRNAs and their target genes would provide important insight into the regulation mechanism of immune responses. Here, we describe the detailed experimental procedure using Illumina sequencing to analyze the expression profiles of smRNAs and mRNAs in Arabidopsis. We focus on a newly developed pathosystem using Phytophthora capsici as the pathogen and include the treatment of Arabidopsis leaves with pathogen-associated molecular patterns (PAMPs) of Phytophthora.

  1. Arabidopsis mRNA polyadenylation machinery: comprehensive analysis of protein-protein interactions and gene expression profiling

    Mo Min

    2008-05-01

    Full Text Available Abstract Background The polyadenylation of mRNA is one of the critical processing steps during expression of almost all eukaryotic genes. It is tightly integrated with transcription, particularly its termination, as well as other RNA processing events, i.e. capping and splicing. The poly(A tail protects the mRNA from unregulated degradation, and it is required for nuclear export and translation initiation. In recent years, it has been demonstrated that the polyadenylation process is also involved in the regulation of gene expression. The polyadenylation process requires two components, the cis-elements on the mRNA and a group of protein factors that recognize the cis-elements and produce the poly(A tail. Here we report a comprehensive pairwise protein-protein interaction mapping and gene expression profiling of the mRNA polyadenylation protein machinery in Arabidopsis. Results By protein sequence homology search using human and yeast polyadenylation factors, we identified 28 proteins that may be components of Arabidopsis polyadenylation machinery. To elucidate the protein network and their functions, we first tested their protein-protein interaction profiles. Out of 320 pair-wise protein-protein interaction assays done using the yeast two-hybrid system, 56 (~17% showed positive interactions. 15 of these interactions were further tested, and all were confirmed by co-immunoprecipitation and/or in vitro co-purification. These interactions organize into three distinct hubs involving the Arabidopsis polyadenylation factors. These hubs are centered around AtCPSF100, AtCLPS, and AtFIPS. The first two are similar to complexes seen in mammals, while the third one stands out as unique to plants. When comparing the gene expression profiles extracted from publicly available microarray datasets, some of the polyadenylation related genes showed tissue-specific expression, suggestive of potential different polyadenylation complex configurations. Conclusion An

  2. MicroRNA expression profiles of multiple system atrophy from formalin-fixed paraffin-embedded samples.

    Wakabayashi, Koichi; Mori, Fumiaki; Kakita, Akiyoshi; Takahashi, Hitoshi; Tanaka, Shinya; Utsumi, Jun; Sasaki, Hidenao

    2016-12-02

    MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression. Recently, we have shown that informative miRNA data can be derived from archived formalin-fixed paraffin-embedded (FFPE) samples from postmortem cases of amyotrophic lateral sclerosis and normal controls. miRNA analysis has now been performed on FFPE samples from affected brain regions in patients with multiple system atrophy (MSA) and the same areas in neurologically normal controls. We evaluated 50 samples from patients with MSA (n=13) and controls (n=13). Twenty-six samples were selected for miRNA analysis on the basis of the criteria reported previously: (i) a formalin fixation time of less than 4 weeks, (ii) a total RNA yield per sample of more than 500ng, and (iii) sufficient quality of the RNA electrophoresis pattern. These included 11 cases of MSA and 5 controls. Thus, the success rate for analysis of RNA from FFPE samples was 52% (26 of 50). For MSA, a total of 395 and 383 miRNAs were identified in the pons and cerebellum, respectively; 5 were up-regulated and 33 were down-regulated in the pons and 5 were up-regulated and 18 were down-regulated in the cerebellum. Several miRNAs down-regulated in the pons (miR-129-2-3p and miR-129-5p) and cerebellum (miR-129-2-3p, miR-129-5p and miR-132-3p) had already been identified in frozen cerebellum from MSA patients. These findings suggest that archived FFPE postmortem samples can be a valuable source for miRNA profiling in MSA. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  3. Whole transcriptome analysis of Acinetobacter baumannii assessed by RNA-sequencing reveals different mRNA expression profiles in biofilm compared to planktonic cells.

    Soraya Rumbo-Feal

    Full Text Available Acinetobacterbaumannii has emerged as a dangerous opportunistic pathogen, with many strains able to form biofilms and thus cause persistent infections. The aim of the present study was to use high-throughput sequencing techniques to establish complete transcriptome profiles of planktonic (free-living and sessile (biofilm forms of A. baumannii ATCC 17978 and thereby identify differences in their gene expression patterns. Collections of mRNA from planktonic (both exponential and stationary phase cultures and sessile (biofilm cells were sequenced. Six mRNA libraries were prepared following the mRNA-Seq protocols from Illumina. Reads were obtained in a HiScanSQ platform and mapped against the complete genome to describe the complete mRNA transcriptomes of planktonic and sessile cells. The results showed that the gene expression pattern of A. baumannii biofilm cells was distinct from that of planktonic cells, including 1621 genes over-expressed in biofilms relative to stationary phase cells and 55 genes expressed only in biofilms. These differences suggested important changes in amino acid and fatty acid metabolism, motility, active transport, DNA-methylation, iron acquisition, transcriptional regulation, and quorum sensing, among other processes. Disruption or deletion of five of these genes caused a significant decrease in biofilm formation ability in the corresponding mutant strains. Among the genes over-expressed in biofilm cells were those in an operon involved in quorum sensing. One of them, encoding an acyl carrier protein, was shown to be involved in biofilm formation as demonstrated by the significant decrease in biofilm formation by the corresponding knockout strain. The present work serves as a basis for future studies examining the complex network systems that regulate bacterial biofilm formation and maintenance.

  4. Higgs mass from neutrino-messenger mixing

    Byakti, Pritibhajan; Khosa, Charanjit K.; Mummidi, V.S.; Vempati, Sudhir K.

    2017-01-01

    The discovery of the Higgs particle at 125 GeV has put strong constraints on minimal messenger models of gauge mediation, pushing the stop masses into the multi-TeV regime. Extensions of these models with matter-messenger mixing terms have been proposed to generate a large trilinear parameter, A t , relaxing these constraints. The detailed survey of these models (DOI: 10.1007/JHEP05(2013)055; 10.1007/JHEP08(2013)093 ) so far considered messenger mixings with only MSSM superfields. In the present work, we extend the survey to MSSM with inverse-seesaw mechanism. The neutrino-sneutrino corrections to the Higgs mass in the inverse seesaw model are not significant in the minimal gauge mediation model, unless one considers messenger-matter interaction terms. We classify all possible models with messenger-matter interactions and perform thorough numerical analysis to find out the promising models. We found that out of the 17 possible models 9 of them can lead to Higgs mass within the observed value without raising the sfermion masses significantly. The successful models have stop masses ∼1.5 TeV with small or negligible mixing and yet a light CP even Higgs at 125 GeV.

  5. Higgs mass from neutrino-messenger mixing

    Byakti, Pritibhajan [Center for High Energy Physics, Indian Institute of Science,C.V. Raman Ave, Bangalore 560012 (India); Department of Theoretical Physics, Indian Association for the Cultivation of Science,2A & 2B Raja S.C. Mullick Road, Kolkata 700 032 (India); Khosa, Charanjit K. [Center for High Energy Physics, Indian Institute of Science,C.V. Raman Ave, Bangalore 560012 (India); Mummidi, V.S. [Harish-Chandra Research Institute,Chhatnag Road, Jhusi, Allahabad 211019 (India); Vempati, Sudhir K. [Center for High Energy Physics, Indian Institute of Science,C.V. Raman Ave, Bangalore 560012 (India)

    2017-03-06

    The discovery of the Higgs particle at 125 GeV has put strong constraints on minimal messenger models of gauge mediation, pushing the stop masses into the multi-TeV regime. Extensions of these models with matter-messenger mixing terms have been proposed to generate a large trilinear parameter, A{sub t}, relaxing these constraints. The detailed survey of these models (DOI: 10.1007/JHEP05(2013)055; 10.1007/JHEP08(2013)093 ) so far considered messenger mixings with only MSSM superfields. In the present work, we extend the survey to MSSM with inverse-seesaw mechanism. The neutrino-sneutrino corrections to the Higgs mass in the inverse seesaw model are not significant in the minimal gauge mediation model, unless one considers messenger-matter interaction terms. We classify all possible models with messenger-matter interactions and perform thorough numerical analysis to find out the promising models. We found that out of the 17 possible models 9 of them can lead to Higgs mass within the observed value without raising the sfermion masses significantly. The successful models have stop masses ∼1.5 TeV with small or negligible mixing and yet a light CP even Higgs at 125 GeV.

  6. Epigallocatechin Gallate-Mediated Alteration of the MicroRNA Expression Profile in 5α-Dihydrotestosterone-Treated Human Dermal Papilla Cells.

    Shin, Shanghun; Kim, Karam; Lee, Myung Joo; Lee, Jeongju; Choi, Sungjin; Kim, Kyung-Suk; Ko, Jung-Min; Han, Hyunjoo; Kim, Su Young; Youn, Hae Jeong; Ahn, Kyu Joong; An, In-Sook; An, Sungkwan; Cha, Hwa Jun

    2016-06-01

    Dihydrotestosterone (DHT) induces androgenic alopecia by shortening the hair follicle growth phase, resulting in hair loss. We previously demonstrated how changes in the microRNA (miRNA) expression profile influenced DHT-mediated cell death, cell cycle arrest, cell viability, the generation of reactive oxygen species (ROS), and senescence. Protective effects against DHT have not, however, been elucidated at the genome level. We showed that epigallocatechin gallate (EGCG), a major component of green tea, protects DHT-induced cell death by regulating the cellular miRNA expression profile. We used a miRNA microarray to identify miRNA expression levels in human dermal papilla cells (DPCs). We investigated whether the miRNA expression influenced the protective effects of EGCG against DHT-induced cell death, growth arrest, intracellular ROS levels, and senescence. EGCG protected against the effects of DHT by altering the miRNA expression profile in human DPCs. In addition, EGCG attenuated DHT-mediated cell death and growth arrest and decreased intracellular ROS levels and senescence. A bioinformatics analysis elucidated the relationship between the altered miRNA expression and EGCG-mediated protective effects against DHT. Overall, our results suggest that EGCG ameliorates the negative effects of DHT by altering the miRNA expression profile in human DPCs.

  7. [Expression profiles and bioinformatic analysis of miRNA in human dental pulp cells during endothelial differentiation].

    Gong, Qimei; Jiang, Hongwei; Wang, Jinming; Ling, Junqi

    2014-05-01

    To investigate the differential expression profile and bioinformatic analysis of microRNA (miRNA) in human dental pulp cells (DPC) during endothelial differentiation. DPC were cultured in endothelial induction medium (50 µg/L vascular endothelial growth factor, 10 µg/L basic fibroblast growth factor and 2% fetal calf serum) for 7 days. Meanwhile non-induced DPC were used as control.Quantitative real-time PCR (qRT-PCR) was applied to detect vascular endothelial marker genes [CD31, von Willebrand factor (vWF) and vascular endothelial-cadherin (VE-cadherin)] and in vitro tube formation on matrigel was used to analyze the angiogenic ability of differentiated cells. And then miRNA expression profiles of DPC were examined using miRNA microarray and then the differentially expressed miRNA were validated by qRT-PCR. Furthermore, bioinformatic analysis was employed to predict the target genes of miRNA and to analyze the possible biological functions and signaling pathways that were involved in DPC after induction. The relative mRNA level of CD31, vWF and VE-cadherin in the control group were (3.48 ± 0.22) ×10(-4), (3.13 ± 0.31) ×10(-4) and (39.60 ± 2.36) ×10(-4), and (19.57 ± 2.20) ×10(-4), (48.13 ± 0.54) ×10(-4) and (228.00 ± 8.89) ×10(-4) in the induced group. The expressions of CD31, vWF and VE-cadherin were increased significantly in endothelial induced DPC compared to the control group (P functions, such as the regulation of transcription, cell motion, blood vessel morphogenesis, angiogenesis and cytoskeletal protein, and signaling pathways including the mitogen-activated protein kinase (MAPK) and the Wnt signaling pathway. The differential miRNA expression identified in this study may be involved in governing DPC endothelial differentiation, thus contributing to the future research on regulatory mechanisms in dental pulp angiogenesis.

  8. RNA profiles of porcine embryos during genome activation reveal complex metabolic switch sensitive to in vitro conditions

    Østrup, Olga; Olbricht, Gayla; Østrup, Esben

    2013-01-01

    produced in vitro. Overall, our data are in good accordance with previously published, genome-wide profiling data in other species. Moreover, comparison with mouse and human embryos showed striking overlap in functional annotation of transcripts during the EGA, suggesting conserved basic mechanisms...... a handful of reports characterize changing transcriptome profiles and resulting metabolic changes in cleavage stage embryos. The aims of the current study were to investigate RNA profiles of in vivo developed (ivv) and in vitro produced (ivt) porcine embryos before (2-cell stage) and after (late 4-cell...... from oocyte and are imposed either before oocyte aspiration or during in vitro maturation. IVT embryos have altered content of apoptotic factors, cell cycle regulation factors and spindle components, and transcription factors, which all may contribute to reduced developmental competence of embryos...

  9. MicroRNA expression profile in head and neck cancer: HOX-cluster embedded microRNA-196a and microRNA-10b dysregulation implicated in cell proliferation

    Severino, Patricia; Mathor, Monica Beatriz; Nunes, Fabio Daumas; Ragoussis, Jiannis; Tajara, Eloiza Helena; Brüggemann, Holger; Andreghetto, Flavia Maziero; Camps, Carme; Klingbeil, Maria de Fatima Garrido; Pereira, Welbert Oliveira de; Soares, Renata Machado; Moyses, Raquel; Wünsch-Filho, Victor

    2013-01-01

    Current evidence implicates aberrant microRNA expression patterns in human malignancies; measurement of microRNA expression may have diagnostic and prognostic applications. Roles for microRNAs in head and neck squamous cell carcinomas (HNSCC) are largely unknown. HNSCC, a smoking-related cancer, is one of the most common malignancies worldwide but reliable diagnostic and prognostic markers have not been discovered so far. Some studies have evaluated the potential use of microRNA as biomarkers with clinical application in HNSCC. MicroRNA expression profile of oral squamous cell carcinoma samples was determined by means of DNA microarrays. We also performed gain-of-function assays for two differentially expressed microRNA using two squamous cell carcinoma cell lines and normal oral keratinocytes. The effect of the over-expression of these molecules was evaluated by means of global gene expression profiling and cell proliferation assessment. Altered microRNA expression was detected for a total of 72 microRNAs. Among these we found well studied molecules, such as the miR-17-92 cluster, comprising potent oncogenic microRNA, and miR-34, recently found to interact with p53. HOX-cluster embedded miR-196a/b and miR-10b were up- and down-regulated, respectively, in tumor samples. Since validated HOX gene targets for these microRNAs are not consistently deregulated in HNSCC, we performed gain-of-function experiments, in an attempt to outline their possible role. Our results suggest that both molecules interfere in cell proliferation through distinct processes, possibly targeting a small set of genes involved in cell cycle progression. Functional data on miRNAs in HNSCC is still scarce. Our data corroborate current literature and brings new insights into the role of microRNAs in HNSCC. We also show that miR-196a and miR-10b, not previously associated with HNSCC, may play an oncogenic role in this disease through the deregulation of cell proliferation. The study of microRNA

  10. Multiple sclerosis: microRNA expression profiles accurately differentiate patients with relapsing-remitting disease from healthy controls.

    Andreas Keller

    Full Text Available Multiple sclerosis (MS is a chronic inflammatory demyelinating disease of the central nervous system, which is heterogenous with respect to clinical manifestations and response to therapy. Identification of biomarkers appears desirable for an improved diagnosis of MS as well as for monitoring of disease activity and treatment response. MicroRNAs (miRNAs are short non-coding RNAs, which have been shown to have the potential to serve as biomarkers for different human diseases, most notably cancer. Here, we analyzed the expression profiles of 866 human miRNAs. In detail, we investigated the miRNA expression in blood cells of 20 patients with relapsing-remitting MS (RRMS and 19 healthy controls using a human miRNA microarray and the Geniom Real Time Analyzer (GRTA platform. We identified 165 miRNAs that were significantly up- or downregulated in patients with RRMS as compared to healthy controls. The best single miRNA marker, hsa-miR-145, allowed discriminating MS from controls with a specificity of 89.5%, a sensitivity of 90.0%, and an accuracy of 89.7%. A set of 48 miRNAs that was evaluated by radial basis function kernel support vector machines and 10-fold cross validation yielded a specificity of 95%, a sensitivity of 97.6%, and an accuracy of 96.3%. While 43 of the 165 miRNAs deregulated in patients with MS have previously been related to other human diseases, the remaining 122 miRNAs are so far exclusively associated with MS. The implications of our study are twofold. The miRNA expression profiles in blood cells may serve as a biomarker for MS, and deregulation of miRNA expression may play a role in the pathogenesis of MS.

  11. Mechanistic profiling of the siRNA delivery dynamics of lipid-polymer hybrid nanoparticles.

    Colombo, Stefano; Cun, Dongmei; Remaut, Katrien; Bunker, Matt; Zhang, Jianxin; Martin-Bertelsen, Birte; Yaghmur, Anan; Braeckmans, Kevin; Nielsen, Hanne M; Foged, Camilla

    2015-03-10

    Understanding the delivery dynamics of nucleic acid nanocarriers is fundamental to improve their design for therapeutic applications. We investigated the carrier structure-function relationship of lipid-polymer hybrid nanoparticles (LPNs) consisting of poly(DL-lactic-co-glycolic acid) (PLGA) nanocarriers modified with the cationic lipid dioleoyltrimethyl-ammoniumpropane (DOTAP). A library of siRNA-loaded LPNs was prepared by systematically varying the nitrogen-to-phosphate (N/P) ratio. Atomic force microscopy (AFM) and cryo-transmission electron microscopy (cryo-TEM) combined with small angle X-ray scattering (SAXS) and confocal laser scanning microscopy (CLSM) studies suggested that the siRNA-loaded LPNs are characterized by a core-shell structure consisting of a PLGA matrix core coated with lamellar DOTAP structures with siRNA localized both in the core and in the shell. Release studies in buffer and serum-containing medium combined with in vitro gene silencing and quantification of intracellular siRNA suggested that this self-assembling core-shell structure influences the siRNA release kinetics and the delivery dynamics. A main delivery mechanism appears to be mediated via the release of transfection-competent siRNA-DOTAP lipoplexes from the LPNs. Based on these results, we suggest a model for the nanostructural characteristics of the LPNs, in which the siRNA is organized in lamellar superficial assemblies and/or as complexes entrapped in the polymeric matrix. Copyright © 2015 Elsevier B.V. All rights reserved.

  12. Alteration of human macrophages microRNA expression profile upon infection with Mycobacterium tuberculosis

    Lucinda Furci

    2013-01-01

    Conclusions: This study signifies the miRNA host response upon intracellular mycobacterial infection in macrophages, providing new aspects of regulation in host-pathogen interactions, at post-transcriptional levels.

  13. Systematic Expression Profiling Analysis Identifies Specific MicroRNA-Gene Interactions that May Differentiate between Active and Latent Tuberculosis Infection

    Lawrence Shih-Hsin Wu

    2014-01-01

    Full Text Available Tuberculosis (TB is the second most common cause of death from infectious diseases. About 90% of those infected are asymptomatic—the so-called latent TB infections (LTBI, with a 10% lifetime chance of progressing to active TB. To further understand the molecular pathogenesis of TB, several molecular studies have attempted to compare the expression profiles between healthy controls and active TB or LTBI patients. However, the results vary due to diverse genetic backgrounds and study designs and the inherent complexity of the disease process. Thus, developing a sensitive and efficient method for the detection of LTBI is both crucial and challenging. For the present study, we performed a systematic analysis of the gene and microRNA profiles of healthy individuals versus those affected with TB or LTBI. Combined with a series of in silico analysis utilizing publicly available microRNA knowledge bases and published literature data, we have uncovered several microRNA-gene interactions that specifically target both the blood and lungs. Some of these molecular interactions are novel and may serve as potential biomarkers of TB and LTBI, facilitating the development for a more sensitive, efficient, and cost-effective diagnostic assay for TB and LTBI for the Taiwanese population.

  14. Systematic expression profiling analysis identifies specific microRNA-gene interactions that may differentiate between active and latent tuberculosis infection.

    Wu, Lawrence Shih-Hsin; Lee, Shih-Wei; Huang, Kai-Yao; Lee, Tzong-Yi; Hsu, Paul Wei-Che; Weng, Julia Tzu-Ya

    2014-01-01

    Tuberculosis (TB) is the second most common cause of death from infectious diseases. About 90% of those infected are asymptomatic--the so-called latent TB infections (LTBI), with a 10% lifetime chance of progressing to active TB. To further understand the molecular pathogenesis of TB, several molecular studies have attempted to compare the expression profiles between healthy controls and active TB or LTBI patients. However, the results vary due to diverse genetic backgrounds and study designs and the inherent complexity of the disease process. Thus, developing a sensitive and efficient method for the detection of LTBI is both crucial and challenging. For the present study, we performed a systematic analysis of the gene and microRNA profiles of healthy individuals versus those affected with TB or LTBI. Combined with a series of in silico analysis utilizing publicly available microRNA knowledge bases and published literature data, we have uncovered several microRNA-gene interactions that specifically target both the blood and lungs. Some of these molecular interactions are novel and may serve as potential biomarkers of TB and LTBI, facilitating the development for a more sensitive, efficient, and cost-effective diagnostic assay for TB and LTBI for the Taiwanese population.

  15. Translational profiling in childhood acute lymphoblastic leukemia: no evidence for glucocorticoid regulation of mRNA translation.

    Aneichyk, Tatsiana; Bindreither, Daniel; Mantinger, Christine; Grazio, Daniela; Goetsch, Katrin; Kofler, Reinhard; Rainer, Johannes

    2013-12-01

    Glucocorticoids (GCs) are natural stress induced steroid hormones causing cell cycle arrest and cell death in lymphoid tissues. Therefore they are the central component in the treatment of lymphoid malignancies, in particular childhood acute lymphoblastic leukemia (chALL). GCs act mainly via regulating gene transcription, which has been intensively studied by us and others. GC control of mRNA translation has also been reported but has never been assessed systematically. In this study we investigate the effect of GCs on mRNA translation on a genome-wide scale. Childhood T- (CCRF-CEM) and precursor B-ALL (NALM6) cells were exposed to GCs and subjected to "translational profiling", a technique combining sucrose-gradient fractionation followed by Affymetrix Exon microarray analysis of mRNA from different fractions, to assess the translational efficiency of the expressed genes. Analysis of GC regulation in ribosome-bound fractions versus transcriptional regulation revealed no significant differences, i.e., GC did not entail a significant shift between ribosomal bound and unbound mRNAs. In the present study we analyzed for the first time possible effects of GC on the translational efficiency of expressed genes in two chALL model systems employing whole genome polysome profiling. Our results did not reveal significant differences in translational efficiency of expressed genes thereby arguing against a potential widespread regulatory effect of GCs on translation at least in the investigated in vitro systems.

  16. RNA Profiling for Biomarker Discovery: Practical Considerations for Limiting Sample Sizes

    Danny J. Kelly

    2005-01-01

    Full Text Available We have compared microarray data generated on Affymetrix™ chips from standard (8 micrograms or low (100 nanograms amounts of total RNA. We evaluated the gene signals and gene fold-change estimates obtained from the two methods and validated a subset of the results by real time, polymerase chain reaction assays. The correlation of low RNA derived gene signals to gene signals obtained from standard RNA was poor for less to moderately abundant genes. Genes with high abundance showed better correlation in signals between the two methods. The signal correlation between the low RNA and standard RNA methods was improved by including a reference sample in the microarray analysis. In contrast, the fold-change estimates for genes were better correlated between the two methods regardless of the magnitude of gene signals. A reference sample based method is suggested for studies that would end up comparing gene signal data from a combination of low and standard RNA templates; no such referencing appears to be necessary when comparing fold-changes of gene expression between standard and low template reactions.

  17. Annotating RNA motifs in sequences and alignments.

    Gardner, Paul P; Eldai, Hisham

    2015-01-01

    RNA performs a diverse array of important functions across all cellular life. These functions include important roles in translation, building translational machinery and maturing messenger RNA. More recent discoveries include the miRNAs and bacterial sRNAs that regulate gene expression, the thermosensors, riboswitches and other cis-regulatory elements that help prokaryotes sense their environment and eukaryotic piRNAs that suppress transposition. However, there can be a long period between the initial discovery of a RNA and determining its function. We present a bioinformatic approach to characterize RNA motifs, which are critical components of many RNA structure-function relationships. These motifs can, in some instances, provide researchers with functional hypotheses for uncharacterized RNAs. Moreover, we introduce a new profile-based database of RNA motifs--RMfam--and illustrate some applications for investigating the evolution and functional characterization of RNA. All the data and scripts associated with this work are available from: https://github.com/ppgardne/RMfam. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

  18. Small RNA fragments in complex culture media cause alterations in protein profiles of three species of bacteria.

    Pavankumar, Asalapuram R; Ayyappasamy, Sudalaiyadum Perumal; Sankaran, Krishnan

    2012-03-01

    Efforts to delineate the basis for variations in protein profiles of different membrane fractions from various bacterial pathogens led to the finding that even the same medium [e.g., Luria Bertani (LB) broth] purchased from different commercial sources generates remarkably dissimilar protein profiles despite similar growth characteristics. Given the pervasive roles small RNAs play in regulating gene expression, we inquired if these source-specific differences due to media arise from disparities in the presence of small RNAs. Indeed, LB media components from two different commercial suppliers contained varying, yet significant, amounts of 10-80 bp small RNAs. Removal of small RNA from LB using RNaseA during media preparation resulted in significant changes in bacterial protein expression profiles. Our studies underscore the fact that seemingly identical growth media can lead to dramatic alterations in protein expression patterns, highlighting the importance of utilizing media free of small RNA during bacteriological studies. Finally, these results raise the intriguing possibility that similar pools of small RNAs in the environment can influence bacterial adaptation.

  19. Integrative RNA- and miRNA-Profile Analysis Reveals a Likely Role of BR and Auxin Signaling in Branch Angle Regulation of B. napus

    Hongtao Cheng

    2017-05-01

    Full Text Available Oilseed rape (Brassica napus L. is the second largest oilseed crop worldwide and one of the most important oil crops in China. As a component of plant architecture, branch angle plays an important role in yield performance, especially under high-density planting conditions. However, the mechanisms underlying the regulation of branch angle are still largely not understood. Two oilseed rape lines with significantly different branch angles were used to conduct RNA- and miRNA-profiling at two developmental stages, identifying differential expression of a large number of genes involved in auxin- and brassinosteroid (BR-related pathways. Many auxin response genes, including AUX1, IAA, GH3, and ARF, were enriched in the compact line. However, a number of genes involved in BR signaling transduction and biosynthesis were down-regulated. Differentially expressed miRNAs included those involved in auxin signaling transduction. Expression patterns of most target genes were fine-tuned by related miRNAs, such as miR156, miR172, and miR319. Some miRNAs were found to be differentially expressed at both developmental stages, including three miR827 members. Our results provide insight that auxin- and BR-signaling may play a pivotal role in branch angle regulation.

  20. Circular RNA expression profiles in hippocampus from mice with perinatal glyphosate exposure.

    Yu, Ning; Tong, Yun; Zhang, Danni; Zhao, Shanshan; Fan, Xinli; Wu, Lihui; Ji, Hua

    2018-05-19

    Glyphosate is the active ingredient in numerous herbicide formulations. The roles of glyphosate in embryo-toxicity and neurotoxicity have been reported in human and animal models. Recently, several studies have reported evidence linking neurodevelopmental disorders (NDDs) with gestational glyphosate exposure. However, the role of glyphosate in neuronal development is still not fully understood. Our previous study found that perinatal glyphosate exposure resulted in differential microRNA expression in the prefrontal cortex of mouse offspring. However, the mechanism of glyphosate-induced neurotoxicity in the developing brain is still not fully understood. Considering the pivotal role of Circular RNAs (circRNAs) in the regulation of gene expression, a circRNA microarray method was used in this study to investigate circRNA expression changes in the hippocampus of mice with perinatal glyphosate exposure. The circRNA microarrays revealed that 663 circRNAs were significantly altered in the perinatal glyphosate exposure group compared with the control group. Among them, 330 were significantly upregulated, and the other 333 were downregulated. Furthermore, the relative expression levels of mmu-circRNA-014015, mmu-circRNA-28128 and mmu-circRNA-29837 were verified using quantitative real-time polymerase chain reaction (qRT-PCR). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses demonstrated that stress-associated steroid metabolism pathways, such as aldosterone synthesis and secretion pathways, may be involved in the neurotoxicity of glyphosate. These results showed that circRNAs are aberrantly expressed in the hippocampus of mice with perinatal glyphosate exposure and play potential roles in glyphosate-induced neurotoxicity. Copyright © 2018 Elsevier Inc. All rights reserved.

  1. MicroRNA Expression Profiles in Cultured Human Fibroblasts in Space

    Wu, Honglu; Lu, Tao; Jeevarajan, John; Rohde, Larry; Zhang, Ye

    2014-01-01

    Microgravity, or an altered gravity environment from the static 1g, has been shown to influence global gene expression patterns and protein levels in living organisms. However, it is unclear how these changes in gene and protein expressions are related to each other or are related to other factors regulating such changes. A different class of RNA, the small non-coding microRNA (miRNA), can have a broad effect on gene expression networks by mainly inhibiting the translation process. Previously, we investigated changes in the expression of miRNA and related genes under simulated microgravity conditions on the ground using the NASA invented bioreactor. In comparison to static 1 g, simulated microgravity altered a number of miRNAs in human lymphoblastoid cells. Pathway analysis with the altered miRNAs and RNA expressions revealed differential involvement of cell communication and catalytic activity, as well as immune response signaling and NGF activation of NF-kB pathways under simulated microgravity condition. The network analysis also identified several projected networks with c- Rel, ETS1 and Ubiquitin C as key factors. In a flight experiment on the International Space Station (ISS), we will investigate the effects of actual spaceflight on miRNA expressions in nondividing human fibroblast cells in mostly G1 phase of the cell cycle. A fibroblast is a type of cell that synthesizes the extracellular matrix and collagen, the structural framework for tissues, and plays a critical role in wound healing and other functions. In addition to miRNA expressions, we will investigate the effects of spaceflight on the cellular response to DNA damages from bleomycin treatment.

  2. Profiling the nucleobase and structure selectivity of anticancer drugs and other DNA alkylating agents by RNA sequencing.

    Gillingham, Dennis; Sauter, Basilius

    2018-05-06

    Drugs that covalently modify DNA are components of most chemotherapy regimens, often serving as first-line treatments. Classically the chemical reactivity of DNA alkylators has been determined in vitro with short oligonucleotides. Here we use next generation RNA sequencing to report on the chemoselectivity of alkylating agents. We develop the method with the well-known clinically used DNA modifiying drugs streptozotocin and temozolomide, and then apply the technique to profile RNA modification with uncharacterized alkylation reactions such as with powerful electrophiles like trimethylsilyldiazomethane. The multiplexed and massively parallel format of NGS offers analyses of chemical reactivity in nucleic acids to be accomplished in less time with greater statistical power. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. The mRNA expression profile of metabolic genes relative to MHC isoform pattern in human skeletal muscles

    Plomgaard, Peter; Penkowa, Milena; Leick, Lotte

    2006-01-01

    The metabolic profile of rodent muscle is generally reflected in the myosin heavy chain (MHC) fiber-type composition. The present study was conducted to test the hypothesis that metabolic gene expression is not tightly coupled with MHC fiber-type composition for all genes in human skeletal muscle....... Triceps brachii, vastus lateralis quadriceps, and soleus muscle biopsies were obtained from normally physically active, healthy, young male volunteers, because these muscles are characterized by different fiber-type compositions. As expected, citrate synthase and 3-hydroxyacyl dehydrogenase activity...... of a broad range of metabolic genes. The triceps muscle had two- to fivefold higher MHC IIa, phosphofructokinase, and LDH A mRNA content and two- to fourfold lower MHC I, lipoprotein lipase, CD36, hormone-sensitive lipase, and LDH B and hexokinase II mRNA than vastus lateralis or soleus. Interestingly...

  4. Progressive changes in non-coding RNA profile in leucocytes with age

    Muñoz-Culla, Maider; Irizar, Haritz; Gorostidi, Ana; Alberro, Ainhoa; Osorio-Querejeta, Iñaki; Ruiz-Martínez, Javier; Olascoaga, Javier; de Munain, Adolfo López; Otaegui, David

    2017-01-01

    It has been observed that immune cell deterioration occurs in the elderly, as well as a chronic low-grade inflammation called inflammaging. These cellular changes must be driven by numerous changes in gene expression and in fact, both protein-coding and non-coding RNA expression alterations have been observed in peripheral blood mononuclear cells from elder people. In the present work we have studied the expression of small non-coding RNA (microRNA and small nucleolar RNA -snoRNA-) from healthy individuals from 24 to 79 years old. We have observed that the expression of 69 non-coding RNAs (56 microRNAs and 13 snoRNAs) changes progressively with chronological age. According to our results, the age range from 47 to 54 is critical given that it is the period when the expression trend (increasing or decreasing) of age-related small non-coding RNAs is more pronounced. Furthermore, age-related miRNAs regulate genes that are involved in immune, cell cycle and cancer-related processes, which had already been associated to human aging. Therefore, human aging could be studied as a result of progressive molecular changes, and different age ranges should be analysed to cover the whole aging process. PMID:28448962

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

    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.

  6. Specific autoantibody profiles and disease subgroups correlate with circulating micro-RNA in systemic sclerosis

    Wuttge, D. M.; Carlsen, A. L.; Teku, G.

    2015-01-01

    Objective. To evaluate the expression profiles of cell-free plasma miRNAs in SSc and to characterize their correlation with disease subgroups (lcSSc and dcSSc) and with autoantibody profiles. Methods. Using quantitative RT-PCR, the abundance of 45 mature miRNAs in plasma was determined in 95 pati...

  7. Multi-Messenger Astronomy with Gravitational Waves

    Sound + images show Bailey was out in the India-Australia match on 12 Jan 2016. Image credit: Rediff / Fox news / Twitter. Page 10. Electromagnetic follow up: the Indian context. Page 11. Multi-Messenger Astronomy with Gravitational Waves | LIGO-G1601377-v2. Varun Bhalerao (IUCAA) | 1 July 2016. 11. 20 – 60 keV:.

  8. Mobile MSN Messenger: Still a Complement?

    Marcus Nyberg

    2008-10-01

    Full Text Available In order to understand how mobile instant messaging services can fit into the users’ current communication behavior, Ericsson Research performed a qualitative user study in Sweden in May 2007. The results showed that the respondents were positive towards (free of charge mobile MSN Messenger and perceived it as an ex¬tension of the computer-based version that could be used anywhere. However, although MSN Messenger on the com¬puter definitely was considered as a ‘must-have’ application, the mobile version was only perceived as a ‘nice-to-have’ application and a complement to text mes¬saging (SMS. Almost one year later, in April 2008, Ericsson Research performed a short qualita¬tive follow-up study with the same set of respondents to un¬derstand if and how the mobile MSN Messenger usage had changed. The results actually revealed that none of the re¬spondents used mobile MSN Messenger anymore as the application no longer was free of charge. On a general level, the study highlights important considera¬tions when intro¬ducing computer-based concepts and Internet services in a mo¬bile environment.

  9. 12 CFR 7.1012 - Messenger service.

    2010-01-01

    ... service” means any service, such as a courier service or armored car service, used by a national bank and... service do not advertise, or otherwise represent, that the bank itself is providing the service, although the bank may advertise that its customers may use one or more third party messenger services to...

  10. What history tells us XLV. The 'instability' of messenger RNA

    Michel Morange

    2018-04-23

    Apr 23, 2018 ... By using heavy isotopes, Rudolph Schoenheimer observed at the ... firmed the relation existing between the instability of ... was problematic is eliminated. ... Harris H 1963 Rapidly labelled ribonucleic acid in the cell nucleus.

  11. Circuit Formation by Spatio-Temporal Control of Messenger RNA ...

    The connections inside the brain need to be wired in a precise manner during development to ensure its proper function. This project will provide insight into circuit formation to help us understand how axon regeneration can improve clinical outcomes. Brain wiring, damage, and developmental defects Researchers have ...

  12. Characterization and Profiling of Liver microRNAs by RNA-sequencing in Cattle Divergently Selected for Residual Feed Intake

    Wijdan Al-Husseini

    2016-10-01

    Full Text Available MicroRNAs (miRNAs are short non-coding RNAs that post-transcriptionally regulate expression of mRNAs in many biological pathways. Liver plays an important role in the feed efficiency of animals and high and low efficient cattle demonstrated different gene expression profiles by microarray. Here we report comprehensive miRNAs profiles by next-gen deep sequencing in Angus cattle divergently selected for residual feed intake (RFI and identify miRNAs related to feed efficiency in beef cattle. Two microRNA libraries were constructed from pooled RNA extracted from livers of low and high RFI cattle, and sequenced by Illumina genome analyser. In total, 23,628,103 high quality short sequence reads were obtained and more than half of these reads were matched to the bovine genome (UMD 3.1. We identified 305 known bovine miRNAs. Bta-miR-143, bta-miR-30, bta-miR-122, bta-miR-378, and bta-let-7 were the top five most abundant miRNAs families expressed in liver, representing more than 63% of expressed miRNAs. We also identified 52 homologous miRNAs and 10 novel putative bovine-specific miRNAs, based on precursor sequence and the secondary structure and utilizing the miRBase (v. 21. We compared the miRNAs profile between high and low RFI animals and ranked the most differentially expressed bovine known miRNAs. Bovine miR-143 was the most abundant miRNA in the bovine liver and comprised 20% of total expressed mapped miRNAs. The most highly expressed miRNA in liver of mice and humans, miR-122, was the third most abundant in our cattle liver samples. We also identified 10 putative novel bovine-specific miRNA candidates. Differentially expressed miRNAs between high and low RFI cattle were identified with 18 miRNAs being up-regulated and 7 other miRNAs down-regulated in low RFI cattle. Our study has identified comprehensive miRNAs expressed in bovine liver. Some of the expressed miRNAs are novel in cattle. The differentially expressed miRNAs between high and low RFI

  13. High-throughput miRNA profiling of human melanoma blood samples

    Rass Knuth

    2010-06-01

    Full Text Available Abstract Background MicroRNA (miRNA signatures are not only found in cancer tissue but also in blood of cancer patients. Specifically, miRNA detection in blood offers the prospect of a non-invasive analysis tool. Methods Using a microarray based approach we screened almost 900 human miRNAs to detect miRNAs that are deregulated in their expression in blood cells of melanoma patients. We analyzed 55 blood samples, including 20 samples of healthy individuals, 24 samples of melanoma patients as test set, and 11 samples of melanoma patients as independent validation set. Results A hypothesis test based approch detected 51 differentially regulated miRNAs, including 21 miRNAs that were downregulated in blood cells of melanoma patients and 30 miRNAs that were upregulated in blood cells of melanoma patients as compared to blood cells of healthy controls. The tets set and the independent validation set of the melanoma samples showed a high correlation of fold changes (0.81. Applying hierarchical clustering and principal component analysis we found that blood samples of melanoma patients and healthy individuals can be well differentiated from each other based on miRNA expression analysis. Using a subset of 16 significant deregulated miRNAs, we were able to reach a classification accuracy of 97.4%, a specificity of 95% and a sensitivity of 98.9% by supervised analysis. MiRNA microarray data were validated by qRT-PCR. Conclusions Our study provides strong evidence for miRNA expression signatures of blood cells as useful biomarkers for melanoma.

  14. Pulmonary microRNA profiling: implications in upper lobe predominant lung disease.

    Armstrong, David A; Nymon, Amanda B; Ringelberg, Carol S; Lesseur, Corina; Hazlett, Haley F; Howard, Louisa; Marsit, Carmen J; Ashare, Alix

    2017-01-01

    Numerous pulmonary diseases manifest with upper lobe predominance including cystic fibrosis, smoking-related chronic obstructive pulmonary disease, and tuberculosis. Zonal hypoxia, characteristic of these pulmonary maladies, and oxygen stress in general is known to exert profound effects on various important aspects of cell biology. Lung macrophages are major participants in the pulmonary innate immune response and regional differences in macrophage responsiveness to hypoxia may contribute in the development of lung disease. MicroRNAs are ubiquitous regulators of human biology and emerging evidence indicates altered microRNA expression modulates respiratory disease processes. The objective of this study is to gain insight into the epigenetic and cellular mechanisms influencing regional differences in lung disease by investigating effect of hypoxia on regional microRNA expression in the lung. All studies were performed using primary alveolar macrophages ( n  = 10) or bronchoalveolar lavage fluid ( n  = 16) isolated from human subjects. MicroRNA was assayed via the NanoString nCounter microRNA assay. Divergent molecular patterns of microRNA expression were observed in alternate lung lobes, specifically noted was disparate expression of miR-93 and miR-4454 in alveolar macrophages along with altered expression of miR-451a and miR-663a in bronchoalveolar lavage fluid. Gene ontology was used to identify potential downstream targets of divergent microRNAs. Targets include cytokines and matrix metalloproteinases, molecules that could have a significant impact on pulmonary inflammation and fibrosis. Our findings show variant regional microRNA expression associated with hypoxia in alveolar macrophages and BAL fluid in the lung-upper vs lower lobe. Future studies should address whether these specific microRNAs may act intracellularly, in a paracrine/endocrine manner to direct the innate immune response or may ultimately be involved in pulmonary host-to-pathogen trans

  15. Small RNA profiling of influenza A virus-infected cells identifies miR-449b as a regulator of histone deacetylase 1 and interferon beta.

    William A Buggele

    Full Text Available The mammalian antiviral response relies on the alteration of cellular gene expression, to induce the production of antiviral effectors and regulate their activities. Recent research has indicated that virus infections can induce the accumulation of cellular microRNA (miRNA species that influence the stability of host mRNAs and their protein products. To determine the potential for miRNA regulation of cellular responses to influenza A virus infection, small RNA profiling was carried out using next generation sequencing. Comparison of miRNA expression profiles in uninfected human A549 cells to cells infected with influenza A virus strains A/Udorn/72 and A/WSN/33, revealed virus-induced changes in miRNA abundance. Gene expression analysis identified mRNA targets for a cohort of highly inducible miRNAs linked to diverse cellular functions. Experiments demonstrate that the histone deacetylase, HDAC1, can be regulated by influenza-inducible miR-449b, resulting in altered mRNA and protein levels. Expression of miR-449b enhances virus and poly(I:C activation of the IFNβ promoter, a process known to be negatively regulated by HDAC1. These findings demonstrate miRNA induction by influenza A virus infection and elucidate an example of miRNA control of antiviral gene expression in human cells, defining a role for miR-449b in regulation of HDAC1 and antiviral cytokine signaling.

  16. MicroRNA profiling reveals dysregulated microRNAs and their target gene regulatory networks in cemento-ossifying fibroma.

    Pereira, Thaís Dos Santos Fontes; Brito, João Artur Ricieri; Guimarães, André Luiz Sena; Gomes, Carolina Cavaliéri; de Lacerda, Júlio Cesar Tanos; de Castro, Wagner Henriques; Coimbra, Roney Santos; Diniz, Marina Gonçalves; Gomez, Ricardo Santiago

    2018-01-01

    Cemento-ossifying fibroma (COF) is a benign fibro-osseous neoplasm of uncertain pathogenesis, and its treatment results in morbidity. MicroRNAs (miRNA) are small non-coding RNAs that regulate gene expression and may represent therapeutic targets. The purpose of the study was to generate a comprehensive miRNA profile of COF compared to normal bone. Additionally, the most relevant pathways and target genes of differentially expressed miRNA were investigated by in silico analysis. Nine COF and ten normal bone samples were included in the study. miRNA profiling was carried out by using TaqMan® OpenArray® Human microRNA panel containing 754 validated human miRNAs. We identified the most relevant miRNAs target genes through the leader gene approach, using STRING and Cytoscape software. Pathways enrichment analysis was performed using DIANA-miRPath. Eleven miRNAs were downregulated (hsa-miR-95-3p, hsa-miR-141-3p, hsa-miR-205-5p, hsa-miR-223-3p, hsa-miR-31-5p, hsa-miR-944, hsa-miR-200b-3p, hsa-miR-135b-5p, hsa-miR-31-3p, hsa-miR-223-5p and hsa-miR-200c-3p), and five were upregulated (hsa-miR-181a-5p, hsa-miR-181c-5p, hsa-miR-149-5p, hsa-miR-138-5p and hsa-miR-199a-3p) in COF compared to normal bone. Eighteen common target genes were predicted, and the leader genes approach identified the following genes involved in human COF: EZH2, XIAP, MET and TGFBR1. According to the biology of bone and COF, the most relevant KEGG pathways revealed by enrichment analysis were proteoglycans in cancer, miRNAs in cancer, pathways in cancer, p53-, PI3K-Akt-, FoxO- and TGF-beta signalling pathways, which were previously found to be differentially regulated in bone neoplasms, odontogenic tumours and osteogenesis. miRNA dysregulation occurs in COF, and EZH2, XIAP, MET and TGFBR1 are potential targets for functional analysis validation. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  17. 5-FU and ixabepilone modify the microRNA expression profiles in MDA-MB-453 triple-negative breast cancer cells

    YAO, YONGSHAN; CHEN, SHENGHAN; ZHOU, XIN; XIE, LI; CHEN, AIJUN

    2013-01-01

    This study aimed to discover new potential mechanisms of chemotherapy with drugs used in the treatment of luminal androgen receptor (LAR)-type triple-negative breast cancer (TNBC). We examined the microRNA (miRNA) expression profiles of LAR-type TNBC in vitro, and explored the variation in miRNA expression profiles in cells when treated with the chemotherapy drugs capecitabine and ixabepilone. The present study revealed that the expression levels of the three antitumor miRNAs, miR-122a, miR-1...

  18. Aging-associated changes in microRNA expression profile of internal anal sphincter smooth muscle: Role of microRNA-133a

    Singh, Jagmohan; Boopathi, Ettickan; Addya, Sankar; Phillips, Benjamin; Rigoutsos, Isidore; Penn, Raymond B.

    2016-01-01

    A comprehensive genomic and proteomic, computational, and physiological approach was employed to examine the (previously unexplored) role of microRNAs (miRNAs) as regulators of internal anal sphincter (IAS) smooth muscle contractile phenotype and basal tone. miRNA profiling, genome-wide expression, validation, and network analyses were employed to assess changes in mRNA and miRNA expression in IAS smooth muscles from young vs. aging rats. Multiple miRNAs, including rno-miR-1, rno-miR-340-5p, rno-miR-185, rno-miR-199a-3p, rno-miR-200c, rno-miR-200b, rno-miR-31, rno-miR-133a, and rno-miR-206, were found to be upregulated in aging IAS. qPCR confirmed the upregulated expression of these miRNAs and downregulation of multiple, predicted targets (Eln, Col3a1, Col1a1, Zeb2, Myocd, Srf, Smad1, Smad2, Rhoa/Rock2, Fn1, Tagln v2, Klf4, and Acta2) involved in regulation of smooth muscle contractility. Subsequent studies demonstrated an aging-associated increase in the expression of miR-133a, corresponding decreases in RhoA, ROCK2, MYOCD, SRF, and SM22α protein expression, RhoA-signaling, and a decrease in basal and agonist [U-46619 (thromboxane A2 analog)]-induced increase in the IAS tone. Moreover, in vitro transfection of miR-133a caused a dose-dependent increase of IAS tone in strips, which was reversed by anti-miR-133a. Last, in vivo perianal injection of anti-miR-133a reversed the loss of IAS tone associated with age. This work establishes the important regulatory effect of miRNA-133a on basal and agonist-stimulated IAS tone. Moreover, reversal of age-associated loss of tone via anti-miR delivery strongly implicates miR dysregulation as a causal factor in the aging-associated decrease in IAS tone and suggests that miR-133a is a feasible therapeutic target in aging-associated rectoanal incontinence. PMID:27634012

  19. MicroRNA Expression Profile in the Prenatal Amniotic Fluid Samples of Pregnant Women with Down Syndrome.

    Karaca, Emin; Aykut, Ayça; Ertürk, Biray; Durmaz, Burak; Güler, Ahmet; Büke, Barış; Yeniel, Ahmet Özgür; Ergenoğlu, Ahmet Mete; Özkınay, Ferda; Özeren, Mehmet; Kazandı, Mert; Akercan, Fuat; Sağol, Sermet; Gündüz, Cumhur; Çoğulu, Özgür

    2018-03-15

    Down syndrome, which is the most common human chromosomal anomaly that can affect people of any race and age, can be diagnosed prenatally in most cases. Prenatal diagnosis via culture method is time-consuming; thus, genetic analysis has thus been introduced and is continually being developed for rapid prenatal diagnosis. For this reason, the effective use of microRNA profiling for the rapid analysis of prenatal amniotic fluid samples for the diagnosis of Down syndrome was investigated. To evaluate the expression levels of 14 microRNAs encoded by chromosome 21 in amniotic fluid samples and their utility for prenatal diagnosis of Down syndrome. Case-control study. We performed invasive prenatal testing for 56 pregnant women; 23 carried fetuses with Down syndrome, and 33 carried fetuses with a normal karyotype. Advanced maternal age and increased risk for Down syndrome in the screening tests were indications for invasive prenatal testing. The age of gestation in the study and control groups ranged between 17 and 18 weeks. The expression levels of microRNA were measured by real-time polymerase chain reaction. The expression levels of microRNA-125b-2, microRNA-155 , and microRNA-3156 were significantly higher in the study group than in the control group. The presence of significantly dysregulated microRNAs may be associated with either the phenotype or the result of abnormal development. Further large-scale comparative studies conducted in a variety of conditions may bring novel insights in the field of abnormal prenatal conditions.

  20. Large-scale expression analysis reveals distinct microRNA profiles at different stages of human neurodevelopment.

    Brandon Smith

    Full Text Available BACKGROUND: MicroRNAs (miRNAs are short non-coding RNAs predicted to regulate one third of protein coding genes via mRNA targeting. In conjunction with key transcription factors, such as the repressor REST (RE1 silencing transcription factor, miRNAs play crucial roles in neurogenesis, which requires a highly orchestrated program of gene expression to ensure the appropriate development and function of diverse neural cell types. Whilst previous studies have highlighted select groups of miRNAs during neural development, there remains a need for amenable models in which miRNA expression and function can be analyzed over the duration of neurogenesis. PRINCIPAL FINDINGS: We performed large-scale expression profiling of miRNAs in human NTera2/D1 (NT2 cells during retinoic acid (RA-induced transition from progenitors to fully differentiated neural phenotypes. Our results revealed dynamic changes of miRNA patterns, resulting in distinct miRNA subsets that could be linked to specific neurodevelopmental stages. Moreover, the cell-type specific miRNA subsets were very similar in NT2-derived differentiated cells and human primary neurons and astrocytes. Further analysis identified miRNAs as putative regulators of REST, as well as candidate miRNAs targeted by REST. Finally, we confirmed the existence of two predicted miRNAs; pred-MIR191 and pred-MIR222 associated with SLAIN1 and FOXP2, respectively, and provided some evidence of their potential co-regulation. CONCLUSIONS: In the present study, we demonstrate that regulation of miRNAs occurs in precise patterns indicative of their roles in cell fate commitment, progenitor expansion and differentiation into neurons and glia. Furthermore, the similarity between our NT2 system and primary human cells suggests their roles in molecular pathways critical for human in vivo neurogenesis.

  1. Integration of miRNA and protein profiling reveals coordinated neuroadaptations in the alcohol-dependent mouse brain.

    Giorgio Gorini

    Full Text Available The molecular mechanisms underlying alcohol dependence involve different neurochemical systems and are brain region-dependent. Chronic Intermittent Ethanol (CIE procedure, combined with a Two-Bottle Choice voluntary drinking paradigm, represents one of the best available animal models for alcohol dependence and relapse drinking. MicroRNAs, master regulators of the cellular transcriptome and proteome, can regulate their targets in a cooperative, combinatorial fashion, ensuring fine tuning and control over a large number of cellular functions. We analyzed cortex and midbrain microRNA expression levels using an integrative approach to combine and relate data to previous protein profiling from the same CIE-subjected samples, and examined the significance of the data in terms of relative contribution to alcohol consumption and dependence. MicroRNA levels were significantly altered in CIE-exposed dependent mice compared with their non-dependent controls. More importantly, our integrative analysis identified modules of coexpressed microRNAs that were highly correlated with CIE effects and predicted target genes encoding differentially expressed proteins. Coexpressed CIE-relevant proteins, in turn, were often negatively correlated with specific microRNA modules. Our results provide evidence that microRNA-orchestrated translational imbalances are driving the behavioral transition from alcohol consumption to dependence. This study represents the first attempt to combine ex vivo microRNA and protein expression on a global scale from the same mammalian brain samples. The integrative systems approach used here will improve our understanding of brain adaptive changes in response to drug abuse and suggests the potential therapeutic use of microRNAs as tools to prevent or compensate multiple neuroadaptations underlying addictive behavior.

  2. Integrative Analysis of miRNA and mRNA Profiles in Response to Ethylene in Rose Petals during Flower Opening

    Pei, Haixia; Ma, Nan; Chen, Jiwei; Zheng, Yi; Tian, Ji; Li, Jing; Zhang, Shuai; Fei, Zhangjun; Gao, Junping

    2013-01-01

    MicroRNAs play an important role in plant development and plant responses to various biotic and abiotic stimuli. As one of the most important ornamental crops, rose (Rosa hybrida) possesses several specific morphological and physiological features, including recurrent flowering, highly divergent flower shapes, colors and volatiles. Ethylene plays an important role in regulating petal cell expansion during rose flower opening. Here, we report the population and expression profiles of miRNAs in rose petals during flower opening and in response to ethylene based on high throughput sequencing. We identified a total of 33 conserved miRNAs, as well as 47 putative novel miRNAs were identified from rose petals. The conserved and novel targets to those miRNAs were predicted using the rose floral transcriptome database. Expression profiling revealed that expression of 28 known (84.8% of known miRNAs) and 39 novel (83.0% of novel miRNAs) miRNAs was substantially changed in rose petals during the earlier opening period. We also found that 28 known and 22 novel miRNAs showed expression changes in response to ethylene treatment. Furthermore, we performed integrative analysis of expression profiles of miRNAs and their targets. We found that ethylene-caused expression changes of five miRNAs (miR156, miR164, miR166, miR5139 and rhy-miRC1) were inversely correlated to those of their seven target genes. These results indicate that these miRNA/target modules might be regulated by ethylene and were involved in ethylene-regulated petal growth. PMID:23696879

  3. Diagnostic microRNA profiling in cutaneous T-cell lymphoma (CTCL)

    Ralfkiaer, Ulrik; Hagedorn, Peter; Bangsgaard, Nannie

    2011-01-01

    from benign inflammation, we studied miRNA expression levels in 198 patients with CTCL, peripheral T-cell lymphoma (PTL), and benign skin diseases (psoriasis and dermatitis). Using microarrays, we show that the most induced (miR-326, miR-663b, and miR-711) and repressed (miR-203 and miR-205) mi...

  4. Circulating microRNA expression profiles associated with systemic lupus erythematosus

    Carlsen, Anting Liu; Schetter, Aaron J; Nielsen, Christoffer

    2013-01-01

    OBJECTIVE: To evaluate the specificity of expression patterns of cell-free, circulating microRNAs in systemic lupus erythematosus (SLE). METHODS: Total RNA was purified from plasma and 45 different specific mature microRNAs were determined using quantitative reverse transcription polymerase chain...

  5. miRNA profiles in cerebrospinal fluid from patients with central hypersomnias

    Holm, Anja; Bang-Berthelsen, Claus Heiner; Knudsen, Stine

    2014-01-01

    addressed whether miRNA levels are altered in the cerebrospinal fluid (CSF) of patients with central hypersomnias. We conducted high-throughput analyses of miRNAs in CSF from patients using quantitative real-time polymerase chain reaction panels. We identified 13, 9, and 11 miRNAs with a more than two...

  6. MicroRNA expression profiles associated with pancreatic adenocarcinoma and ampullary adenocarcinoma

    Schultz, Nicolai A; Werner, Jens; Willenbrock, Hanni

    2012-01-01

    MicroRNAs have potential as diagnostic cancer biomarkers. The aim of this study was (1) to define microRNA expression patterns in formalin-fixed parafin-embedded tissue from pancreatic ductal adenocarcinoma, ampullary adenocarcinoma, normal pancreas and chronic pancreatitis without using micro-di...

  7. Profile of microRNA in Giant Panda Blood: A Resource for Immune-Related and Novel microRNAs.

    Yang, Mingyu; Du, Lianming; Li, Wujiao; Shen, Fujun; Fan, Zhenxin; Jian, Zuoyi; Hou, Rong; Shen, Yongmei; Yue, Bisong; Zhang, Xiuyue

    2015-01-01

    The giant panda (Ailuropoda melanoleuca) is one of the world's most beloved endangered mammals. Although the draft genome of this species had been assembled, little was known about the composition of its microRNAs (miRNAs) or their functional profiles. Recent studies demonstrated that changes in the expression of miRNAs are associated with immunity. In this study, miRNAs were extracted from the blood of four healthy giant pandas and sequenced by Illumina next generation sequencing technology. As determined by miRNA screening, a total of 276 conserved miRNAs and 51 novel putative miRNAs candidates were detected. After differential expression analysis, we noticed that the expressions of 7 miRNAs were significantly up-regulated in young giant pandas compared with that of adults. Moreover, 2 miRNAs were up-regulated in female giant pandas and 1 in the male individuals. Target gene prediction suggested that the miRNAs of giant panda might be relevant to the expressions of 4,602 downstream genes. Subseuqently, the predicted target genes were conducted to KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis and we found that these genes were mainly involved in host immunity, including the Ras signaling pathway, the PI3K-Akt signaling pathway, and the MAPK signaling pathway. In conclusion, our results provide the first miRNA profiles of giant panda blood, and the predicted functional analyses may open an avenue for further study of giant panda immunity.

  8. Rhea: a transparent and modular R pipeline for microbial profiling based on 16S rRNA gene amplicons.

    Lagkouvardos, Ilias; Fischer, Sandra; Kumar, Neeraj; Clavel, Thomas

    2017-01-01

    The importance of 16S rRNA gene amplicon profiles for understanding the influence of microbes in a variety of environments coupled with the steep reduction in sequencing costs led to a surge of microbial sequencing projects. The expanding crowd of scientists and clinicians wanting to make use of sequencing datasets can choose among a range of multipurpose software platforms, the use of which can be intimidating for non-expert users. Among available pipeline options for high-throughput 16S rRNA gene analysis, the R programming language and software environment for statistical computing stands out for its power and increased flexibility, and the possibility to adhere to most recent best practices and to adjust to individual project needs. Here we present the Rhea pipeline, a set of R scripts that encode a series of well-documented choices for the downstream analysis of Operational Taxonomic Units (OTUs) tables, including normalization steps, alpha - and beta -diversity analysis, taxonomic composition, statistical comparisons, and calculation of correlations. Rhea is primarily a straightforward starting point for beginners, but can also be a framework for advanced users who can modify and expand the tool. As the community standards evolve, Rhea will adapt to always represent the current state-of-the-art in microbial profiles analysis in the clear and comprehensive way allowed by the R language. Rhea scripts and documentation are freely available at https://lagkouvardos.github.io/Rhea.

  9. Rhea: a transparent and modular R pipeline for microbial profiling based on 16S rRNA gene amplicons

    Ilias Lagkouvardos

    2017-01-01

    Full Text Available The importance of 16S rRNA gene amplicon profiles for understanding the influence of microbes in a variety of environments coupled with the steep reduction in sequencing costs led to a surge of microbial sequencing projects. The expanding crowd of scientists and clinicians wanting to make use of sequencing datasets can choose among a range of multipurpose software platforms, the use of which can be intimidating for non-expert users. Among available pipeline options for high-throughput 16S rRNA gene analysis, the R programming language and software environment for statistical computing stands out for its power and increased flexibility, and the possibility to adhere to most recent best practices and to adjust to individual project needs. Here we present the Rhea pipeline, a set of R scripts that encode a series of well-documented choices for the downstream analysis of Operational Taxonomic Units (OTUs tables, including normalization steps, alpha- and beta-diversity analysis, taxonomic composition, statistical comparisons, and calculation of correlations. Rhea is primarily a straightforward starting point for beginners, but can also be a framework for advanced users who can modify and expand the tool. As the community standards evolve, Rhea will adapt to always represent the current state-of-the-art in microbial profiles analysis in the clear and comprehensive way allowed by the R language. Rhea scripts and documentation are freely available at https://lagkouvardos.github.io/Rhea.

  10. Discovery and small RNA profile of Pecan mosaic-associated virus, a novel potyvirus of pecan trees.

    Su, Xiu; Fu, Shuai; Qian, Yajuan; Zhang, Liqin; Xu, Yi; Zhou, Xueping

    2016-05-26

    A novel potyvirus was discovered in pecan (Carya illinoensis) showing leaf mosaic symptom through the use of deep sequencing of small RNAs. The complete genome of this virus was determined to comprise of 9,310 nucleotides (nt), and shared 24.0% to 58.9% nucleotide similarities with that of other Potyviridae viruses. The genome was deduced to encode a single open reading frame (polyprotein) on the plus strand. Phylogenetic analysis based on the whole genome sequence and coat protein amino acid sequence showed that this virus is most closely related to Lettuce mosaic virus. Using electron microscopy, the typical Potyvirus filamentous particles were identified in infected pecan leaves with mosaic symptoms. Our results clearly show that this virus is a new member of the genus Potyvirus in the family Potyviridae. The virus is tentatively named Pecan mosaic-associated virus (PMaV). Additionally, profiling of the PMaV-derived small RNA (PMaV-sRNA) showed that the most abundant PMaV-sRNAs were 21-nt in length. There are several hotspots for small RNA production along the PMaV genome; two 21-nt PMaV-sRNAs starting at 811 nt and 610 nt of the minus-strand genome were highly repeated.

  11. Circular RNA profiling reveals that circular RNAs from ANXA2 can be used as new biomarkers for multiple sclerosis.

    Iparraguirre, Leire; Muñoz-Culla, Maider; Prada-Luengo, Iñigo; Castillo-Triviño, Tamara; Olascoaga, Javier; Otaegui, David

    2017-09-15

    Multiple sclerosis is an autoimmune disease, with higher prevalence in women, in whom the immune system is dysregulated. This dysregulation has been shown to correlate with changes in transcriptome expression as well as in gene-expression regulators, such as non-coding RNAs (e.g. microRNAs). Indeed, some of these have been suggested as biomarkers for multiple sclerosis even though few biomarkers have reached the clinical practice. Recently, a novel family of non-coding RNAs, circular RNAs, has emerged as a new player in the complex network of gene-expression regulation. MicroRNA regulation function through a 'sponge system' and a RNA splicing regulation function have been proposed for the circular RNAs. This regulating role together with their high stability in biofluids makes them seemingly good candidates as biomarkers. Given the dysregulation of both protein-coding and non-coding transcriptome that have been reported in multiple sclerosis patients, we hypothesised that circular RNA expression may also be altered. Therefore, we carried out expression profiling of 13.617 circular RNAs in peripheral blood leucocytes from multiple sclerosis patients and healthy controls finding 406 differentially expressed (P-value  1.5) and demonstrate after validation that, circ_0005402 and circ_0035560 are underexpressed in multiple sclerosis patients and could be used as biomarkers of the disease. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  12. Changes in miRNA expression profile of space-flown Caenorhabditis elegans during Shenzhou-8 mission

    Xu, Dan; Gao, Ying; Huang, Lei; Sun, Yeqing

    2014-04-01

    Recent advances in the field of molecular biology have demonstrated that small non-coding microRNAs (miRNAs) have a broad effect on gene expression networks and play a key role in biological responses to environmental stressors. However, little is known about how space radiation exposure and altered gravity affect miRNA expression. The "International Space Biological Experiments" project was carried out in November 2011 by an international collaboration between China and Germany during the Shenzhou-8 (SZ-8) mission. To study the effects of spaceflight on Caenorhabditis elegans (C. elegans), we explored the expression profile miRNA changes in space-flown C. elegans. Dauer C. elegans larvae were taken by SZ-8 spacecraft and experienced the 16.5-day shuttle spaceflight. We performed miRNA microarray analysis, and the results showed that 23 miRNAs were altered in a complex space environment and different expression patterns were observed in the space synthetic and radiation environments. Most putative target genes of the altered miRNAs in the space synthetic environment were predicted to be involved in developmental processes instead of in the regulation of transcription, and the enrichment of these genes was due to space radiation. Furthermore, integration analysis of the miRNA and mRNA expression profiles confirmed that twelve genes were differently regulated by seven miRNAs. These genes may be involved in embryonic development, reproduction, transcription factor activity, oviposition in a space synthetic environment, positive regulation of growth and body morphogenesis in a space radiation environment. Specifically, we found that cel-miR-52, -55, and -56 of the miR-51 family were sensitive to space environmental stressors and could regulate biological behavioural responses and neprilysin activity through the different isoforms of T01C4.1 and F18A12.8. These findings suggest that C. elegans responded to spaceflight by altering the expression of miRNAs and some target

  13. microRNA expression profile in human coronary smooth muscle cell-derived microparticles is a source of biomarkers.

    de Gonzalo-Calvo, David; Cenarro, Ana; Civeira, Fernando; Llorente-Cortes, Vicenta

    2016-01-01

    microRNA (miRNA) expression profile of extracellular vesicles is a potential tool for clinical practice. Despite the key role of vascular smooth muscle cells (VSMC) in cardiovascular pathology, there is limited information about the presence of miRNAs in microparticles secreted by this cell type, including human coronary artery smooth muscle cells (HCASMC). Here, we tested whether HCASMC-derived microparticles contain miRNAs and the value of these miRNAs as biomarkers. HCASMC and explants from atherosclerotic or non-atherosclerotic areas were obtained from coronary arteries of patients undergoing heart transplant. Plasma samples were collected from: normocholesterolemic controls (N=12) and familial hypercholesterolemia (FH) patients (N=12). Both groups were strictly matched for age, sex and cardiovascular risk factors. Microparticle (0.1-1μm) isolation and characterization was performed using standard techniques. VSMC-enriched miRNAs expression (miR-21-5p, -143-3p, -145-5p, -221-3p and -222-3p) was analyzed using RT-qPCR. Total RNA isolated from HCASMC-derived microparticles contained small RNAs, including VSMC-enriched miRNAs. Exposition of HCASMC to pathophysiological conditions, such as hypercholesterolemia, induced a decrease in the expression level of miR-143-3p and miR-222-3p in microparticles, not in cells. Expression levels of miR-222-3p were lower in circulating microparticles from FH patients compared to normocholesterolemic controls. Microparticles derived from atherosclerotic plaque areas showed a decreased level of miR-143-3p and miR-222-3p compared to non-atherosclerotic areas. We demonstrated for the first time that microparticles secreted by HCASMC contain microRNAs. Hypercholesterolemia alters the microRNA profile of HCASMC-derived microparticles. The miRNA signature of HCASMC-derived microparticles is a source of cardiovascular biomarkers. Copyright © 2016 Sociedad Española de Arteriosclerosis. Publicado por Elsevier España, S.L.U. All rights

  14. The effect of dys-1 mutation on miRNA expression profile in Caenorhabditis elegans during Shenzhou-8 mission

    Xu, Dan; Sun, Yeqing; Gao, Ying; Xing, Yanfang

    microRNAs (miRNAs) is reported to be sensitive to radiation exposure and altered gravity, involved in a variety of biological processes through negative regulation of gene expression. Dystrophin-like dys-1 gene is expressed and required in muscle tissue, which plays a vital role in mechanical transduction when gravity varies. In the present study, we investigated the effect of dys-1 mutation on miRNA expression profile in Caenorhabditis elegans (C. elegans) under space radiation associated with microgravity (R+M) and radiation alone (R) environment during Shenzhou-8 mission. We performed miRNA microarray analysis in dys-1 mutant and wide-type (WT) of dauer larvae and found that 27 miRNAs changed in abundance after spaceflight. Compared with WT, there was different miRNA expression pattern in different treatments in dys-1 mutant. Cel-miR-796 and miR-124 were reversely expressed under R+M and R environment in WT and dys-1 mutant, respectively, indicating they might be affected by microgravity. Mutation of dys-1 remarkably reduced the number of altered miRNAs under space environment, resulting in the decrease of genes in biological categories of “body morphogenesis”, “behavior”, “cell adhesion” and so on. Particularly, we found that those genes controlling regulation of locomotion in WT were lost in dys-1 mutant, while genes in positive regulation of developmental process only existed in dys-1 mutant. miR-796 was predicted to target genes ace-1 and dyc-1 that are functionally linked to dys-1. Integration analysis of miRNA and mRNA expression profile revealed that miR-56 and miR-124 were involved in behavior and locomotion by regulating different target genes under space environment, among which nep-11, deb-1, C07H4.1 and F11H8.2 might be associated with neuromuscular system. Our findings suggest that dys-1 could cause alteration of miRNAs and target genes, involved in regulating the response of C. elegans to space microgravity in neuromuscular system. This

  15. Specific Regional and Age-Related Small Noncoding RNA Expression Patterns Within Superior Temporal Gyrus of Typical Human Brains Are Less Distinct in Autism Brains.

    Stamova, Boryana; Ander, Bradley P; Barger, Nicole; Sharp, Frank R; Schumann, Cynthia M

    2015-12-01

    Small noncoding RNAs play a critical role in regulating messenger RNA throughout brain development and when altered could have profound effects leading to disorders such as autism spectrum disorders (ASD). We assessed small noncoding RNAs, including microRNA and small nucleolar RNA, in superior temporal sulcus association cortex and primary auditory cortex in typical and ASD brains from early childhood to adulthood. Typical small noncoding RNA expression profiles were less distinct in ASD, both between regions and changes with age. Typical micro-RNA coexpression associations were absent in ASD brains. miR-132, miR-103, and miR-320 micro-RNAs were dysregulated in ASD and have previously been associated with autism spectrum disorders. These diminished region- and age-related micro-RNA expression profiles are in line with previously reported findings of attenuated messenger RNA and long noncoding RNA in ASD brain. This study demonstrates alterations in superior temporal sulcus in ASD, a region implicated in social impairment, and is the first to demonstrate molecular alterations in the primary auditory cortex. © The Author(s) 2015.

  16. Comparison of miRNA and gene expression profiles between metastatic and primary prostate cancer.

    Guo, Kaimin; Liang, Zuowen; Li, Fubiao; Wang, Hongliang

    2017-11-01

    The present study aimed to identify the regulatory mechanisms associated with the metastasis of prostate cancer (PC). The microRNA (miRNA/miR) microarray dataset GSE21036 and gene transcript dataset GSE21034 were downloaded from the Gene Expression Omnibus database. Following pre-processing, differentially expressed miRNAs (DEMs) and differentially expressed genes (DEGs) between samples from patients with primary prostate cancer (PPC) and metastatic prostate cancer (MPC) with |log 2 fold change (FC)| >1 and a false discovery rate terms (36 terms), followed by miR-494 (24 terms), miR-30d (18 terms), miR-181a (15 terms), hsa-miR-196a (8 terms), miR-708 (7 terms) and miR-486-5p (2 terms). Therefore, these miRNAs may serve roles in the metastasis of PC cells via downregulation of their corresponding target DEGs.

  17. Global microRNA profiles and signaling pathways in the development of cardiac hypertrophy

    Feng, H.J.; Ouyang, W.; Liu, J.H.; Sun, Y.G.; Hu, R.; Huang, L.H.; Xian, J.L. [Southern Medical University, Department of Nuclear Medicine, Zhujiang Hospital, Guangzhou, China, Department of Nuclear Medicine, Zhujiang Hospital, Southern Medical University, Guangzhou (China); Jing, C.F.; Zhou, M.J. [Sun Yat-Sen University, South China Sea Marine Biotechnology, National Engineering Research Center, Guangzhou, China, National Engineering Research Center, South China Sea Marine Biotechnology, Sun Yat-Sen University, Guangzhou (China)

    2014-04-11

    Hypertrophy is a major predictor of progressive heart disease and has an adverse prognosis. MicroRNAs (miRNAs) that accumulate during the course of cardiac hypertrophy may participate in the process. However, the nature of any interaction between a hypertrophy-specific signaling pathway and aberrant expression of miRNAs remains unclear. In this study, Spague Dawley male rats were treated with transverse aortic constriction (TAC) surgery to mimic pathological hypertrophy. Hearts were isolated from TAC and sham operated rats (n=5 for each group at 5, 10, 15, and 20 days after surgery) for miRNA microarray assay. The miRNAs dysexpressed during hypertrophy were further analyzed using a combination of bioinformatics algorithms in order to predict possible targets. Increased expression of the target genes identified in diverse signaling pathways was also analyzed. Two sets of miRNAs were identified, showing different expression patterns during hypertrophy. Bioinformatics analysis suggested the miRNAs may regulate multiple hypertrophy-specific signaling pathways by targeting the member genes and the interaction of miRNA and mRNA might form a network that leads to cardiac hypertrophy. In addition, the multifold changes in several miRNAs suggested that upregulation of rno-miR-331*, rno-miR-3596b, rno-miR-3557-5p and downregulation of rno-miR-10a, miR-221, miR-190, miR-451 could be seen as biomarkers of prognosis in clinical therapy of heart failure. This study described, for the first time, a potential mechanism of cardiac hypertrophy involving multiple signaling pathways that control up- and downregulation of miRNAs. It represents a first step in the systematic discovery of miRNA function in cardiovascular hypertrophy.

  18. Delineating miRNA profile induced by chewing tobacco in oral keratinocytes

    Mohd Younis Bhat

    2017-10-01

    Full Text Available The major established etiologic risk factor for oral cancer is tobacco (chewed, smoked and snuffed forms. Chewing form of tobacco is predominantly used in India making it the leading cause of oral cancer. Despite being one of the leading causes of oral cancer, the molecular alterations induced by chewing tobacco remains largely unclear. Carcinogenic effect of chewing tobacco is through chronic and not acute exposure. To understand the molecular alterations induced by chewing tobacco, we developed a cell line model where non-neoplastic oral keratinocytes were chronically exposed to chewing tobacco for a period of 6 months. This resulted in increased cellular proliferation and invasive ability of normal oral keratinocytes. Using this cellular model we studied the differential expression of miRNAs associated with chewing tobacco and the altered signaling pathways through which the aberrantly expressed miRNAs affect tumorigenesis. miRNA sequencing  was carried out using Illumina HiSeq 2500 platform  which resulted in the identification of 427 annotated miRNAs of which 10 were significantly dysregulated (≥ 4 fold; p-value ≤ 0.05 in tobacco exposed cells compared to untreated parental cells. To study the altered signaling in oral keratinocytes chronically exposed to chewing tobacco, we employed quantitative proteomics to characterize the dysregulated proteins. Integration of miRNA sequencing data with proteomic data resulted in identification of 36 proven protein targets which (≥1.5 fold; p-value ≤ 0.05 showed expression correlation with the 10 significantly dysregulated miRNAs. Pathway analysis of the dysregulated targets revealed enrichment of interferon signaling and mRNA processing related pathways in the chewing tobacco exposed cells. In addition, we also identified 6 novel miRNA in oral keratinocytes chronically exposed to chewing tobacco extract. Our study provides a framework to understand the oncogenic transformation induced by

  19. MicroRNA Expression Profiling Identifies Molecular Diagnostic Signatures for Anaplastic Large Cell Lymphoma

    Liu, Cuiling; Iqbal, Javeed; Teruya-Feldstein, Julie

    2013-01-01

    distinct clustering of ALCL, PTCL-NOS, and the AITL subtype of PTCL. Cases of ALK(+) ALCL and ALK(-) ALCL were interspersed in unsupervised analysis, suggesting a close relationship at the molecular level. We identified an miRNA signature of 7 miRNAs (5 upregulated: miR-512-3p, miR-886-5p, miR-886-3p, mi...

  20. CoverageAnalyzer (CAn: A Tool for Inspection of Modification Signatures in RNA Sequencing Profiles

    Ralf Hauenschild

    2016-11-01

    Full Text Available Combination of reverse transcription (RT and deep sequencing has emerged as a powerful instrument for the detection of RNA modifications, a field that has seen a recent surge in activity because of its importance in gene regulation. Recent studies yielded high-resolution RT signatures of modified ribonucleotides relying on both sequence-dependent mismatch patterns and reverse transcription arrests. Common alignment viewers lack specialized functionality, such as filtering, tailored visualization, image export and differential analysis. Consequently, the community will profit from a platform seamlessly connecting detailed visual inspection of RT signatures and automated screening for modification candidates. CoverageAnalyzer (CAn was developed in response to the demand for a powerful inspection tool. It is freely available for all three main operating systems. With SAM file format as standard input, CAn is an intuitive and user-friendly tool that is generally applicable to the large community of biomedical users, starting from simple visualization of RNA sequencing (RNA-Seq data, up to sophisticated modification analysis with significance-based modification candidate calling.

  1. Mechanistic profiling of the siRNA delivery dynamics of lipid-polymer hybrid nanoparticles

    Colombo, Stefano; Cun, Dongmei; Remaut, Katrien

    2015-01-01

    Understanding the delivery dynamics of nucleic acid nanocarriers is fundamental to improve their design for therapeutic applications. We investigated the carrier structure-function relationship of lipid-polymer hybrid nanoparticles (LPNs) consisting of poly(dl-lactic-co-glycolic acid) (PLGA) nano...... of transfection-competent siRNA-DOTAP lipoplexes from the LPNs. Based on these results, we suggest a model for the nanostructural characteristics of the LPNs, in which the siRNA is organized in lamellar superficial assemblies and/or as complexes entrapped in the polymeric matrix.......Understanding the delivery dynamics of nucleic acid nanocarriers is fundamental to improve their design for therapeutic applications. We investigated the carrier structure-function relationship of lipid-polymer hybrid nanoparticles (LPNs) consisting of poly(dl-lactic-co-glycolic acid) (PLGA......) nanocarriers modified with the cationic lipid dioleoyltrimethyl-ammoniumpropane (DOTAP). A library of siRNA-loaded LPNs was prepared by systematically varying the nitrogen-to-phosphate (N/P) ratio. Atomic force microscopy (AFM) and cryo-transmission electron microscopy (cryo-TEM) combined with small angle X...

  2. Integrated analysis of microRNA and gene expression profiles reveals a functional regulatory module associated with liver fibrosis.

    Chen, Wei; Zhao, Wenshan; Yang, Aiting; Xu, Anjian; Wang, Huan; Cong, Min; Liu, Tianhui; Wang, Ping; You, Hong

    2017-12-15

    -receptor interaction" were remarked significant (adjusted pfunctional miRNA-gene regulatory module that contains 7 miRNAs, 22 genes and 42 miRNA-gene connections. Gene interaction analysis based on String database revealed that 8 out of 22 genes were highly clustered. Finally, we experimentally confirmed a functional regulatory module containing 5 miRNAs (miR-130b-3p, miR-148a-3p, miR-345-5p, miR-378a-3p, and miR-422a) and 6 genes (COL6A1, COL6A2, COL6A3, PIK3R3, COL1A1, CCND2) associated with liver fibrosis. Our integrated analysis of miRNA and gene expression profiles highlighted a functional miRNA-gene regulatory module associated with liver fibrosis, which, to some extent, may provide important clues to better understand the underlying pathogenesis of liver fibrosis. Copyright © 2017. Published by Elsevier B.V.

  3. RNA sequencing of the human milk fat layer transcriptome reveals distinct gene expression profiles at three stages of lactation.

    Danielle G Lemay

    Full Text Available Aware of the important benefits of human milk, most U.S. women initiate breastfeeding but difficulties with milk supply lead some to quit earlier than intended. Yet, the contribution of maternal physiology to lactation difficulties remains poorly understood. Human milk fat globules, by enveloping cell contents during their secretion into milk, are a rich source of mammary cell RNA. Here, we pair this non-invasive mRNA source with RNA-sequencing to probe the milk fat layer transcriptome during three stages of lactation: colostral, transitional, and mature milk production. The resulting transcriptomes paint an exquisite portrait of human lactation. The resulting transcriptional profiles cluster not by postpartum day, but by milk Na:K ratio, indicating that women sampled during similar postpartum time frames could be at markedly different stages of gene expression. Each stage of lactation is characterized by a dynamic range (10(5-fold in transcript abundances not previously observed with microarray technology. We discovered that transcripts for isoferritins and cathepsins are strikingly abundant during colostrum production, highlighting the potential importance of these proteins for neonatal health. Two transcripts, encoding β-casein (CSN2 and α-lactalbumin (LALBA, make up 45% of the total pool of mRNA in mature lactation. Genes significantly expressed across all stages of lactation are associated with making, modifying, transporting, and packaging milk proteins. Stage-specific transcripts are associated with immune defense during the colostral stage, up-regulation of the machinery needed for milk protein synthesis during the transitional stage, and the production of lipids during mature lactation. We observed strong modulation of key genes involved in lactose synthesis and insulin signaling. In particular, protein tyrosine phosphatase, receptor type, F (PTPRF may serve as a biomarker linking insulin resistance with insufficient milk supply. This

  4. Combining RNA-seq and proteomic profiling to identify seminal fluid proteins in the migratory grasshopper Melanoplus sanguinipes (F).

    Bonilla, Martha L; Todd, Christopher; Erlandson, Martin; Andres, Jose

    2015-12-22

    Seminal fluid proteins control many aspects of fertilization and in turn, they play a key role in post-mating sexual selection and possibly reproductive isolation. Because effective proteome profiling relies on the availability of high-quality DNA reference databases, our knowledge of these proteins is still largely limited to model organisms with ample genetic resources. New advances in sequencing technology allow for the rapid characterization of transcriptomes at low cost. By combining high throughput RNA-seq and shotgun proteomic profiling, we have characterized the seminal fluid proteins secreted by the primary male accessory gland of the migratory grasshopper (Melanoplus sanguinipes), one of the main agricultural pests in central North America. Using RNA sequencing, we characterized the transcripts of ~ 8,100 genes expressed in the long hyaline tubules (LHT) of the accessory glands. Proteomic profiling identified 353 proteins expressed in the long hyaline tubules (LHT). Of special interest are seminal fluid proteins (SFPs), such as EJAC-SP, ACE and prostaglandin synthetases, which are known to regulate female oviposition in insects. Our study provides new insights into the proteomic components of male ejaculate in Orthopterans, and highlights several important patterns. First, the presence of proteins that lack predicted classical secretory tags in accessory gland proteomes is common in male accessory glands. Second, the products of a few highly expressed genes dominate the accessory gland secretions. Third, accessory gland transcriptomes are enriched for novel transcripts. Fourth, there is conservation of SFPs' functional classes across distantly related taxonomic groups with very different life histories, mating systems and sperm transferring mechanisms. The identified SFPs may serve as targets of future efforts to develop species- specific genetic control strategies.

  5. Messengers of the universe: Session IV Summary

    Bernardini, Elisa; Serpico, Pasquale Dario

    2013-01-01

    Being stable, light and neutral weakly interacting particles, neutrinos are ideal messengers of the deep universe and a channel of choice in particular to explore the very high energy Galactic and Extragalactic sky, playing a synergic role most notably with gamma-ray observations. Neutrino astronomy—long after the SN1987A detection in the MeV range—is mature enough for decisive tests of astrophysical paradigms. Its current status constitutes one of the two big pillars of the “Messengers of the universe” session of the Neutrino Oscillation Workshop 2012. Neutrinos may also play a role in some cosmological contexts, such as the early universe and the dark matter problem. We review both aspects in this session summary report

  6. Bacterial nucleotide-based second messengers.

    Pesavento, Christina; Hengge, Regine

    2009-04-01

    In all domains of life nucleotide-based second messengers transduce signals originating from changes in the environment or in intracellular conditions into appropriate cellular responses. In prokaryotes cyclic di-GMP has emerged as an important and ubiquitous second messenger regulating bacterial life-style transitions relevant for biofilm formation, virulence, and many other bacterial functions. This review describes similarities and differences in the architecture of the cAMP, (p)ppGpp, and c-di-GMP signaling systems and their underlying signaling principles. Moreover, recent advances in c-di-GMP-mediated signaling will be presented and the integration of c-di-GMP signaling with other nucleotide-based signaling systems will be discussed.

  7. An Enumerative Combinatorics Model for Fragmentation Patterns in RNA Sequencing Provides Insights into Nonuniformity of the Expected Fragment Starting-Point and Coverage Profile.

    Prakash, Celine; Haeseler, Arndt Von

    2017-03-01

    RNA sequencing (RNA-seq) has emerged as the method of choice for measuring the expression of RNAs in a given cell population. In most RNA-seq technologies, sequencing the full length of RNA molecules requires fragmentation into smaller pieces. Unfortunately, the issue of nonuniform sequencing coverage across a genomic feature has been a concern in RNA-seq and is attributed to biases for certain fragments in RNA-seq library preparation and sequencing. To investigate the expected coverage obtained from fragmentation, we develop a simple fragmentation model that is independent of bias from the experimental method and is not specific to the transcript sequence. Essentially, we enumerate all configurations for maximal placement of a given fragment length, F, on transcript length, T, to represent every possible fragmentation pattern, from which we compute the expected coverage profile across a transcript. We extend this model to incorporate general empirical attributes such as read length, fragment length distribution, and number of molecules of the transcript. We further introduce the fragment starting-point, fragment coverage, and read coverage profiles. We find that the expected profiles are not uniform and that factors such as fragment length to transcript length ratio, read length to fragment length ratio, fragment length distribution, and number of molecules influence the variability of coverage across a transcript. Finally, we explore a potential application of the model where, with simulations, we show that it is possible to correctly estimate the transcript copy number for any transcript in the RNA-seq experiment.

  8. The Energy Messenger, Number 1, Volume 4

    Stancil, J.

    1995-01-01

    'The Energy Messenger' is a Department of Energy publication on energy activities of interest to American Indians. The first issue of 1995 (in a magazine format) includes articles on: tribes winning grants to develop energy resources, recruiting of internships for DOE, information about Title XXVI-Indian Energy Resources, American Indian Heritage Month, tribal perspective on DOE actions, joint ventures between tribes and the DOE, and brief description of recent DOE activities

  9. The Energy Messenger, Number 1, Volume 4

    Stancil, J. [ed.

    1995-01-01

    `The Energy Messenger` is a Department of Energy publication on energy activities of interest to American Indians. The first issue of 1995 (in a magazine format) includes articles on: tribes winning grants to develop energy resources, recruiting of internships for DOE, information about Title XXVI-Indian Energy Resources, American Indian Heritage Month, tribal perspective on DOE actions, joint ventures between tribes and the DOE, and brief description of recent DOE activities.

  10. A probabilistic approach for the interpretation of RNA profiles as cell type evidence

    de Zoete, J.; Curran, J.; Sjerps, M.

    2016-01-01

    DNA profiles can be used as evidence to distinguish between possible donors of a crime stain. In some cases, both the prosecution and the defence claim that the cell material was left by the suspect but they dispute which cell type was left behind. For example, in sexual offense cases the

  11. Holographic gauge mediation via strongly coupled messengers

    McGuirk, Paul; Shiu, Gary; Sumitomo, Yoske

    2010-01-01

    We consider a relative of semidirect gauge mediation where the hidden sector exists at large 't Hooft coupling. Such scenarios can be difficult to describe using perturbative field theory methods but may fall into the class of holographic gauge mediation scenarios, meaning that they are amenable to the techniques of gauge/gravity duality. We use a recently found gravity solution to examine one such case, where the hidden sector is a cascading gauge theory resulting in a confinement scale not much smaller than the messenger mass. In the original construction of holographic gauge mediation, as in other examples of semidirect gauge mediation at strong coupling, the primary contributions to visible sector soft terms come from weakly coupled messenger mesons. In contrast to these examples, we describe the dual of a gauge theory where there are significant contributions from scales in which the strongly coupled messenger quarks are the effective degrees of freedom. In this regime, the visible sector gaugino mass can be calculated entirely from holography.

  12. Analysis of RNA metabolism in fission yeast

    Wise, Jo Ann; Nielsen, Olaf

    2017-01-01

    Here we focus on the biogenesis and function of messenger RNA (mRNA) in fission yeast cells. Following a general introduction that also briefly touches on other classes of RNA, we provide an overview of methods used to analyze mRNAs throughout their life cycles....

  13. Regulatory RNAs derived from transfer RNA?

    Pederson, Thoru

    2010-10-01

    Four recent studies suggest that cleavages of transfer RNAs generate products with microRNA-like features, with some evidence of function. If their regulatory functions were to be confirmed, these newly revealed RNAs would add to the expanding repertoire of small noncoding RNAs and would also provide new perspectives on the coevolution of transfer RNA and messenger RNA.

  14. Identifying airway sensitizers: cytokine mRNA profiles induced by various anhydrides

    Plitnick, L.M.; Loveless, S.E.; Ladics, G.S.; Holsapple, M.P.; Smialowicz, R.J.; Woolhiser, M.R.; Anderson, P.K.; Smith, C.; Selgrade, M.J.K.

    2003-01-01

    Exposure to low molecular weight (LMW) chemicals in the workplace has been linked to a variety of respiratory effects. Within the LMW chemicals, one of the major classes involved in these effects are the acid anhydrides. The immunological basis of respiratory hypersensitivity involves CD4+ cells. By virtue of their induction of cytokines typical of CD4+ T-helper type 2 (Th2) cells--interleukin (IL)-4, 10, and 13--respiratory sensitizers may be identified and differentiated from contact sensitizers which induce Th1 cytokines (IL-2 and IFN-γ). Our previous work suggested that the ribonuclease protection assay (RPA) was useful in identifying the respiratory sensitizer, trimellitic anhydride (TMA), based on quantitative differences in Th2 cytokine mRNA as compared to the contact sensitizer dinitrochlorobenzene (DNCB). Therefore, the purpose of the studies described in this report was to expand the chemicals tested in the RPA. To this end, four acid anhydrides with known respiratory sensitization potential, TMA, maleic anhydride (MA), phthalic anhydride (PA) and hexahydrophthalic anhydride (HHPA), were tested. Although previously determined to induce immunologically equivalent responses in a local lymph node assay (LLNA), the initial dose chosen (2.5%) failed to induce Th2 cytokine mRNA expression. To determine if the lack of cytokine expression was related to dose, LLNAs were conducted at higher doses for each of the anhydrides. The highest doses evaluated (four- to six-fold higher than those used in the initial RPA) gave equivalent proliferative responses for the various anhydrides and were used for subsequent RPA testing. At these higher doses, significant increases in Th2 versus Th1 cytokine mRNA were observed for all anhydrides tested. These results suggest that the RPA has the potential to serve as a screen for the detection of LMW airway sensitizing chemicals. However, the basis for selecting immunologically equivalent doses may require some modification

  15. Comprehensive microRNA profiling in B-cells of human centenarians by massively parallel sequencing

    Gombar Saurabh

    2012-07-01

    Full Text Available Abstract Background MicroRNAs (miRNAs are small, non-coding RNAs that regulate gene expression and play a critical role in development, homeostasis, and disease. Despite their demonstrated roles in age-associated pathologies, little is known about the role of miRNAs in human aging and longevity. Results We employed massively parallel sequencing technology to identify miRNAs expressed in B-cells from Ashkenazi Jewish centenarians, i.e., those living to a hundred and a human model of exceptional longevity, and younger controls without a family history of longevity. With data from 26.7 million reads comprising 9.4 × 108 bp from 3 centenarian and 3 control individuals, we discovered a total of 276 known miRNAs and 8 unknown miRNAs ranging several orders of magnitude in expression levels, a typical characteristics of saturated miRNA-sequencing. A total of 22 miRNAs were found to be significantly upregulated, with only 2 miRNAs downregulated, in centenarians as compared to controls. Gene Ontology analysis of the predicted and validated targets of the 24 differentially expressed miRNAs indicated enrichment of functional pathways involved in cell metabolism, cell cycle, cell signaling, and cell differentiation. A cross sectional expression analysis of the differentially expressed miRNAs in B-cells from Ashkenazi Jewish individuals between the 50th and 100th years of age indicated that expression levels of miR-363* declined significantly with age. Centenarians, however, maintained the youthful expression level. This result suggests that miR-363* may be a candidate longevity-associated miRNA. Conclusion Our comprehensive miRNA data provide a resource for further studies to identify genetic pathways associated with aging and longevity in humans.

  16. Structural profiles of human miRNA families from pairwise clustering

    Kaczkowski, Bogumil; Þórarinsson, Elfar; Reiche, Kristin

    2009-01-01

    secondary structure already predicted, little is known about the patterns of structural conservation among pre-miRNAs. We address this issue by clustering the human pre-miRNA sequences based on pairwise, sequence and secondary structure alignment using FOLDALIGN, followed by global multiple alignment...... of obtained clusters by WAR. As a result, the common secondary structure was successfully determined for four FOLDALIGN clusters: the RF00027 structural family of the Rfam database and three clusters with previously undescribed consensus structures. Availability: http://genome.ku.dk/resources/mirclust...

  17. RNA-Seq profiling reveals novel hepatic gene expression pattern in aflatoxin B1 treated rats.

    B Alex Merrick

    Full Text Available Deep sequencing was used to investigate the subchronic effects of 1 ppm aflatoxin B1 (AFB1, a potent hepatocarcinogen, on the male rat liver transcriptome prior to onset of histopathological lesions or tumors. We hypothesized RNA-Seq would reveal more differentially expressed genes (DEG than microarray analysis, including low copy and novel transcripts related to AFB1's carcinogenic activity compared to feed controls (CTRL. Paired-end reads were mapped to the rat genome (Rn4 with TopHat and further analyzed by DESeq and Cufflinks-Cuffdiff pipelines to identify differentially expressed transcripts, new exons and unannotated transcripts. PCA and cluster analysis of DEGs showed clear separation between AFB1 and CTRL treatments and concordance among group replicates. qPCR of eight high and medium DEGs and three low DEGs showed good comparability among RNA-Seq and microarray transcripts. DESeq analysis identified 1,026 differentially expressed transcripts at greater than two-fold change (p<0.005 compared to 626 transcripts by microarray due to base pair resolution of transcripts by RNA-Seq, probe placement within transcripts or an absence of probes to detect novel transcripts, splice variants and exons. Pathway analysis among DEGs revealed signaling of Ahr, Nrf2, GSH, xenobiotic, cell cycle, extracellular matrix, and cell differentiation networks consistent with pathways leading to AFB1 carcinogenesis, including almost 200 upregulated transcripts controlled by E2f1-related pathways related to kinetochore structure, mitotic spindle assembly and tissue remodeling. We report 49 novel, differentially-expressed transcripts including confirmation by PCR-cloning of two unique, unannotated, hepatic AFB1-responsive transcripts (HAfT's on chromosomes 1.q55 and 15.q11, overexpressed by 10 to 25-fold. Several potentially novel exons were found and exon refinements were made including AFB1 exon-specific induction of homologous family members, Ugt1a6 and Ugt1a7c

  18. RNA-Seq profiling reveals novel hepatic gene expression pattern in aflatoxin B1 treated rats.

    Merrick, B Alex; Phadke, Dhiral P; Auerbach, Scott S; Mav, Deepak; Stiegelmeyer, Suzy M; Shah, Ruchir R; Tice, Raymond R

    2013-01-01

    Deep sequencing was used to investigate the subchronic effects of 1 ppm aflatoxin B1 (AFB1), a potent hepatocarcinogen, on the male rat liver transcriptome prior to onset of histopathological lesions or tumors. We hypothesized RNA-Seq would reveal more differentially expressed genes (DEG) than microarray analysis, including low copy and novel transcripts related to AFB1's carcinogenic activity compared to feed controls (CTRL). Paired-end reads were mapped to the rat genome (Rn4) with TopHat and further analyzed by DESeq and Cufflinks-Cuffdiff pipelines to identify differentially expressed transcripts, new exons and unannotated transcripts. PCA and cluster analysis of DEGs showed clear separation between AFB1 and CTRL treatments and concordance among group replicates. qPCR of eight high and medium DEGs and three low DEGs showed good comparability among RNA-Seq and microarray transcripts. DESeq analysis identified 1,026 differentially expressed transcripts at greater than two-fold change (p<0.005) compared to 626 transcripts by microarray due to base pair resolution of transcripts by RNA-Seq, probe placement within transcripts or an absence of probes to detect novel transcripts, splice variants and exons. Pathway analysis among DEGs revealed signaling of Ahr, Nrf2, GSH, xenobiotic, cell cycle, extracellular matrix, and cell differentiation networks consistent with pathways leading to AFB1 carcinogenesis, including almost 200 upregulated transcripts controlled by E2f1-related pathways related to kinetochore structure, mitotic spindle assembly and tissue remodeling. We report 49 novel, differentially-expressed transcripts including confirmation by PCR-cloning of two unique, unannotated, hepatic AFB1-responsive transcripts (HAfT's) on chromosomes 1.q55 and 15.q11, overexpressed by 10 to 25-fold. Several potentially novel exons were found and exon refinements were made including AFB1 exon-specific induction of homologous family members, Ugt1a6 and Ugt1a7c. We find the

  19. Profiling of Ribose Methylations in RNA by High-Throughput Sequencing

    Birkedal, Ulf; Christensen-Dalsgaard, Mikkel; Krogh, Nicolai

    2015-01-01

    (RiboMeth-seq) and its application to yeast ribosomes, presently the best-studied eukaryotic model system. We demonstrate detection of the known as well as new modifications, reveal partial modifications and unexpected communication between modification events, and determine the order of modification...... at several sites during ribosome biogenesis. Surprisingly, the method also provides information on a subset of other modifications. Hence, RiboMeth-seq enables a detailed evaluation of the importance of RNA modifications in the cells most sophisticated molecular machine. RiboMeth-seq can be adapted to other...

  20. Novel mRNA-specific effects of ribosome drop-off on translation rate and polysome profile.

    Pierre Bonnin

    2017-05-01

    Full Text Available The well established phenomenon of ribosome drop-off plays crucial roles in translational accuracy and nutrient starvation responses during protein translation. When cells are under stress conditions, such as amino acid starvation or aminoacyl-tRNA depletion due to a high level of recombinant protein expression, ribosome drop-off can substantially affect the efficiency of protein expression. Here we introduce a mathematical model that describes the effects of ribosome drop-off on the ribosome density along the mRNA and on the concomitant protein synthesis rate. Our results show that ribosome premature termination may lead to non-intuitive ribosome density profiles, such as a ribosome density which increases from the 5' to the 3' end. Importantly, the model predicts that the effects of ribosome drop-off on the translation rate are mRNA-specific, and we quantify their resilience to drop-off, showing that the mRNAs which present ribosome queues are much less affected by ribosome drop-off than those which do not. Moreover, among those mRNAs that do not present ribosome queues, resilience to drop-off correlates positively with the elongation rate, so that sequences using fast codons are expected to be less affected by ribosome drop-off. This result is consistent with a genome-wide analysis of S. cerevisiae, which reveals that under favourable growth conditions mRNAs coding for proteins involved in the translation machinery, known to be highly codon biased and using preferentially fast codons, are highly resilient to ribosome drop-off. Moreover, in physiological conditions, the translation rate of mRNAs coding for regulatory, stress-related proteins, is less resilient to ribosome drop-off. This model therefore allows analysis of variations in the translational efficiency of individual mRNAs by accounting for the full range of known ribosome behaviours, as well as explaining mRNA-specific variations in ribosome density emerging from ribosome profiling

  1. Clinical implication of long noncoding RNA 91H expression profile in osteosarcoma patients

    Xia WK

    2016-07-01

    Full Text Available Wen-Kai Xia,1 Qing-Feng Lin,2 Dong Shen,2 Zhi-Li Liu,2 Jun Su,2 Wei-Dong Mao2 1Department of Nephrology, 2Department of Oncology, The Affiliated Jiangyin Hospital, School of Medicine, Southeast University, Jiangyin, Jiangsu, People’s Republic of China Abstract: Long noncoding RNAs have been documented as having widespread roles in carcinogenesis and cancer progression. However, roles of long noncoding RNAs in osteosarcoma remain unclear. This study is to investigate the clinical relevance and biological functions of long noncoding RNA 91H in osteosarcoma. Herein, we confirmed that 91H expression was notably increased in osteosarcoma patients and cell lines compared to healthy controls and normal human bone cell lines. High expression of 91H was significantly correlated with advanced clinical stage, chemotherapy after surgery, and tumor size >5 cm. Furthermore, 91H was an independent prognostic factor for overall survival in osteosarcoma patients after treatments. Additionally, the knockdown of 91H expression inhibited osteosarcoma cells’ proliferation and promoted their apoptosis in vitro. In summary, these findings indicate that 91H may be a novel biomarker for risk prognostication and also provide a clue to the molecular etiology of osteosarcoma. Keywords: long noncoding RNA, 91H, osteosarcoma, survival

  2. Amniotic fluid RNA gene expression profiling provides insights into the phenotype of Turner syndrome.

    Massingham, Lauren J; Johnson, Kirby L; Scholl, Thomas M; Slonim, Donna K; Wick, Heather C; Bianchi, Diana W

    2014-09-01

    Turner syndrome is a sex chromosome aneuploidy with characteristic malformations. Amniotic fluid, a complex biological material, could contribute to the understanding of Turner syndrome pathogenesis. In this pilot study, global gene expression analysis of cell-free RNA in amniotic fluid supernatant was utilized to identify specific genes/organ systems that may play a role in Turner syndrome pathophysiology. Cell-free RNA from amniotic fluid of five mid-trimester Turner syndrome fetuses and five euploid female fetuses matched for gestational age was extracted, amplified, and hybridized onto Affymetrix(®) U133 Plus 2.0 arrays. Significantly differentially regulated genes were identified using paired t tests. Biological interpretation was performed using Ingenuity Pathway Analysis and BioGPS gene expression atlas. There were 470 statistically significantly differentially expressed genes identified. They were widely distributed across the genome. XIST was significantly down-regulated (p Turner syndrome transcriptome from other aneuploidies we previously studied. Manual curation of the differentially expressed gene list identified genes of possible pathologic significance, including NFATC3, IGFBP5, and LDLR. Transcriptomic differences in the amniotic fluid of Turner syndrome fetuses are due to genome-wide dysregulation. The hematologic/immune system differences may play a role in early-onset autoimmune dysfunction. Other genes identified with possible pathologic significance are associated with cardiac and skeletal systems, which are known to be affected in females with Turner syndrome. The discovery-driven approach described here may be useful in elucidating novel mechanisms of disease in Turner syndrome.

  3. Profiling and bioinformatic analysis of circular RNA expression regulated by c-Myc.

    Gou, Qiheng; Wu, Ke; Zhou, Jian-Kang; Xie, Yuxin; Liu, Lunxu; Peng, Yong

    2017-09-22

    The c-Myc transcription factor is involved in cell proliferation, cell cycle and apoptosis by activating or repressing transcription of multiple genes. Circular RNAs (circRNAs) are widely expressed non-coding RNAs participating in the regulation of gene expression. Using a high-throughput microarray assay, we showed that Myc regulates the expression of certain circRNAs. A total of 309 up- and 252 down-regulated circRNAs were identified. Among them, randomly selected 8 circRNAs were confirmed by real-time PCR. Subsequently, Myc-binding sites were found to generally exist in the promoter regions of differentially expressed circRNAs. Based on miRNA sponge mechanism, we constructed circRNAs/miRNAs network regulated by Myc, suggesting that circRNAs may widely regulate protein expression through miRNA sponge mechanism. Lastly, we took advantage of Gene Ontology and KEGG analyses to point out that Myc-regulated circRNAs could impact cell proliferation through affecting Ras signaling pathway and pathways in cancer. Our study for the first time demonstrated that Myc transcription factor regulates the expression of circRNAs, adding a novel component of the Myc tumorigenic program and opening a window to investigate the function of certain circRNAs in tumorigenesis.

  4. Comprehensive microRNA profiling reveals potential augmentation of the IL1 pathway in rheumatic heart valve disease.

    Lu, Qiyu; Sun, Yi; Duan, Yuyin; Li, Bin; Xia, Jianming; Yu, Songhua; Zhang, Guimin

    2018-03-16

    Valvular heart disease is a leading cause of cardiovascular mortality, especially in China. More than a half of valvular heart diseases are caused by acute rheumatic fever. microRNA is involved in many physiological and pathological processes. However, the miRNA profile of the rheumatic valvular heart disease is unknown. This research is to discuss microRNAs and their target gene pathways involved in rheumatic heart valve disease. Serum miRNA from one healthy individual and four rheumatic heart disease patients were sequenced. Specific differentially expressed miRNAs were quantified by Q-PCR in 40 patients, with 20 low-to-moderate rheumatic mitral valve stenosis patients and 20 severe mitral valve stenosis patients. The target relationship between certain miRNA and predicted target genes were analysis by Luciferase reporter assay. The IL-1β and IL1R1 expression levels were analyzed by immunohistochemistry and western blot in the mitral valve from surgery of mitral valve replacement. The results showed that 13 and 91 miRNAs were commonly upregulated or downregulated in all four patients. Nine miRNAs, 1 upregulated and 8 downregulated, that had a similar fold change in all 4 patients were selected for quantitative PCR verification. The results showed similar results from miRNA sequencing. Within these 9 tested miRNAs, hsa-miR-205-3p and hsa-miR-3909 showed a low degree of dispersion between the members of each group. Hsa miR-205-3p and hsa-miR-3909 were predicted to target the 3'UTR of IL-1β and IL1R1 respectively. This was verified by luciferase reporter assays. Immunohistochemistry and Western blot results showed that the mitral valve from rheumatic valve heart disease showed higher levels of IL- 1β and IL1R1 expression compared with congenital heart valve disease. This suggested a difference between rheumatic heart valve disease and other types of heart valve diseases, with more inflammatory responses in the former. In the present study, by next generation

  5. Micro-RNA profile and proteins in peritoneal fluid from women with endometriosis: their relationship with sterility.

    Marí-Alexandre, Josep; Barceló-Molina, Moisés; Belmonte-López, Elisa; García-Oms, Javier; Estellés, Amparo; Braza-Boïls, Aitana; Gilabert-Estellés, Juan

    2018-04-01

    To define the microRNA (miRNA) profile and its relationship with cytokines content in peritoneal fluid (PF) from endometriosis patients. Case-control study. University hospital, research institute. One hundred twenty-six women with endometriosis (EPF) and 45 control women (CPF). MiRNA arrays were prepared from six EPF and six CPF. Quantitative reverse transcription-polymerase chain reaction validation of nine selected miRNAs (miR-29c-3p, -106b-3p, -130a-3p, -150-5p, -185-5p, -195-5p, -451a, -486-5p, and -1343-5p) was performed. Vascular endothelial growth factor-A (VEGF-A), thrombospondin-1 (TSP-1), urokinase plasminogen activator (uPA), plasminogen activator inhibitor-1 (PAI-1), matrix metalloproteinase-3 (MMP3), tissue inhibitor of metalloproteinases type 1 (TIMP-1), interleukin (IL)-6, IL-8, IL-17A, macrophage inflammatory protein 1β (MIP1beta), platelet-derived growth factor α-polypeptide A, and regulated on activation, normal T cell expressed and secreted (RANTES) were quantified by ELISA and MILLIPLEX. MiRNA arrays showed 126 miRNAs differentially expressed (fold change ±1.2) (78 down-regulated, 48 up-regulated) in EPF. Validation showed higher levels of miR-106b-3p, -451a, -486-5p, IL-6, IL-8, uPA, and TIMP-1 in EPF. In menstrual phase, EPF presented up-regulation of miR-106b-3p, -130a-3p, -150-5p, -185-5p, -451a, -486-5p, VEGF-A, IL-8, MIF 1β, uPA, and PAI-1 compared with other phases; however, CPF did not. MiRNA-486-5p was up-regulated in sterile EPF compared with sterile controls, and VEGF-A, IL-8, and TIMP-1 were increased in sterile and fertile EPF compared with fertile CPF. MiRNAs seem to be involved in the peritoneal alterations in endometriosis, suggesting new mechanisms by which ectopic lesions could implant in endometriosis patients; and to serve as biomarkers for fertility outcome prediction. Copyright © 2017 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  6. Differential plasma microRNA profiles in HBeAg positive and HBeAg negative children with chronic hepatitis B

    Winther, Thilde Nordmann; Bang-Berthelsen, Claus Heiner; Heiberg, Ida Louise

    2013-01-01

    Children chronically infected with hepatitis B virus (HBV) are at high risk of progressive liver disease. However, no treatment is available that is consistently effective in curing chronic hepatitis B (CHB) in children. Improved understanding of the natural course of disease is warranted....... Identification of specific microRNA (miRNA) profiles in children chronically infected with HBV may provide insight into the pathogenesis of CHB and lead to advances in the management of children with CHB....

  7. Soil DNA extraction procedure influences protist 18S rRNA gene community profiling outcome

    Santos, Susana S.; Nunes, Ines Marques; Nielsen, Tue K.

    2017-01-01

    Advances in sequencing technologies allow deeper studies of the soil protist diversity and function. However, little attention has been given to the impact of the chosen soil DNA extraction procedure to the overall results. We examined the effect of three acknowledged DNA recovery methods, two...... manual methods (ISOm-11063, GnS-GII) and one commercial kit (MoBio), on soil protist community structures obtained from different sites with different land uses. Results from 18S rRNA gene amplicon sequencing suggest that DNA extraction method significantly affect the replicate homogeneity, the total...... number of operational taxonomic units (OTUs) recovered and the overall taxonomic structure and diversity of soil protist communities. However, DNA extraction effects did not overwhelm the natural variation among samples, as the community data still strongly grouped by geographical location...

  8. Early second-trimester serum miRNA profiling predicts gestational diabetes mellitus.

    Chun Zhao

    Full Text Available BACKGROUND: Gestational diabetes mellitus (GDM is one type of diabetes that presents during pregnancy and significantly increases the risk of a number of adverse consequences for the fetus and mother. The microRNAs (miRNA have recently been demonstrated to abundantly and stably exist in serum and to be potentially disease-specific. However, no reported study investigates the associations between serum miRNA and GDM. METHODOLOGY/PRINCIPAL FINDINGS: We systematically used the TaqMan Low Density Array followed by individual quantitative reverse transcription polymerase chain reaction assays to screen miRNAs in serum collected at 16-19 gestational weeks. The expression levels of three miRNAs (miR-132, miR-29a and miR-222 were significantly decreased in GDM women with respect to the controls in similar gestational weeks in our discovery evaluation and internal validation, and two miRNAs (miR-29a and miR-222 were also consistently validated in two-centric external validation sample sets. In addition, the knockdown of miR-29a could increase Insulin-induced gene 1 (Insig1 expression level and subsequently the level of Phosphoenolpyruvate Carboxy Kinase2 (PCK2 in HepG2 cell lines. CONCLUSIONS/SIGNIFICANCE: Serum miRNAs are differentially expressed between GDM women and controls and could be candidate biomarkers for predicting GDM. The utility of miR-29a, miR-222 and miR-132 as serum-based non-invasive biomarkers warrants further evaluation and optimization.

  9. Differential MicroRNA Expression Profile in Myxomatous Mitral Valve Prolapse and Fibroelastic Deficiency Valves

    Yei-Tsung Chen

    2016-05-01

    Full Text Available Myxomatous mitral valve prolapse (MMVP and fibroelastic deficiency (FED are two common variants of degenerative mitral valve disease (DMVD, which is a leading cause of mitral regurgitation worldwide. While pathohistological studies have revealed differences in extracellular matrix content in MMVP and FED, the molecular mechanisms underlying these two disease entities remain to be elucidated. By using surgically removed valvular specimens from MMVP and FED patients that were categorized on the basis of echocardiographic, clinical and operative findings, a cluster of microRNAs that expressed differentially were identified. The expressions of has-miR-500, -3174, -17, -1193, -646, -1273e, -4298, -203, -505, and -939 showed significant differences between MMVP and FED after applying Bonferroni correction (p < 0.002174. The possible involvement of microRNAs in the pathogenesis of DMVD were further suggested by the presences of in silico predicted target sites on a number of genes reported to be involved in extracellular matrix homeostasis and marker genes for cellular composition of mitral valves, including decorin (DCN, aggrecan (ACAN, fibromodulin (FMOD, α actin 2 (ACTA2, extracellular matrix protein 2 (ECM2, desmin (DES, endothelial cell specific molecule 1 (ESM1, and platelet/ endothelial cell adhesion molecule 1 (PECAM1, as well as inverse correlations of selected microRNA and mRNA expression in MMVP and FED groups. Our results provide evidence that distinct molecular mechanisms underlie MMVP and FED. Moreover, the microRNAs identified may be targets for the future development of diagnostic biomarkers and therapeutics.

  10. Profile of microRNA in Giant Panda Blood: A Resource for Immune-Related and Novel microRNAs.

    Mingyu Yang

    Full Text Available The giant panda (Ailuropoda melanoleuca is one of the world's most beloved endangered mammals. Although the draft genome of this species had been assembled, little was known about the composition of its microRNAs (miRNAs or their functional profiles. Recent studies demonstrated that changes in the expression of miRNAs are associated with immunity. In this study, miRNAs were extracted from the blood of four healthy giant pandas and sequenced by Illumina next generation sequencing technology. As determined by miRNA screening, a total of 276 conserved miRNAs and 51 novel putative miRNAs candidates were detected. After differential expression analysis, we noticed that the expressions of 7 miRNAs were significantly up-regulated in young giant pandas compared with that of adults. Moreover, 2 miRNAs were up-regulated in female giant pandas and 1 in the male individuals. Target gene prediction suggested that the miRNAs of giant panda might be relevant to the expressions of 4,602 downstream genes. Subseuqently, the predicted target genes were conducted to KEGG (Kyoto Encyclopedia of Genes and Genomes pathway analysis and we found that these genes were mainly involved in host immunity, including the Ras signaling pathway, the PI3K-Akt signaling pathway, and the MAPK signaling pathway. In conclusion, our results provide the first miRNA profiles of giant panda blood, and the predicted functional analyses may open an avenue for further study of giant panda immunity.

  11. PPARalpha siRNA-treated expression profiles uncover the causal sufficiency network for compound-induced liver hypertrophy.

    Xudong Dai

    2007-03-01

    Full Text Available Uncovering pathways underlying drug-induced toxicity is a fundamental objective in the field of toxicogenomics. Developing mechanism-based toxicity biomarkers requires the identification of such novel pathways and the order of their sufficiency in causing a phenotypic response. Genome-wide RNA interference (RNAi phenotypic screening has emerged as an effective tool in unveiling the genes essential for specific cellular functions and biological activities. However, eliciting the relative contribution of and sufficiency relationships among the genes identified remains challenging. In the rodent, the most widely used animal model in preclinical studies, it is unrealistic to exhaustively examine all potential interactions by RNAi screening. Application of existing computational approaches to infer regulatory networks with biological outcomes in the rodent is limited by the requirements for a large number of targeted permutations. Therefore, we developed a two-step relay method that requires only one targeted perturbation for genome-wide de novo pathway discovery. Using expression profiles in response to small interfering RNAs (siRNAs against the gene for peroxisome proliferator-activated receptor alpha (Ppara, our method unveiled the potential causal sufficiency order network for liver hypertrophy in the rodent. The validity of the inferred 16 causal transcripts or 15 known genes for PPARalpha-induced liver hypertrophy is supported by their ability to predict non-PPARalpha-induced liver hypertrophy with 84% sensitivity and 76% specificity. Simulation shows that the probability of achieving such predictive accuracy without the inferred causal relationship is exceedingly small (p < 0.005. Five of the most sufficient causal genes have been previously disrupted in mouse models; the resulting phenotypic changes in the liver support the inferred causal roles in liver hypertrophy. Our results demonstrate the feasibility of defining pathways mediating drug

  12. High-Throughput Data of Circular RNA Profiles in Human Temporal Cortex Tissue Reveals Novel Insights into Temporal Lobe Epilepsy.

    Li, Jiaxin; Lin, Haijun; Sun, Zhenrong; Kong, Guanyi; Yan, Xu; Wang, Yujiao; Wang, Xiaoxuan; Wen, Yanhua; Liu, Xiang; Zheng, Hongkun; Jia, Mei; Shi, Zhongfang; Xu, Rong; Yang, Shaohua; Yuan, Fang

    2018-01-01

    Circular RNAs (circRNAs) are a class of long noncoding RNAs with a closed loop structure that regulate gene expression as microRNA sponges. CircRNAs are more enriched in brain tissue, but knowledge of the role of circRNAs in temporal lobe epilepsy (TLE) has remained limited. This study is the first to identify the global expression profiles and characteristics of circRNAs in human temporal cortex tissue from TLE patients. Temporal cortices were collected from 17 TLE patients and 17 non-TLE patients. Total RNA was isolated, and high-throughput sequencing was used to profile the transcriptome of dysregulated circRNAs. Quantitative PCR was performed for the validation of changed circRNAs. In total, 78983 circRNAs, including 15.29% known and 84.71% novel circRNAs, were detected in this study. Intriguingly, 442 circRNAs were differentially expressed between the TLE and non-TLE groups (fold change≥2.0 and FDR≤0.05). Of these circRNAs, 188 were up-regulated, and 254 were down-regulated in the TLE patient group. Eight circRNAs were validated by real-time PCR. Remarkably, circ-EFCAB2 was intensely up-regulated, while circ-DROSHA expression was significantly lower in the TLE group than in the non-TLE group (P<0.05). Bioinformatic analysis revealed that circ-EFCAB2 binds to miR-485-5p to increase the expression level of the ion channel CLCN6, while circ-DROSHA interacts with miR-1252-5p to decrease the expression level of ATP1A2. The dysregulations of circRNAs may reflect the pathogenesis of TLE and circ-EFCAB2 and circ-DROSHA might be potential therapeutic targets and biomarkers in TLE patients. © 2018 The Author(s). Published by S. Karger AG, Basel.

  13. A powerful method for transcriptional profiling of specific cell types in eukaryotes: laser-assisted microdissection and RNA sequencing.

    Marc W Schmid

    Full Text Available The acquisition of distinct cell fates is central to the development of multicellular organisms and is largely mediated by gene expression patterns specific to individual cells and tissues. A spatially and temporally resolved analysis of gene expression facilitates the elucidation of transcriptional networks linked to cellular identity and function. We present an approach that allows cell type-specific transcriptional profiling of distinct target cells, which are rare and difficult to access, with unprecedented sensitivity and resolution. We combined laser-assisted microdissection (LAM, linear amplification starting from <1 ng of total RNA, and RNA-sequencing (RNA-Seq. As a model we used the central cell of the Arabidopsis thaliana female gametophyte, one of the female gametes harbored in the reproductive organs of the flower. We estimated the number of expressed genes to be more than twice the number reported previously in a study using LAM and ATH1 microarrays, and identified several classes of genes that were systematically underrepresented in the transcriptome measured with the ATH1 microarray. Among them are many genes that are likely to be important for developmental processes and specific cellular functions. In addition, we identified several intergenic regions, which are likely to be transcribed, and describe a considerable fraction of reads mapping to introns and regions flanking annotated loci, which may represent alternative transcript isoforms. Finally, we performed a de novo assembly of the transcriptome and show that the method is suitable for studying individual cell types of organisms lacking reference sequence information, demonstrating that this approach can be applied to most eukaryotic organisms.

  14. Simultaneous RNA-seq based transcriptional profiling of intracellular Brucella abortus and B. abortus-infected murine macrophages.

    Hop, Huynh Tan; Arayan, Lauren Togonon; Reyes, Alisha Wehdnesday Bernardo; Huy, Tran Xuan Ngoc; Min, WonGi; Lee, Hu Jang; Son, Jee Soo; Kim, Suk

    2017-12-01

    Brucella is a zoonotic pathogen that survives within macrophages; however the replicative mechanisms involved are not fully understood. We describe the isolation of sufficient Brucella abortus RNA from primary host cell environment using modified reported methods for RNA-seq analysis, and simultaneously characterize the transcriptional profiles of intracellular B. abortus and bone marrow-derived macrophages (BMM) from BALB/c mice at 24 h (replicative phase) post-infection. Our results revealed that 25.12% (801/3190) and 16.16% (515/3190) of the total B. abortus genes were up-regulated and down-regulated at >2-fold, respectively as compared to the free-living B. abortus. Among >5-fold differentially expressed genes, the up-regulated genes are mostly involved in DNA, RNA manipulations as well as protein biosynthesis and secretion while the down-regulated genes are mainly involved in energy production and metabolism. On the other hand, the host responses during B. abortus infection revealed that 14.01% (6071/43,346) of BMM genes were reproducibly transcribed at >5-fold during infection. Transcription of cytokines, chemokines and transcriptional factors, such as tumor necrosis factor (Tnf), interleukin-1α (Il1α), interleukin-1β (Il1β), interleukin-6 (Il6), interleukin-12 (Il12), chemokine C-X-C motif (CXCL) family, nuclear factor kappa B (Nf-κb), signal transducer and activator of transcription 1 (Stat1), that may contribute to host defense were markedly induced while transcription of various genes involved in cell proliferation and metabolism were suppressed upon B. abortus infection. In conclusion, these data suggest that Brucella modulates gene expression in hostile intracellular environment while simultaneously alters the host pathways that may lead to the pathogen's intracellular survival and infection. Copyright © 2017 Elsevier Ltd. All rights reserved.

  15. A comparison of microRNA expression profiles from splenic hemangiosarcoma, splenic nodular hyperplasia, and normal spleens of dogs.

    Grimes, Janet A; Prasad, Nripesh; Levy, Shawn; Cattley, Russell; Lindley, Stephanie; Boothe, Harry W; Henderson, Ralph A; Smith, Bruce F

    2016-12-03

    Splenic masses are common in older dogs; yet diagnosis preceding splenectomy and histopathology remains elusive. MicroRNAs (miRNAs) are short, non-coding RNAs that play a role in post-transcriptional regulation, and differential expression of miRNAs between normal and tumor tissue has been used to diagnose neoplastic diseases. The objective of this study was to determine differential expression of miRNAs by use of RNA-sequencing in canine spleens that were histologically confirmed as hemangiosarcoma, nodular hyperplasia, or normal. Twenty-two miRNAs were found to be differentially expressed in hemangiosarcoma samples (4 between hemangiosarcoma and both nodular hyperplasia and normal spleen and 18 between hemangiosarcoma and normal spleen only). In particular, mir-26a, mir-126, mir-139, mir-140, mir-150, mir-203, mir-424, mir-503, mir-505, mir-542, mir-30e, mir-33b, mir-365, mir-758, mir-22, and mir-452 are of interest in the pathogenesis of hemangiosarcoma. Findings of this study confirm the hypothesis that miRNA expression profiles are different between canine splenic hemangiosarcoma, nodular hyperplasia, and normal spleens. A large portion of the differentially expressed miRNAs have roles in angiogenesis, with an additional group of miRNAs being dysregulated in vascular disease processes. Two other miRNAs have been implicated in cancer pathways such as PTEN and cell cycle checkpoints. The finding of multiple miRNAs with roles in angiogenesis and vascular disease is important, as hemangiosarcoma is a tumor of endothelial cells, which are driven by angiogenic stimuli. This study shows that miRNA dysregulation is a potential player in the pathogenesis of canine splenic hemangiosarcoma.

  16. Expression profiling of c-kit and its impact after esiRNA silencing during gonadal development in catfish.

    Laldinsangi, C; Senthilkumaran, B

    2018-04-03

    C-kit receptor is a member of a family of growth factor receptors that have tyrosine kinase activity, and are involved in the transduction of growth regulatory signals across plasma membrane by activation of its ligand, kitl/scf. The present study analysed mRNA and protein expression profiles of c-kit in the gonads of catfish, Clarias gariepinus, using real time PCR, in situ hybridization and immunohistochemistry. Tissue distribution analysis revealed higher expression mainly in the catfish gonads. Ontogeny studies showed minimal expression during early developmental stages and highest during 50-75 days post hatch, and the dimorphic expression in gonads decreased gradually till adulthood, which might suggest an important role for this gene around later stages of sex differentiation and gonadal development. Expression of C-kit was analysed at various phases of gonadal cycle in both male and female, which showed minimal expression during the resting phase, and higher expression in male compared to females during the pre-spawning phase. In vitro and in vivo induction using human chorionic gonadotropin elevated the expression of c-kit indicating the regulatory influence of hypothalamo-hypophyseal axis. In vivo transient gene silencing using c-kit-esiRNA in adult catfish during gonadal recrudescence showed a decrease in c-kit expression, which affected the expression level of germ cell meiotic marker sycp3, as well as several factors and steroidogenic enzyme genes involved in germ cell development. Decrease in the levels of serum 11-KT and T were also observed after esiRNA silencing. The findings of this study suggest that c-kit has an important role in the process of germ cell proliferation, development and maturation during gonadal development and recrudescence in catfish. Copyright © 2018. Published by Elsevier Inc.

  17. Zika virus alters the microRNA expression profile and elicits an RNAi response in Aedes aegypti mosquitoes.

    Miguel A Saldaña

    2017-07-01

    Full Text Available Zika virus (ZIKV, a flavivirus transmitted primarily by Aedes aegypti, has recently spread globally in an unprecedented fashion, yet we have a poor understanding of host-microbe interactions in this system. To gain insights into the interplay between ZIKV and the mosquito, we sequenced the small RNA profiles in ZIKV-infected and non-infected Ae. aegypti mosquitoes at 2, 7 and 14 days post-infection. ZIKA induced an RNAi response in the mosquito with virus-derived short interfering RNAs and PIWI-interacting RNAs dramatically increased in abundance post-infection. Further, we found 17 host microRNAs (miRNAs that were modulated by ZIKV infection at all time points. Strikingly, many of these regulated miRNAs have been reported to have their expression altered by dengue and West Nile viruses, while the response was divergent from that induced by the alphavirus Chikungunya virus in mosquitoes. This suggests that conserved miRNA responses occur within mosquitoes in response to flavivirus infection. This study expands our understanding of ZIKV-vector interactions and provides potential avenues to be further investigated to target ZIKV in the mosquito host.

  18. Classification of follicular cell-derived thyroid cancer by global RNA profiling

    Rossing, Maria

    2013-01-01

    The incidence of thyroid cancer is increasing worldwide and thyroid nodules are a frequent clinical finding. Diagnosing follicular cell-derived cancers is, however, challenging both histopathologically and especially cytopathologically. The advent of high-throughput molecular technologies has...... profiling of follicular cell-derived thyroid cancers....... prompted many researchers to explore the transcriptome and, in recent years, also the miRNome in order to generate new molecular classifiers capable of classifying thyroid tumours more accurately than by conventional cytopathological and histopathological methods. This has led to a number of molecular...

  19. Cytokine mRNA profiles in pigs exposed prenatally and postnatally to Schistosoma japonicum

    Techau, Michala E.; Johansen, Maria V.; Aasted, Bent

    2007-01-01

    of septal fibrosis were significantly higher in the postnatal group compared to the prenatal group (P prenatally infected animals compared to the control...... group (P prenatal group showed higher levels of TGF-beta 1 in the liver compared with the postnatally infected group (P control group (P prenatally exposed pigs.......The pig is a natural host for Schistosoma japonicum and a useful animal model of human infection. The aim of the present study was to assess the differences between the cytokine profiles in prenatally or postnatally S. japonicum exposed pigs. Seven prenatally exposed pigs, 7 postnatally exposed...

  20. Transcriptional Profiling and Identification of Heat-Responsive Genes in Perennial Ryegrass by RNA-Sequencing

    Kehua Wang

    2017-06-01

    Full Text Available Perennial ryegrass (Lolium perenne is one of the most widely used forage and turf grasses in the world due to its desirable agronomic qualities. However, as a cool-season perennial grass species, high temperature is a major factor limiting its performance in warmer and transition regions. In this study, a de novo transcriptome was generated using a cDNA library constructed from perennial ryegrass leaves subjected to short-term heat stress treatment. Then the expression profiling and identification of perennial ryegrass heat response genes by digital gene expression analyses was performed. The goal of this work was to produce expression profiles of high temperature stress responsive genes in perennial ryegrass leaves and further identify the potentially important candidate genes with altered levels of transcript, such as those genes involved in transcriptional regulation, antioxidant responses, plant hormones and signal transduction, and cellular metabolism. The de novo assembly of perennial ryegrass transcriptome in this study obtained more total and annotated unigenes compared to previously published ones. Many DEGs identified were genes that are known to respond to heat stress in plants, including HSFs, HSPs, and antioxidant related genes. In the meanwhile, we also identified four gene candidates mainly involved in C4 carbon fixation, and one TOR gene. Their exact roles in plant heat stress response need to dissect further. This study would be important by providing the gene resources for improving heat stress tolerance in both perennial ryegrass and other cool-season perennial grass plants.

  1. Pathophysiological implications of the chemical messengers

    Blazquez Fernandez, E.

    2009-01-01

    To maintain a physical organization and a different composition of its surroundings environment, living beings use a great part of the energy that they produce. Vital processes require an elevated number of reactions which are regulated and integrated by chemical messengers. They use autocrine, paracrine, endocrine and synaptic signals through receptors of cell surface, nuclear or associated with ionic channels, enzymes, trim eric G proteins and to intracellular kinases. Through these mechanisms pheromones play an important role in the relationships between different individuals, and hormones are able to regulate the integrative functions of our organism. In the nervous system, neurotransmitters, neuromodulators, sensors and receptors between other messengers, play functions of great relevance, while growth factors stimulate cell proliferation and cytokines have many effects but the most important is the ones related with the control of the immflamatory process. Alterations of these messengers permit us a better understanding of the diseases and possibly of its treatments in a near future. Modifications of the expression of genes from the nuclear and mitochondrial genomes are responsible of monogenic, polygenic and mitochondrial diseases, while alterations in the activities of dopamine and serotonin neurotransmitters are related with schizophrenia, Parkinson disease and depression, respectively. Other example is the hyperthyroidism of the Graves-Bassedow disease due to the competitive interference of the LATS immunoglobulin with TSH at the level of the follicular cells producing thyroid hormones Twenty five years ago in the reviews on the mechanisms of insulin action, there was presentations in which the insulin receptor was located in the plasma membrane of the target cells while in the cytoplasm only a big interrogative was observed, that at present is replaced by chemical mediators cascades responsible of the multiple effects of insulin. This finding is similar

  2. Mercury's Na Exosphere from MESSENGER Data

    Killen, Rosemary M.; Burger, M. H.; Cassidy, T. A.; Sarantos, M.; Vervack, R. J.; McClintock, W. El; Merkel, A. W.; Sprague, A. L.; Solomon, S. C.

    2012-01-01

    MESSENGER entered orbit about Mercury on March 18, 2011. Since then, the Ultraviolet and Visible Spectrometer (UWS) channel of MESSENGER's Mercury Atmospheric and Surface Composition Spectrometer (MASCS) has been observing Mercury's exosphere nearly continuously. Daily measurements of Na brightness were fitted with non-uniform exospheric models. With Monte Carlo sampling we traced the trajectories of a representative number of test particles, generally one million per run per source process, until photoionization, escape from the gravitational well, or permanent sticking at the surface removed the atom from the simulation. Atoms were assumed to partially thermally accommodate on each encounter with the surface with accommodation coefficient 0.25. Runs for different assumed source processes are run separately, scaled and co-added. Once these model results were saved onto a 3D grid, we ran lines of sight from the MESSENGER spacecraft :0 infinity using the SPICE kernels and we computed brightness integrals. Note that only particles that contribute to the measurement can be constrained with our method. Atoms and molecules produced on the nightside must escape the shadow in order to scatter light if the excitation process is resonant-light scattering, as assumed here. The aggregate distribution of Na atoms fits a 1200 K gas, with a PSD distribution, along with a hotter component. Our models constrain the hot component, assumed to be impact vaporization, to be emitted with a 2500 K Maxwellian. Most orbits show a dawnside enhancement in the hot component broadly spread over the leading hemisphere. However, on some dates there is no dawn/dusk asymmetry. The portion of the hot/cold source appears to be highly variable.

  3. Monocytic microRNA profile associated with coronary collateral artery function in chronic total occlusion patients.

    Hakimzadeh, Nazanin; Elias, Joëlle; Wijntjens, Gilbert W M; Theunissen, Ruud; van Weert, Angela; Smulders, Martijn W; van den Akker, Nynke; Moerland, Perry D; Verberne, Hein J; Hoebers, Loes P; Henriques, Jose P S; van der Laan, Anja M; Ilhan, Mustafa; Post, Mark; Bekkers, Sebastiaan C A M; Piek, Jan J

    2017-05-08

    An expansive collateral artery network is correlated with improved survival in case of adverse cardiac episodes. We aimed to identify cellular microRNAs (miRNA; miR) important for collateral artery growth. Chronic total occlusion (CTO) patients (n = 26) were dichotomized using pressure-derived collateral flow index (CFI p ) measurements; high collateral capacity (CFI p  > 0.39; n = 14) and low collateral (CFI p  collateral capacity patients. Validation by real-time polymerase chain reaction demonstrated significantly decreased expression of miR339-5p in all stimulated monocyte phenotypes of low collateral capacity patients. MiR339-5p showed significant correlation with CFI p values in stimulated monocytes. Ingenuity pathway analysis of predicted gene targets of miR339-5p and differential gene expression data from high versus low CFI p patients (n = 20), revealed significant association with STAT3 pathway, and also suggested a possible regulatory role for this signaling pathway. These results identify a novel association between miR339-5p and coronary collateral function. Future work examining modulation of miR339-5p and downstream effects on the STAT3 pathway and subsequent collateral vessel growth are warranted.

  4. Gravitational Waves and Multi-Messenger Astronomy

    Centrella, Joan M.

    2010-01-01

    Gravitational waves are produced by a wide variety of sources throughout the cosmos, including the mergers of black hole and neutron star binaries/compact objects spiraling into central black holes in galactic nuclei, close compact binaries/and phase transitions and quantum fluctuations in the early universe. Observing these signals can bring new, and often very precise, information about their sources across vast stretches of cosmic time. In this talk we will focus on thee opening of this gravitational-wave window on the universe, highlighting new opportunities for discovery and multi-messenger astronomy.

  5. Change in circulating microRNA profile of obese children indicates future risk of adult diabetes.

    Cui, Xianwei; You, Lianghui; Zhu, Lijun; Wang, Xing; Zhou, Yahui; Li, Yun; Wen, Juan; Xia, Yankai; Wang, Xinru; Ji, Chenbo; Guo, Xirong

    2018-01-01

    Childhood obesity increases susceptibility to type 2 diabetes (T2D) in adults. Circulating microRNAs (miRNAs) in serum have been proposed as potential diagnostic biomarkers, and they may contribute to the progression toward T2D. Here, we investigated the possibility of predicting the future risk of adult T2D in obese children by using circulating miRNAs. We performed miRNA high-throughput sequencing to screen relevant circulating miRNAs in obese children. The expression patterns of targeted miRNAs were further explored in obese children and adults with T2D. To investigate the underlying contributions of these miRNAs to the development of T2D, we detected the impacts of the candidate miRNAs on preadipocyte proliferation, insulin secretion by pancreatic β-cell, and glucose uptake by skeletal muscle cells. Three miRNAs (miR-486, miR-146b and miR-15b), whose expression in the circulation was most dramatically augmented in obese children and adult T2D patients, were selected for further investigation. Of these 3 miRNAs, miR-486 was implicated in accelerating preadipocyte proliferation and myotube glucose intolerance, miR-146b and miR-15b were engaged in the suppression of high concentration glucose-induced pancreatic insulin secretion, and they all contributed to the pathological processes of obesity and T2D. Our results provide a better understanding of the role of circulating miRNAs, particularly miR-486, miR-146b and miR-15b, in predicting the future risk of T2D in obese children. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  6. Characterization and comparative profiling of the small RNA transcriptomes in the Hemipteran insect Nilaparvata lugens.

    Zha, Wenjun; Zhou, Lei; Li, Sanhe; Liu, Kai; Yang, Guocai; Chen, Zhijun; Liu, Kai; Xu, Huashan; Li, Peide; Hussain, Saddam; You, Aiqing

    2016-12-20

    MicroRNAs (miRNAs) are a group of small RNAs involved in various biological processes through negative regulation of mRNAs at the post-transcriptional level. The brown planthopper (BPH), Nilaparvata lugens (Stål), is one of the most serious and destructive insect pests of rice. In the present study, two small RNA libraries of virulent N. lugens populations (Biotype I survives on susceptive rice variety TN1 and Biotype Y survives on moderately resistant rice variety YHY15) were constructed and sequenced using the high-throughput sequencing technology in order to identify the relationship between miRNAs of N.lugens and adaptation of BPH pests to rice resistance. In total 15,758,632 and 11,442,592 reads, corresponding to 3,144,026 and 2,550,049 unique sequences, were obtained in the two libraries (BPH-TN1 and BPH-YHY15 libraries), respectively. A total of 41 potential novel miRNAs were predicted in the two libraries, and 26 miRNAs showed significantly differential expression between two libraries. All miRNAs were significantly up-regulated in the BPH-TN1 library. Target genes likely regulated by these differentially expressed miRNAs were predicted using computational prediction. The functional annotation of target genes performed by Gene Ontology enrichment (GO) and Kyoto Encyclopedia of Genes and Genomes pathway analysis (KEGG) indicated that a majority of differential miRNAs were involved in "Metabolism" pathway. These results provided an understanding of the role of miRNAs in BPH to adaptability of BPH on rice resistance, and will be useful in developing new control strategies for host defense against BPH. Copyright © 2016 Elsevier B.V. All rights reserved.

  7. Nutritional status affects the microRNA profile of the hypothalamus of female sheep.

    Yang, Heng; Lin, Shan; Lei, Xiaoping; Yuan, Cong; Yu, Yaosheng; Zhao, Zongsheng; Chen, Jingbo

    2018-01-25

    Recent studies on the seasonal regulation of the oestrous cycle in sheep have focussed mainly on the responses to photoperiod. However, the brain systems that control reproductive activity also respond to nutritional inputs, although the molecular mechanisms involved are not completely understood. One possibility is that small, non-coding RNAs, such as micro-RNAs (miRNAs), have significant influence. In the present study, the amounts and characteristics of miRNAs in hypothalamus from oestrous and anestrous ewes, fed low- or high-nutrient diets, were compared using Illumina HiSeq sequencing technology. In total, 398 miRNAs, including 261 novel miRNAs, were identified in ewes with an enhanced nutritional status (HEN), whereas 384 miRNAs, including 247 novel miRNAs, were identified in the ewes with a lesser nutritional status (HAN). There were eight conserved and 140 novel miRNAs expressed differentially between the two libraries. Based on quantitative real-time polymerase chain reaction, six miRNAs were assessed to verify the accuracy of the library database. Moreover, the correlation between the miRNA target and several upstream and downstream genes in the oestrus-related pathways were also verified in hypothalamus nerve cells. According to the results, nutritional status plays an important role in oestrous regulation in sheep, and the hypothalamic processes and pathways induced by nutritional signals (folic acid and tyrosine) are different from those induced by photoperiodic regulation of oestrus. We have expanded the repertoire of sheep miRNAs that could contribute to the molecular mechanisms that regulate the initiation of oestrous cycles in anestrous ewes in response to the influence of nutritional status.

  8. Long non-coding RNA expression profile in cervical cancer tissues

    Zhu, Hua; Chen, Xiangjian; Hu, Yan; Shi, Zhengzheng; Zhou, Qing; Zheng, Jingjie; Wang, Yifeng

    2017-01-01

    Cervical cancer (CC), one of the most common types of cancer of the female population, presents an enormous challenge in diagnosis and treatment. Long non-coding (lnc)RNAs, non-coding (nc)RNAs with length >200 nucleotides, have been identified to be associated with multiple types of cancer, including CC. This class of nc transcripts serves an important role in tumor suppression and oncogenic signaling pathways. In the present study, the microarray method was used to obtain the expression profile of lncRNAs and protein-coding mRNAs and to compare the expression of lncRNAs between CC tissues and corresponding adjacent non-cancerous tissues in order to screen potential lncRNAs for associations with CC. Overall, 3356 lncRNAs with significantly different expression pattern in CC tissues compared with adjacent non-cancerous tissues were identified, while 1,857 of them were upregulated. These differentially expressed lncRNAs were additionally classified into 5 subgroups. Reverse transcription quantitative polymerase chain reactions were performed to validate the expression pattern of 5 random selected lncRNAs, and 2lncRNAs were identified to have significantly different expression in CC samples compared with adjacent non-cancerous tissues. This finding suggests that those lncRNAs with different expression may serve important roles in the development of CC, and the expression data may provide information for additional study on the involvement of lncRNAs in CC. PMID:28789353

  9. Peripheral blood RNA gene expression profiling in illicit methcathinone users reveals effect on immune system

    Katrin eSikk

    2011-08-01

    Full Text Available Methcathinone (ephedrone is relatively easily accessible for abuse. Its users develop an extrapyramidal syndrome and it is not known if this is caused by methcathinone itself, by side-ingredients (manganese, or both. In the present study we aimed to clarify molecular mechanisms underlying this condition. We analyzed whole genome gene expression patterns of peripheral blood from 20 methcathinone users and 20 matched controls. Gene expression profile data was analyzed by Bayesian modelling and functional annotation. In order to verify the genechip results we performed quantitative real-time (RT PCR in selected genes. 326 out of analyzed 28,869 genes showed statistically significant differential expression with FDR adjusted p-values below 0.05. Quantitative RT-PCR confirmed differential expression for the most of selected genes. Functional annotation and network analysis indicated that most of the genes were related to activation immunological disease, cellular movement and cardiovascular disease gene network (enrichment score 42. As HIV and HCV infections were confounding factors, we performed additional stratification of patients. A similar functional activation of the immunological disease pathway was evident when we compared patients according to the injection status (past versus current users, balanced for HIV and HCV infection. However, this difference was not large therefore the major effect was related to the HIV status of the patients. Mn-methcathinone abusers have blood transcriptional patterns mostly caused by their HIV and HCV infections.

  10. Multi-Messenger Astronomy and Dark Matter

    Bergström, Lars

    This chapter presents the elaborated lecture notes on Multi-Messenger Astronomy and Dark Matter given by Lars Bergström at the 40th Saas-Fee Advanced Course on "Astrophysics at Very High Energies". One of the main problems of astrophysics and astro-particle physics is that the nature of dark matter remains unsolved. There are basically three complementary approaches to try to solve this problem. One is the detection of new particles with accelerators, the second is the observation of various types of messengers from radio waves to gamma-ray photons and neutrinos, and the third is the use of ingenious experiments for direct detection of dark matter particles. After giving an introduction to the particle universe, the author discusses the relic density of particles, basic cross sections for neutrinos and gamma-rays, supersymmetric dark matter, detection methods for neutralino dark matter, particular dark matter candidates, the status of dark matter detection, a detailled calculation on an hypothetical "Saas-Fee Wimp", primordial black holes, and gravitational waves.

  11. mRNA profiling reveals determinants of trastuzumab efficiency in HER2-positive breast cancer.

    Silvia von der Heyde

    Full Text Available Intrinsic and acquired resistance to the monoclonal antibody drug trastuzumab is a major problem in the treatment of HER2-positive breast cancer. A deeper understanding of the underlying mechanisms could help to develop new agents. Our intention was to detect genes and single nucleotide polymorphisms (SNPs affecting trastuzumab efficiency in cell culture. Three HER2-positive breast cancer cell lines with different resistance phenotypes were analyzed. We chose BT474 as model of trastuzumab sensitivity, HCC1954 as model of intrinsic resistance, and BTR50, derived from BT474, as model of acquired resistance. Based on RNA-Seq data, we performed differential expression analyses on these cell lines with and without trastuzumab treatment. Differentially expressed genes between the resistant cell lines and BT474 are expected to contribute to resistance. Differentially expressed genes between untreated and trastuzumab treated BT474 are expected to contribute to drug efficacy. To exclude false positives from the candidate gene set, we removed genes that were also differentially expressed between untreated and trastuzumab treated BTR50. We further searched for SNPs in the untreated cell lines which could contribute to trastuzumab resistance. The analysis resulted in 54 differentially expressed candidate genes that might be connected to trastuzumab efficiency. 90% of 40 selected candidates were validated by RT-qPCR. ALPP, CALCOCO1, CAV1, CYP1A2 and IGFBP3 were significantly higher expressed in the trastuzumab treated than in the untreated BT474 cell line. GDF15, IL8, LCN2, PTGS2 and 20 other genes were significantly higher expressed in HCC1954 than in BT474, while NCAM2, COLEC12, AFF3, TFF3, NRCAM, GREB1 and TFF1 were significantly lower expressed. Additionally, we inferred SNPs in HCC1954 for CAV1, PTGS2, IL8 and IGFBP3. The latter also had a variation in BTR50. 20% of the validated subset have already been mentioned in literature. For half of them we

  12. Hepatitis C virus (HCV) RNA profiles among chronic HIV/HCV-coinfected individuals in ESPRIT; spontaneous HCV RNA clearance observed in nine individuals

    Grint, D; Tedaldi, Ellen; Peters, L

    2017-01-01

    OBJECTIVES: Studies have shown that hepatitis C virus (HCV) RNA levels remain stable over time in HIV/HCV-coinfected individuals taking combination antiretroviral therapy (cART), while spontaneous clearance of HCV RNA during the persistent infection phase has been documented only rarely among tho...

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

    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.

  14. Characterization of MicroRNA Expression Profiles and Identification of Potential Biomarkers in Leprosy.

    Jorge, Karina T O S; Souza, Renan P; Assis, Marieta T A; Araújo, Marcelo G; Locati, Massimo; Jesus, Amélia M R; Dias Baptista, Ida M F; Lima, Cristiano X; Teixeira, Antônio L; Teixeira, Mauro M; Soriani, Frederico M

    2017-05-01

    Leprosy is an important cause of disability in the developing world. Early diagnosis is essential to allow for cure and to interrupt transmission of this infection. MicroRNAs (miRNAs) are important factors for host-pathogen interaction and they have been identified as biomarkers for various infectious diseases. The expression profile of 377 microRNAs were analyzed by TaqMan low-density array (TLDA) in skin lesions of tuberculoid and lepromatous leprosy patients as well as skin specimens from healthy controls. In a second step, 16 microRNAs were selected for validation experiments with reverse transcription-quantitative PCR (qRT-PCR) in skin samples from new individuals. Principal-component analysis followed by logistic regression model and receiver operating characteristic (ROC) curve analyses were performed to evaluate the diagnostic potential of selected miRNAs. Four patterns of differential expression were identified in the TLDA experiment, suggesting a diagnostic potential of miRNAs in leprosy. After validation experiments, a combination of four miRNAs (miR-101, miR-196b, miR-27b, and miR-29c) was revealed as able to discriminate between healthy control and leprosy patients with 80% sensitivity and 91% specificity. This set of miRNAs was also able to discriminate between lepromatous and tuberculoid patients with a sensitivity of 83% and 80% specificity. In this work, it was possible to identify a set of miRNAs with good diagnostic potential for leprosy. Copyright © 2017 American Society for Microbiology.

  15. Unravelling the complexity of microRNA-mediated gene regulation in black pepper (Piper nigrum L.) using high-throughput small RNA profiling.

    Asha, Srinivasan; Sreekumar, Sweda; Soniya, E V

    2016-01-01

    Analysis of high-throughput small RNA deep sequencing data, in combination with black pepper transcriptome sequences revealed microRNA-mediated gene regulation in black pepper ( Piper nigrum L.). Black pepper is an important spice crop and its berries are used worldwide as a natural food additive that contributes unique flavour to foods. In the present study to characterize microRNAs from black pepper, we generated a small RNA library from black pepper leaf and sequenced it by Illumina high-throughput sequencing technology. MicroRNAs belonging to a total of 303 conserved miRNA families were identified from the sRNAome data. Subsequent analysis from recently sequenced black pepper transcriptome confirmed precursor sequences of 50 conserved miRNAs and four potential novel miRNA candidates. Stem-loop qRT-PCR experiments demonstrated differential expression of eight conserved miRNAs in black pepper. Computational analysis of targets of the miRNAs showed 223 potential black pepper unigene targets that encode diverse transcription factors and enzymes involved in plant development, disease resistance, metabolic and signalling pathways. RLM-RACE experiments further mapped miRNA-mediated cleavage at five of the mRNA targets. In addition, miRNA isoforms corresponding to 18 miRNA families were also identified from black pepper. This study presents the first large-scale identification of microRNAs from black pepper and provides the foundation for the future studies of miRNA-mediated gene regulation of stress responses and diverse metabolic processes in black pepper.

  16. Unique gene expression profile of the proliferating Xenopus tadpole tail blastema cells deciphered by RNA-sequencing analysis.

    Hiroshi Tsujioka

    Full Text Available Organ regenerative ability depends on the animal species and the developmental stage. The molecular bases for variable organ regenerative ability, however, remain unknown. Previous studies have identified genes preferentially expressed in the blastema tissues in various animals, but transcriptome analysis of the isolated proliferating blastema cells has not yet been reported. In the present study, we used RNA-sequencing analysis to analyze the gene expression profile of isolated proliferating blastema cells of regenerating Xenopus laevis tadpole tails. We used flow cytometry to isolate proliferating cells, and non-proliferating blastema cells, from regenerating tadpole tails as well as proliferating tail bud cells from tail bud embryos, the latter two of which were used as control cells, based on their DNA content. Among the 28 candidate genes identified by RNA-sequencing analysis, quantitative reverse transcription-polymerase chain reaction identified 10 genes whose expression was enriched in regenerating tadpole tails compared with non-regenerating tadpole tails or tails from the tail bud embryos. Among them, whole mount in situ hybridization revealed that chromosome segregation 1-like and interleukin 11 were expressed in the broad area of the tail blastema, while brevican, lysyl oxidase, and keratin 18 were mainly expressed in the notochord bud in regenerating tails. We further combined whole mount in situ hybridization with immunohistochemistry for the incorporated 5-bromo-2-deoxyuridine to confirm that keratin 18 and interleukin 11 were expressed in the proliferating tail blastema cells. Based on the proposed functions of their homologs in other animal species, these genes might have roles in the extracellular matrix formation in the notochord bud (brevican and lysyl oxidase, cell proliferation (chromosome segregation 1-like and keratin 18, and in the maintenance of the differentiation ability of proliferating blastema cells (interleukin 11

  17. Analysis of microRNA Expression Profiles Induced by Yiqifumai Injection in Rats with Chronic Heart Failure

    Yu Zhao

    2018-02-01

    Full Text Available Background: Yiqifumai Injection (YQFM is clinically used to treat various cardiovascular diseases including chronic heart failure (CHF. The efficacy of YQFM for treating heart failure has been suggested, but the mechanism of action for pharmacological effects of YQFM is unclear.Methods: Echocardiography detection, left ventricular intubation evaluation, histopathology and immunohistochemical examination were performed in CHF rats to evaluate the cardioprotective effect of YQFM. Rat miRNA microarray and bioinformatics analysis were employed to investigate the differentially expressed microRNAs. In vitro models of AngII-induced hypertrophy and t-BHP induced oxidative stress in H9c2 myocardial cells were used to validate the anti-hypertrophy and anti-apoptosis effects of YQFM. Measurement of cell surface area, ATP content and cell viability, Real-time PCR and Western blot were performed.Results: YQFM significantly improved the cardiac function of CHF rats by increasing left ventricular ejection fraction and fractional shortening, decreasing left ventricular internal diameter and enhancing cardiac output. Seven microRNAs which have a reversible regulation by YQFM treatment were found. Among them, miR-21-3p and miR-542-3p are related to myocardial hypertrophy and cell proliferation, respectively and were further verified by RT-PCR. Target gene network was established and potential related signaling pathways were predicted. YQFM could significantly alleviate AngII induced hypertrophy in cellular model. It also significantly increased cell viabilities and ATP content in t-BHP induced apoptotic cell model. Western blot analysis showed that YQFM could increase the phosphorylation of Akt.Conclusion: Our findings provided scientific evidence to uncover the mechanism of action of YQFM on miRNAs regulation against CHF by miRNA expression profile technology. The results indicated that YQFM has a potential effect on alleviate cardiac hypertrophy and apoptosis

  18. Deep RNA-Seq profile reveals biodiversity, plant-microbe interactions and a large family of NBS-LRR resistance genes in walnut (Juglans regia) tissues.

    Chakraborty, Sandeep; Britton, Monica; Martínez-García, P J; Dandekar, Abhaya M

    2016-03-01

    Deep RNA-Seq profiling, a revolutionary method used for quantifying transcriptional levels, often includes non-specific transcripts from other co-existing organisms in spite of stringent protocols. Using the recently published walnut genome sequence as a filter, we present a broad analysis of the RNA-Seq derived transcriptome profiles obtained from twenty different tissues to extract the biodiversity and possible plant-microbe interactions in the walnut ecosystem in California. Since the residual nature of the transcripts being analyzed does not provide sufficient information to identify the exact strain, inferences made are constrained to the genus level. The presence of the pathogenic oomycete Phytophthora was detected in the root through the presence of a glyceraldehyde-3-phosphate dehydrogenase. Cryptococcus, the causal agent of cryptococcosis, was found in the catkins and vegetative buds, corroborating previous work indicating that the plant surface supported the sexual cycle of this human pathogen. The RNA-Seq profile revealed several species of the endophytic nitrogen fixing Actinobacteria. Another bacterial species implicated in aerobic biodegradation of methyl tert-butyl ether (Methylibium petroleiphilum) is also found in the root. RNA encoding proteins from the pea aphid were found in the leaves and vegetative buds, while a serine protease from mosquito with significant homology to a female reproductive tract protease from Drosophila mojavensis in the vegetative bud suggests egg-laying activities. The comprehensive analysis of RNA-seq data present also unraveled detailed, tissue-specific information of ~400 transcripts encoded by the largest family of resistance (R) genes (NBS-LRR), which possibly rationalizes the resistance of the specific walnut plant to the pathogens detected. Thus, we elucidate the biodiversity and possible plant-microbe interactions in several walnut (Juglans regia) tissues in California using deep RNA-Seq profiling.

  19. Selective 2′-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP) for direct, versatile, and accurate RNA structure analysis

    Smola, Matthew J.; Rice, Greggory M.; Busan, Steven; Siegfried, Nathan A.; Weeks, Kevin M.

    2016-01-01

    SHAPE chemistries exploit small electrophilic reagents that react with the 2′-hydroxyl group to interrogate RNA structure at single-nucleotide resolution. Mutational profiling (MaP) identifies modified residues based on the ability of reverse transcriptase to misread a SHAPE-modified nucleotide and then counting the resulting mutations by massively parallel sequencing. The SHAPE-MaP approach measures the structure of large and transcriptome-wide systems as accurately as for simple model RNAs. This protocol describes the experimental steps, implemented over three days, required to perform SHAPE probing and construct multiplexed SHAPE-MaP libraries suitable for deep sequencing. These steps include RNA folding and SHAPE structure probing, mutational profiling by reverse transcription, library construction, and sequencing. Automated processing of MaP sequencing data is accomplished using two software packages. ShapeMapper converts raw sequencing files into mutational profiles, creates SHAPE reactivity plots, and provides useful troubleshooting information, often within an hour. SuperFold uses these data to model RNA secondary structures, identify regions with well-defined structures, and visualize probable and alternative helices, often in under a day. We illustrate these algorithms with the E. coli thiamine pyrophosphate riboswitch, E. coli 16S rRNA, and HIV-1 genomic RNAs. SHAPE-MaP can be used to make nucleotide-resolution biophysical measurements of individual RNA motifs, rare components of complex RNA ensembles, and entire transcriptomes. The straightforward MaP strategy greatly expands the number, length, and complexity of analyzable RNA structures. PMID:26426499

  20. MicroRNA profiling of the bovine alveolar macrophage response to Mycobacterium bovis infection suggests pathogen survival is enhanced by microRNA regulation of endocytosis and lysosome trafficking

    BRADLEY, DANIEL

    2015-01-01

    PUBLISHED Mycobacterium bovis, the causative agent of bovine tuberculosis, a major problem for global agriculture, spreads via an airborne route and is taken up by alveolar macrophages (AM) in the lung. Here, we describe the first next-generation sequencing (RNA-seq) approach to temporally profile miRNA expression in primary bovine AMs post-infection with M. bovis. One, six, and forty miRNAs were identified as significantly differentially expressed at 2, 24 and 48 h post-infection, respect...

  1. Analysis of plasma microRNA expression profiles in male textile workers with noise-induced hearing loss.

    Ding, Lu; Liu, Jing; Shen, Huan-Xi; Pan, Li-Ping; Liu, Qing-Dong; Zhang, Heng-Dong; Han, Lei; Shuai, Li-Guo; Ding, En-Min; Zhao, Qiu-Ni; Wang, Bo-Shen; Zhu, Bao-Li

    2016-03-01

    Circulating microRNAs (miRNAs) have attracted interests as non-invasive biomarkers of physiological and pathological conditions, which may be applied in noise-induced hearing loss (NIHL). However, no epidemiology studies have yet examined the potential effects of NIHL or noise exposure on miRNA expression profiles. We sought to identify permanent NIHL-related miRNAs and to predict the biological functions of the putative genes encoding the indicated miRNAs. In the discovery stage, we used a microarray assay to detect the miRNA expression profiles between pooled plasma samples from 10 noise-exposed individuals with normal hearing and 10 NIHL patients. In addition, we conducted a preliminary validation of six candidate miRNAs in the same 20 workers. Subsequently, three miRNAs were selected for expanded validation in 23 non-exposed individuals with normal hearing and 46 noise-exposed textile workers which including 23 noise-exposed workers with normal hearing and 23 NIHL patients. Moreover, we predicted the biological functions of the putative target genes using a Gene Ontology (GO) function enrichment analysis. In the discovery stage, compared with the noise exposures with normal hearing, 73 miRNAs demonstrated at least a 1.5-fold differential expression in the NIHL patients. In the preliminary validation, compared with the noise exposures, the plasma levels of miR-16-5p, miR-24-3p, miR-185-5p and miR-451a were all upregulated (P < 0.001) in the NIHL patients. In the expanded validation stage, compared with the non-exposures, the plasma levels of miR-24, miR-185-5p and miR-451a were all significantly downregulated (P < 0.001) in the exposures. And compared with the noise exposures, the plasma levels of miR-185-5p and miR-451a were slightly elevated (P < 0.001) in the NIHL patients, which were consistent with the results of preliminary validation and microarray analysis. The two indicated plasma miRNAs may be biomarkers of indicating responses to noise exposure

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

    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. Analysis of Biobanked Serum from a Mycobacterium avium subsp paratuberculosis Bovine Infection Model Confirms the Remarkable Stability of Circulating miRNA Profiles and Defines a Bovine Serum miRNA Repertoire.

    Ronan G Shaughnessy

    Full Text Available Johne's Disease (JD is a chronic enteritis of ruminants caused by Mycobacterium avium subspecies paratuberculosis (MAP. Current disease control strategies are hampered by the lack of sensitive and specific diagnostic modalities. Therefore, novel diagnostic and prognostic tools are needed, and circulating microRNAs (miRNAs may hold potential in this area. The aims of this study were twofold: (i to address the stability of miRNA in bovine sera from biobanked samples, and (ii to assess the potential of miRNAs as biomarkers for JD disease progression. To address these aims we used bovine sera from an experimental MAP infection model that had been stored at -20°C for over a decade, allowing us to also assess the stability of miRNA profiles in biobanked serum samples through comparison with fresh sera. Approximately 100-200 intact miRNAs were identified in each sample with 83 of these being consistently detected across all 57 samples. The miRNA profile of the biobanked sera stored at -20°C for over 10 years was highly similar to the profile of <1 year-old sera stored at -80°C, with an overlap of 73 shared miRNAs. IsomiR analysis also indicated a distinct bovine serum-specific isomiR profile as compared to previously reported bovine macrophage miRNA profiles. To explore the prognostic potential of miRNA profiles cattle defined as seropositive for anti-MAP antibodies (n = 5 were compared against seronegative cattle (n = 7. No significant differential expressed miRNAs were detected at either the early (6 months or late (43, 46 and 49 months intervals (FDR≤0.05, fold-change≥1.5 across seropositive or seronegative animals. However, comparing pre-infection sera to the early and late time-points identified increased miR-29a and miR-92b abundance (2-fold that may be due to blood-cell population changes over time (P<0.001. In conclusion our study has demonstrated that bovine circulating miRNAs retain their integrity under long-term sub-optimal storage

  4. Long non-coding RNA profile in mantle cell lymphoma identifies a functional lncRNA ROR1-AS1 associated with EZH2/PRC2 complex

    Hu, Guangzhen; Gupta, Shiv K.; Troska, Tammy P.; Nair, Asha; Gupta, Mamta

    2017-01-01

    Mantle cell lymphoma (MCL) is an aggressive B-cell lymphoma characterized by rapid disease progression. The needs for new therapeutic strategies for MCL patients call for further understanding on the molecular mechanisms of pathogenesis of MCL. Recently, long noncoding RNAs (lncRNAs) have been recognized as key regulators of gene expression and disease development, however, the role of lncRNAs in non-Hodgkin lymphoma and specifically in MCL is still unknown. Next generation RNA-sequencing was carried out on MCL patient samples along with normal controls and data was analyzed. As a result, several novel lncRNAs were found significantly overexpressed in the MCL samples with lncRNA ROR1-AS1 the most significant one. We cloned the ROR1-AS1 lncRNA in expression vector and ectopically transfected in MCL cell lines. Results showed that overexpression of ROR1-AS1 lncRNA promoted growth of MCL cells while decreased sensitivity to the treatment with drugs ibrutinib and dexamethasone. ROR-AS1 overexpression also decreased the mRNA expression of P16 (P = 0.21), and SOX11 (p = 0.017), without much effect on P53, ATM and P14 mRNA. RNA-immunoprecipitation assays demonstrated high affinity binding of lncRNA ROR1-AS1 with EZH2 and SUZ12 proteins of the polycomb repressive complex-2 (PRC2). Suppressing EZH2 activity with pharmacological inhibitor GSK343 abolished binding of ROR1-AS1 with EZH2. Taken together, this study identified a functional lncRNA ROR-AS1 involved with regulation of gene transcription via associating with PRC2 complex, and may serve as a novel biomarker in MCL patients. PMID:29113297

  5. Natural RNA circles function as efficient microRNA sponges

    Hansen, Thomas Birkballe; Jensen, Trine I; Clausen, Bettina Hjelm

    2013-01-01

    MicroRNAs (miRNAs) are important post-transcriptional regulators of gene expression that act by direct base pairing to target sites within untranslated regions of messenger RNAs. Recently, miRNA activity has been shown to be affected by the presence of miRNA sponge transcripts, the so-called comp......MicroRNAs (miRNAs) are important post-transcriptional regulators of gene expression that act by direct base pairing to target sites within untranslated regions of messenger RNAs. Recently, miRNA activity has been shown to be affected by the presence of miRNA sponge transcripts, the so......-called competing endogenous RNA in humans and target mimicry in plants. We previously identified a highly expressed circular RNA (circRNA) in human and mouse brain. Here we show that this circRNA acts as a miR-7 sponge; we term this circular transcript ciRS-7 (circular RNA sponge for miR-7). ciRS-7 contains more...... sponge, suggesting that miRNA sponge effects achieved by circRNA formation are a general phenomenon. This study serves as the first, to our knowledge, functional analysis of a naturally expressed circRNA....

  6. Sustained miRNA-mediated Knockdown of Mutant AAT With Simultaneous Augmentation of Wild-type AAT Has Minimal Effect on Global Liver miRNA Profiles

    2013-01-01

    α-1 antitrypsin (AAT) deficiency can exhibit two pathologic states: a lung disease that is primarily due to the loss of AAT's antiprotease function, and a liver disease resulting from a toxic gain-of-function of the PiZ-AAT (Z-AAT) mutant protein. We have developed several recombinant adeno-associated virus (rAAV) vectors that incorporate microRNA (miRNA) sequences targeting the AAT gene while also driving the expression of miRNA-resistant wild-type AAT-PiM (M-AAT) gene, thus achieving concom...

  7. Mercury's Interior from MESSENGER Radio Science Data

    Genova, A.; Mazarico, E.; Goossens, S. J.; Lemoine, F. G.; Neumann, G. A.; Smith, D. E.; Zuber, M. T.

    2017-12-01

    The MErcury Surface, Space ENvironment, GEochemistry, and Ranging (MESSENGER) spacecraft provided precise radio tracking data in orbit about Mercury for more than 4 years, from March 2011 to April 2015. These geodetic measurements enable us to investigate the interior structure of the planet from the inner core to the crust. The first three years of radio data allowed us to determine the gravity field of Mercury with a resolution of 150 km in the northern hemisphere (degree and order 50 in spherical harmonics) since the periapsis was located at higher latitudes (>65˚N) and 200-500 km altitudes. The comparison of this gravity solution with Mercury's topography, which was retrieved by using over 25 million individual measurements of the Mercury Laser Altimeter (MLA), resulted in a preliminary map of the crustal thickness of the planet. However, those results were limited by the resolution of the gravity field since the topography was defined in spherical harmonics up to degree and order 125. The last year of the MESSENGER extended mission was dedicated to a low-altitude campaign, where the spacecraft periapsis was maintained at altitudes between 25 and 100 km. The radio data collected during this mission phase allowed us to significantly improve the resolution of the gravity field locally in the northern hemisphere up to degree and order 100 in spherical harmonics. We present the gravity anomalies and crustal thickness maps that lead to a better understanding on the formation and evolution of specific regions. We present our estimated orientation model, which slightly differs from the solutions that were obtained by using Earth-based radar measurements and the co-registration of MESSENGER imaging and altimetry data. These previous estimates provide a direct measurement of the surface response, whereas the orientation model from gravity is more sensitive to the inner and outer core. A discrepancy between core and surface obliquities may provide fundamental

  8. Genome-wide dynamic transcriptional profiling in clostridium beijerinckii NCIMB 8052 using single-nucleotide resolution RNA-Seq

    Wang Yi

    2012-03-01

    Full Text Available Abstract Background Clostridium beijerinckii is a prominent solvent-producing microbe that has great potential for biofuel and chemical industries. Although transcriptional analysis is essential to understand gene functions and regulation and thus elucidate proper strategies for further strain improvement, limited information is available on the genome-wide transcriptional analysis for C. beijerinckii. Results The genome-wide transcriptional dynamics of C. beijerinckii NCIMB 8052 over a batch fermentation process was investigated using high-throughput RNA-Seq technology. The gene expression profiles indicated that the glycolysis genes were highly expressed throughout the fermentation, with comparatively more active expression during acidogenesis phase. The expression of acid formation genes was down-regulated at the onset of solvent formation, in accordance with the metabolic pathway shift from acidogenesis to solventogenesis. The acetone formation gene (adc, as a part of the sol operon, exhibited highly-coordinated expression with the other sol genes. Out of the > 20 genes encoding alcohol dehydrogenase in C. beijerinckii, Cbei_1722 and Cbei_2181 were highly up-regulated at the onset of solventogenesis, corresponding to their key roles in primary alcohol production. Most sporulation genes in C. beijerinckii 8052 demonstrated similar temporal expression patterns to those observed in B. subtilis and C. acetobutylicum, while sporulation sigma factor genes sigE and sigG exhibited accelerated and stronger expression in C. beijerinckii 8052, which is consistent with the more rapid forespore and endspore development in this strain. Global expression patterns for specific gene functional classes were examined using self-organizing map analysis. The genes associated with specific functional classes demonstrated global expression profiles corresponding to the cell physiological variation and metabolic pathway switch. Conclusions The results from this

  9. siRNAs targeted to certain polyadenylation sites promote specific, RISC-independent degradation of messenger RNAs.

    Vickers, Timothy A; Crooke, Stanley T

    2012-07-01

    While most siRNAs induce sequence-specific target mRNA cleavage and degradation in a process mediated by Ago2/RNA-induced silencing complex (RISC), certain siRNAs have also been demonstrated to direct target RNA reduction through deadenylation and subsequent degradation of target transcripts in a process which involves Ago1/RISC and P-bodies. In the current study, we present data suggesting that a third class of siRNA exist, which are capable of promoting target RNA reduction that is independent of both Ago and RISC. These siRNAs bind the target messenger RNA at the polyA signal and are capable of redirecting a small amount of polyadenylation to downstream polyA sites when present, however, the majority of the activity appears to be due to inhibition of polyadenylation or deadenylation of the transcript, followed by exosomal degradation of the immature mRNA.

  10. Transcription Profiling Demonstrates Epigenetic Control of Non-retroviral RNA Virus-Derived Elements in the Human Genome

    Kozue Sofuku

    2015-09-01

    Full Text Available Endogenous bornavirus-like nucleoprotein elements (EBLNs are DNA sequences in vertebrate genomes formed by the retrotransposon-mediated integration of ancient bornavirus sequence. Thus, EBLNs evidence a mechanism of retrotransposon-mediated RNA-to-DNA information flow from environment to animals. Although EBLNs are non-transposable, they share some features with retrotransposons. Here, to test whether hosts control the expression of EBLNs similarly to retrotransposons, we profiled the transcription of all Homo sapiens EBLNs (hsEBLN-1 to hsEBLN-7. We could detect transcription of all hsEBLNs in at least one tissue. Among them, hsEBLN-1 is transcribed almost exclusively in the testis. In most tissues, expression from the hsEBLN-1 locus is silenced epigenetically. Finally, we showed the possibility that hsEBLN-1 integration at this locus affects the expression of a neighboring gene. Our results suggest that hosts regulate the expression of endogenous non-retroviral virus elements similarly to how they regulate the expression of retrotransposons, possibly contributing to new transcripts and regulatory complexity to the human genome.

  11. Ribosome profiling-guided depletion of an mRNA increases cell growth rate and protein secretion

    Beuchert Kallehauge, Thomas; Li, Shangzhong; Pedersen, Lasse Ebdrup

    2017-01-01

    Recombinant protein production coopts the host cell machinery to provide high protein yields of industrial enzymes or biotherapeutics. However, since protein translation is energetically expensive and tightly controlled, it is unclear if highly expressed recombinant genes are translated as effici......Recombinant protein production coopts the host cell machinery to provide high protein yields of industrial enzymes or biotherapeutics. However, since protein translation is energetically expensive and tightly controlled, it is unclear if highly expressed recombinant genes are translated...... as efficiently as host genes. Furthermore, it is unclear how the high expression impacts global translation. Here, we present the first genome-wide view of protein translation in an IgG-producing CHO cell line, measured with ribosome profiling. Through this we found that our recombinant mRNAs were translated...... as efficiently as the host cell transcriptome, and sequestered up to 15% of the total ribosome occupancy. During cell culture, changes in recombinant mRNA translation were consistent with changes in transcription, demonstrating that transcript levels influence specific productivity. Using this information, we...

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

    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.

  13. Application of RNA interference in treating human diseases

    ference than either strand individually. After injection into ... antisense strand to messenger RNAs (mRNAs) that bear ... processing of longer dsRNA and stem loop precursors (Nov- ... RNAi has several applications in biomedical research,.

  14. Deep sequencing uncovers commonality in small RNA profiles between transgene-induced and naturally occurring RNA silencing of chalcone synthase-A gene in petunia.

    Kasai, Megumi; Matsumura, Hideo; Yoshida, Kentaro; Terauchi, Ryohei; Taneda, Akito; Kanazawa, Akira

    2013-01-30

    Introduction of a transgene that transcribes RNA homologous to an endogenous gene in the plant genome can induce silencing of both genes, a phenomenon termed cosuppression. Cosuppression was first discovered in transgenic petunia plants transformed with the CHS-A gene encoding chalcone synthase, in which nonpigmented sectors in flowers or completely white flowers are produced. Some of the flower-color patterns observed in transgenic petunias having CHS-A cosuppression resemble those in existing nontransgenic varieties. Although the mechanism by which white sectors are generated in nontransgenic petunia is known to be due to RNA silencing of the CHS-A gene as in cosuppression, whether the same trigger(s) and/or pattern of RNA degradation are involved in these phenomena has not been known. Here, we addressed this question using deep-sequencing and bioinformatic analyses of small RNAs. We analyzed short interfering RNAs (siRNAs) produced in nonpigmented sectors of petal tissues in transgenic petunia plants that have CHS-A cosuppression and a nontransgenic petunia variety Red Star, that has naturally occurring CHS-A RNA silencing. In both silencing systems, 21-nt and 22-nt siRNAs were the most and the second-most abundant size classes, respectively. CHS-A siRNA production was confined to exon 2, indicating that RNA degradation through the RNA silencing pathway occurred in this exon. Common siRNAs were detected in cosuppression and naturally occurring RNA silencing, and their ranks based on the number of siRNAs in these plants were correlated with each other. Noticeably, highly abundant siRNAs were common in these systems. Phased siRNAs were detected in multiple phases at multiple sites, and some of the ends of the regions that produced phased siRNAs were conserved. The features of siRNA production found to be common to cosuppression and naturally occurring silencing of the CHS-A gene indicate mechanistic similarities between these silencing systems especially in the

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

    Grace Truong

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

  16. The adipokine leptin increases skeletal muscle mass and significantly alters skeletal muscle miRNA expression profile in aged mice

    Hamrick, Mark W.; Herberg, Samuel; Arounleut, Phonepasong; He, Hong-Zhi; Shiver, Austin; Qi, Rui-Qun; Zhou, Li; Isales, Carlos M.

    2010-01-01

    Research highlights: → Aging is associated with muscle atrophy and loss of muscle mass, known as the sarcopenia of aging. → We demonstrate that age-related muscle atrophy is associated with marked changes in miRNA expression in muscle. → Treating aged mice with the adipokine leptin significantly increased muscle mass and the expression of miRNAs involved in muscle repair. → Recombinant leptin therapy may therefore be a novel approach for treating age-related muscle atrophy. -- Abstract: Age-associated loss of muscle mass, or sarcopenia, contributes directly to frailty and an increased risk of falls and fractures among the elderly. Aged mice and elderly adults both show decreased muscle mass as well as relatively low levels of the fat-derived hormone leptin. Here we demonstrate that loss of muscle mass and myofiber size with aging in mice is associated with significant changes in the expression of specific miRNAs. Aging altered the expression of 57 miRNAs in mouse skeletal muscle, and many of these miRNAs are now reported to be associated specifically with age-related muscle atrophy. These include miR-221, previously identified in studies of myogenesis and muscle development as playing a role in the proliferation and terminal differentiation of myogenic precursors. We also treated aged mice with recombinant leptin, to determine whether leptin therapy could improve muscle mass and alter the miRNA expression profile of aging skeletal muscle. Leptin treatment significantly increased hindlimb muscle mass and extensor digitorum longus fiber size in aged mice. Furthermore, the expression of 37 miRNAs was altered in muscles of leptin-treated mice. In particular, leptin treatment increased the expression of miR-31 and miR-223, miRNAs known to be elevated during muscle regeneration and repair. These findings suggest that aging in skeletal muscle is associated with marked changes in the expression of specific miRNAs, and that nutrient-related hormones such as leptin

  17. The adipokine leptin increases skeletal muscle mass and significantly alters skeletal muscle miRNA expression profile in aged mice

    Hamrick, Mark W., E-mail: mhamrick@mail.mcg.edu [Department of Cellular Biology and Anatomy, Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, GA (United States); Department of Orthopaedic Surgery, Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, GA (United States); Herberg, Samuel; Arounleut, Phonepasong [Department of Cellular Biology and Anatomy, Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, GA (United States); Department of Orthopaedic Surgery, Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, GA (United States); He, Hong-Zhi [Henry Ford Immunology Program, Henry Ford Health System, Detroit, MI (United States); Department of Dermatology, Henry Ford Health System, Detroit, MI (United States); Shiver, Austin [Department of Cellular Biology and Anatomy, Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, GA (United States); Department of Orthopaedic Surgery, Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, GA (United States); Qi, Rui-Qun [Henry Ford Immunology Program, Henry Ford Health System, Detroit, MI (United States); Department of Dermatology, Henry Ford Health System, Detroit, MI (United States); Zhou, Li [Henry Ford Immunology Program, Henry Ford Health System, Detroit, MI (United States); Department of Dermatology, Henry Ford Health System, Detroit, MI (United States); Department of Internal Medicine, Henry Ford Health System, Detroit, MI (United States); Isales, Carlos M. [Department of Cellular Biology and Anatomy, Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, GA (United States); Department of Orthopaedic Surgery, Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, GA (United States); others, and

    2010-09-24

    Research highlights: {yields} Aging is associated with muscle atrophy and loss of muscle mass, known as the sarcopenia of aging. {yields} We demonstrate that age-related muscle atrophy is associated with marked changes in miRNA expression in muscle. {yields} Treating aged mice with the adipokine leptin significantly increased muscle mass and the expression of miRNAs involved in muscle repair. {yields} Recombinant leptin therapy may therefore be a novel approach for treating age-related muscle atrophy. -- Abstract: Age-associated loss of muscle mass, or sarcopenia, contributes directly to frailty and an increased risk of falls and fractures among the elderly. Aged mice and elderly adults both show decreased muscle mass as well as relatively low levels of the fat-derived hormone leptin. Here we demonstrate that loss of muscle mass and myofiber size with aging in mice is associated with significant changes in the expression of specific miRNAs. Aging altered the expression of 57 miRNAs in mouse skeletal muscle, and many of these miRNAs are now reported to be associated specifically with age-related muscle atrophy. These include miR-221, previously identified in studies of myogenesis and muscle development as playing a role in the proliferation and terminal differentiation of myogenic precursors. We also treated aged mice with recombinant leptin, to determine whether leptin therapy could improve muscle mass and alter the miRNA expression profile of aging skeletal muscle. Leptin treatment significantly increased hindlimb muscle mass and extensor digitorum longus fiber size in aged mice. Furthermore, the expression of 37 miRNAs was altered in muscles of leptin-treated mice. In particular, leptin treatment increased the expression of miR-31 and miR-223, miRNAs known to be elevated during muscle regeneration and repair. These findings suggest that aging in skeletal muscle is associated with marked changes in the expression of specific miRNAs, and that nutrient

  18. Molecular risk assessment of BIG 1-98 participants by expression profiling using RNA from archival tissue

    Antonov, Janine; Altermatt, Hans Jörg; Aebi, Stefan; Jaggi, Rolf; Popovici, Vlad; Delorenzi, Mauro; Wirapati, Pratyaksha; Baltzer, Anna; Oberli, Andrea; Thürlimann, Beat; Giobbie-Hurder, Anita; Viale, Giuseppe

    2010-01-01

    The purpose of the work reported here is to test reliable molecular profiles using routinely processed formalin-fixed paraffin-embedded (FFPE) tissues from participants of the clinical trial BIG 1-98 with a median follow-up of 60 months. RNA from fresh frozen (FF) and FFPE tumor samples of 82 patients were used for quality control, and independent FFPE tissues of 342 postmenopausal participants of BIG 1-98 with ER-positive cancer were analyzed by measuring prospectively selected genes and computing scores representing the functions of the estrogen receptor (eight genes, ER-8), the progesterone receptor (five genes, PGR-5), Her2 (two genes, HER2-2), and proliferation (ten genes, PRO-10) by quantitative reverse transcription PCR (qRT-PCR) on TaqMan Low Density Arrays. Molecular scores were computed for each category and ER-8, PGR-5, HER2-2, and PRO-10 scores were combined into a RISK-25 score. Pearson correlation coefficients between FF- and FFPE-derived scores were at least 0.94 and high concordance was observed between molecular scores and immunohistochemical data. The HER2-2, PGR-5, PRO-10 and RISK-25 scores were significant predictors of disease free-survival (DFS) in univariate Cox proportional hazard regression. PRO-10 and RISK-25 scores predicted DFS in patients with histological grade II breast cancer and in lymph node positive disease. The PRO-10 and PGR-5 scores were independent predictors of DFS in multivariate Cox regression models incorporating clinical risk indicators; PRO-10 outperformed Ki-67 labeling index in multivariate Cox proportional hazard analyses. Scores representing the endocrine responsiveness and proliferation status of breast cancers were developed from gene expression analyses based on RNA derived from FFPE tissues. The validation of the molecular scores with tumor samples of participants of the BIG 1-98 trial demonstrates that such scores can serve as independent prognostic factors to estimate disease free survival (DFS) in

  19. Comparative profiling of miRNA expression in developing seeds of high linoleic and high oleic safflower (Carthamus tinctorius L. plants

    Shijiang eCao

    2013-12-01

    Full Text Available Vegetable oils high in oleic acid are considered to be advantageous because of their better nutritional value and potential industrial applications. The oleic acid content in the classic safflower oil is normally 10-15% while a natural mutant (ol accumulates elevated oleic acid up to 70% in seed oil. As a part of our investigation into the molecular features of the high oleic (HO trait in safflower we have profiled the microRNA (miRNA populations in developing safflower seeds expressing the ol allele in comparison to the wild type high linoleic (HL safflower using deep sequencing technology. The small RNA populations of the mid-maturity developing embryos of homozygous ol HO and wild type HL safflower had a very similar size distribution pattern, however, only ~16.5% of the unique small RNAs were overlapping in these two genotypes. From these two small RNA populations we have found 55 known miRNAs and identified two candidate novel miRNA families to be likely unique to the developing safflower seeds. Target genes with conserved as well as novel functions were predicted for the conserved miRNAs. We have also identified 13 miRNAs differentially expressed between the HO and HL safflower genotypes. The results may lay a foundation for unravelling the miRNA-mediated molecular processes that regulate oleic acid accumulation in the HO safflower mutant and developmental processes in safflower embryos in general.

  20. Exosomes as divine messengers: are they the Hermes of modern molecular oncology?

    Braicu, C; Tomuleasa, C; Monroig, P; Cucuianu, A; Berindan-Neagoe, I; Calin, G A

    2015-01-01

    Exosomes are cell-derived vesicles that convey key elements with the potential to modulate intercellular communication. They are known to be secreted from all types of cells, and are crucial messengers that can regulate cellular processes by ‘trafficking' molecules from cells of one tissue to another. The exosomal content has been shown to be broad, composed of different types of cytokines, growth factors, proteins, or nucleic acids. Besides messenger RNA (mRNA) they can also contain noncoding transcripts such as microRNAs (miRNAs), which are small endogenous cellular regulators of protein expression. In diseases such as cancer, exosomes can facilitate tumor progression by altering their vesicular content and supplying the tumor niche with molecules that favor the progression of oncogenic processes such as proliferation, invasion and metastasis, or even drug resistance. The packaging of their molecular content is known to be tissue specific, a fact that makes them interesting tools in clinical diagnostics and ideal candidates for biomarkers. In the current report, we describe the main properties of exosomes and explain their involvement in processes such as cell differentiation and cell death. Furthermore, we emphasize the need of developing patient-targeted treatments by applying the conceptualization of exosomal-derived miRNA-based therapeutics. PMID:25236394

  1. MESSENGER MERCURY RSS/MLA LEVEL 5 DERIVED DATA V1.0

    National Aeronautics and Space Administration — This data set contains archival results from radio science investigations conducted during the MESSENGER mission. Radio measurements were made using the MESSENGER...

  2. HLA-G allelic variants are associated with differences in the HLA-G mRNA isoform profile and HLA-G mRNA levels

    Hviid, Thomas Vauvert F; Hylenius, Sine; Rørbye, Christina

    2003-01-01

    between mother and fetus in several ways. Finally, the expression of membrane-bound HLA-G and soluble HLA-G has been proposed to influence the outcome of pregnancy, and an aberrant HLA-G expression in pre-eclamptic placentas and spontaneous abortions has been reported. Here, an association between certain...... HLA-G polymorphisms and the mRNA levels of the different alternatively spliced HLA-G isoforms in first trimester trophoblast cell populations is reported. Several alternatively spliced HLA-G mRNA isoforms, including a 14-bp polymorphism in the 3'UTR end (exon 8) of the HLA-G gene, are expressed...

  3. RNA Localization in Astrocytes

    Thomsen, Rune

    2012-01-01

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

  4. Comparison of transcriptional profiles of Clostridium thermocellum grown on cellobiose and pretreated yellow poplar using RNA-Seq

    Hui eWei

    2014-04-01

    Full Text Available The anaerobic, thermophilic bacterium, Clostridium thermocellum, secretes multi-protein enzyme complexes, termed cellulosomes, which synergistically interact with the microbial cell surface and efficiently disassemble plant cell wall biomass. C. thermocellum has also been considered a potential consolidated bioprocessing (CBP organism due to its ability to produce the biofuel products, hydrogen and ethanol. We found that C. thermocellum fermentation of pretreated yellow poplar (PYP produced 30% and 39% of ethanol and hydrogen product concentrations, respectively, compared to fermentation of cellobiose. RNA-seq was used to analyze the transcriptional profiles of these cells. The PYP-grown cells taken for analysis at the late stationary phase showed 1211 genes up-regulated and 314 down-regulated by more than 2-fold compared to the cellobiose-grown cells. These affected genes cover a broad spectrum of specific functional categories. The transcriptional analysis was further validated by sub-proteomics data taken from the literature; as well as by quantitative reverse transcription-PCR (qRT-PCR analyses of selected genes. Specifically, 47 cellulosomal protein-encoding genes, genes for 4 pairs of SigI-RsgI for polysaccharide sensing, 7 cellodextrin ABC transporter genes, and a set of NAD(PH hydogenase and alcohol dehydrogenase genes were up-regulated for cells growing on PYP compared to cellobiose. These genes could be potential candidates for future studies aimed at gaining insight into the regulatory mechanism of this organism as well as for improvement of C. thermocellum in its role as a CBP organism.

  5. Effects of clofibric acid on mRNA expression profiles in primary cultures of rat, mouse and human hepatocytes

    Richert, Lysiane; Lamboley, Christelle; Viollon-Abadie, Catherine; Grass, Peter; Hartmann, Nicole; Laurent, Stephane; Heyd, Bruno; Mantion, Georges; Chibout, Salah-Dine; Staedtler, Frank

    2003-01-01

    The mRNA expression profile in control and clofibric acid (CLO)-treated mouse, rat, and human hepatocytes was analyzed using species-specific oligonucleotide DNA microarrays (Affymetrix). A statistical empirical Bayes procedure was applied in order to select the significantly differentially expressed genes. Treatment with the peroxisome proliferator CLO induced up-regulation of genes involved in peroxisome proliferation and in cell proliferation as well as down-regulation of genes involved in apoptosis in hepatocytes of rodent but not of human origin. CLO treatment induced up-regulation of microsomal cytochrome P450 4a genes in rodent hepatocytes and in two of six human hepatocyte cultures. In addition, genes encoding phenobarbital-inducible cytochrome P450s were also up-regulated by CLO in rodent and human hepatocyte cultures. Up-regulation of phenobarbital-inducible UDP-glucuronosyl-transferase genes by CLO was observed in both rat and human but not in mouse hepatocytes. CLO treatment induced up-regulation of L-fatty acid binding protein (L-FABP) gene in hepatocytes of both rodent and human origin. However, while genes of the cytosolic, microsomal, and mitochondrial pathways involved in fatty acid transport and metabolism were up-regulated by CLO in both rodent and human hepatocyte cultures, genes of the peroxisomal pathway of lipid metabolism were up-regulated in rodents only. An up-regulation of hepatocyte nuclear factor 1α (HNF1α) by CLO was observed only in human hepatocyte cultures, suggesting that this trans-activating factor may play a key role in the regulation of fatty acid metabolism in human liver as well as in the nonresponsiveness of human liver to CLO-induced regulation of cell proliferation and apoptosis

  6. Effects of clofibric acid on mRNA expression profiles in primary cultures of rat, mouse and human hepatocytes.

    Richert, Lysiane; Lamboley, Christelle; Viollon-Abadie, Catherine; Grass, Peter; Hartmann, Nicole; Laurent, Stephane; Heyd, Bruno; Mantion, Georges; Chibout, Salah-Dine; Staedtler, Frank

    2003-09-01

    The mRNA expression profile in control and clofibric acid (CLO)-treated mouse, rat, and human hepatocytes was analyzed using species-specific oligonucleotide DNA microarrays (Affymetrix). A statistical empirical Bayes procedure was applied in order to select the significantly differentially expressed genes. Treatment with the peroxisome proliferator CLO induced up-regulation of genes involved in peroxisome proliferation and in cell proliferation as well as down-regulation of genes involved in apoptosis in hepatocytes of rodent but not of human origin. CLO treatment induced up-regulation of microsomal cytochrome P450 4a genes in rodent hepatocytes and in two of six human hepatocyte cultures. In addition, genes encoding phenobarbital-inducible cytochrome P450s were also up-regulated by CLO in rodent and human hepatocyte cultures. Up-regulation of phenobarbital-inducible UDP-glucuronosyl-transferase genes by CLO was observed in both rat and human but not in mouse hepatocytes. CLO treatment induced up-regulation of L-fatty acid binding protein (L-FABP) gene in hepatocytes of both rodent and human origin. However, while genes of the cytosolic, microsomal, and mitochondrial pathways involved in fatty acid transport and metabolism were up-regulated by CLO in both rodent and human hepatocyte cultures, genes of the peroxisomal pathway of lipid metabolism were up-regulated in rodents only. An up-regulation of hepatocyte nuclear factor 1alpha (HNF1alpha) by CLO was observed only in human hepatocyte cultures, suggesting that this trans-activating factor may play a key role in the regulation of fatty acid metabolism in human liver as well as in the nonresponsiveness of human liver to CLO-induced regulation of cell proliferation and apoptosis.

  7. Dynamic transcriptome profiling of the floral buds in the dioecious cucurbit Coccinia grandis using RNA-Seq

    Jatindra Nath Mohanty

    2017-10-01

    Full Text Available Angiosperms exhibits diversified sexual systems encompassing bisexual, monoecious and dioecious conditions. Dioecy offers opportunities to explore separately, the male and female systems giving an insight into the evolutionary, developmental and molecular processes of sex expression in plants. Coccinia grandis (Family: Cucurbitaceae with small genome size and heteromorphic sex chromosomes is often considered a model dioecious plant for sex evolution. However, the information relating to its genetic orientation, physical state and sex determining factors is highly ambiguous and limited. In the present study we have attempted to identify the molecular basis of sex determination in C. grandis through genome wide transcriptome profiling of the floral buds. About 75 million clean reads were generated resulting in 72,479 unigenes for male library and 63,308 unigenes for female library with a mean length of 736 bp. 1410 unigenes were differentially expressed (DEGs between the male and female buds as identified from the RNA-Seq pattern and qRT-PCR validation. Functional annotation using BLAST2GO and KEGG revealed high enrichment of DEGs in phytohormone biosynthesis, hormone signaling and transduction, transcriptional regulation and methyl transferase activity. Manifold up-regulation of genes phytohormone responsive genes such as ARF6, ACC synthase1, SNRK2 and BRI1-associated receptor kinase 1 (BAK1 suggest that a signaling crosstalk is implicated in the sex determination of this species. Besides, a wide range of transcription factors including zinc fingers, homeodomain leucine zippers and MYBs were recognized as major determinants of male specific expression in the dioecious plant. Additionally, C. grandis transcriptome revealed 48 target genes for many miRNAs sequences with established role in floral development and sex determination. Overall, our study resulted in the identification of a large amount of molecular resources that could be critical to

  8. MicroRNA expression profile associated with response to neoadjuvant chemoradiotherapy in locally advanced rectal cancer patients

    Svoboda, Marek; Sana, Jiri; Fabian, Pavel; Kocakova, Ilona; Gombosova, Jana; Nekvindova, Jana; Radova, Lenka; Vyzula, Rostislav; Slaby, Ondrej

    2012-01-01

    Rectal cancer accounts for approximately one third of all colorectal cancers (CRC), which belong among leading causes of cancer deaths worldwide. Standard treatment for locally advanced rectal cancer (cT3/4 and/or cN+) includes neoadjuvant chemoradiotherapy with fluoropyrimidines (capecitabine or 5-fluorouracil) followed by radical surgical resection. Unfortunately, a significant proportion of tumors do not respond enough to the neoadjuvant treatment and these patients are at risk of relapse. MicroRNAs (miRNAs) are small non-coding RNAs playing significant roles in the pathogenesis of many cancers including rectal cancer. MiRNAs could present the new predictive biomarkers for rectal cancer patients. We selected 20 patients who underwent neoadjuvant chemoradiotherapy for advanced rectal cancer and whose tumors were classified as most sensitive or resistant to the treatment. These two groups were compared using large-scale miRNA expression profiling. Expression levels of 8 miRNAs significantly differed between two groups. MiR-215, miR-190b and miR-29b-2* have been overexpressed in non-responders, and let-7e, miR-196b, miR-450a, miR-450b-5p and miR-99a* have shown higher expression levels in responders. Using these miRNAs 9 of 10 responders and 9 of 10 non-responders (p < 0.05) have been correctly classified. Our pilot study suggests that miRNAs are part of the mechanisms that are involved in response of rectal cancer to the chemoradiotherapy and that miRNAs may be promising predictive biomarkers for such patients. In most miRNAs we identified (miR-215, miR-99a*, miR-196b, miR-450b-5p and let-7e), the connection between their expression and radioresistance or chemoresistance to inhibitors of thymidylate synthetase was already established

  9. Alterations in mRNA profiles of trastuzumab‑resistant Her‑2‑positive breast cancer.

    Zhao, Bin; Zhao, Yang; Sun, Yan; Niu, Haitao; Sheng, Long; Huang, Dongfang; Li, Li

    2018-05-07

    Breast cancer is one of the most common malignancies in women. Neoadjuvant trastuzumab therapy improves the prognosis of certain Her‑2‑positive breast cancer patients, however around two‑thirds of patients with Her‑2‑positive breast cancer do not benefit from Her‑2‑targeted therapy. To investigate the key mechanisms in trastuzumab resistance, potential biomarkers for neoadjuvant trastuzumab sensitivity were investigated using the gene expression omnibus (GEO) database for mRNA microarray data of Her‑2‑positive breast cancer patients who received neoadjuvant trastuzumab therapy. GEO profiles of 22 patients with a complete response and 48 patients with a partial response were identified in the GSE22358, GSE62327 and GSE66305 datasets. A total of 2,376, 1,000 and 1,152 differentially expressed genes in GSE22358, GSE62327 and GSE66305 datasets were demonstrated, respectively, utilizing GEO2R software. Furthermore, enriched gene ontology terms and Kyoto Encyclopedia of Genes and Genomes pathways were analyzed using the Database for Annotation, Visualization and Integrated Discovery software. Subsequently, a protein‑protein interaction network was established using STRING software. The results demonstrated that low sex‑determining region Y‑box 11 and high Bcl‑2 expression may be employed as markers for neoadjuvant trastuzumab therapy for Her‑2‑positive breast cancer. More importantly, phosphoinositide 3‑kinase/Akt and angiogenesis pathways, which are known to be the key targets of trastuzumab, were activated at a lower level in the partial response patients, while the Wnt and estrogen receptor signaling pathways were activated in these patients. Therefore, combination therapy of trastuzumab and anti‑Wnt or hormone therapy may be a promising treatment modality and should be tested in further studies.

  10. Human glioblastoma-associated microglia/monocytes express a distinct RNA profile compared to human control and murine samples.

    Szulzewsky, Frank; Arora, Sonali; de Witte, Lot; Ulas, Thomas; Markovic, Darko; Schultze, Joachim L; Holland, Eric C; Synowitz, Michael; Wolf, Susanne A; Kettenmann, Helmut

    2016-08-01

    Glioblastoma (GBM) is the most aggressive brain tumor in adults. It is strongly infiltrated by microglia and peripheral monocytes that support tumor growth. In the present study we used RNA sequencing to compare the expression profile of CD11b(+) human glioblastoma-associated microglia/monocytes (hGAMs) to CD11b(+) microglia isolated from non-tumor samples. Hierarchical clustering and principal component analysis showed a clear separation of the two sample groups and we identified 334 significantly regulated genes in hGAMs. In comparison to human control microglia hGAMs upregulated genes associated with mitotic cell cycle, cell migration, cell adhesion, and extracellular matrix organization. We validated the expression of several genes associated with extracellular matrix organization in samples of human control microglia, hGAMs, and the hGAMs-depleted fraction via qPCR. The comparison to murine GAMs (mGAMs) showed that both cell populations share a significant fraction of upregulated transcripts compared with their respective controls. These genes were mostly related to mitotic cell cycle. However, in contrast to murine cells, human GAMs did not upregulate genes associated to immune activation. Comparison of human and murine GAMs expression data to several data sets of in vitro-activated human macrophages and murine microglia showed that, in contrast to mGAMs, hGAMs share a smaller overlap to these data sets in general and in particular to cells activated by proinflammatory stimulation with LPS + INFγ or TNFα. Our findings provide new insights into the biology of human glioblastoma-associated microglia/monocytes and give detailed information about the validity of murine experimental models. GLIA 2016 GLIA 2016;64:1416-1436. © 2016 Wiley Periodicals, Inc.

  11. Serum microRNA expression profile distinguishes enterovirus 71 and coxsackievirus 16 infections in patients with hand-foot-and-mouth disease.

    Lunbiao Cui

    Full Text Available Altered circulating microRNA (miRNA profiles have been noted in patients with microbial infections. We compared host serum miRNA levels in patients with hand-foot-and-mouth disease (HFMD caused by enterovirus 71 (EV71 and coxsackievirus 16 (CVA16 as well as in other microbial infections and in healthy individuals. Among 664 different miRNAs analyzed using a miRNA array, 102 were up-regulated and 26 were down-regulated in sera of patients with enteroviral infections. Expression levels of ten candidate miRNAs were further evaluated by quantitative real-time PCR assays. A receiver operating characteristic (ROC curve analysis revealed that six miRNAs (miR-148a, miR-143, miR-324-3p, miR-628-3p, miR-140-5p, and miR-362-3p were able to discriminate patients with enterovirus infections from healthy controls with area under curve (AUC values ranged from 0.828 to 0.934. The combined six miRNA using multiple logistic regression analysis provided not only a sensitivity of 97.1% and a specificity of 92.7% but also a unique profile that differentiated enterovirial infections from other microbial infections. Expression levels of five miRNAs (miR-148a, miR-143, miR-324-3p, miR-545, and miR-140-5p were significantly increased in patients with CVA16 versus those with EV71 (p<0.05. Combination of miR-545, miR-324-3p, and miR-143 possessed a moderate ability to discrimination between CVA16 and EV71 with an AUC value of 0.761. These data indicate that sera from patients with different subtypes of enteroviral infection express unique miRNA profiles. Serum miRNA expression profiles may provide supplemental biomarkers for diagnosing and subtyping enteroviral HFMD infections.

  12. Calcium in Mercury's Exosphere: Modeling MESSENGER Data

    Burger, Matthew H.; Killen, Rosemary M.; McClintock, William E.; Merkel, Aimee; Vervack, Ronald J.; Sarantos, Menelaos; Sprague, Ann L.

    2011-01-01

    Mercury is surrounded by a surface-bounded exosphere comprised of atomic species including hydrogen, sodium, potassium, calcium, magnesium, and likely oxygen. Because it is collisionless. the exosphere's composition represents a balance of the active source and loss processes. The Mercury Atmospheric and Surface Composition Spectrometer (MASCS) on the MErcury Surface. Space ENvironment. GEochemistry. and Ranging (MESSENGER) spacecraft has made high spatial-resolution observations of sodium, calcium, and magnesium near Mercury's surface and in the extended, anti-sunward direction. The most striking feature of these data has been the substantial differences in the spatial distribution of each species, Our modeling demonstrates that these differences cannot be due to post-ejection dynamics such as differences in photo-ionization rate and radiation pressure. but instead point to differences in the source mechanisms and regions on the surface from which each is ejected. The observations of calcium have revealed a strong dawn/dusk asymmetry. with the abundance over the dawn hemisphere significantly greater than over the dusk. To understand this asymmetry, we use a Monte Carlo model of Mercury's exosphere that we developed to track the motions of exospheric neutrals under the influence of gravity and radiation pressure. Ca atoms can be ejected directly from the surface or produced in a molecular exosphere (e.g., one consisting of CaO). Particles are removed from the system if they stick to the surface or escape from the model region of interest (within 15 Mercury radii). Photoionization reduces the final weighting given to each particle when simulating the Ca radiance. Preliminary results suggest a high temperature ( I-2x 10(exp 4) K) source of atomic Ca concentrated over the dawn hemisphere. The high temperature is consistent with the dissociation of CaO in a near-surface exosphere with scale height <= 100 km, which imparts 2 eV to the freshly produced Ca atom. This

  13. Genomic GC-content affects the accuracy of 16S rRNA gene sequencing bsed microbial profiling due to PCR bias

    Laursen, Martin F.; Dalgaard, Marlene Danner; Bahl, Martin Iain

    2017-01-01

    Profiling of microbial community composition is frequently performed by partial 16S rRNA gene sequencing on benchtop platforms following PCR amplification of specific hypervariable regions within this gene. Accuracy and reproducibility of this strategy are two key parameters to consider, which may...... be influenced during all processes from sample collection and storage, through DNA extraction and PCR based library preparation to the final sequencing. In order to evaluate both the reproducibility and accuracy of 16S rRNA gene based microbial profiling using the Ion Torrent PGM platform, we prepared libraries...... be explained partly by premature read truncation, but to larger degree their genomic GC-content, which correlated negatively with the observed relative abundances, suggesting a PCR bias against GC-rich species during library preparation. Increasing the initial denaturation time during the PCR amplification...

  14. miRNA expression profiles in cerebrospinal fluid and blood of patients with Alzheimer's disease and other types of dementia - an exploratory study

    Sørensen, Sofie Sølvsten; Hillig, Ann-Britt Nygaard; Christensen, Thomas

    2016-01-01

    . The purpose of this exploratory investigation was to analyze the expression of miRNAs in CSF and blood of patients with Alzheimer's disease (AD) and other neurodegenerative disorders in order to identify potential miRNA biomarker candidates able to separate AD from other types of dementia. METHODS: CSF...... was collected by lumbar puncture performed on 10 patients diagnosed with AD and 10 patients diagnosed with either vascular dementia, frontotemporal dementia or dementia with Lewy bodies. Blood samples were taken immediately after. Total RNA was extracted from cell free fractions of CSF and plasma...... significantly up-regulated and miR-194-5p was significantly down-regulated in AD patients compared to controls. CONCLUSIONS: Detection of miRNA expression profiles in blood and in particular CSF of patients diagnosed with different types of dementia is feasible and it seems that several expressional differences...

  15. Sustained miRNA-mediated knockdown of mutant AAT with simultaneous augmentation of wild-type AAT has minimal effect on global liver miRNA profiles.

    Mueller, Christian; Tang, Qiushi; Gruntman, Alisha; Blomenkamp, Keith; Teckman, Jeffery; Song, Lina; Zamore, Phillip D; Flotte, Terence R

    2012-03-01

    α-1 antitrypsin (AAT) deficiency can exhibit two pathologic states: a lung disease that is primarily due to the loss of AAT's antiprotease function, and a liver disease resulting from a toxic gain-of-function of the PiZ-AAT (Z-AAT) mutant protein. We have developed several recombinant adeno-associated virus (rAAV) vectors that incorporate microRNA (miRNA) sequences targeting the AAT gene while also driving the expression of miRNA-resistant wild-type AAT-PiM (M-AAT) gene, thus achieving concomitant Z-AAT knockdown in the liver and increased expression of M-AAT. Transgenic mice expressing the human PiZ allele treated with dual-function rAAV9 vectors showed that serum PiZ was stably and persistently reduced by an average of 80%. Treated animals showed knockdown of Z-AAT in liver and serum with concomitant increased serum M-AAT as determined by allele-specific enzyme-linked immunosorbent assays (ELISAs). In addition, decreased globular accumulation of misfolded Z-AAT in hepatocytes and a reduction in inflammatory infiltrates in the liver was observed. Results from microarray studies demonstrate that endogenous miRNAs were minimally affected by this treatment. These data suggests that miRNA mediated knockdown does not saturate the miRNA pathway as has been seen with viral vector expression of short hairpin RNAs (shRNAs). This safe dual-therapy approach can be applied to other disorders such as amyotrophic lateral sclerosis, Huntington disease, cerebral ataxia, and optic atrophies.

  16. Messenger Observations of Mercury's Bow Shock and Magnetopause

    Slavin J. A.; Acuna, M. H.; Anderson, B. J.; Benna, M.; Gloeckler, G.; Krimigis, S. M.; Raines, M.; Schriver, D.; Travnicek, P.; Zurbuchen, T. H.

    2008-01-01

    The MESSENGER spacecraft made the first of three flybys of Mercury on January 14.2008 (1). New observations of solar wind interaction with Mercury were made with MESSENGER'S Magnetometer (MAG) (2.3) and Energetic Particle and Plasma Spectrometer (EPPS) - composed of the Energetic Particle Spectrometer (EPS) and Fast Imaging Plasma Spectrometer (FIPS) (3,4). These MESSENGER observations show that Mercury's magnetosphere has a large-scale structure that is distinctly Earth-like, but it is immersed in a comet-like cloud of planetary ions [5]. Fig. 1 provides a schematic view of the coupled solar wind - magnetosphere - neutral atmosphere - solid planet system at Mercury.

  17. Prospective validation of a blood-based 9-miRNA profile for early detection of breast cancer in a cohort of women examined by clinical mammography

    Lyng, Maria B; Kodahl, Annette R; Binder, Harald

    2016-01-01

    developed a 9-miRNA profile using serum and LNA-based qPCR that effectively stratified patients with early stage breast cancer vs. healthy women. To further develop the test into routine clinical practice, we collected serum of women examined by clinical mammography (N = 197) according to standard...... is significantly different between women with breast cancer and controls (p-value within one year despite being categorized as clinically healthy...

  18. Comparison of microRNA profiles between benign and malignant salivary gland tumors in tissue, blood and saliva samples: a prospective, case-control study

    Cinpolat, Ovgu; Unal, Zeynep Nil; Ismi, Onur; Gorur, Aysegul; Unal, Murat

    2017-01-01

    Abstract Introduction: Salivary gland tumors (SGTs) are rare head and neck malignancies consisting of a spectrum of tumors with different biological behaviors. Objective: In this study we aimed to find out differential expression of microRNA profiles between benign and malignant SGTs. Methods: We investigated the possible role of 95 microRNAs in the 20 patients with salivary gland tumors with comparison of 17 patients without malignancy or salivary gland diseases. Sixteen of the tumors wer...

  19. Profiles

    2004-01-01

    Profiles is a synthetic overview of more than 100 national energy markets in the world, providing insightful facts and key energy statistics. A Profile is structured around 6 main items and completed by key statistics: Ministries, public agencies, energy policy are concerned; main companies in the oil, gas, electricity and coal sectors, status, shareholders; reserve, production, imports and exports, electricity and refining capacities; deregulation of prices, subsidies, taxes; consumption trends by sector, energy market shares; main energy projects, production and consumption prospects. Statistical Profiles are present in about 3 pages the main data and indicators on oil, gas, coal and electricity. (A.L.B.)

  20. Selective 2'-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP) for direct, versatile and accurate RNA structure analysis.

    Smola, Matthew J; Rice, Greggory M; Busan, Steven; Siegfried, Nathan A; Weeks, Kevin M

    2015-11-01

    Selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) chemistries exploit small electrophilic reagents that react with 2'-hydroxyl groups to interrogate RNA structure at single-nucleotide resolution. Mutational profiling (MaP) identifies modified residues by using reverse transcriptase to misread a SHAPE-modified nucleotide and then counting the resulting mutations by massively parallel sequencing. The SHAPE-MaP approach measures the structure of large and transcriptome-wide systems as accurately as can be done for simple model RNAs. This protocol describes the experimental steps, implemented over 3 d, that are required to perform SHAPE probing and to construct multiplexed SHAPE-MaP libraries suitable for deep sequencing. Automated processing of MaP sequencing data is accomplished using two software packages. ShapeMapper converts raw sequencing files into mutational profiles, creates SHAPE reactivity plots and provides useful troubleshooting information. SuperFold uses these data to model RNA secondary structures, identify regions with well-defined structures and visualize probable and alternative helices, often in under 1 d. SHAPE-MaP can be used to make nucleotide-resolution biophysical measurements of individual RNA motifs, rare components of complex RNA ensembles and entire transcriptomes.

  1. Semiautomated improvement of RNA alignments

    Andersen, Ebbe Sloth; Lind-Thomsen, Allan; Knudsen, Bjarne

    2007-01-01

    connects to external tools to provide a flexible semiautomatic editing environment. A new method, Pcluster, is introduced for dividing the sequences of an RNA alignment into subgroups with secondary structure differences. Pcluster was used to evaluate 574 seed alignments obtained from the Rfam database...... and we identified 71 alignments with significant prediction of inconsistent base pairs and 102 alignments with significant prediction of novel base pairs. Four RNA families were used to illustrate how SARSE can be used to manually or automatically correct the inconsistent base pairs detected by Pcluster......: the mir-399 RNA, vertebrate telomase RNA (vert-TR), bacterial transfer-messenger RNA (tmRNA), and the signal recognition particle (SRP) RNA. The general use of the method is illustrated by the ability to accommodate pseudoknots and handle even large and divergent RNA families. The open architecture...

  2. MicroRNA profiling in ocular adnexal lymphoma: a role for MYC and NFKB1 mediated dysregulation of microRNA expression in aggressive disease

    Hother, Christoffer; Rasmussen, Peter Kristian; Joshi, Tejal

    2013-01-01

    Purpose. Ocular adnexal lymphoma (i.e., lymphoma with involvement of the orbit, eyelids, conjunctiva, lacrimal gland, and lacrimal sac), although rare, is common among malignant tumors involving the ocular adnexal region. The main subtypes are low-grade extranodal marginal zone lymphoma (EMZL) an...... that fundamental differences in miRNA expression exist between ocular adnexal EMZL and DLBCL, mainly due to differences in MYC and NF-ĸB regulatory pathways...

  3. Quantitative analyses of postmortem heat shock protein mRNA profiles in the occipital lobes of human cerebral cortices: implications in cause of death.

    Chung, Ukhee; Seo, Joong-Seok; Kim, Yu-Hoon; Son, Gi Hoon; Hwang, Juck-Joon

    2012-11-01

    Quantitative RNA analyses of autopsy materials to diagnose the cause and mechanism of death are challenging tasks in the field of forensic molecular pathology. Alterations in mRNA profiles can be induced by cellular stress responses during supravital reactions as well as by lethal insults at the time of death. Here, we demonstrate that several gene transcripts encoding heat shock proteins (HSPs), a gene family primarily responsible for cellular stress responses, can be differentially expressed in the occipital region of postmortem human cerebral cortices with regard to the cause of death. HSPA2 mRNA levels were higher in subjects who died due to mechanical asphyxiation (ASP), compared with those who died by traumatic injury (TI). By contrast, HSPA7 and A13 gene transcripts were much higher in the TI group than in the ASP and sudden cardiac death (SCD) groups. More importantly, relative abundances between such HSP mRNA species exhibit a stronger correlation to, and thus provide more discriminative information on, the death process than does routine normalization to a housekeeping gene. Therefore, the present study proposes alterations in HSP mRNA composition in the occipital lobe as potential forensic biological markers, which may implicate the cause and process of death.

  4. Short-term calorie restriction feminizes the mRNA profiles of drug metabolizing enzymes and transporters in livers of mice

    Fu, Zidong Donna; Klaassen, Curtis D.

    2014-01-01

    Calorie restriction (CR) is one of the most effective anti-aging interventions in mammals. A modern theory suggests that aging results from a decline in detoxification capabilities and thus accumulation of damaged macromolecules. The present study aimed to determine how short-term CR alters mRNA profiles of genes that encode metabolism and detoxification machinery in the liver. Male C57BL/6 mice were fed CR (0, 15, 30, or 40%) diets for one month, followed by mRNA quantification of 98 xenobiotic processing genes (XPGs) in the liver, including 7 uptake transporters, 39 phase-I enzymes, 37 phase-II enzymes, 10 efflux transporters, and 5 transcription factors. In general, 15% CR did not alter mRNAs of most XPGs, whereas 30 and 40% CR altered over half of the XPGs (32 increased and 29 decreased). CR up-regulated some phase-I enzymes (fold increase), such as Cyp4a14 (12), Por (2.3), Nqo1 (1.4), Fmo2 (5.4), and Fmo3 (346), and numerous number of phase-II enzymes, such as Sult1a1 (1.2), Sult1d1 (2.0), Sult1e1 (33), Sult3a1 (2.2), Gsta4 (1.3), Gstm2 (1.3), Gstm3 (1.7), and Mgst3 (2.2). CR feminized the mRNA profiles of 32 XPGs in livers of male mice. For instance, CR decreased the male-predominantly expressed Oatp1a1 (97%) and increased the female-predominantly expressed Oatp1a4 (11). In conclusion, short-term CR alters the mRNA levels of over half of the 98 XPGs quantified in livers of male mice, and over half of these alterations appear to be due to feminization of the liver. - Highlights: • Utilized a graded CR model in male mice • The mRNA profiles of xenobiotic processing genes (XPGs) in liver were investigated. • CR up-regulates many phase-II enzymes. • CR tends to feminize the mRNA profiles of XPGs

  5. Short-term calorie restriction feminizes the mRNA profiles of drug metabolizing enzymes and transporters in livers of mice

    Fu, Zidong Donna [Department of Pharmacology, Toxicology, and Therapeutics, University of Kansas Medical Center, Kansas City, KS 66160 (United States); Klaassen, Curtis D., E-mail: cklaasse@kumc.edu [Department of Internal Medicine, University of Kansas Medical Center, Kansas City, KS 66160 (United States)

    2014-01-01

    Calorie restriction (CR) is one of the most effective anti-aging interventions in mammals. A modern theory suggests that aging results from a decline in detoxification capabilities and thus accumulation of damaged macromolecules. The present study aimed to determine how short-term CR alters mRNA profiles of genes that encode metabolism and detoxification machinery in the liver. Male C57BL/6 mice were fed CR (0, 15, 30, or 40%) diets for one month, followed by mRNA quantification of 98 xenobiotic processing genes (XPGs) in the liver, including 7 uptake transporters, 39 phase-I enzymes, 37 phase-II enzymes, 10 efflux transporters, and 5 transcription factors. In general, 15% CR did not alter mRNAs of most XPGs, whereas 30 and 40% CR altered over half of the XPGs (32 increased and 29 decreased). CR up-regulated some phase-I enzymes (fold increase), such as Cyp4a14 (12), Por (2.3), Nqo1 (1.4), Fmo2 (5.4), and Fmo3 (346), and numerous number of phase-II enzymes, such as Sult1a1 (1.2), Sult1d1 (2.0), Sult1e1 (33), Sult3a1 (2.2), Gsta4 (1.3), Gstm2 (1.3), Gstm3 (1.7), and Mgst3 (2.2). CR feminized the mRNA profiles of 32 XPGs in livers of male mice. For instance, CR decreased the male-predominantly expressed Oatp1a1 (97%) and increased the female-predominantly expressed Oatp1a4 (11). In conclusion, short-term CR alters the mRNA levels of over half of the 98 XPGs quantified in livers of male mice, and over half of these alterations appear to b