Gene expression profiling via LongSAGE in a non-model plant species: a case study in seeds of Brassica napus

PubMed Central

Obermeier, Christian; Hosseini, Bashir; Friedt, Wolfgang; Snowdon, Rod

2009-01-01

Background Serial analysis of gene expression (LongSAGE) was applied for gene expression profiling in seeds of oilseed rape (Brassica napus ssp. napus). The usefulness of this technique for detailed expression profiling in a non-model organism was demonstrated for the highly complex, neither fully sequenced nor annotated genome of B. napus by applying a tag-to-gene matching strategy based on Brassica ESTs and the annotated proteome of the closely related model crucifer A. thaliana. Results Transcripts from 3,094 genes were detected at two time-points of seed development, 23 days and 35 days after pollination (DAP). Differential expression showed a shift from gene expression involved in diverse developmental processes including cell proliferation and seed coat formation at 23 DAP to more focussed metabolic processes including storage protein accumulation and lipid deposition at 35 DAP. The most abundant transcripts at 23 DAP were coding for diverse protease inhibitor proteins and proteases, including cysteine proteases involved in seed coat formation and a number of lipid transfer proteins involved in embryo pattern formation. At 35 DAP, transcripts encoding napin, cruciferin and oleosin storage proteins were most abundant. Over both time-points, 18.6% of the detected genes were matched by Brassica ESTs identified by LongSAGE tags in antisense orientation. This suggests a strong involvement of antisense transcript expression in regulatory processes during B. napus seed development. Conclusion This study underlines the potential of transcript tagging approaches for gene expression profiling in Brassica crop species via EST matching to annotated A. thaliana genes. Limits of tag detection for low-abundance transcripts can today be overcome by ultra-high throughput sequencing approaches, so that tag-based gene expression profiling may soon become the method of choice for global expression profiling in non-model species. PMID:19575793

RNAi-Mediated Knock-Down of transformer and transformer 2 to Generate Male-Only Progeny in the Oriental Fruit Fly, Bactrocera dorsalis (Hendel)

PubMed Central

Li, Jianwei; Zhang, Guifen; Wan, Fanghao

2015-01-01

The transformer (tra) gene appears to act as the genetic switch that promotes female development by interaction with the transformer2 (tra-2) gene in several dipteran species including the Medfly, housefly and Drosophila melanogaster. In this study, we describe the isolation, expression and function of tra and tra-2 in the economically important agricultural pest, the oriental fruit fly, Bactrocera dorsalis (Hendel). Bdtra and Bdtra-2 are similar to their homologs from other tephritid species. Bdtra demonstrated sex-specific transcripts: one transcript in females and two transcripts in males. In contrast, Bdtra-2 only had one transcript that was common to males and females, which was transcribed continuously in different adult tissues and developmental stages. Bdtra-2 and the female form of Bdtra were maternally inherited in eggs, whereas the male form of Bdtra was not detectable until embryos of 1 and 2 h after egg laying. Function analyses of Bdtra and Bdtra-2 indicated that both were indispensable for female development, as nearly 100% males were obtained with embryonic RNAi against either Bdtra or Bdtra-2. The fertility of these RNAi-generated males was subsequently tested. More than 80% of RNAi-generated males could mate and the mated females could lay eggs, but only 40-48.6% males gave rise to progeny. In XX-reversed males and intersex individuals, no clear female gonadal morphology was observed after dissection. These results shed light on the development of a genetic sexing system with male-only release for this agricultural pest. PMID:26057559

RNAi-Mediated Knock-Down of transformer and transformer 2 to Generate Male-Only Progeny in the Oriental Fruit Fly, Bactrocera dorsalis (Hendel).

PubMed

Liu, Guiqing; Wu, Qiang; Li, Jianwei; Zhang, Guifen; Wan, Fanghao

2015-01-01

The transformer (tra) gene appears to act as the genetic switch that promotes female development by interaction with the transformer2 (tra-2) gene in several dipteran species including the Medfly, housefly and Drosophila melanogaster. In this study, we describe the isolation, expression and function of tra and tra-2 in the economically important agricultural pest, the oriental fruit fly, Bactrocera dorsalis (Hendel). Bdtra and Bdtra-2 are similar to their homologs from other tephritid species. Bdtra demonstrated sex-specific transcripts: one transcript in females and two transcripts in males. In contrast, Bdtra-2 only had one transcript that was common to males and females, which was transcribed continuously in different adult tissues and developmental stages. Bdtra-2 and the female form of Bdtra were maternally inherited in eggs, whereas the male form of Bdtra was not detectable until embryos of 1 and 2 h after egg laying. Function analyses of Bdtra and Bdtra-2 indicated that both were indispensable for female development, as nearly 100% males were obtained with embryonic RNAi against either Bdtra or Bdtra-2. The fertility of these RNAi-generated males was subsequently tested. More than 80% of RNAi-generated males could mate and the mated females could lay eggs, but only 40-48.6% males gave rise to progeny. In XX-reversed males and intersex individuals, no clear female gonadal morphology was observed after dissection. These results shed light on the development of a genetic sexing system with male-only release for this agricultural pest.

Changes in transcriptional orientation are associated with increases in evolutionary rates of enterobacterial genes.

PubMed

Lin, Chieh-Hua; Lian, Chun-Yi; Hsiung, Chao Agnes; Chen, Feng-Chi

2011-10-05

Changes in transcriptional orientation ("CTOs") occur frequently in prokaryotic genomes. Such changes usually result from genomic inversions, which may cause a conflict between the directions of replication and transcription and an increase in mutation rate. However, CTOs do not always lead to the replication-transcription confrontation. Furthermore, CTOs may cause deleterious disruptions of operon structure and/or gene regulations. The currently existing CTOs may indicate relaxation of selection pressure. Therefore, it is of interest to investigate whether CTOs have an independent effect on the evolutionary rates of the affected genes, and whether these genes are subject to any type of selection pressure in prokaryotes. Three closely related enterbacteria, Escherichia coli, Klebsiella pneumoniae and Salmonella enterica serovar Typhimurium, were selected for comparisons of synonymous (dS) and nonsynonymous (dN) substitution rate between the genes that have experienced changes in transcriptional orientation (changed-orientation genes, "COGs") and those that do not (same-orientation genes, "SOGs"). The dN/dS ratio was also derived to evaluate the selection pressure on the analyzed genes. Confounding factors in the estimation of evolutionary rates, such as gene essentiality, gene expression level, replication-transcription confrontation, and decreased dS at gene terminals were controlled in the COG-SOG comparisons. We demonstrate that COGs have significantly higher dN and dS than SOGs when a series of confounding factors are controlled. However, the dN/dS ratios are similar between the two gene groups, suggesting that the increase in dS can sufficiently explain the increase in dN in COGs. Therefore, the increases in evolutionary rates in COGs may be mainly mutation-driven. Here we show that CTOs can increase the evolutionary rates of the affected genes. This effect is independent of the replication-transcription confrontation, which is suggested to be the major cause of inversion-associated evolutionary rate increases. The real cause of such evolutionary rate increases remains unclear but is worth further explorations.

Transcriptional Coupling of Neighboring Genes and Gene Expression Noise: Evidence that Gene Orientation and Noncoding Transcripts Are Modulators of Noise

PubMed Central

Wang, Guang-Zhong; Lercher, Martin J.; Hurst, Laurence D.

2011-01-01

Abstract How is noise in gene expression modulated? Do mechanisms of noise control impact genome organization? In yeast, the expression of one gene can affect that of a very close neighbor. As the effect is highly regionalized, we hypothesize that genes in different orientations will have differing degrees of coupled expression and, in turn, different noise levels. Divergently organized gene pairs, in particular those with bidirectional promoters, have close promoters, maximizing the likelihood that expression of one gene affects the neighbor. With more distant promoters, the same is less likely to hold for gene pairs in nondivergent orientation. Stochastic models suggest that coupled chromatin dynamics will typically result in low abundance-corrected noise (ACN). Transcription of noncoding RNA (ncRNA) from a bidirectional promoter, we thus hypothesize to be a noise-reduction, expression-priming, mechanism. The hypothesis correctly predicts that protein-coding genes with a bidirectional promoter, including those with a ncRNA partner, have lower ACN than other genes and divergent gene pairs uniquely have correlated ACN. Moreover, as predicted, ACN increases with the distance between promoters. The model also correctly predicts ncRNA transcripts to be often divergently transcribed from genes that a priori would be under selection for low noise (essential genes, protein complex genes) and that the latter genes should commonly reside in divergent orientation. Likewise, that genes with bidirectional promoters are rare subtelomerically, cluster together, and are enriched in essential gene clusters is expected and observed. We conclude that gene orientation and transcription of ncRNAs are candidate modulators of noise. PMID:21402863

Stereochemical analysis of the functional significance of the conserved inverted CCAAT and TATA elements in the rat bone sialoprotein gene promoter.

PubMed

Su, Ming; Lee, Daniel; Ganss, Bernhard; Sodek, Jaro

2006-04-14

Basal transcription of the bone sialoprotein gene is mediated by highly conserved inverted CCAAT (ICE; ATTGG) and TATA elements (TTTATA) separated by precisely 21 nucleotides. Here we studied the importance of the relative position and orientation of the CCAAT and TATA elements in the proximal promoter by measuring the transcriptional activity of a series of mutated reporter constructs in transient transfection assays. Whereas inverting the TTTATA (wild type) to a TATAAA (consensus TATA) sequence increased transcription slightly, transcription was reduced when the flanking dinucleotides were also inverted. In contrast, reversing the ATTGG (wild type; ICE) to a CCAAT (RICE) sequence caused a marked reduction in transcription, whereas both transcription and NF-Y binding were progressively increased with the simultaneous inversion of flanking nucleotides (f-RICE-f). Reducing the distance between the ICE and TATA elements produced cyclical changes in transcriptional activity that correlated with progressive alterations in the relative positions of the CCAAT and TATA elements on the face of the DNA helix. Minimal transcription was observed after 5 nucleotides were deleted (equivalent to approximately one half turn of the helix), whereas transcription was fully restored after deleting 10 nucleotides (approximately one full turn of the DNA helix), transcriptional activity being progressively lost with deletions beyond 10 nucleotides. In comparison, when deletions were made with the ICE in the reversed (f-RICE-f) orientation transcriptional activity was progressively lost with no recovery. These results show that, although transcription can still occur when the CCAAT box is reversed and/or displaced relative to the TATA box, the activity is dependent upon the flexibility of the intervening DNA helix needed to align the NF-Y complex on the CCAAT box with preinitiation complex proteins that bind to the TATA box. Thus, the precise location and orientation of the CCAAT element is necessary for optimizing basal transcription of the bone sialoprotein gene.

Characteristics of functional enrichment and gene expression level of human putative transcriptional target genes.

PubMed

Osato, Naoki

2018-01-19

Transcriptional target genes show functional enrichment of genes. However, how many and how significantly transcriptional target genes include functional enrichments are still unclear. To address these issues, I predicted human transcriptional target genes using open chromatin regions, ChIP-seq data and DNA binding sequences of transcription factors in databases, and examined functional enrichment and gene expression level of putative transcriptional target genes. Gene Ontology annotations showed four times larger numbers of functional enrichments in putative transcriptional target genes than gene expression information alone, independent of transcriptional target genes. To compare the number of functional enrichments of putative transcriptional target genes between cells or search conditions, I normalized the number of functional enrichment by calculating its ratios in the total number of transcriptional target genes. With this analysis, native putative transcriptional target genes showed the largest normalized number of functional enrichments, compared with target genes including 5-60% of randomly selected genes. The normalized number of functional enrichments was changed according to the criteria of enhancer-promoter interactions such as distance from transcriptional start sites and orientation of CTCF-binding sites. Forward-reverse orientation of CTCF-binding sites showed significantly higher normalized number of functional enrichments than the other orientations. Journal papers showed that the top five frequent functional enrichments were related to the cellular functions in the three cell types. The median expression level of transcriptional target genes changed according to the criteria of enhancer-promoter assignments (i.e. interactions) and was correlated with the changes of the normalized number of functional enrichments of transcriptional target genes. Human putative transcriptional target genes showed significant functional enrichments. Functional enrichments were related to the cellular functions. The normalized number of functional enrichments of human putative transcriptional target genes changed according to the criteria of enhancer-promoter assignments and correlated with the median expression level of the target genes. These analyses and characters of human putative transcriptional target genes would be useful to examine the criteria of enhancer-promoter assignments and to predict the novel mechanisms and factors such as DNA binding proteins and DNA sequences of enhancer-promoter interactions.

Building a minimal and generalizable model of transcription factor-based biosensors: Showcasing flavonoids.

PubMed

Trabelsi, Heykel; Koch, Mathilde; Faulon, Jean-Loup

2018-05-07

Progress in synthetic biology tools has transformed the way we engineer living cells. Applications of circuit design have reached a new level, offering solutions for metabolic engineering challenges that include developing screening approaches for libraries of pathway variants. The use of transcription-factor-based biosensors for screening has shown promising results, but the quantitative relationship between the sensors and the sensed molecules still needs more rational understanding. Herein, we have successfully developed a novel biosensor to detect pinocembrin based on a transcriptional regulator. The FdeR transcription factor (TF), known to respond to naringenin, was combined with a fluorescent reporter protein. By varying the copy number of its plasmid and the concentration of the biosensor TF through a combinatorial library, different responses have been recorded and modeled. The fitted model provides a tool to understand the impact of these parameters on the biosensor behavior in terms of dose-response and time curves and offers guidelines to build constructs oriented to increased sensitivity and or ability of linear detection at higher titers. Our model, the first to explicitly take into account the impact of plasmid copy number on biosensor sensitivity using Hill-based formalism, is able to explain uncharacterized systems without extensive knowledge of the properties of the TF. Moreover, it can be used to model the response of the biosensor to different compounds (here naringenin and pinocembrin) with minimal parameter refitting. © 2018 Wiley Periodicals, Inc.

To Your Health: NLM update transcript - Health and wealth loss

MedlinePlus

... https://medlineplus.gov/podcast/transcript062518.html To Your Health: NLM update Transcript Health and wealth loss : 06/25/2018 To use ... is what's new this week in To Your Health, a consumer health-oriented podcast from NLM, that ...

Effects of Gene Orientation and Use of Multiple Promoters on the Expression of XYL1 and XYL2 in Saccharomyces cerevisiae

NASA Astrophysics Data System (ADS)

Bae, Ju Yun; Laplaza, José; Jeffries, Thomas W.

Orientation of adjacent genes has been reported to affect their expression in eukaryotic systems, and metabolic engineering also often makes repeated use of a few promoters to obtain high expression. To improve transcriptional control in heterologous expression, we examined how these factors affect gene expression and enzymatic activity in Saccharomyces cerevisiae. We assembled d-xylose reductase (XYL1) and d-xylitol dehydrogenase (XYL2) in four ways. Each pair of genes was placed in two different tandem (l→2→ or √1√2), convergent (1→√2), and divergent (√1 2→) orientations in autonomous plasmids. The TEF1 promoter was used to drive XYL1 and the TDH3 promoter to drive XYL2 in each of the constructs. The effects of gene orientation on growth, transcription, and enzyme activity were analyzed. The transcription level as measured by quantitative PCR (q-PCR) correlated with enzyme activities, but our data did not show a significant effect of gene orientation. To test the possible dilution of promoter strength due to multiple use of the same promoter, we examined the level of expression of XYL1 driven by either the TEF1 or TDH3 promoter when carried on a single copy plasmid. We then coexpressed XYL2 from either a single or multicopy plasmid, which was also driven by the same promoter. XYL2 transcript and enzyme expression increased with plasmid copy number, while the expression of XYLl was constant regardless of the number of other TEF1 or TDH3 promoters present in the cell. According to our data, there is no significant effect of gene orientation or multiple promoter use on gene transcription and translation when genes are expressed from plasmids; however, other factors could affect expression of adjacent genes in chromosomes.

Changes in transcriptional orientation are associated with increases in evolutionary rates of enterobacterial genes

PubMed Central

2011-01-01

Background Changes in transcriptional orientation (“CTOs”) occur frequently in prokaryotic genomes. Such changes usually result from genomic inversions, which may cause a conflict between the directions of replication and transcription and an increase in mutation rate. However, CTOs do not always lead to the replication-transcription confrontation. Furthermore, CTOs may cause deleterious disruptions of operon structure and/or gene regulations. The currently existing CTOs may indicate relaxation of selection pressure. Therefore, it is of interest to investigate whether CTOs have an independent effect on the evolutionary rates of the affected genes, and whether these genes are subject to any type of selection pressure in prokaryotes. Methods Three closely related enterbacteria, Escherichia coli, Klebsiella pneumoniae and Salmonella enterica serovar Typhimurium, were selected for comparisons of synonymous (dS) and nonsynonymous (dN) substitution rate between the genes that have experienced changes in transcriptional orientation (changed-orientation genes, “COGs”) and those that do not (same-orientation genes, “SOGs”). The dN/dS ratio was also derived to evaluate the selection pressure on the analyzed genes. Confounding factors in the estimation of evolutionary rates, such as gene essentiality, gene expression level, replication-transcription confrontation, and decreased dS at gene terminals were controlled in the COG-SOG comparisons. Results We demonstrate that COGs have significantly higher dN and dS than SOGs when a series of confounding factors are controlled. However, the dN/dS ratios are similar between the two gene groups, suggesting that the increase in dS can sufficiently explain the increase in dN in COGs. Therefore, the increases in evolutionary rates in COGs may be mainly mutation-driven. Conclusions Here we show that CTOs can increase the evolutionary rates of the affected genes. This effect is independent of the replication-transcription confrontation, which is suggested to be the major cause of inversion-associated evolutionary rate increases. The real cause of such evolutionary rate increases remains unclear but is worth further explorations. PMID:22152004

The divergently transcribed genes encoding yeast ribosomal proteins L46 and S24 are activated by shared RPG-boxes.

PubMed Central

Kraakman, L S; Mager, W H; Maurer, K T; Nieuwint, R T; Planta, R J

1989-01-01

Transcription of the majority of the ribosomal protein (rp) genes in yeast is activated through common cis-acting elements, designated RPG-boxes. These elements have been shown to act as specific binding sites for the protein factor TUF/RAP1/GRF1 in vitro. Two such elements occur in the intergenic region separating the divergently transcribed genes encoding L46 and S24. To investigate whether the two RPG-boxes mediate transcription activation of both the L46 and S24 gene, two experimental strategies were followed: cloning of the respective genes on multicopy vectors and construction of fusion genes. Cloning of the L46 + S24 gene including the intergenic region in a multicopy yeast vector indicated that both genes are transcriptionally active. Using constructs in which only the S24 or the L46 gene is present, with or without the intergenic region, we obtained evidence that the intergenic region is indispensable for transcription activation of either gene. To demarcate the element(s) responsible for this activation, fusions of the intergenic region in either orientation to the galK reporter gene were made. Northern analysis of the levels of hybrid mRNA demonstrated that the intergenic region can serve as an heterologous promoter when it is in the 'S24-orientation'. Surprisingly, however, when fused in the reverse orientation the intergenic region did hardly confer transcription activity on the fusion gene. Furthermore, a 274 bp FnuDII-FnuDII fragment from the intergenic region that contains the RPG-boxes, could replace the naturally occurring upstream activation site (UASrpg) of the L25 rp-gene only when inserted in the 'S24-orientation'. Removal of 15 bp from the FnuDII fragment appeared to be sufficient to obtain transcription activation in the 'L46 orientation' as well. Analysis of a construct in which the RPG-boxes were selectively deleted from the promoter region of the L46 gene indicated that the RPG-boxes are needed for efficient transcriptional activation of the L46 gene. We conclude that all promoter elements for the S24 gene are located within the intergenic region, where the RPG-boxes are the most likely UAS-elements. However, the intergenic region (including the RPG-boxes) is required but not sufficient to confer transcription activity on the L46 gene. Images PMID:2602141

The divergently transcribed genes encoding yeast ribosomal proteins L46 and S24 are activated by shared RPG-boxes.

PubMed

Kraakman, L S; Mager, W H; Maurer, K T; Nieuwint, R T; Planta, R J

1989-12-11

Transcription of the majority of the ribosomal protein (rp) genes in yeast is activated through common cis-acting elements, designated RPG-boxes. These elements have been shown to act as specific binding sites for the protein factor TUF/RAP1/GRF1 in vitro. Two such elements occur in the intergenic region separating the divergently transcribed genes encoding L46 and S24. To investigate whether the two RPG-boxes mediate transcription activation of both the L46 and S24 gene, two experimental strategies were followed: cloning of the respective genes on multicopy vectors and construction of fusion genes. Cloning of the L46 + S24 gene including the intergenic region in a multicopy yeast vector indicated that both genes are transcriptionally active. Using constructs in which only the S24 or the L46 gene is present, with or without the intergenic region, we obtained evidence that the intergenic region is indispensable for transcription activation of either gene. To demarcate the element(s) responsible for this activation, fusions of the intergenic region in either orientation to the galK reporter gene were made. Northern analysis of the levels of hybrid mRNA demonstrated that the intergenic region can serve as an heterologous promoter when it is in the 'S24-orientation'. Surprisingly, however, when fused in the reverse orientation the intergenic region did hardly confer transcription activity on the fusion gene. Furthermore, a 274 bp FnuDII-FnuDII fragment from the intergenic region that contains the RPG-boxes, could replace the naturally occurring upstream activation site (UASrpg) of the L25 rp-gene only when inserted in the 'S24-orientation'. Removal of 15 bp from the FnuDII fragment appeared to be sufficient to obtain transcription activation in the 'L46 orientation' as well. Analysis of a construct in which the RPG-boxes were selectively deleted from the promoter region of the L46 gene indicated that the RPG-boxes are needed for efficient transcriptional activation of the L46 gene. We conclude that all promoter elements for the S24 gene are located within the intergenic region, where the RPG-boxes are the most likely UAS-elements. However, the intergenic region (including the RPG-boxes) is required but not sufficient to confer transcription activity on the L46 gene.

G-Boxes, Bigfoot Genes, and Environmental Response: Characterization of Intragenomic Conserved Noncoding Sequences in Arabidopsis[W

PubMed Central

Freeling, Michael; Rapaka, Lakshmi; Lyons, Eric; Pedersen, Brent; Thomas, Brian C.

2007-01-01

A tetraploidy left Arabidopsis thaliana with 6358 pairs of homoeologs that, when aligned, generated 14,944 intragenomic conserved noncoding sequences (CNSs). Our previous work assembled these phylogenetic footprints into a database. We show that known transcription factor (TF) binding motifs, including the G-box, are overrepresented in these CNSs. A total of 254 genes spanning long lengths of CNS-rich chromosomes (Bigfoot) dominate this database. Therefore, we made subdatabases: one containing Bigfoot genes and the other containing genes with three to five CNSs (Smallfoot). Bigfoot genes are generally TFs that respond to signals, with their modal CNS positioned 3.1 kb 5′ from the ATG. Smallfoot genes encode components of signal transduction machinery, the cytoskeleton, or involve transcription. We queried each subdatabase with each possible 7-nucleotide sequence. Among hundreds of hits, most were purified from CNSs, and almost all of those significantly enriched in CNSs had no experimental history. The 7-mers in CNSs are not 5′- to 3′-oriented in Bigfoot genes but are often oriented in Smallfoot genes. CNSs with one G-box tend to have two G-boxes. CNSs were shared with the homoeolog only and with no other gene, suggesting that binding site turnover impedes detection. Bigfoot genes may function in adaptation to environmental change. PMID:17496117

G-boxes, bigfoot genes, and environmental response: characterization of intragenomic conserved noncoding sequences in Arabidopsis.

PubMed

Freeling, Michael; Rapaka, Lakshmi; Lyons, Eric; Pedersen, Brent; Thomas, Brian C

2007-05-01

A tetraploidy left Arabidopsis thaliana with 6358 pairs of homoeologs that, when aligned, generated 14,944 intragenomic conserved noncoding sequences (CNSs). Our previous work assembled these phylogenetic footprints into a database. We show that known transcription factor (TF) binding motifs, including the G-box, are overrepresented in these CNSs. A total of 254 genes spanning long lengths of CNS-rich chromosomes (Bigfoot) dominate this database. Therefore, we made subdatabases: one containing Bigfoot genes and the other containing genes with three to five CNSs (Smallfoot). Bigfoot genes are generally TFs that respond to signals, with their modal CNS positioned 3.1 kb 5' from the ATG. Smallfoot genes encode components of signal transduction machinery, the cytoskeleton, or involve transcription. We queried each subdatabase with each possible 7-nucleotide sequence. Among hundreds of hits, most were purified from CNSs, and almost all of those significantly enriched in CNSs had no experimental history. The 7-mers in CNSs are not 5'- to 3'-oriented in Bigfoot genes but are often oriented in Smallfoot genes. CNSs with one G-box tend to have two G-boxes. CNSs were shared with the homoeolog only and with no other gene, suggesting that binding site turnover impedes detection. Bigfoot genes may function in adaptation to environmental change.

Segments, Letters and Gestures: Thoughts on Doing and Teaching Phonetics and Transcription

ERIC Educational Resources Information Center

Muller, Nicole; Papakyritsis, Ioannis

2011-01-01

This brief article reflects on some pitfalls inherent in the learning and teaching of segmental phonetic transcription. We suggest that a gestural interpretation to disordered speech data, in conjunction with segmental phonetic transcription, can add valuable insight into patterns of disordered speech, and that a gestural orientation should form…

RNAP II Processivity is a Limiting Step for HIV-1 Transcription Independent of Orientation to and Activity of Endogenous Neighboring Promoters

PubMed Central

Michaels, Katarzyna Kaczmarek; Wolschendorf, Frank; Schiralli Lester, Gillian M.; Natarajan, Malini; Kutsch, Olaf; Henderson, Andrew J.

2015-01-01

Since HIV-1 has a propensity to integrate into actively expressed genes, transcriptional interference from neighboring host promoters has been proposed to contribute to the establishment and maintenance HIV-1 latency. To gain insights into how endogenous promoters influence HIV-1 transcription we utilized a set of inducible T cell lines and characterized whether there were correlations between expression of endogenous genes, provirus and long terminal repeat architecture. We show that neighboring promoters are active but have minimal impact on HIV-1 transcription, in particular, expression of the endogenous gene did not prevent expression of HIV-1 following induction of latent provirus. We also demonstrate that releasing paused RNAP II by diminishing negative elongation factor (NELF) is sufficient to reactivate transcriptionally repressed HIV-1 provirus regardless of the integration site and orientation of the provirus suggesting that NELF-mediated RNAP II pausing is a common mechanism of maintaining HIV-1 latency. PMID:26379089

Promoter selection in human mitochondria involves binding of a transcription factor to orientation-independent upstream regulatory elements.

PubMed

Fisher, R P; Topper, J N; Clayton, D A

1987-07-17

Selective transcription of human mitochondrial DNA requires a transcription factor (mtTF) in addition to an essentially nonselective RNA polymerase. Partially purified mtTF is able to sequester promoter-containing DNA in preinitiation complexes in the absence of mitochondrial RNA polymerase, suggesting a DNA-binding mechanism for factor activity. Functional domains, required for positive transcriptional regulation by mtTF, are identified within both major promoters of human mtDNA through transcription of mutant promoter templates in a reconstituted in vitro system. These domains are essentially coextensive with DNA sequences protected from nuclease digestion by mtTF-binding. Comparison of the sequences of the two mtTF-responsive elements reveals significant homology only when one sequence is inverted; the binding sites are in opposite orientations with respect to the predominant direction of transcription. Thus mtTF may function bidirectionally, requiring additional protein-DNA interactions to dictate transcriptional polarity. The mtTF-responsive elements are arrayed as direct repeats, separated by approximately 80 bp within the displacement-loop region of human mitochondrial DNA; this arrangement may reflect duplication of an ancestral bidirectional promoter, giving rise to separate, unidirectional promoters for each strand.

A systematic evaluation of expression of HERV-W elements; influence of genomic context, viral structure and orientation

PubMed Central

2011-01-01

Background One member of the W family of human endogenous retroviruses (HERV) appears to have been functionally adopted by the human host. Nevertheless, a highly diversified and regulated transcription from a range of HERV-W elements has been observed in human tissues and cells. Aberrant expression of members of this family has also been associated with human disease such as multiple sclerosis (MS) and schizophrenia. It is not known whether this broad expression of HERV-W elements represents transcriptional leakage or specific transcription initiated from the retroviral promoter in the long terminal repeat (LTR) region. Therefore, potential influences of genomic context, structure and orientation on the expression levels of individual HERV-W elements in normal human tissues were systematically investigated. Results Whereas intronic HERV-W elements with a pseudogene structure exhibited a strong anti-sense orientation bias, intronic elements with a proviral structure and solo LTRs did not. Although a highly variable expression across tissues and elements was observed, systematic effects of context, structure and orientation were also observed. Elements located in intronic regions appeared to be expressed at higher levels than elements located in intergenic regions. Intronic elements with proviral structures were expressed at higher levels than those elements bearing hallmarks of processed pseudogenes or solo LTRs. Relative to their corresponding genes, intronic elements integrated on the sense strand appeared to be transcribed at higher levels than those integrated on the anti-sense strand. Moreover, the expression of proviral elements appeared to be independent from that of their corresponding genes. Conclusions Intronic HERV-W provirus integrations on the sense strand appear to have elicited a weaker negative selection than pseudogene integrations of transcripts from such elements. Our current findings suggest that the previously observed diversified and tissue-specific expression of elements in the HERV-W family is the result of both directed transcription (involving both the LTR and internal sequence) and leaky transcription of HERV-W elements in normal human tissues. PMID:21226900

Latency of Epstein-Barr virus is stabilized by antisense-mediated control of the viral immediate-early gene BZLF-1.

PubMed

Prang, N; Wolf, H; Schwarzmann, F

1999-12-01

The ability of the Epstein-Barr virus (EBV) to avoid lytic replication and to establish a latent infection in B-lymphocytes is fundamental for its lifelong persistence and the pathogenesis of various EBV-associated diseases. The viral immediate-early gene BZLF-1 plays a key role for the induction of lytic replication and its activity is strictly regulated on different levels of gene expression. Recently, it was demonstrated that BZLF-1 is also controlled by a posttranscriptional mechanism. Transient synthesis of a mutated competitor RNA saturated this mechanism and caused both expression of the BZLF-1 protein and the induction of lytic viral replication. Using short overlapping fragments of the competitor, it is shown that this control acts on the unspliced primary transcript. RT-PCR demonstrated unspliced BZLF-1 RNA in latently infected B-lymphocytes in the absence of BZLF-1 protein. Due to the complementarity of the gene BZLF-1 and the latency-associated gene EBNA-1 on the opposite strand of the genome, we propose an antisense-mediated mechanism. RNase protection assays demonstrated transcripts in antisense orientation to the BZLF-1 transcript during latency, which comprise a comparable constellation to other herpesviruses. A combined RNAse protection/RT-PCR assay detected the double-stranded hybrid RNA, consisting of the unspliced BZLF-1 transcript and a noncoding intron of the EBNA-1 gene. Binding of BZLF-1 transcripts is suggested to be an important backup control mechanism in addition to transcriptional regulation, stabilizing latency and preventing inappropriate lytic viral replication in vivo. Copyright 1999 Wiley-Liss, Inc.

Upstream regulatory elements are necessary and sufficient for transcription of a U6 RNA gene by RNA polymerase III.

PubMed Central

Das, G; Henning, D; Wright, D; Reddy, R

1988-01-01

Whereas the genes coding for trimethyl guanosine-capped snRNAs are transcribed by RNA polymerase II, the U6 RNA genes are transcribed by RNA polymerase III. In this study, we have analyzed the cis-regulatory elements involved in the transcription of a mouse U6 snRNA gene in vitro and in frog oocytes. Transcriptional analysis of mutant U6 gene constructs showed that, unlike most known cases of polymerase III transcription, intragenic sequences except the initiation nucleotide are dispensable for efficient and accurate transcription of U6 gene in vitro. Transcription of 5' deletion mutants in vitro and in frog oocytes showed that the upstream region, within 79 bp from the initiation nucleotide, contains elements necessary for U6 gene transcription. Transcription studies were carried out in frog oocytes with U6 genes containing 5' distal sequence; these studies revealed that the distal element acts as an orientation-dependent enhancer when present upstream to the gene, while it is orientation-independent but distance-dependent enhancer when placed down-stream to the U6 gene. Analysis of 3' deletion mutants showed that the transcription termination of U6 RNA is dependent on a T cluster present on the 3' end of the gene, thus providing further support to other lines of evidence that U6 genes are transcribed by RNA polymerase III. These observations suggest the involvement of a composite of components of RNA polymerase II and III transcription machineries in the transcription of U6 genes by RNA polymerase III. Images PMID:3366121

Identification of a novel transcript disrupted by a balanced translocation associated with DiGeorge syndrome

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sutherland, H.F.; Wadey, R.; McKie, J.M.

1996-07-01

Most cases of DiGeorge syndrome (DGS) and related abnormalities are associated with deletions within 22q11. Shortest region on deletion overlap (SRO) mapping previously identified a critical region (the DGCR) of 500 kb, which was presumed to contain a gene or genes of major effect in the haploinsufficiency syndromes. The DGCR also contains sequences disrupted by a balanced translocation that is associated with DGS - the ADU breakpoint. We have cloned sequences at the breakpoint and screened for novel genes in its vicinity. A series of alternatively spliced transcripts expressed during human and murine embryogenesis, but with no obvious protein encodingmore » potential, were identified. The gene encoding these RNAs has been named DGCR5 and it is disrupted by the patient ADU breakpoint. DGCR5 is distinct from the DGCR3 open reading frame (ORF) previously shown to be interrupted by the ADU translocation, although DGCR3 is embedded within a DGCR5 intron and in the same (predicted) transcriptional orientation. No mutations of DGCR5 have yet been detected. By analogy to other loci encoding conserved, nontranslated RNAs, it is possible that DGCR5 originates from a cis-acting transcriptional control element in the vicinity of the ADU/VDU breakpoint. Disruption of such an element would result in altered transcription of neighboring genes secondary to a position effect, a hypothesis in keeping with recent refinement of the SRO placing the ADU breakpoint outside the DGCR. 38 refs., 3 figs., 1 tab.« less

Involvement of the C-terminal extension of the alpha polypeptide and of the PucC protein in LH2 complex biosynthesis in Rubrivivax gelatinosus.

PubMed

Steunou, Anne-Soisig; Ouchane, Soufian; Reiss-Husson, Françoise; Astier, Chantal

2004-05-01

The facultative phototrophic nonsulfur bacterium Rubrivivax gelatinosus exhibits several differences from other species of purple bacteria in the organization of its photosynthetic genes. In particular, the puc operon contains only the pucB and pucA genes encoding the beta and alpha polypeptides of the light-harvesting 2 (LH2) complex. Downstream of the pucBA operon is the pucC gene in the opposite transcriptional orientation. The transcription of pucBA and pucC has been studied. No pucC transcript was detected either by Northern blotting or by reverse transcription-PCR analysis. The initiation site of pucBA transcription was determined by primer extension, and Northern blot analysis revealed the presence of two transcripts of 0.8 and 0.65 kb. The half-lives of both transcripts are longer in cells grown semiaerobically than in photosynthetically grown cells, and the small transcript is the less stable. It was reported that the alpha polypeptide, encoded by the pucA gene, presents a C-terminal extension which is not essential for LH2 function in vitro. The biological role of this alanine- and proline-rich C-terminal extension in vivo has been investigated. Two mutants with C-terminal deletions of 13 and 18 residues have been constructed. Both present the two pucBA transcripts, while their phenotypes are, respectively, LH2+ and LH2-, suggesting that a minimal length of the C-terminal extension is required for LH2 biogenesis. Another important factor involved in the LH2 biogenesis is the PucC protein. To gain insight into the function of this protein in R. gelatinosus, we constructed and characterized a PucC mutant. The mutant is devoid of LH2 complex under semiaerobiosis but still produces a small amount of these antennae under photosynthetic growth conditions. This conditional phenotype suggests the involvement of another factor in LH2 biogenesis.

Imaging, object detection, and change detection with a polarized multistatic GPR array

DOE Office of Scientific and Technical Information (OSTI.GOV)

Beer, N. Reginald; Paglieroni, David W.

A polarized detection system performs imaging, object detection, and change detection factoring in the orientation of an object relative to the orientation of transceivers. The polarized detection system may operate on one of several modes of operation based on whether the imaging, object detection, or change detection is performed separately for each transceiver orientation. In combined change mode, the polarized detection system performs imaging, object detection, and change detection separately for each transceiver orientation, and then combines changes across polarizations. In combined object mode, the polarized detection system performs imaging and object detection separately for each transceiver orientation, and thenmore » combines objects across polarizations and performs change detection on the result. In combined image mode, the polarized detection system performs imaging separately for each transceiver orientation, and then combines images across polarizations and performs object detection followed by change detection on the result.« less

Genome assembly with in vitro proximity ligation data and whole-genome triplication in lettuce

PubMed Central

Reyes-Chin-Wo, Sebastian; Wang, Zhiwen; Yang, Xinhua; Kozik, Alexander; Arikit, Siwaret; Song, Chi; Xia, Liangfeng; Froenicke, Lutz; Lavelle, Dean O.; Truco, María-José; Xia, Rui; Zhu, Shilin; Xu, Chunyan; Xu, Huaqin; Xu, Xun; Cox, Kyle; Korf, Ian; Meyers, Blake C.; Michelmore, Richard W.

2017-01-01

Lettuce (Lactuca sativa) is a major crop and a member of the large, highly successful Compositae family of flowering plants. Here we present a reference assembly for the species and family. This was generated using whole-genome shotgun Illumina reads plus in vitro proximity ligation data to create large superscaffolds; it was validated genetically and superscaffolds were oriented in genetic bins ordered along nine chromosomal pseudomolecules. We identify several genomic features that may have contributed to the success of the family, including genes encoding Cycloidea-like transcription factors, kinases, enzymes involved in rubber biosynthesis and disease resistance proteins that are expanded in the genome. We characterize 21 novel microRNAs, one of which may trigger phasiRNAs from numerous kinase transcripts. We provide evidence for a whole-genome triplication event specific but basal to the Compositae. We detect 26% of the genome in triplicated regions containing 30% of all genes that are enriched for regulatory sequences and depleted for genes involved in defence. PMID:28401891

Transcriptome analysis by strand-specific sequencing of complementary DNA

PubMed Central

Parkhomchuk, Dmitri; Borodina, Tatiana; Amstislavskiy, Vyacheslav; Banaru, Maria; Hallen, Linda; Krobitsch, Sylvia; Lehrach, Hans; Soldatov, Alexey

2009-01-01

High-throughput complementary DNA sequencing (RNA-Seq) is a powerful tool for whole-transcriptome analysis, supplying information about a transcript's expression level and structure. However, it is difficult to determine the polarity of transcripts, and therefore identify which strand is transcribed. Here, we present a simple cDNA sequencing protocol that preserves information about a transcript's direction. Using Saccharomyces cerevisiae and mouse brain transcriptomes as models, we demonstrate that knowing the transcript's orientation allows more accurate determination of the structure and expression of genes. It also helps to identify new genes and enables studying promoter-associated and antisense transcription. The transcriptional landscapes we obtained are available online. PMID:19620212

Transcriptome analysis by strand-specific sequencing of complementary DNA.

PubMed

Parkhomchuk, Dmitri; Borodina, Tatiana; Amstislavskiy, Vyacheslav; Banaru, Maria; Hallen, Linda; Krobitsch, Sylvia; Lehrach, Hans; Soldatov, Alexey

2009-10-01

High-throughput complementary DNA sequencing (RNA-Seq) is a powerful tool for whole-transcriptome analysis, supplying information about a transcript's expression level and structure. However, it is difficult to determine the polarity of transcripts, and therefore identify which strand is transcribed. Here, we present a simple cDNA sequencing protocol that preserves information about a transcript's direction. Using Saccharomyces cerevisiae and mouse brain transcriptomes as models, we demonstrate that knowing the transcript's orientation allows more accurate determination of the structure and expression of genes. It also helps to identify new genes and enables studying promoter-associated and antisense transcription. The transcriptional landscapes we obtained are available online.

Coupled transcription and processing of mouse ribosomal RNA in a cell-free system.

PubMed Central

Mishima, Y; Mitsuma, T; Ogata, K

1985-01-01

An in vitro processing system of mouse rRNA was achieved using an RNA polymerase I-specific transcription system, (S100) and recombinant plasmids consisting of mouse rRNA gene (rDNA) segments containing the transcription initiation and 5'-terminal region of 18S (or 41S) rRNA. Pulse-chase experiments showed that a specific processing occurred with transcripts of the plasmid DNAs when the direction of transcription was the correct orientation relative to the 18S rRNA coding sequence, but not with transcripts of the DNA templates in which this coding sequence was in the opposite orientation. From the S1 nuclease protection analyses, we concluded that there are several steps of endonucleolytic cleavage including one 105 nucleotides upstream from the 5' end of 18S rRNA. Intermediates cleaved at this site were identified in in vivo processing of rRNA. This result indicates that endonucleolytic cleavage takes place 105 nucleotides upstream from the 5' terminus of 18S rRNA prior to the formation of mature 18S rRNA. Trimming or cleavage of the 105 nucleotides may be involved in the formation of the 5' terminus of mature 18S rRNA. Images Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. PMID:3004977

Real-time detection with AdaBoost-svm combination in various face orientation

NASA Astrophysics Data System (ADS)

Fhonna, R. P.; Nasution, M. K. M.; Tulus

2018-03-01

Most of the research has used algorithm AdaBoost-SVM for face detection. However, to our knowledge so far there is no research has been facing detection on real-time data with various orientations using the combination of AdaBoost and Support Vector Machine (SVM). Characteristics of complex and diverse face variations and real-time data in various orientations, and with a very complex application will slow down the performance of the face detection system this becomes a challenge in this research. Face orientation performed on the detection system, that is 900, 450, 00, -450, and -900. This combination method is expected to be an effective and efficient solution in various face orientations. The results showed that the highest average detection rate is on the face detection oriented 00 and the lowest detection rate is in the face orientation 900.

AtmiRNET: a web-based resource for reconstructing regulatory networks of Arabidopsis microRNAs.

PubMed

Chien, Chia-Hung; Chiang-Hsieh, Yi-Fan; Chen, Yi-An; Chow, Chi-Nga; Wu, Nai-Yun; Hou, Ping-Fu; Chang, Wen-Chi

2015-01-01

Compared with animal microRNAs (miRNAs), our limited knowledge of how miRNAs involve in significant biological processes in plants is still unclear. AtmiRNET is a novel resource geared toward plant scientists for reconstructing regulatory networks of Arabidopsis miRNAs. By means of highlighted miRNA studies in target recognition, functional enrichment of target genes, promoter identification and detection of cis- and trans-elements, AtmiRNET allows users to explore mechanisms of transcriptional regulation and miRNA functions in Arabidopsis thaliana, which are rarely investigated so far. High-throughput next-generation sequencing datasets from transcriptional start sites (TSSs)-relevant experiments as well as five core promoter elements were collected to establish the support vector machine-based prediction model for Arabidopsis miRNA TSSs. Then, high-confidence transcription factors participate in transcriptional regulation of Arabidopsis miRNAs are provided based on statistical approach. Furthermore, both experimentally verified and putative miRNA-target interactions, whose validity was supported by the correlations between the expression levels of miRNAs and their targets, are elucidated for functional enrichment analysis. The inferred regulatory networks give users an intuitive insight into the pivotal roles of Arabidopsis miRNAs through the crosstalk between miRNA transcriptional regulation (upstream) and miRNA-mediate (downstream) gene circuits. The valuable information that is visually oriented in AtmiRNET recruits the scant understanding of plant miRNAs and will be useful (e.g. ABA-miR167c-auxin signaling pathway) for further research. Database URL: http://AtmiRNET.itps.ncku.edu.tw/ © The Author(s) 2015. Published by Oxford University Press.

Global effects of the CSR-1 RNA interference pathway on the transcriptional landscape.

PubMed

Cecere, Germano; Hoersch, Sebastian; O'Keeffe, Sean; Sachidanandam, Ravi; Grishok, Alla

2014-04-01

Argonaute proteins and their small RNA cofactors short interfering RNAs are known to inhibit gene expression at the transcriptional and post-transcriptional levels. In Caenorhabditis elegans, the Argonaute CSR-1 binds thousands of endogenous siRNAs (endo-siRNAs) that are antisense to germline transcripts. However, its role in gene expression regulation remains controversial. Here we used genome-wide profiling of nascent RNA transcripts and found that the CSR-1 RNA interference pathway promoted sense-oriented RNA polymerase II transcription. Moreover, a loss of CSR-1 function resulted in global increase in antisense transcription and ectopic transcription of silent chromatin domains, which led to reduced chromatin incorporation of centromere-specific histone H3. On the basis of these findings, we propose that the CSR-1 pathway helps maintain the directionality of active transcription, thereby propagating the distinction between transcriptionally active and silent genomic regions.

The Wnt-1 (int-1) oncogene promoter and its mechanism of activation by insertion of proviral DNA of the mouse mammary tumor virus.

PubMed Central

Nusse, R; Theunissen, H; Wagenaar, E; Rijsewijk, F; Gennissen, A; Otte, A; Schuuring, E; van Ooyen, A

1990-01-01

Wnt-1 (int-1) is a cellular oncogene often activated by insertion of proviral DNA of the mouse mammary tumor virus. We have mapped the 5' end and the promoter area of the Wnt-1 gene by nuclease protection and primer extension assays. In differentiating P19 embryonal carcinoma cells, in which Wnt-1 is naturally expressed, two start sites of transcription were found, one preceded by two TATA boxes and one preceded by several GC boxes. In P19 cells, a 1-kilobase upstream sequence of Wnt-1 was able to confer differentiation-specific expression on a heterologous gene. We have investigated how Wnt-1 transcription was affected by mouse mammary tumor virus proviral integrations in various configurations near the promoters of the gene. One provirus has been inserted in the 5' nontranslated part of Wnt-1, in the same transcriptional orientation, and has functionally replaced the Wnt-1 promoters. Wnt-1 transcription in this tumor starts in the right long terminal repeat of the provirus, with considerable readthrough transcription from the left long terminal repeat. Another provirus has been inserted in the orientation opposite that of Wnt-1 into a GC box, disrupting the first Wnt-1 transcription start site but not the downstream start site. Most insertions have not structurally altered the Wnt-1 transcripts and have enhanced the activity of the normal two promoters. Images PMID:1695322

Characterization of a Vibrio vulnificus LysR homologue, HupR, which regulates expression of the haem uptake outer membrane protein, HupA.

PubMed

Litwin, C M; Quackenbush, J

2001-12-01

In Vibrio vulnificus, the ability to acquire iron from the host has been shown to correlate with virulence. Here, we show that the DNA upstream of hupA (haem uptake receptor) in V. vulnificus encodes a protein in the inverse orientation to hupA (named hupR). HupR shares homology with the LysR family of positive transcriptional activators. A hupA-lacZ fusion contained on a plasmid was transformed into Fur(-), Fur(+)and HupR(-)strains of V. vulnificus. The beta-galactosidase assays and Northern blot analysis showed that transcription of hupA is negatively regulated by iron and the Fur repressor in V. vulnificus. Under low-iron conditions with added haemin, the expression of hupA in the hupR mutant was significantly lower than in the wild-type. This diminished response to haem was detected by both Northern blot and hupA-lacZ fusion analysis. The haem response of hupA in the hupR mutant was restored to wild-type levels when complemented with hupR in trans. These studies suggest that HupR may act as a positive regulator of hupA transcription under low-iron conditions in the presence of haemin. Copyright 2001 Academic Press.

Poly A- transcripts expressed in HeLa cells.

PubMed

Wu, Qingfa; Kim, Yeong C; Lu, Jian; Xuan, Zhenyu; Chen, Jun; Zheng, Yonglan; Zhou, Tom; Zhang, Michael Q; Wu, Chung-I; Wang, San Ming

2008-07-30

Transcripts expressed in eukaryotes are classified as poly A+ transcripts or poly A- transcripts based on the presence or absence of the 3' poly A tail. Most transcripts identified so far are poly A+ transcripts, whereas the poly A- transcripts remain largely unknown. We developed the TRD (Total RNA Detection) system for transcript identification. The system detects the transcripts through the following steps: 1) depleting the abundant ribosomal and small-size transcripts; 2) synthesizing cDNA without regard to the status of the 3' poly A tail; 3) applying the 454 sequencing technology for massive 3' EST collection from the cDNA; and 4) determining the genome origins of the detected transcripts by mapping the sequences to the human genome reference sequences. Using this system, we characterized the cytoplasmic transcripts from HeLa cells. Of the 13,467 distinct 3' ESTs analyzed, 24% are poly A-, 36% are poly A+, and 40% are bimorphic with poly A+ features but without the 3' poly A tail. Most of the poly A- 3' ESTs do not match known transcript sequences; they have a similar distribution pattern in the genome as the poly A+ and bimorphic 3' ESTs, and their mapped intergenic regions are evolutionarily conserved. Experiments confirmed the authenticity of the detected poly A- transcripts. Our study provides the first large-scale sequence evidence for the presence of poly A- transcripts in eukaryotes. The abundance of the poly A- transcripts highlights the need for comprehensive identification of these transcripts for decoding the transcriptome, annotating the genome and studying biological relevance of the poly A- transcripts.

Ovarian-specific expression of a new gene regulated by the goat PIS region and transcribed by a FOXL2 bidirectional promoter.

PubMed

Pannetier, Maëlle; Renault, Lauriane; Jolivet, Geneviève; Cotinot, Corinne; Pailhoux, Eric

2005-06-01

Studies on XX sex reversal in polled goats (PIS mutation: polled intersex syndrome) have led to the discovery of a female-specific locus crucial for ovarian differentiation. This genomic region is composed of at least two genes, FOXL2 and PISRT1, sharing a common transcriptional regulatory region, PIS. In this paper, we describe a third gene, PFOXic (promoter FOXL2 inverse complementary), located near FOXL2 in the opposite orientation. This gene composed of five exons encodes a 1723-bp cDNA, enclosing two repetitive elements in its 3' end. PFOXic mRNA encodes a putative protein of 163 amino acids with no homologies in any of the databases tested. The transcriptional expression of PFOXic is driven by a bidirectional promoter also enhancing FOXL2 transcription. In goats, PFOXic is expressed in developing ovaries, from 36 days postcoitum until adulthood. Ovarian-specific expression of PFOXic is regulated by the PIS region. PFOXic is found conserved only in Bovidae. But, a human gene located in the opposite orientation relative to FOXL2 can be considered a human PFOXic. Finally, we discuss evidence arguing for regulation of the level of FOXL2 transcription via the bidirectional promoter and the level of transcription of PFOXic.

Poly A- Transcripts Expressed in HeLa Cells

PubMed Central

Lu, Jian; Xuan, Zhenyu; Chen, Jun; Zheng, Yonglan; Zhou, Tom; Zhang, Michael Q.; Wu, Chung-I; Wang, San Ming

2008-01-01

Background Transcripts expressed in eukaryotes are classified as poly A+ transcripts or poly A- transcripts based on the presence or absence of the 3′ poly A tail. Most transcripts identified so far are poly A+ transcripts, whereas the poly A- transcripts remain largely unknown. Methodology/Principal Findings We developed the TRD (Total RNA Detection) system for transcript identification. The system detects the transcripts through the following steps: 1) depleting the abundant ribosomal and small-size transcripts; 2) synthesizing cDNA without regard to the status of the 3′ poly A tail; 3) applying the 454 sequencing technology for massive 3′ EST collection from the cDNA; and 4) determining the genome origins of the detected transcripts by mapping the sequences to the human genome reference sequences. Using this system, we characterized the cytoplasmic transcripts from HeLa cells. Of the 13,467 distinct 3′ ESTs analyzed, 24% are poly A-, 36% are poly A+, and 40% are bimorphic with poly A+ features but without the 3′ poly A tail. Most of the poly A- 3′ ESTs do not match known transcript sequences; they have a similar distribution pattern in the genome as the poly A+ and bimorphic 3′ ESTs, and their mapped intergenic regions are evolutionarily conserved. Experiments confirmed the authenticity of the detected poly A- transcripts. Conclusion/Significance Our study provides the first large-scale sequence evidence for the presence of poly A- transcripts in eukaryotes. The abundance of the poly A- transcripts highlights the need for comprehensive identification of these transcripts for decoding the transcriptome, annotating the genome and studying biological relevance of the poly A- transcripts. PMID:18665230

RAP: RNA-Seq Analysis Pipeline, a new cloud-based NGS web application.

PubMed

D'Antonio, Mattia; D'Onorio De Meo, Paolo; Pallocca, Matteo; Picardi, Ernesto; D'Erchia, Anna Maria; Calogero, Raffaele A; Castrignanò, Tiziana; Pesole, Graziano

2015-01-01

The study of RNA has been dramatically improved by the introduction of Next Generation Sequencing platforms allowing massive and cheap sequencing of selected RNA fractions, also providing information on strand orientation (RNA-Seq). The complexity of transcriptomes and of their regulative pathways make RNA-Seq one of most complex field of NGS applications, addressing several aspects of the expression process (e.g. identification and quantification of expressed genes and transcripts, alternative splicing and polyadenylation, fusion genes and trans-splicing, post-transcriptional events, etc.). In order to provide researchers with an effective and friendly resource for analyzing RNA-Seq data, we present here RAP (RNA-Seq Analysis Pipeline), a cloud computing web application implementing a complete but modular analysis workflow. This pipeline integrates both state-of-the-art bioinformatics tools for RNA-Seq analysis and in-house developed scripts to offer to the user a comprehensive strategy for data analysis. RAP is able to perform quality checks (adopting FastQC and NGS QC Toolkit), identify and quantify expressed genes and transcripts (with Tophat, Cufflinks and HTSeq), detect alternative splicing events (using SpliceTrap) and chimeric transcripts (with ChimeraScan). This pipeline is also able to identify splicing junctions and constitutive or alternative polyadenylation sites (implementing custom analysis modules) and call for statistically significant differences in genes and transcripts expression, splicing pattern and polyadenylation site usage (using Cuffdiff2 and DESeq). Through a user friendly web interface, the RAP workflow can be suitably customized by the user and it is automatically executed on our cloud computing environment. This strategy allows to access to bioinformatics tools and computational resources without specific bioinformatics and IT skills. RAP provides a set of tabular and graphical results that can be helpful to browse, filter and export analyzed data, according to the user needs.

Global effects of the CSR-1 RNA interference pathway on transcriptional landscape

PubMed Central

Cecere, Germano; Hoersch, Sebastian; O’Keeffe, Sean; Sachidanandam, Ravi; Grishok, Alla

2014-01-01

Argonaute proteins and their small RNA co-factors short interfering RNAs (siRNAs) are known to inhibit gene expression at the transcriptional and post-transcriptional levels. In Caenorhabditis elegans, the Argonaute CSR-1 binds thousands of endogenous siRNAs (endo-siRNAs) antisense to germline transcripts and associates with chromatin in a siRNA-dependent manner. However, its role in gene expression regulation remains controversial. Here, we used a genome-wide profiling of nascent RNA transcripts to demonstrate that the CSR-1 RNAi pathway promotes sense-oriented Pol II transcription. Moreover, a loss of CSR-1 function resulted in global increase in antisense transcription and ectopic transcription of silent chromatin domains, which led to reduced chromatin incorporation of centromere-specific histone H3. Based on these findings, we propose that the CSR-1 pathway has a role in maintaining the directionality of active transcription thereby propagating the distinction between transcriptionally active and silent genomic regions. PMID:24681887

Characterization of OfWRKY3, a transcription factor that positively regulates the carotenoid cleavage dioxygenase gene OfCCD4 in Osmanthus fragrans.

PubMed

Han, Yuanji; Wu, Miao; Cao, Liya; Yuan, Wangjun; Dong, Meifang; Wang, Xiaohui; Chen, Weicai; Shang, Fude

2016-07-01

The sweet osmanthus carotenoid cleavage dioxygenase 4 (OfCCD4) cleaves carotenoids such as β-carotene and zeaxanthin to yield β-ionone. OfCCD4 is a member of the CCD gene family, and its promoter contains a W-box palindrome with two reversely oriented TGAC repeats, which are the proposed binding sites of WRKY transcription factors. We isolated three WRKY cDNAs from the petal of Osmanthus fragrans. One of them, OfWRKY3, encodes a protein containing two WRKY domains and two zinc finger motifs. OfWRKY3 and OfCCD4 had nearly identical expression profile in petals of 'Dangui' and 'Yingui' at different flowering stages and showed similar expression patterns in petals treated by salicylic acid, jasmonic acid and abscisic acid. Activation of OfCCD4pro:GUS by OfWRKY3 was detected in coinfiltrated tobacco leaves and very weak GUS activity was detected in control tissues, indicating that OfWRKY3 can interact with the OfCCD4 promoter. Yeast one-hybrid and electrophoretic mobility shift assay showed that OfWRKY3 was able to bind to the W-box palindrome motif present in the OfCCD4 promoter. These results suggest that OfWRKY3 is a positive regulator of the OfCCD4 gene, and might partly account for the biosynthesis of β-ionone in sweet osmanthus.

The yeast DNA ligase gene CDC9 is controlled by six orientation specific upstream activating sequences that respond to cellular proliferation but which alone cannot mediate cell cycle regulation.

PubMed Central

White, J H; Johnson, A L; Lowndes, N F; Johnston, L H

1991-01-01

By fusing the CDC9 structural gene to the PGK upstream sequences and the CDC9 upstream to lacZ, we showed that the cell cycle expression of CDC9 is largely due to transcriptional regulation. To investigate the role of six ATGATT upstream repeats in CDC9 regulation, synthetic copies of the sequence were attached to a heterologous gene. The repeats stimulated transcription strongly and additively, but, unlike conventional yeast UAS elements, only when present in one orientation. Transcription driven by the repeats declines in cells held at START of the cell cycle or in stationary phase, as occurs with CDC9. However, the repeats by themselves cannot impart cell cycle regulation to a heterologous gene. CDC9 may therefore be controlled by an activating system operating through the repeats that is sensitive to cellular proliferation and a separate mechanism that governs the periodic expression in the cell cycle. Images PMID:1901644

New Partner Orientation

EPA Pesticide Factsheets

This EPA presentation provides information on the SmartWay Transport Partnership Program, including key information about EPA, Partners' roles, benefits, tools, partner recognition, awards, and brand value. Transcript available.

Control of male sexual behavior and sexual orientation in Drosophila by the fruitless gene.

PubMed

Ryner, L C; Goodwin, S F; Castrillon, D H; Anand, A; Villella, A; Baker, B S; Hall, J C; Taylor, B J; Wasserman, S A

1996-12-13

Sexual orientation and courtship behavior in Drosophila are regulated by fruitless (fru), the first gene in a branch of the sex-determination hierarchy functioning specifically in the central nervous system (CNS). The phenotypes of new fru mutants encompass nearly all aspects of male sexual behavior. Alternative splicing of fru transcripts produces sex-specific proteins belonging to the BTB-ZF family of transcriptional regulators. The sex-specific fru products are produced in only about 500 of the 10(5) neurons that comprise the CNS. The properties of neurons expressing these fru products suggest that fru specifies the fates or activities of neurons that carry out higher order control functions to elicit and coordinate the activities comprising male courtship behavior.

A Goal Orientation Analysis of Teachers' Motivations to Participate in the School Self-Assessment Processes of a Quality Assurance System in Chile

ERIC Educational Resources Information Center

Montecinos, Carmen; Madrid, Romina; Fernández, María Beatriz; Ahumada, Luis

2014-01-01

The current study examined the goal orientations that could be inferred from how teachers from six municipal schools in Chile described their understandings, emotions, and behaviors during their participation in the assessment phase of the School Management Quality Assurance System. Content analysis of focus group interview transcripts evidenced…

Direct activation of a notochord cis-regulatory module by Brachyury and FoxA in the ascidian Ciona intestinalis.

PubMed

Passamaneck, Yale J; Katikala, Lavanya; Perrone, Lorena; Dunn, Matthew P; Oda-Ishii, Izumi; Di Gregorio, Anna

2009-11-01

The notochord is a defining feature of the chordate body plan. Experiments in ascidian, frog and mouse embryos have shown that co-expression of Brachyury and FoxA class transcription factors is required for notochord development. However, studies on the cis-regulatory sequences mediating the synergistic effects of these transcription factors are complicated by the limited knowledge of notochord genes and cis-regulatory modules (CRMs) that are directly targeted by both. We have identified an easily testable model for such investigations in a 155-bp notochord-specific CRM from the ascidian Ciona intestinalis. This CRM contains functional binding sites for both Ciona Brachyury (Ci-Bra) and FoxA (Ci-FoxA-a). By combining point mutation analysis and misexpression experiments, we demonstrate that binding of both transcription factors to this CRM is necessary and sufficient to activate transcription. To gain insights into the cis-regulatory criteria controlling its activity, we investigated the organization of the transcription factor binding sites within the 155-bp CRM. The 155-bp sequence contains two Ci-Bra binding sites with identical core sequences but opposite orientations, only one of which is required for enhancer activity. Changes in both orientation and spacing of these sites substantially affect the activity of the CRM, as clusters of identical sites found in the Ciona genome with different arrangements are unable to activate transcription in notochord cells. This work presents the first evidence of a synergistic interaction between Brachyury and FoxA in the activation of an individual notochord CRM, and highlights the importance of transcription factor binding site arrangement for its function.

Effects of gene orientation and use of multiple promoters on the expression of XYL1 and XYL2 in Saccharomyces cerevisiae

Treesearch

Ju Yun Bae; Jose Laplaza; Thomas W. Jeffries

2008-01-01

Orientation of adjacent genes has been reported to affect their expression in eukaryotic systems, and metabolic engineering also often makes repeated use of a few promoters to obtain high expression. To improve transcriptional control in heterologous expression, we examined how these factors affect gene expression and enzymatic activity in Saccharomyces cerevisiae. We...

An empirical strategy to detect bacterial transcript structure from directional RNA-seq transcriptome data.

PubMed

Wang, Yejun; MacKenzie, Keith D; White, Aaron P

2015-05-07

As sequencing costs are being lowered continuously, RNA-seq has gradually been adopted as the first choice for comparative transcriptome studies with bacteria. Unlike microarrays, RNA-seq can directly detect cDNA derived from mRNA transcripts at a single nucleotide resolution. Not only does this allow researchers to determine the absolute expression level of genes, but it also conveys information about transcript structure. Few automatic software tools have yet been established to investigate large-scale RNA-seq data for bacterial transcript structure analysis. In this study, 54 directional RNA-seq libraries from Salmonella serovar Typhimurium (S. Typhimurium) 14028s were examined for potential relationships between read mapping patterns and transcript structure. We developed an empirical method, combined with statistical tests, to automatically detect key transcript features, including transcriptional start sites (TSSs), transcriptional termination sites (TTSs) and operon organization. Using our method, we obtained 2,764 TSSs and 1,467 TTSs for 1331 and 844 different genes, respectively. Identification of TSSs facilitated further discrimination of 215 putative sigma 38 regulons and 863 potential sigma 70 regulons. Combining the TSSs and TTSs with intergenic distance and co-expression information, we comprehensively annotated the operon organization in S. Typhimurium 14028s. Our results show that directional RNA-seq can be used to detect transcriptional borders at an acceptable resolution of ±10-20 nucleotides. Technical limitations of the RNA-seq procedure may prevent single nucleotide resolution. The automatic transcript border detection methods, statistical models and operon organization pipeline that we have described could be widely applied to RNA-seq studies in other bacteria. Furthermore, the TSSs, TTSs, operons, promoters and unstranslated regions that we have defined for S. Typhimurium 14028s may constitute valuable resources that can be used for comparative analyses with other Salmonella serotypes.

Characterization of virulence factor regulation by SrrAB, a two-component system in Staphylococcus aureus.

PubMed

Pragman, Alexa A; Yarwood, Jeremy M; Tripp, Timothy J; Schlievert, Patrick M

2004-04-01

Workers in our laboratory have previously identified the staphylococcal respiratory response AB (SrrAB), a Staphylococcus aureus two-component system that acts in the global regulation of virulence factors. This system down-regulates production of agr RNAIII, protein A, and toxic shock syndrome toxin 1 (TSST-1), particularly under low-oxygen conditions. In this study we investigated the localization and membrane orientation of SrrA and SrrB, transcription of the srrAB operon, the DNA-binding properties of SrrA, and the effect of SrrAB expression on S. aureus virulence. We found that SrrA is localized to the S. aureus cytoplasm, while SrrB is localized to the membrane and is properly oriented to function as a histidine kinase. srrAB has one transcriptional start site which results in either an srrA transcript or a full-length srrAB transcript; srrB must be cotranscribed with srrA. Gel shift assays of the agr P2, agr P3, protein A (spa), TSST-1 (tst), and srr promoters revealed SrrA binding at each of these promoters. Analysis of SrrAB-overexpressing strains by using the rabbit model of bacterial endocarditis demonstrated that overexpression of SrrAB decreased the virulence of the organisms compared to the virulence of isogenic strains that do not overexpress SrrAB. We concluded that SrrAB is properly localized and oriented to function as a two-component system. Overexpression of SrrAB, which represses agr RNAIII, TSST-1, and protein A in vitro, decreases virulence in the rabbit endocarditis model. Repression of these virulence factors is likely due to a direct interaction between SrrA and the agr, tst, and spa promoters.

NCLscan: accurate identification of non-co-linear transcripts (fusion, trans-splicing and circular RNA) with a good balance between sensitivity and precision.

PubMed

Chuang, Trees-Juen; Wu, Chan-Shuo; Chen, Chia-Ying; Hung, Li-Yuan; Chiang, Tai-Wei; Yang, Min-Yu

2016-02-18

Analysis of RNA-seq data often detects numerous 'non-co-linear' (NCL) transcripts, which comprised sequence segments that are topologically inconsistent with their corresponding DNA sequences in the reference genome. However, detection of NCL transcripts involves two major challenges: removal of false positives arising from alignment artifacts and discrimination between different types of NCL transcripts (trans-spliced, circular or fusion transcripts). Here, we developed a new NCL-transcript-detecting method ('NCLscan'), which utilized a stepwise alignment strategy to almost completely eliminate false calls (>98% precision) without sacrificing true positives, enabling NCLscan outperform 18 other publicly-available tools (including fusion- and circular-RNA-detecting tools) in terms of sensitivity and precision, regardless of the generation strategy of simulated dataset, type of intragenic or intergenic NCL event, read depth of coverage, read length or expression level of NCL transcript. With the high accuracy, NCLscan was applied to distinguishing between trans-spliced, circular and fusion transcripts on the basis of poly(A)- and nonpoly(A)-selected RNA-seq data. We showed that circular RNAs were expressed more ubiquitously, more abundantly and less cell type-specifically than trans-spliced and fusion transcripts. Our study thus describes a robust pipeline for the discovery of NCL transcripts, and sheds light on the fundamental biology of these non-canonical RNA events in human transcriptome. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Expression of cathepsin S antisense transcripts by adenovirus in retinal pigment epithelial cells.

PubMed

Rakoczy, P E; Lai, M C; Baines, M G; Spilsbury, K; Constable, I J

1998-10-01

To show the production of sense or antisense transcripts by recombinant adenoviruses, to investigate whether the transcripts produced were suitable for downregulating the expression of the targeted gene, cathepsin S (CatS), and to examine the effect of antisense transcript production on the biologic function of retinal pigment epithelial (RPE) cells, including the regulation of endogenous aspartic protease expression. Ad.MLP.CatSAS, Ad.RSV.CatSAS, and Ad.MLP.CatSS recombinant viruses were produced by homologous recombination. The recombinant viruses were tested by restriction enzyme digestion to confirm the orientation of the inserts. The expression of antisense transcripts was tested by northern blot analysis. Western blot analysis was used to study the regulation of the endogenous CatS protein in ARPE19 cells. The biologic effect of CatS downregulation in ARPE19 cells was tested by proliferation and phagocytosis assays, de novo cathepsin D (CatD) synthesis, and measurement of aspartic protease activity. After characterization of the recombinant adenovirus constructs, the production of antisense and sense CatS transcripts was shown in ARPE19 cells. The transcripts appeared at approximately 1.9 kb 48 hours after transduction, and the expression of the antisense transcripts was similar in constructs carrying either the MLP or the RSV promoter. Western blot analysis showed that ARPE19 cells transduced with Ad.MLP.CatSAS and Ad.RSV.CatSAS had no detectable CatS. In contrast, there was a strong signal appearing at 24 kDa in ARPE19 cells transduced with Ad.MLP.CatSS. ARPE19 cells were transduced to a high level. The transduction of ARPE19 cells with the recombinant adenoviruses did not affect the morphologic appearance of the cells, their proliferation, or their phagocytosing ability. However, ARPE19 cells transduced by Ad.MLP.CatSAS recombinant adenovirus showed a significant downregulation of de novo CatD synthesis and a twofold decrease in aspartic protease activity. Recombinant adenoviruses were shown to be suitable for producing antisense CatS transcripts to modulate endogenous CatS expression in RPE cells. It is proposed that CatS may play an important role, directly or indirectly, in the lysosomal digestion of outer segments through the regulation of other lysosomal enzyme activity, such as the expression of CatD.

Contour symmetry detection: the influence of axis orientation and number of objects.

PubMed

Friedenberg, J; Bertamini, M

2000-09-01

Participants discriminated symmetrical from random contours connected by straight lines to form part of one- or two-objects. In experiment one, symmetrical contours were translated or reflected and presented at vertical, horizontal, and oblique axis orientations with orientation constant within blocks. Translated two-object contours were detected more easily than one, replicating a "lock-and-key" effect obtained previously for vertical orientations only [M. Bertamini, J.D. Friedenberg, M. Kubovy, Acta Psychologica, 95 (1997) 119-140]. A second experiment extended these results to a wider variety of axis orientations under mixed block conditions. The pattern of performance for translation and reflection at different orientations corresponded in both experiments, suggesting that orientation is processed similarly in the detection of these symmetries.

Variation in bat detections due to detector orientation in a forest.

Treesearch

Theodore J. Weller; Zabel Cynthia J.

2002-01-01

Bat detectors are widely used to compare bat activity among habitats. We placed 8 Anabat II detectors at 2 heights. 3 directions and 2 angles with respect to horizontal to evaluate the effect of detector orientation on the number of bat detections received. The orientation receiving the maximum number of detections had 70% more detections than the mean of the 7...

Global analyses of TetR family transcriptional regulators in mycobacteria indicates conservation across species and diversity in regulated functions.

PubMed

Balhana, Ricardo J C; Singla, Ashima; Sikder, Mahmudul Hasan; Withers, Mike; Kendall, Sharon L

2015-06-27

Mycobacteria inhabit diverse niches and display high metabolic versatility. They can colonise both humans and animals and are also able to survive in the environment. In order to succeed, response to environmental cues via transcriptional regulation is required. In this study we focused on the TetR family of transcriptional regulators (TFTRs) in mycobacteria. We used InterPro to classify the entire complement of transcriptional regulators in 10 mycobacterial species and these analyses showed that TFTRs are the most abundant family of regulators in all species. We identified those TFTRs that are conserved across all species analysed and those that are unique to the pathogens included in the analysis. We examined genomic contexts of 663 of the conserved TFTRs and observed that the majority of TFTRs are separated by 200 bp or less from divergently oriented genes. Analyses of divergent genes indicated that the TFTRs control diverse biochemical functions not limited to efflux pumps. TFTRs typically bind to palindromic motifs and we identified 11 highly significant novel motifs in the upstream regions of divergently oriented TFTRs. The C-terminal ligand binding domain from the TFTR complement in M. tuberculosis showed great diversity in amino acid sequence but with an overall architecture common to other TFTRs. This study suggests that mycobacteria depend on TFTRs for the transcriptional control of a number of metabolic functions yet the physiological role of the majority of these regulators remain unknown.

Illegitimate transcription: transcription of any gene in any cell type.

PubMed Central

Chelly, J; Concordet, J P; Kaplan, J C; Kahn, A

1989-01-01

Using in vitro amplification of cDNA by the polymerase chain reaction, we have detected spliced transcripts of various tissue-specific genes (genes for anti-Müllerian hormone, beta-globin, aldolase A, and factor VIIIc) in human nonspecific cells, such as fibroblasts, hepatoma cells, and lymphoblasts. In rats, erythroid- and liver-type pyruvate kinase transcripts were also detected in brain, lung, and muscle. The abundance of these "illegitimate" transcripts is very low; yet, their existence and the possibility of amplifying them by the cDNA polymerase chain reaction provide a powerful tool to analyze pathological transcripts of any tissue-specific gene by using any accessible cell. Images PMID:2495532

De novo characterization of microRNAs in oriental fruit moth Grapholita molesta and selection of reference genes for normalization of microRNA expression

PubMed Central

Zhang, Jing; Zhang, Qingwen; Liu, Xiaoxia; Li, Zhen

2017-01-01

MicroRNAs (miRNAs) are a group of endogenous non-coding small RNAs that have critical regulatory functions in almost all known biological processes at the post-transcriptional level in a variety of organisms. The oriental fruit moth Grapholita molesta is one of the most serious pests in orchards worldwide and threatens the production of Rosacea fruits. In this study, a de novo small RNA library constructed from mixed stages of G. molesta was sequenced through Illumina sequencing platform and a total of 536 mature miRNAs consisting of 291 conserved and 245 novel miRNAs were identified. Most of the conserved and novel miRNAs were detected with moderate abundance. The miRNAs in the same cluster normally showed correlated expressional profiles. A comparative analysis of the 79 conserved miRNA families within 31 arthropod species indicated that these miRNA families were more conserved among insects and within orders of closer phylogenetic relationships. The KEGG pathway analysis and network prediction of target genes indicated that the complex composed of miRNAs, clock genes and developmental regulation genes may play vital roles to regulate the developmental circadian rhythm of G. molesta. Furthermore, based on the sRNA library of G. molesta, suitable reference genes were selected and validated for study of miRNA transcriptional profile in G. molesta under two biotic and six abiotic experimental conditions. This study systematically documented the miRNA profile in G. molesta, which could lay a foundation for further understanding of the regulatory roles of miRNAs in the development and metabolism in this pest and might also suggest clues to the development of genetic-based techniques for agricultural pest control. PMID:28158242

Detection of rearrangements and transcriptional up-regulation of ALK in FFPE lung cancer specimens using a novel, sensitive, quantitative reverse transcription polymerase chain reaction assay.

PubMed

Gruber, Kim; Horn, Heike; Kalla, Jörg; Fritz, Peter; Rosenwald, Andreas; Kohlhäufl, Martin; Friedel, Godehard; Schwab, Matthias; Ott, German; Kalla, Claudia

2014-03-01

The approved dual-color fluorescence in situ hybridization (FISH) test for the detection of anaplastic lymphoma receptor tyrosine kinase (ALK) gene rearrangements in non-small-cell lung cancer (NSCLC) is complex and represents a low-throughput assay difficult to use in daily diagnostic practice. We devised a sensitive and robust routine diagnostic test for the detection of rearrangements and transcriptional up-regulation of ALK. We developed a quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay adapted to RNA isolated from routine formalin-fixed, paraffin-embedded material and applied it to 652 NSCLC specimens. The reliability of this technique to detect ALK dysregulation was shown by comparison with FISH and immunohistochemistry. qRT-PCR analysis detected unbalanced ALK expression indicative of a gene rearrangement in 24 (4.6%) and full-length ALK transcript expression in six (1.1%) of 523 interpretable tumors. Among 182 tumors simultaneously analyzed by FISH and qRT-PCR, the latter accurately typed 97% of 19 rearranged and 158 nonrearranged tumors and identified ALK deregulation in two cases with insufficient FISH. Six tumors expressing full-length ALK transcripts did not show rearrangements of the gene. Immunohistochemistry detected ALK protein overexpression in tumors with gene fusions and transcriptional up-regulation, but did not distinguish between the two. One case with full-length ALK expression carried a heterozygous point mutation (S1220Y) within the kinase domain potentially interfering with kinase activity and/or inhibitor binding. Our qRT-PCR assay reliably identifies and distinguishes ALK rearrangements and full-length transcript expression in formalin-fixed, paraffin-embedded material. It is an easy-to-perform, cost-effective, and high-throughput tool for the diagnosis of ALK activation. The expression of full-length ALK transcripts may be relevant for ALK inhibitor therapy in NSCLC.

No apparent influence of psychometrically-defined schizotypy on orientation-dependent contextual modulation of visual contrast detection.

PubMed

Mannion, Damien J; Donkin, Chris; Whitford, Thomas J

2017-01-01

We investigated the relationship between psychometrically-defined schizotypy and the ability to detect a visual target pattern. Target detection is typically impaired by a surrounding pattern (context) with an orientation that is parallel to the target, relative to a surrounding pattern with an orientation that is orthogonal to the target (orientation-dependent contextual modulation). Based on reports that this effect is reduced in those with schizophrenia, we hypothesised that there would be a negative relationship between the relative score on psychometrically-defined schizotypy and the relative effect of orientation-dependent contextual modulation. We measured visual contrast detection thresholds and scores on the Oxford-Liverpool Inventory of Feelings and Experiences (O-LIFE) from a non-clinical sample ( N = 100). Contrary to our hypothesis, we find an absence of a monotonic relationship between the relative magnitude of orientation-dependent contextual modulation of visual contrast detection and the relative score on any of the subscales of the O-LIFE. The apparent difference of this result with previous reports on those with schizophrenia suggests that orientation-dependent contextual modulation may be an informative condition in which schizophrenia and psychometrically-defined schizotypy are dissociated. However, further research is also required to clarify the strength of orientation-dependent contextual modulation in those with schizophrenia.

Mechanisms of radiation-induced gene responses

DOE Office of Scientific and Technical Information (OSTI.GOV)

Woloschak, G.E.; Paunesku, T.

1996-10-01

In the process of identifying genes differentially expressed in cells exposed ultraviolet radiation, we have identified a transcript having a 26-bp region that is highly conserved in a variety of species including Bacillus circulans, yeast, pumpkin, Drosophila, mouse, and man. When the 5` region (flanking region or UTR) of a gene, the sequence is predominantly in +/+ orientation with respect to the coding DNA strand; while in the coding region and the 3` region (UTR), the sequence is most frequently in the +/-orientation with respect to the coding DNA strand. In two genes, the element is split into two parts;more » however, in most cases, it is found only once but with a minimum of 11 consecutive nucleotides precisely depicting the original sequence. The element is found in a large number of different genes with diverse functions (from human ras p21 to B. circulans chitonase). Gel shift assays demonstrated the presence of a protein in HeLa cell extracts that binds to the sense and antisense single-stranded consensus oligomers, as well as to the double- stranded oligonucleotide. When double-stranded oligomer was used, the size shift demonstrated as additional protein-oligomer complex larger than the one bound to either sense or antisense single-stranded consensus oligomers alone. It is speculated either that this element binds to protein(s) important in maintaining DNA is a single-stranded orientation for transcription or, alternatively that this element is important in the transcription-coupled DNA repair process.« less

Bovine papilloma virus contains an activator of gene expression at the distal end of the early transcription unit.

PubMed Central

Lusky, M; Berg, L; Weiher, H; Botchan, M

1983-01-01

Bovine papilloma virus (BPV) contains a cis-acting DNA element which can enhance transcription of distal promoters. Utilizing both direct and indirect transient transfection assays, we showed that a 59-base-pair DNA sequence from the BPV genome could activate the simian virus 40 promoter from distances exceeding 2.5 kilobases and in an orientation-independent manner. In contrast to the promoter 5'-proximal localization of other known viral activators, this element was located immediately 3' to the early polyadenylation signal in the BPV genome. Deletion of these sequences from the BPV genome inactivated the transforming ability of BPV recombinant plasmids. Orientation-independent reinsertion of this 59-base-pair sequence, or alternatively of activator DNA sequences from simian virus 40 or polyoma virus, restored the transforming activity of the BPV recombinant plasmids. Furthermore, the stable transformation frequency of the herpes simplex virus type 1 thymidine kinase gene was enhanced when linked to restriction fragments of BPV DNA which included the defined activator element. This enhancement was orientation independent with respect to the thymidine kinase promoter. The enhancement also appeared to be unrelated to the establishment of the recombinant plasmids as episomes, since in transformed cells these sequences are found linked to high-molecular-weight DNA. We propose that the enhancement of stable transformation frequencies and the activation of transcription units are in this case alternate manifestations of the same biochemical events. Images PMID:6308425

Co-regulation analysis of co-expressed modules under cold and pathogen stress conditions in tomato.

PubMed

Abedini, Davar; Rashidi Monfared, Sajad

2018-06-01

A primary mechanism for controlling the development of multicellular organisms is transcriptional regulation, which carried out by transcription factors (TFs) that recognize and bind to their binding sites on promoter region. The distance from translation start site, order, orientation, and spacing between cis elements are key factors in the concentration of active nuclear TFs and transcriptional regulation of target genes. In this study, overrepresented motifs in cold and pathogenesis responsive genes were scanned via Gibbs sampling method, this method is based on detection of overrepresented motifs by means of a stochastic optimization strategy that searches for all possible sets of short DNA segments. Then, identified motifs were checked by TRANSFAC, PLACE and Soft Berry databases in order to identify putative TFs which, interact to the motifs. Several cis/trans regulatory elements were found using these databases. Moreover, cross-talk between cold and pathogenesis responsive genes were confirmed. Statistical analysis was used to determine distribution of identified motifs on promoter region. In addition, co-regulation analysis results, illustrated genes in pathogenesis responsive module are divided into two main groups. Also, promoter region was crunched to six subareas in order to draw the pattern of distribution of motifs in promoter subareas. The result showed the majority of motifs are concentrated on 700 nucleotides upstream of the translational start site (ATG). In contrast, this result isn't true in another group. In other words, there was no difference between total and compartmentalized regions in cold responsive genes.

Learning Oriented Region-based Convolutional Neural Networks for Building Detection in Satellite Remote Sensing Images

NASA Astrophysics Data System (ADS)

Chen, C.; Gong, W.; Hu, Y.; Chen, Y.; Ding, Y.

2017-05-01

The automated building detection in aerial images is a fundamental problem encountered in aerial and satellite images analysis. Recently, thanks to the advances in feature descriptions, Region-based CNN model (R-CNN) for object detection is receiving an increasing attention. Despite the excellent performance in object detection, it is problematic to directly leverage the features of R-CNN model for building detection in single aerial image. As we know, the single aerial image is in vertical view and the buildings possess significant directional feature. However, in R-CNN model, direction of the building is ignored and the detection results are represented by horizontal rectangles. For this reason, the detection results with horizontal rectangle cannot describe the building precisely. To address this problem, in this paper, we proposed a novel model with a key feature related to orientation, namely, Oriented R-CNN (OR-CNN). Our contributions are mainly in the following two aspects: 1) Introducing a new oriented layer network for detecting the rotation angle of building on the basis of the successful VGG-net R-CNN model; 2) the oriented rectangle is proposed to leverage the powerful R-CNN for remote-sensing building detection. In experiments, we establish a complete and bran-new data set for training our oriented R-CNN model and comprehensively evaluate the proposed method on a publicly available building detection data set. We demonstrate State-of-the-art results compared with the previous baseline methods.

Redundant CArG Box Cis-motif Activity Mediates SHATTERPROOF2 Transcriptional Regulation during Arabidopsis thaliana Gynoecium Development

PubMed Central

Sehra, Bhupinder; Franks, Robert G.

2017-01-01

In the Arabidopsis thaliana seed pod, pod shatter and seed dispersal properties are in part determined by the development of a longitudinally orientated dehiscence zone (DZ) that derives from cells of the gynoecial valve margin (VM). Transcriptional regulation of the MADS protein encoding transcription factors genes SHATTERPROOF1 (SHP1) and SHATTERPROOF2 (SHP2) are critical for proper VM identity specification and later on for DZ development. Current models of SHP1 and SHP2 regulation indicate that the transcription factors FRUITFULL (FUL) and REPLUMLESS (RPL) repress these SHP genes in the developing valve and replum domains, respectively. Thus the expression of the SHP genes is restricted to the VM. FUL encodes a MADS-box containing transcription factor that is predicted to act through CArG-box containing cis-regulatory motifs. Here we delimit functional modules within the SHP2 cis-regulatory region and examine the functional importance of CArG box motifs within these regulatory regions. We have characterized a 2.2kb region upstream of the SHP2 translation start site that drives early and late medial domain expression in the gynoecium, as well as expression within the VM and DZ. We identified two separable, independent cis-regulatory modules, a 1kb promoter region and a 700bp enhancer region, that are capable of giving VM and DZ expression. Our results argue for multiple independent cis-regulatory modules that support SHP2 expression during VM development and may contribute to the robustness of SHP2 expression in this tissue. Additionally, three closely positioned CArG box motifs located in the SHP2 upstream regulatory region were mutated in the context of the 2.2kb reporter construct. Mutating simultaneously all three CArG boxes caused a moderate de-repression of the SHP2 reporter that was detected within the valve domain, suggesting that these CArG boxes are involved in SHP2 repression in the valve. PMID:29085379

RAP: RNA-Seq Analysis Pipeline, a new cloud-based NGS web application

PubMed Central

2015-01-01

Background The study of RNA has been dramatically improved by the introduction of Next Generation Sequencing platforms allowing massive and cheap sequencing of selected RNA fractions, also providing information on strand orientation (RNA-Seq). The complexity of transcriptomes and of their regulative pathways make RNA-Seq one of most complex field of NGS applications, addressing several aspects of the expression process (e.g. identification and quantification of expressed genes and transcripts, alternative splicing and polyadenylation, fusion genes and trans-splicing, post-transcriptional events, etc.). Moreover, the huge volume of data generated by NGS platforms introduces unprecedented computational and technological challenges to efficiently analyze and store sequence data and results. Methods In order to provide researchers with an effective and friendly resource for analyzing RNA-Seq data, we present here RAP (RNA-Seq Analysis Pipeline), a cloud computing web application implementing a complete but modular analysis workflow. This pipeline integrates both state-of-the-art bioinformatics tools for RNA-Seq analysis and in-house developed scripts to offer to the user a comprehensive strategy for data analysis. RAP is able to perform quality checks (adopting FastQC and NGS QC Toolkit), identify and quantify expressed genes and transcripts (with Tophat, Cufflinks and HTSeq), detect alternative splicing events (using SpliceTrap) and chimeric transcripts (with ChimeraScan). This pipeline is also able to identify splicing junctions and constitutive or alternative polyadenylation sites (implementing custom analysis modules) and call for statistically significant differences in genes and transcripts expression, splicing pattern and polyadenylation site usage (using Cuffdiff2 and DESeq). Results Through a user friendly web interface, the RAP workflow can be suitably customized by the user and it is automatically executed on our cloud computing environment. This strategy allows to access to bioinformatics tools and computational resources without specific bioinformatics and IT skills. RAP provides a set of tabular and graphical results that can be helpful to browse, filter and export analyzed data, according to the user needs. PMID:26046471

Two distinct promoters drive transcription of the human D1A dopamine receptor gene.

PubMed

Lee, S H; Minowa, M T; Mouradian, M M

1996-10-11

The human D1A dopamine receptor gene has a GC-rich, TATA-less promoter located upstream of a small, noncoding exon 1, which is separated from the coding exon 2 by a 116-base pair (bp)-long intron. Serial 3'-deletions of the 5'-noncoding region of this gene, including the intron and 5'-end of exon 2, resulted in 80 and 40% decrease in transcriptional activity of the upstream promoter in two D1A-expressing neuroblastoma cell lines, SK-N-MC and NS20Y, respectively. To investigate the function of this region, the intron and 245 bp at the 5'-end of exon 2 were investigated. Transient expression analyses using various chloramphenicol acetyltransferase constructs showed that the transcriptional activity of the intron is higher than that of the upstream promoter by 12-fold in SK-N-MC cells and by 5.5-fold in NS20Y cells in an orientation-dependent manner, indicating that the D1A intron is a strong promoter. Primer extension and ribonuclease protection assays revealed that transcription driven by the intron promoter is initiated at the junction of intron and exon 2 and at a cluster of nucleotides located 50 bp downstream from this junction. The same transcription start sites are utilized by the chloramphenicol acetyltransferase constructs employed in transfections as well as by the D1A gene expressed within the human caudate. The relative abundance of D1A transcripts originating from the upstream promoter compared with those transcribed from the intron promoter is 1.5-2.9 times in SK-N-MC cells and 2 times in the human caudate. Transcript stability studies in SK-N-MC cells revealed that longer D1A mRNA molecules containing exon 1 are degraded 1.8 times faster than shorter transcripts lacking exon 1. Although gel mobility shift assay could not detect DNA-protein interaction at the D1A intron, competitive co-transfection using the intron as competitor confirmed the presence of trans-acting factors at the intron. These data taken together indicate that the human D1A gene has two functional TATA-less promoters, both in D1A expressing cultured neuroblastoma cells and in the human striatum.

Characterization of a multicopper oxidase gene cluster in Phanerochaete chrysosporium and evidence of altered splicing of the mco transcripts

Treesearch

Luis F. Larrondo; Bernardo Gonzalez; Dan Cullen; Rafael Vicuna

2004-01-01

A cluster of multicopper oxidase genes (mco1, mco2, mco3, mco4) from the lignin-degrading basidiomycete Phanerochaete chrysosporium is described. The four genes share the same transcriptional orientation within a 25 kb region. mco1, mco2 and mco3 are tightly grouped, with intergenic regions of 2.3 and 0.8 kb, respectively, whereas mco4 is located 11 kb upstream of mco1...

Trainable multiscript orientation detection

NASA Astrophysics Data System (ADS)

Van Beusekom, Joost; Rangoni, Yves; Breuel, Thomas M.

2010-01-01

Detecting the correct orientation of document images is an important step in large scale digitization processes, as most subsequent document analysis and optical character recognition methods assume upright position of the document page. Many methods have been proposed to solve the problem, most of which base on ascender to descender ratio computation. Unfortunately, this cannot be used for scripts having no descenders nor ascenders. Therefore, we present a trainable method using character similarity to compute the correct orientation. A connected component based distance measure is computed to compare the characters of the document image to characters whose orientation is known. This allows to detect the orientation for which the distance is lowest as the correct orientation. Training is easily achieved by exchanging the reference characters by characters of the script to be analyzed. Evaluation of the proposed approach showed accuracy of above 99% for Latin and Japanese script from the public UW-III and UW-II datasets. An accuracy of 98.9% was obtained for Fraktur on a non-public dataset. Comparison of the proposed method to two methods using ascender / descender ratio based orientation detection shows a significant improvement.

Transcription-dependent induction of G1 phase during the zebra fish midblastula transition.

PubMed

Zamir, E; Kam, Z; Yarden, A

1997-02-01

The early development of the zebra fish (Danio rerio) embryo is characterized by a series of rapid and synchronous cell cycles with no detectable transcription. This period is followed by the midblastula transition (MBT), during which the cell cycle gradually lengthens, cell synchrony is lost, and zygotic transcription is initially detected. In this work, we examined the changes in the pattern of the cell cycle during MBT in zebra fish and whether these changes are dependent on the initiation of zygotic transcription. To characterize the pattern of the early zebra fish cell cycles, the embryonic DNA content was determined by flow cytometric analysis. We found that G1 phase is below detection levels during the first 10 cleavages and can be initially detected at the onset of MBT. Inhibition of zygotic transcription, by microinjection of actinomycin D, abolished the appearance of G1 phase at MBT. Premature activation of zygotic transcription, by microinjection of nonspecific DNA, induced G1 phase before the onset of MBT, while coinjection of actinomycin D and nonspecific DNA abolished this early appearance of G1 phase. We therefore suggest that during the early development of the zebra fish embryo, G1 phase appears at the onset of MBT and that the activation of transcription at MBT is essential and sufficient for the G1-phase induction.

Square cell packing in the Drosophila embryo through spatiotemporally regulated EGF receptor signaling

PubMed Central

Tamada, Masako; Zallen, Jennifer A.

2015-01-01

Summary Cells display dynamic and diverse morphologies during development, but the strategies by which differentiated tissues achieve precise shapes and patterns are not well understood. Here we identify a developmental program that generates a highly ordered square cell grid in the Drosophila embryo through sequential and spatially regulated cell alignment, oriented cell division, and apicobasal cell elongation. The basic leucine zipper transcriptional regulator Cnc is necessary and sufficient to produce a square cell grid in the presence of a midline signal provided by the EGF receptor ligand, Spitz. Spitz orients cell divisions through a Pins/LGN-dependent spindle positioning mechanism and controls cell shape and alignment through a transcriptional pathway that requires the Pointed ETS domain protein. These results identify a strategy for producing ordered square cell packing configurations in epithelia and reveal a molecular mechanism by which organized tissue structure is generated through spatiotemporally regulated responses to EGF receptor activation. PMID:26506305

Automatic image orientation detection via confidence-based integration of low-level and semantic cues.

PubMed

Luo, Jiebo; Boutell, Matthew

2005-05-01

Automatic image orientation detection for natural images is a useful, yet challenging research topic. Humans use scene context and semantic object recognition to identify the correct image orientation. However, it is difficult for a computer to perform the task in the same way because current object recognition algorithms are extremely limited in their scope and robustness. As a result, existing orientation detection methods were built upon low-level vision features such as spatial distributions of color and texture. Discrepant detection rates have been reported for these methods in the literature. We have developed a probabilistic approach to image orientation detection via confidence-based integration of low-level and semantic cues within a Bayesian framework. Our current accuracy is 90 percent for unconstrained consumer photos, impressive given the findings of a psychophysical study conducted recently. The proposed framework is an attempt to bridge the gap between computer and human vision systems and is applicable to other problems involving semantic scene content understanding.

Rapid detection of Prunus necrotic ringspot virus using magnetic nanoparticle-assisted reverse transcription loop-mediated isothermal amplification.

PubMed

Zong, Xiaojuan; Wang, Wenwen; Wei, Hairong; Wang, Jiawei; Chen, Xin; Xu, Li; Zhu, Dongzi; Tan, Yue; Liu, Qingzhong

2014-11-01

Prunus necrotic ringspot virus (PNRSV) has seriously reduced the yield of Prunus species worldwide. In this study, a highly efficient and specific two-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) was developed to detect PNRSV. Total RNA was extracted from sweet cherry leaf samples using a commercial kit based on a magnetic nanoparticle technique. Transcripts were used as the templates for the assay. The results of this assay can be detected using agarose gel electrophoresis or by assessing in-tube fluorescence after adding SYBR Green I. The assay is highly specific for PNRSV, and it is more sensitive than reverse-transcription polymerase chain reaction (RT-PCR). Restriction enzyme digestion verified further the reliability of this RT-LAMP assay. To our knowledge, this is the first report of the application of RT-LAMP to PNRSV detection in Prunus species. Copyright © 2014 Elsevier B.V. All rights reserved.

Relative Orientation and Position Detections Based on an RGB-D Sensor and Dynamic Cooperation Strategies for Jumping Sensor Nodes Recycling

PubMed Central

Zhang, Jun; Yang, Xi; Song, Guang-Ming; Chen, Tian-Yuan; Zhang, Yong

2015-01-01

This paper presents relative orientation and position detection methods for jumping sensor nodes (JSNs) recycling. The methods are based on motion captures of the JSNs by an RGB-D sensor mounted on a carrier robot and the dynamic cooperation between the carrier and the JSNs. A disc-like label with two different colored sides is mounted on the top of the JSNs. The RGB-D sensor can detect the motion of the label to calculate the orientations and positions of the JSNs and the carrier relative to each other. After the orientations and positions have been detected, the JSNs jump into a cabin mounted on the carrier in dynamic cooperation with the carrier for recycling. The performances of the proposed methods are tested with a prototype system. The results show that the carrier can detect a JSN from up to 2 m away and sense its relative orientation and position successfully. The errors of the JSN’s orientation and position detections relative to the carrier could be reduced to the values smaller than 1° and 1 cm, respectively, by using the dynamic cooperation strategies. The proposed methods in this paper could also be used for other kinds of mobile sensor nodes and multi-robot systems. PMID:26393589

A case for safety leadership team training of hospital managers.

PubMed

Singer, Sara J; Hayes, Jennifer; Cooper, Jeffrey B; Vogt, Jay W; Sales, Michael; Aristidou, Angela; Gray, Garry C; Kiang, Mathew V; Meyer, Gregg S

2011-01-01

Delivering safe patient care remains an elusive goal. Resolving problems in complex organizations like hospitals requires managers to work together. Safety leadership training that encourages managers to exercise learning-oriented, team-based leadership behaviors could promote systemic problem solving and enhance patient safety. Despite the need for such training, few programs teach multidisciplinary groups of managers about specific behaviors that can enhance their role as leadership teams in the realm of patient safety. The aims of this study were to describe a learning-oriented, team-based, safety leadership training program composed of reinforcing exercises and to provide evidence confirming the need for such training and demonstrating behavior change among management groups after training. Twelve groups of managers from an academic medical center based in the Northeast United States were randomly selected to participate in the program and exposed to its customized, experience-based, integrated, multimodal curriculum. We extracted data from transcripts of four training sessions over 15 months with groups of managers about the need for the training in these groups and change in participants' awareness, professional behaviors, and group activity. Training transcripts confirmed the need for safety leadership team training and provided evidence of the potential for training to increase targeted behaviors. The training increased awareness and use of leadership behaviors among many managers and led to new routines and coordinated effort among most management groups. Enhanced learning-oriented leadership often helped promote a learning orientation in managers' work areas. Team-based training that promotes specific learning-oriented leader behaviors can promote behavioral change among multidisciplinary groups of hospital managers.

The Effect of Local Orientation Change on the Detection of Contours Defined by Constant Curvature: Psychophysics and Image Statistics.

PubMed

Khuu, Sieu K; Cham, Joey; Hayes, Anthony

2016-01-01

In the present study, we investigated the detection of contours defined by constant curvature and the statistics of curved contours in natural scenes. In Experiment 1, we examined the degree to which human sensitivity to contours is affected by changing the curvature angle and disrupting contour curvature continuity by varying the orientation of end elements. We find that (1) changing the angle of contour curvature decreased detection performance, while (2) end elements oriented in the direction (i.e., clockwise) of curvature facilitated contour detection regardless of the curvature angle of the contour. In Experiment 2 we further established that the relative effect of end-element orientation on contour detection was not only dependent on their orientation (collinear or cocircular), but also their spatial separation from the contour, and whether the contour shape was curved or not (i.e., C-shaped or S-shaped). Increasing the spatial separation of end-elements reduced contour detection performance regardless of their orientation or the contour shape. However, at small separations, cocircular end-elements facilitated the detection of C-shaped contours, but not S-shaped contours. The opposite result was observed for collinear end-elements, which improved the detection of S- shaped, but not C-shaped contours. These dissociative results confirmed that the visual system specifically codes contour curvature, but the association of contour elements occurs locally. Finally, we undertook an analysis of natural images that mapped contours with a constant angular change and determined the frequency of occurrence of end elements with different orientations. Analogous to our behavioral data, this image analysis revealed that the mapped end elements of constantly curved contours are likely to be oriented clockwise to the angle of curvature. Our findings indicate that the visual system is selectively sensitive to contours defined by constant curvature and that this might reflect the properties of curved contours in natural images.

Micronuclei and other erythrocyte nuclear abnormalities in fishes from the Great Lakes Basin, USA

USGS Publications Warehouse

Braham, Ryan P.; Blazer, Vicki S.; Shaw, Cassidy H.; Mazik, Patricia M.

2017-01-01

Biological markers (biomarkers) sensitive to genotoxic and mutagenic contamination in fishes are widely used to identify exposure effects in aquatic environments. The micronucleus assay was incorporated into a suite of indicators to assess exposure to genotoxic and mutagenic contamination at five Great Lakes Areas of Concern (AOCs), as well as one non-AOC (reference) site. The assay allowed enumeration of micronuclei as well as other nuclear abnormalities for both site and species comparisons. Erythrocyte abnormality data was also compared to skin and liver tumor prevalence and hepatic transcript abundance. Erythrocyte abnormalities were observed at all sites with variable occurrence and severity among sites and species. Benthic-oriented brown bullhead (Ameiurus nebulosus) and white sucker (Catostomus commersonii) expressed lower rates of erythrocyte abnormalities, but higher rates of skin and liver neoplasms, when compared to pelagic-oriented largemouth bass (Micropterus salmoides) or smallmouth bass (Micropterus dolomieu) at the same site. The reduced erythrocyte abnormalities, increased transcript abundance associated with Phase I and II toxicant responsive pathways, and increased neoplastic lesions among benthic-oriented taxa may indicate the development of contaminant resistance of these species to more acute effects.

Development and evaluation of a boat-mounted RFID antenna for monitoring freshwater mussels

USGS Publications Warehouse

Fischer, Jesse R.; Neebling, Travis E.; Quist, Michael C.

2012-01-01

Development of radio frequency identification (RFID) technology and passive integrated transponder (PIT) tags has substantially increased the ability of researchers and managers to monitor populations of aquatic organisms. However, use of transportable RFID antenna systems (i.e., backpack-mounted) is currently limited to wadeable aquatic environments (<1.4 m water depth). We describe the design, construction, and evaluation of a boat-mounted RFID antenna to detect individually PIT-tagged benthic aquatic organisms (mussels). We evaluated the effects of tag orientation on detection distances in water with a 32-mm half-duplex PIT tag. Detection distances up to 50 cm from the antenna coils were obtained, but detection distance was dependent on tag orientation. We also evaluated detection distance of PIT tags beneath the sediment to simulate detection of burrowing mussels with 23- and 32-mm tags. In sand substrate, the maximum detection distance varied from 3.5 cm and 4.5 cm (vertical tag orientation) to 24.7 cm and 39.4 cm (45° tag orientation) for the 23- and 32-mm PIT tags, respectively. Our results suggest a 1.4-m total detection width for tagged mussels on the substrate surface by the boat-mounted antenna system regardless of tag orientation. However, burrowed mussels may require multiple passes to increase detection that would be influenced by depth, tag orientation, and tag size. Construction of the boat-mounted antenna was relatively low in cost (<500 USD) and had several advantages (less labor and time intensive, increased safety) over traditional mussel sampling techniques (diving, snorkeling) in nonwadeable habitats.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kerr, J.M.; Fisher, L.W.; Termine, J.D.

The authors have isolated and partially sequenced the human bone sialoprotein gene (IBSP). IBSP has been sublocalized by in situ hybridization to chromosome 4q38-q31 and is composed of six small exons (51 to 159 bp) and 1 large exon ([approximately]2.6 kb). The intron/exon junctions defined by sequence analysis are of class O, retaining an intact coding triplet. Sequence analysis of the 5[prime] upstream region revealed a TATAA (nucleotides -30 to-25 from the transcriptional start point) and a CCAAT (nucleotides -56 to-52) box, both in the reverse orientation. Intron 1 contains interesting structural elements composed of polypyrimidine repeats followed by amore » poly(AC)[sub n] tract. Both types of structural elements have been detected in promoter regions of other genes and have been implicated in transcriptional regulation. Several differences between the previously published cDNA sequence and the authors' sequence have been identified, most of which are contained within the untranslated exon 1. Three base revisions in the coding region include a G to T (Gly to Val, amino acid 195), T to C (Val to Ala, amino acid 268), and T to A (Glu to Asp, amino acid 270). In conclusion, the genomic organization and potential regulatory elements of human IBSP have been elucidated. 42 refs., 4 figs., 1 tab.« less

Oligosyndactylism Mice Have an Inversion of Chromosome 8

PubMed Central

Wise, Thomas L.; Pravtcheva, Dimitrina D.

2004-01-01

The radiation-induced mutation Oligosyndactylism (Os) is associated with limb and kidney defects in heterozygotes and with mitotic arrest and embryonic lethality in homozygotes. We reported that the cell cycle block in Os and in the 94-A/K transgene-induced mutations is due to disruption of the Anapc10 (Apc10/Doc1) gene. To understand the genetic basis of the limb and kidney abnormalities in Os mice we characterized the structural changes of chromosome 8 associated with this mutation. We demonstrate that the Os chromosome 8 has suffered two breaks that are 5 cM (∼10 Mb) apart and the internal fragment delineated by the breaks is in an inverted orientation on the mutant chromosome. While sequences in proximity to the distal break are present in an abnormal Os-specific Anapc10 hybrid transcript, transcription of these sequences in normal mice is low and difficult to detect. Transfer of the Os mutation onto an FVB/N background indicated that the absence of dominant effects in 94-A/K mice is not due to strain background effects on the mutation. Further analysis of this mutation will determine if a gene interrupted by the break or a long-range effect of the rearrangement on neighboring genes is responsible for the dominant effects of Os. PMID:15611179

Regulation of an antisense RNA with the transition of neonatal to IIb myosin heavy chain during postnatal development and hypothyroidism in rat skeletal muscle

PubMed Central

Jiang, Weihua; Qin, Anqi X.; Bodell, Paul W.; Baldwin, Kenneth M.; Haddad, Fadia

2012-01-01

Postnatal development of fast skeletal muscle is characterized by a transition in expression of myosin heavy chain (MHC) isoforms, from primarily neonatal MHC at birth to primarily IIb MHC in adults, in a tightly coordinated manner. These isoforms are encoded by distinct genes, which are separated by ∼17 kb on rat chromosome 10. The neonatal-to-IIb MHC transition is inhibited by a hypothyroid state. We examined RNA products [mRNA, pre-mRNA, and natural antisense transcript (NAT)] of developmental and adult-expressed MHC genes (embryonic, neonatal, I, IIa, IIx, and IIb) at 2, 10, 20, and 40 days after birth in normal and thyroid-deficient rat neonates treated with propylthiouracil. We found that a long noncoding antisense-oriented RNA transcript, termed bII NAT, is transcribed from a site within the IIb-Neo intergenic region and across most of the IIb MHC gene. NATs have previously been shown to mediate transcriptional repression of sense-oriented counterparts. The bII NAT is transcriptionally regulated during postnatal development and in response to hypothyroidism. Evidence for a regulatory mechanism is suggested by an inverse relationship between IIb MHC and bII NAT in normal and hypothyroid-treated muscle. Neonatal MHC transcription is coordinately expressed with bII NAT. A comparative phylogenetic analysis also suggests that bII NAT-mediated regulation has been a conserved trait of placental mammals for most of the eutherian evolutionary history. The evidence in support of the regulatory model implicates long noncoding antisense RNA as a mechanism to coordinate the transition between neonatal and IIb MHC during postnatal development. PMID:22262309

Regulation of an antisense RNA with the transition of neonatal to IIb myosin heavy chain during postnatal development and hypothyroidism in rat skeletal muscle.

PubMed

Pandorf, Clay E; Jiang, Weihua; Qin, Anqi X; Bodell, Paul W; Baldwin, Kenneth M; Haddad, Fadia

2012-04-01

Postnatal development of fast skeletal muscle is characterized by a transition in expression of myosin heavy chain (MHC) isoforms, from primarily neonatal MHC at birth to primarily IIb MHC in adults, in a tightly coordinated manner. These isoforms are encoded by distinct genes, which are separated by ∼17 kb on rat chromosome 10. The neonatal-to-IIb MHC transition is inhibited by a hypothyroid state. We examined RNA products [mRNA, pre-mRNA, and natural antisense transcript (NAT)] of developmental and adult-expressed MHC genes (embryonic, neonatal, I, IIa, IIx, and IIb) at 2, 10, 20, and 40 days after birth in normal and thyroid-deficient rat neonates treated with propylthiouracil. We found that a long noncoding antisense-oriented RNA transcript, termed bII NAT, is transcribed from a site within the IIb-Neo intergenic region and across most of the IIb MHC gene. NATs have previously been shown to mediate transcriptional repression of sense-oriented counterparts. The bII NAT is transcriptionally regulated during postnatal development and in response to hypothyroidism. Evidence for a regulatory mechanism is suggested by an inverse relationship between IIb MHC and bII NAT in normal and hypothyroid-treated muscle. Neonatal MHC transcription is coordinately expressed with bII NAT. A comparative phylogenetic analysis also suggests that bII NAT-mediated regulation has been a conserved trait of placental mammals for most of the eutherian evolutionary history. The evidence in support of the regulatory model implicates long noncoding antisense RNA as a mechanism to coordinate the transition between neonatal and IIb MHC during postnatal development.

USE OF TRANSCRIPTIONAL COUPLING AND KEGG PATHWAY ANALYSIS OF GLOBAL GENE EXPRESSION TO REVEAL TRANSCRIPTIONAL CHANGES BETWEEN STATIONARY- AND LOG-PHASE SALMONELLA TYPHIMURIUM LT2

EPA Science Inventory

DNA microarray analysis is plagued by a lack of data reproducibility and by limits to the detectability of transcripts by hybridization. To mitigate these limitations, we employed transcriptional coupling within the S. typhimurium genome. This genome has 2664 transcriptionally co...

ExtraTrain: a database of Extragenic regions and Transcriptional information in prokaryotic organisms

PubMed Central

Pareja, Eduardo; Pareja-Tobes, Pablo; Manrique, Marina; Pareja-Tobes, Eduardo; Bonal, Javier; Tobes, Raquel

2006-01-01

Background Transcriptional regulation processes are the principal mechanisms of adaptation in prokaryotes. In these processes, the regulatory proteins and the regulatory DNA signals located in extragenic regions are the key elements involved. As all extragenic spaces are putative regulatory regions, ExtraTrain covers all extragenic regions of available genomes and regulatory proteins from bacteria and archaea included in the UniProt database. Description ExtraTrain provides integrated and easily manageable information for 679816 extragenic regions and for the genes delimiting each of them. In addition ExtraTrain supplies a tool to explore extragenic regions, named Palinsight, oriented to detect and search palindromic patterns. This interactive visual tool is totally integrated in the database, allowing the search for regulatory signals in user defined sets of extragenic regions. The 26046 regulatory proteins included in ExtraTrain belong to the families AraC/XylS, ArsR, AsnC, Cold shock domain, CRP-FNR, DeoR, GntR, IclR, LacI, LuxR, LysR, MarR, MerR, NtrC/Fis, OmpR and TetR. The database follows the InterPro criteria to define these families. The information about regulators includes manually curated sets of references specifically associated to regulator entries. In order to achieve a sustainable and maintainable knowledge database ExtraTrain is a platform open to the contribution of knowledge by the scientific community providing a system for the incorporation of textual knowledge. Conclusion ExtraTrain is a new database for exploring Extragenic regions and Transcriptional information in bacteria and archaea. ExtraTrain database is available at . PMID:16539733

Specific detection of rinderpest virus by real-time reverse transcription-PCR in preclincal and clinical samples of experimentally infected cattle

USDA-ARS?s Scientific Manuscript database

A highly sensitive detection test for Rinderpest virus (RPV), based on a real-time reverse transcription-PCR (RT-PR) system, was developed. Five different RPV genomic targets were examined, and one was selected and optimized to detect viral RNA in infected tissue culture fluid with a level of detec...

Development of duplex SYBR Green I-based real-time quantitative reverse-transcription PCR for detection and discrimination of grapevine viruses

USDA-ARS?s Scientific Manuscript database

A SYBR® Green-based real-time quantitative reverse transcription PCR (qRT-PCR) assay in combination with melt curve analysis (MCA) was developed for the detection of nine grapevine viruses. The detection limits for singleplex qRT-PCR for all nine grapevine viruses were determined to be in the range ...

Characterizing the developmental transcriptome of the oriental fruit fly, Bactrocera dorsalis (Diptera: Tephritidae) through comparative genomic analysis with Drosophila melanogaster utilizing modENCODE datasets.

PubMed

Geib, Scott M; Calla, Bernarda; Hall, Brian; Hou, Shaobin; Manoukis, Nicholas C

2014-10-28

The oriental fruit fly, Bactrocera dorsalis, is an important pest of fruit and vegetable crops throughout Asia, and is considered a high risk pest for establishment in the mainland United States. It is a member of the family Tephritidae, which are the most agriculturally important family of flies, and can be considered an out-group to well-studied members of the family Drosophilidae. Despite their importance as pests and their relatedness to Drosophila, little information is present on B. dorsalis transcripts and proteins. The objective of this paper is to comprehensively characterize the transcripts present throughout the life history of B. dorsalis and functionally annotate and analyse these transcripts relative to the presence, expression, and function of orthologous sequences present in Drosophila melanogaster. We present a detailed transcriptome assembly of B. dorsalis from egg through adult stages containing 20,666 transcripts across 10,799 unigene components. Utilizing data available through Flybase and the modENCODE project, we compared expression patterns of these transcripts to putative orthologs in D. melanogaster in terms of timing, abundance, and function. In addition, temporal expression patterns in B. dorsalis were characterized between stages, to establish the constitutive or stage-specific expression patterns of particular transcripts. A fully annotated transcriptome assembly is made available through NCBI, in addition to corresponding expression data. Through characterizing the transcriptome of B. dorsalis through its life history and comparing the transcriptome of B. dorsalis to the model organism D. melanogaster, a database has been developed that can be used as the foundation to functional genomic research in Bactrocera flies and help identify orthologous genes between B. dorsalis and D. melanogaster. This data provides the foundation for future functional genomic research that will focus on improving our understanding of the physiology and biology of this species at the molecular level. This knowledge can also be applied towards developing improved methods for control, survey, and eradication of this important pest.

A non-isotopic assay uses bromouridine and RNA synthesis to detect DNA damage responses.

PubMed

Hasegawa, Mayu; Iwai, Shigenori; Kuraoka, Isao

2010-06-17

Individuals with inherited xeroderma pigmentosum (XP) disorder and Cockayne syndrome (CS) are deficient in nucleotide excision repair and experience hypersensitivity to sunlight. Although there are several diagnostic assays for these disorders, the recovery of RNA synthesis (RRS) assay that can discriminate between XP cells and CS cells is very laborious. Here, we report on a novel non-radioisotope RRS assay that uses bromouridine (a uridine analog) as an alternative to (3)H-uridine. This assay can easily detect RNA polymerase I transcription in nucleoli and RNA polymerase II transcription in nuclei. The non-RI RSS assay also can rapidly detect normal RRS activity in HeLa cells. Thus, this assay is useful as a novel and easy technique for CS diagnosis. Because RRS is thought to be related to transcription-coupled DNA repair, which is triggered by the blockage of transcriptional machinery by DNA lesions, this assay may be of use for analysis of DNA repair, transcription, and/or genetic toxicity. Copyright 2010 Elsevier B.V. All rights reserved.

Detection of small orientation changes and the precision of visual working memory.

PubMed

Salmela, Viljami R; Saarinen, Jussi

2013-01-14

We investigated the precision of orientation representations with two tasks, change detection and recall. Previously change detection has been measured only with relatively large orientation changes compared to psychophysical thresholds. In the first experiment, we measured the observers' ability (d') to detect small changes in orientation (5-30°) with 1-4 Gabor items. With one item even a 10° change was well detected (average d'=2.5). As the amount of change increased to 30°, the d' increased to 5.2. When the number of items was increased, the d's gradually decreased. In the second experiment, we used a recall task and the observers adjusted the orientation of a probe Gabor to match the orientation of a Gabor held in the memory. The standard deviation (s.d.) of errors was calculated from the Gaussian distribution fitted to the data. As the number of items increased from 1 to 6, the s.d. increased from 8.6° to 19.6°. Even with six items, the observers did not make any random adjustments. The results show a square root relation between the d'/s.d. and the number of items. The d' in change detection is directly proportional to the square root of (1/n) and the orientation change. The increase of the s.d. in recall task is inversely proportional to square root of (1/n). The results suggest that limited resources and precision of representations, without additional assumptions, determine the memory performance. Copyright © 2012 Elsevier Ltd. All rights reserved.

Deciphering principles of transcription regulation in eukaryotic genomes

PubMed Central

Nguyen, Dat H; D'haeseleer, Patrik

2006-01-01

Transcription regulation has been responsible for organismal complexity and diversity in the course of biological evolution and adaptation, and it is determined largely by the context-dependent behavior of cis-regulatory elements (CREs). Therefore, understanding principles underlying CRE behavior in regulating transcription constitutes a fundamental objective of quantitative biology, yet these remain poorly understood. Here we present a deterministic mathematical strategy, the motif expression decomposition (MED) method, for deriving principles of transcription regulation at the single-gene resolution level. MED operates on all genes in a genome without requiring any a priori knowledge of gene cluster membership, or manual tuning of parameters. Applying MED to Saccharomyces cerevisiae transcriptional networks, we identified four functions describing four different ways that CREs can quantitatively affect gene expression levels. These functions, three of which have extrema in different positions in the gene promoter (short-, mid-, and long-range) whereas the other depends on the motif orientation, are validated by expression data. We illustrate how nature could use these principles as an additional dimension to amplify the combinatorial power of a small set of CREs in regulating transcription. PMID:16738557

A distinct subgroup of cardiomyopathy patients characterized by transcriptionally active cardiotropic erythrovirus and altered cardiac gene expression.

PubMed

Kuhl, U; Lassner, D; Dorner, A; Rohde, M; Escher, F; Seeberg, B; Hertel, E; Tschope, C; Skurk, C; Gross, U M; Schultheiss, H-P; Poller, W

2013-09-01

Recent studies have detected erythrovirus genomes in the hearts of cardiomyopathy and cardiac transplant patients. Assessment of the functional status of viruses may provide clinically important information beyond detection of the viral genomes. Here, we report transcriptional activation of cardiotropic erythrovirus to be associated with strongly altered myocardial gene expression in a distinct subgroup of cardiomyopathy patients. Endomyocardial biopsies (EMBs) from 415 consecutive cardiac erythrovirus (B19V)-positive patients with clinically suspected cardiomyopathy were screened for virus-encoded VP1/VP2 mRNA indicating transcriptional activation of the virus, and correlated with cardiac host gene expression patterns in transcriptionally active versus latent infections, and in virus-free control hearts. Transcriptional activity was detected in baseline biopsies of only 66/415 patients (15.9 %) harbouring erythrovirus. At the molecular level, significant differences between cardiac B19V-positive patients with transcriptionally active versus latent virus were revealed by expression profiling of EMBs. Importantly, latent B19V infection was indistinguishable from controls. Genes involved encode proteins of antiviral immune response, B19V receptor complex, and mitochondrial energy metabolism. Thus, functional mapping of erythrovirus allows definition of a subgroup of B19V-infected cardiomyopathy patients characterized by virus-encoded VP1/VP2 transcripts and anomalous host myocardial transcriptomes. Cardiac B19V reactivation from latency, as reported here for the first time, is a key factor required for erythrovirus to induce altered cardiac gene expression in a subgroup of cardiomyopathy patients. Virus genome detection is insufficient to assess pathogenic potential, but additional transcriptional mapping should be incorporated into future pathogenetic and therapeutic studies both in cardiology and transplantation medicine.

Assessment of Anaerobic Toluene Biodegradation Activity by bssA Transcript/Gene Ratios

PubMed Central

Brow, Christina N.; O'Brien Johnson, Reid; Johnson, Richard L.

2013-01-01

Benzylsuccinate synthase (bssA) genes associated with toluene degradation were profiled across a groundwater contaminant plume under nitrate-reducing conditions and were detected in significant numbers throughout the plume. However, differences between groundwater and core sediment samples suggested that microbial transport, rather than local activity, was the underlying cause of the high copy numbers within the downgradient plume. Both gene transcript and reactant concentrations were consistent with this hypothesis. Expression of bssA genes from denitrifying toluene degraders was induced by toluene but only in the presence of nitrate, and transcript abundance dropped rapidly following the removal of either toluene or nitrate. The drop in bssA transcripts following the removal of toluene could be described by an exponential decay function with a half-life on the order of 1 h. Interestingly, bssA transcripts never disappeared completely but were always detected at some level if either inducer was present. Therefore, the detection of transcripts alone may not be sufficient evidence for contaminant degradation. To avoid mistakenly associating basal-level gene expression with actively degrading microbial populations, an integrated approach using the ratio of functional gene transcripts to gene copies is recommended. This approach minimizes the impact of microbial transport on activity assessment and allows reliable assessments of microbial activity to be obtained from water samples. PMID:23811506

A focus on polarity: Investigating the role of orientation cues in mediating student performance on mRNA synthesis tasks in an introductory cell and molecular biology course.

PubMed

Olimpo, Jeffrey T; Quijas, Daniel A; Quintana, Anita M

2017-11-01

The central dogma has served as a foundational model for information flow, exchange, and storage in the biological sciences for several decades. Despite its continued importance, however, recent research suggests that novices in the domain possess several misconceptions regarding the aforementioned processes, including those pertaining specifically to the formation of messenger ribonucleic acid (mRNA) transcripts. In the present study, we sought to expand upon these observations through exploration of the influence of orientation cues on students' aptitude at synthesizing mRNAs from provided deoxyribonucleic acid (DNA) template strands. Data indicated that participants (n = 45) were proficient at solving tasks of this nature when the DNA template strand and the mRNA molecule were represented in an antiparallel orientation. In contrast, participants' performance decreased significantly on items in which the mRNA was depicted in a parallel orientation relative to the DNA template strand. Furthermore, participants' Grade Point Average, self-reported confidence in understanding the transcriptional process, and spatial ability were found to mediate their performance on the mRNA synthesis tasks. Collectively, these data reaffirm the need for future research and pedagogical interventions designed to enhance students' comprehension of the central dogma in a manner that makes transparent its relevance to real-world scientific phenomena. © 2017 by The International Union of Biochemistry and Molecular Biology, 45(6):501-508, 2017. © 2017 The International Union of Biochemistry and Molecular Biology.

SU-E-J-15: Automatically Detect Patient Treatment Position and Orientation in KV Portal Images

DOE Office of Scientific and Technical Information (OSTI.GOV)

Qiu, J; Yang, D

2015-06-15

Purpose: In the course of radiation therapy, the complex information processing workflow will Result in potential errors, such as incorrect or inaccurate patient setups. With automatic image check and patient identification, such errors could be effectively reduced. For this purpose, we developed a simple and rapid image processing method, to automatically detect the patient position and orientation in 2D portal images, so to allow automatic check of positions and orientations for patient daily RT treatments. Methods: Based on the principle of portal image formation, a set of whole body DRR images were reconstructed from multiple whole body CT volume datasets,more » and fused together to be used as the matching template. To identify the patient setup position and orientation shown in a 2D portal image, the 2D portal image was preprocessed (contrast enhancement, down-sampling and couch table detection), then matched to the template image so to identify the laterality (left or right), position, orientation and treatment site. Results: Five day’s clinical qualified portal images were gathered randomly, then were processed by the automatic detection and matching method without any additional information. The detection results were visually checked by physicists. 182 images were correct detection in a total of 200kV portal images. The correct rate was 91%. Conclusion: The proposed method can detect patient setup and orientation quickly and automatically. It only requires the image intensity information in KV portal images. This method can be useful in the framework of Electronic Chart Check (ECCK) to reduce the potential errors in workflow of radiation therapy and so to improve patient safety. In addition, the auto-detection results, as the patient treatment site position and patient orientation, could be useful to guide the sequential image processing procedures, e.g. verification of patient daily setup accuracy. This work was partially supported by research grant from Varian Medical System.« less

Single-Reaction Multiplex Reverse Transcription PCR for Detection of Zika, Chikungunya, and Dengue Viruses

PubMed Central

Waggoner, Jesse J.; Gresh, Lionel; Mohamed-Hadley, Alisha; Ballesteros, Gabriela; Davila, Maria Jose Vargas; Tellez, Yolanda; Sahoo, Malaya K.; Balmaseda, Angel; Harris, Eva

2016-01-01

Clinical manifestations of Zika virus, chikungunya virus, and dengue virus infections can be similar. To improve virus detection, streamline molecular workflow, and decrease test costs, we developed and evaluated a multiplex real-time reverse transcription PCR for these viruses. PMID:27184629

Determining the orientation of depth-rotated familiar objects.

PubMed

Niimi, Ryosuke; Yokosawa, Kazuhiko

2008-02-01

How does the human visual system determine the depth-orientation of familiar objects? We examined reaction times and errors in the detection of 15 degrees differences in the depth orientations of two simultaneously presented familiar objects, which were the same objects (Experiment 1) or different objects (Experiment 2). Detection of orientation differences was best for 0 degrees (front) and 180 degrees (back), while 45 degrees and 135 degrees yielded poorer results, and 90 degrees (side) showed intermediate results, suggesting that the visual system is tuned for front, side and back orientations. We further found that those advantages are due to orientation-specific features such as horizontal linear contours and symmetry, since the 90 degrees advantage was absent for objects with curvilinear contours, and asymmetric object diminished the 0 degrees and 180 degrees advantages. We conclude that the efficiency of visually determining object orientation is highly orientation-dependent, and object orientation may be perceived in favor of front-back axes.

Multiple groups of orientation-selective visual mechanisms underlying rapid orientated-line detection.

PubMed Central

Foster, D H; Westland, S

1998-01-01

Visual search for an edge or line element differing in orientation from a background of other edge or line elements can be performed rapidly and effortlessly. In this study, based on psychophysical measurements with ten human observers, threshold values of the angle between a target and background line elements were obtained as functions of background-element orientation, in brief masked displays. A repeated-loess analysis of the threshold functions suggested the existence of several groups of orientation-selective mechanisms contributing to rapid orientated-line detection; specifically, coarse, intermediate and fine mechanisms with preferred orientations spaced at angles of approximately 90 degrees, 35 degrees, and 10 degrees-25 degrees, respectively. The preferred orientations of coarse and some intermediate mechanisms coincided with the vertical or horizontal of the frontoparallel plane, but the preferred orientations of fine mechanisms varied randomly from observer to observer, possibly reflecting individual variations in neuronal sampling characteristics. PMID:9753784

Regulation of transcriptional activators by DNA-binding domain ubiquitination

PubMed Central

Landré, Vivien; Revi, Bhindu; Mir, Maria Gil; Verma, Chandra; Hupp, Ted R; Gilbert, Nick; Ball, Kathryn L

2017-01-01

Ubiquitin is a key component of the regulatory network that maintains gene expression in eukaryotes, yet the molecular mechanism(s) by which non-degradative ubiquitination modulates transcriptional activator (TA) function is unknown. Here endogenous p53, a stress-activated transcription factor required to maintain health, is stably monoubiquitinated, following pathway activation by IR or Nutlin-3 and localized to the nucleus where it becomes tightly associated with chromatin. Comparative structure–function analysis and in silico modelling demonstrate a direct role for DNA-binding domain (DBD) monoubiquitination in TA activation. When attached to the DBD of either p53, or a second TA IRF-1, ubiquitin is orientated towards, and makes contact with, the DNA. The contact is made between a predominantly cationic surface on ubiquitin and the anionic DNA. Our data demonstrate an unexpected role for ubiquitin in the mechanism of TA-activity enhancement and provides insight into a new level of transcriptional regulation. PMID:28362432

In vivo expression of Salmonella enterica serotype Typhi genes in the blood of patients with typhoid fever in Bangladesh.

PubMed

Sheikh, Alaullah; Charles, Richelle C; Sharmeen, Nusrat; Rollins, Sean M; Harris, Jason B; Bhuiyan, Md Saruar; Arifuzzaman, Mohammad; Khanam, Farhana; Bukka, Archana; Kalsy, Anuj; Porwollik, Steffen; Leung, Daniel T; Brooks, W Abdullah; LaRocque, Regina C; Hohmann, Elizabeth L; Cravioto, Alejandro; Logvinenko, Tanya; Calderwood, Stephen B; McClelland, Michael; Graham, James E; Qadri, Firdausi; Ryan, Edward T

2011-12-01

Salmonella enterica serotype Typhi is the cause of typhoid fever. It is a human-restricted pathogen, and few data exist on S. Typhi gene expression in humans. We applied an RNA capture and amplification technique, Selective Capture of Transcribed Sequences (SCOTS), and microarray hybridization to identify S. Typhi transcripts expressed in the blood of five humans infected with S. Typhi in Bangladesh. In total, we detected the expression of mRNAs for 2,046 S. Typhi genes (44% of the S. Typhi genome) in human blood; expression of 912 genes was detected in all 5 patients, and expression of 1,100 genes was detected in 4 or more patients. Identified transcripts were associated with the virulence-associated PhoP regulon, Salmonella pathogenicity islands, the use of alternative carbon and energy sources, synthesis and transport of iron, thiamine, and biotin, and resistance to antimicrobial peptides and oxidative stress. The most highly represented group were genes currently annotated as encoding proteins designated as hypothetical, unknown, or unclassified. Of the 2,046 detected transcripts, 1,320 (29% of the S. Typhi genome) had significantly different levels of detection in human blood compared to in vitro cultures; detection of 141 transcripts was significantly different in all 5 patients, and detection of 331 transcripts varied in at least 4 patients. These mRNAs encode proteins of unknown function, those involved in energy metabolism, transport and binding, cell envelope, cellular processes, and pathogenesis. We confirmed increased expression of a subset of identified mRNAs by quantitative-PCR. We report the first characterization of bacterial transcriptional profiles in the blood of patients with typhoid fever. S. Typhi is an important global pathogen whose restricted host range has greatly inhibited laboratory studies. Our results suggest that S. Typhi uses a largely uncharacterized genetic repertoire to survive within cells and utilize alternate energy sources during infection.

Identification and characterization of Kaposi's sarcoma-associated herpesvirus open reading frame 11 promotor activation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, Lei

2008-01-01

Open reading frame 11 (ORF11) of Kaposi's sarcoma-associated herpesvirus belongs to a herpesviral homologous protein family shared by some members of the gamma- herpesvirus subfamily. Little is known about this ORF11 homologous protein family. We have characterized an unknown open reading frame, ORF11, located adjacent and in the opposite orientation to a well-characterized viral IL-6 gene. Northern blot analysis reveals that ORF11 is expressed during the KSHV lytic cycle with delayed-early transcription kinetics. We have determined the 5{prime} and 3{prime} untranslated region of the unspliced ORF11 transcript and identified both the transcription start site and the transcription termination site. Coremore » promoter region, representing ORF11 promoter activity, was mapped to a 159nt fragment 5{prime} most proximal to the transcription start site. A functional TATA box was identified in the core promoter region. Interestingly, we found that ORF11 transcriptional activation is not responsive to Rta, the KSHV lytic switch protein. We also discovered that part of the ORF11 promoter region, the 209nt fragment upstream of the transcription start site, was repressed by phorbol esters. Our data help to understand transcription regulation of ORF11 and to elucidate roles of ORF11 in KSHV pathogenesis and life cycle.« less

[Disappearance of residual disease confirmed by RT-PCR following induction chemotherapy in two hypoplastic leukemia patients with t(8;21)].

PubMed

Sawada, M; Tsurumi, H; Yamada, T; Hara, T; Oyama, M; Moriwaki, H

1999-04-01

Reverse transcriptase-polymerase chain reaction (RT-PCR) methods often detect the AML1/MTG8 fusion transcript even in acute myelogenous leukemia (AML) patients with t(8;21) who have been in long-term remission. We encountered 2 hypoplastic leukemia patients with t(8;21) who achieved cytogenetic remission with short-term conventional chemotherapy. Patient 1 was a 42-year-old woman. Chromosomal analysis detected t(8;21) (q22;q22) and PCR analysis (35 cycles PCR amplification; detection limit 1 x 10(-5) cells) detected the AML1/MTG8 fusion transcript. Complete remission was obtained with 1 course of chemotherapy consisting of low-dose cytarabine (20 mg x 14 days) and etoposide (50 mg x 14 days). After 2 courses of consolidation chemotherapy consisting of conventional-dose cytarabine and mitoxantrone, the RT-PCR findings were negative for the AML1/MTG8 fusion transcript. Patient 2 was a 67-year-old man. Cytogenetic analysis detected t(8;21) (q22;q22), and was positive for the AML1/MTG8 fusion transcript. After 2 courses of induction chemotherapy comprising low-dose cytarabine (20 mg x 14 days) and etoposide (50 mg x 14 days), and 3 courses of conventional consolidation chemotherapy, RT-PCR analysis confirmed the disappearance of the AML1/MTG8 fusion transcript.

POU2F2-oriented network promotes human gastric cancer metastasis

PubMed Central

Wang, Si-Meng; Tie, Jun; Wang, Wen-Lan; Hu, Si-Jun; Yin, Ji-Peng; Yi, Xiao-Fang; Tian, Zu-Hong; Zhang, Xiang-Yuan; Li, Meng-Bin; Li, Zeng-Shan; Nie, Yong-Zhan; Wu, Kai-Chun; Fan, Dai-Ming

2016-01-01

Background and aims Aberrant upregulation of POU2F2 expression has been discovered in metastatic gastric cancer (GC). However, the mechanisms underlying the aberrant upregulation and the potential functions of POU2F2 remain uncertain. Design The role and mechanism of POU2F2 in GC metastasis were investigated in gastric epithelial cells, GC cell lines and an experimental metastasis animal model by gain of function and loss of function. Upstream and downstream targets of POU2F2 were selected by bioinformatics and identified by luciferase reporter assay, electrophoretic mobility shift assay and chromatin immunoprecipitation PCR. The influence of miR-218 on its putative target genes (POU2F2, ROBO1 and IKK-β) and GC metastasis was further explored via in vitro and in vivo approaches. Results Increased POU2F2 expression was detected in metastatic GC cell lines and patient samples. POU2F2 was induced by the activation of nuclear factor (NF)-κB and, in turn, regulated ROBO1 transcription, thus functionally contributing to GC metastasis. Finally, miR-218 was found to suppress GC metastasis by simultaneously mediating multiple molecules in the POU2F2-oriented network. Conclusions This study demonstrated that NF-κB and the SLIT2/ROBO1 interaction network with POU2F2 as the central part may exert critical effects on tumour metastasis. Blocking the activation of the POU2F2-oriented metastasis network using miR-218 precursors exemplified a promising approach that sheds light on new strategies for GC treatment. PMID:26019213

An Annotation Agnostic Algorithm for Detecting Nascent RNA Transcripts in GRO-Seq.

PubMed

Azofeifa, Joseph G; Allen, Mary A; Lladser, Manuel E; Dowell, Robin D

2017-01-01

We present a fast and simple algorithm to detect nascent RNA transcription in global nuclear run-on sequencing (GRO-seq). GRO-seq is a relatively new protocol that captures nascent transcripts from actively engaged polymerase, providing a direct read-out on bona fide transcription. Most traditional assays, such as RNA-seq, measure steady state RNA levels which are affected by transcription, post-transcriptional processing, and RNA stability. GRO-seq data, however, presents unique analysis challenges that are only beginning to be addressed. Here, we describe a new algorithm, Fast Read Stitcher (FStitch), that takes advantage of two popular machine-learning techniques, hidden Markov models and logistic regression, to classify which regions of the genome are transcribed. Given a small user-defined training set, our algorithm is accurate, robust to varying read depth, annotation agnostic, and fast. Analysis of GRO-seq data without a priori need for annotation uncovers surprising new insights into several aspects of the transcription process.

Urinary exosomal transcription factors, a new class of biomarkers for renal disease

PubMed Central

Zhou, Hua; Cheruvanky, Anita; Hu, Xuzhen; Matsumoto, Takayuki; Hiramatsu, Noriyuki; Cho, Monique E.; Berger, Alexandra; Leelahavanichkul, Asada; Doi, Kent; Chawla, Lakhmir S.; Illei, Gabor G.; Kopp, Jeffrey B.; Balow, James E.; Austin, Howard A.; Yuen, Peter S.T.; Star, Robert A.

2008-01-01

Urinary exosomes are excreted from all nephron segments and are a rich source of kidney injury biomarkers. Because exosomes contain intracellular proteins, we asked if transcription factors (TF) can be measured in urinary exosomes. We collected urine from two acute kidney injury (AKI) models (cisplatin or ischemia/reperfusion) and two podocyte injury models (puromycin-treated rats and podocin/Vpr transgenic mice). Human urine was obtained from patients with AKI, focal segmental glomerulosclerosis (FSGS), and matched controls. After isolating urine exosomes by differential centrifugation, activating transcription factor 3 (ATF3) and Wilms Tumor 1 (WT-1) were detected by western blot. ATF3 was continuously detected in urine exosomes 2–24 hr after ischemia/reperfusion and in a biphasic pattern after cisplatin. In both models, urinary ATF3 was detected earlier than serum creatinine. Urinary ATF3 was detected in AKI patients but not in normal subjects or patients with chronic kidney disease (CKD). Urinary WT-1 was detected in animal models before significant glomerular sclerosis. Urinary WT-1 was detected in 9/10 FSGS patients, but not in 8 controls. Transcription factors can be detected in urine exosomes, but not in whole urine. Urinary ATF3 may be a novel renal tubular cell injury biomarker for detecting early AKI, whereas urinary WT-1 may detect early podocyte injury. Urinary exosomal TFs represent a new class of biomarkers for acute and chronic renal diseases and may offer insight into cellular regulatory pathways. PMID:18509321

Understanding the molecular aspects of oriental obesity pattern differentiation using DNA microarray.

PubMed

Hong, Sun Woo; Yoo, Jae-Wook; Bose, Shambhunath; Park, Jung-Hyun; Han, Kyungsun; Kim, Soyoun; Lim, Chi-Yeon; Kim, Hojun; Lee, Dong-Ki

2015-10-19

Human constitution, the fundamental basis of oriental medicine, is categorized into different patterns for a particular disease according to the physical, physiological, and clinical characteristics of the individuals. Obesity, a condition of metabolic disorder, is classified according to six patterns in oriental medicine, as follows: spleen deficiency syndrome, phlegm fluid syndrome, yang deficiency syndrome (YDS), food accumulation syndrome (FAS), liver depression syndrome (LDS), and blood stasis syndrome. In oriental medicine, identification of the disease pattern for individual obese patients is performed on the basis of differentiation in obesity syndrome index and, accordingly, personalized treatment is provided to the patients. The aim of the current study was to understand the obesity patterns in oriental medicine from the genomic point of view via determining the gene expression signature of obese patients using peripheral blood mononuclear cells as the samples. The study was conducted in 23 South Korean obese subjects (19 female and four male) with BMI ≥25 kg/m(2). Identification of oriental obesity pattern was based on the software-guided evaluation of the responses of the subjects to a questionnaire developed by the Korean Institute of Oriental Medicine. The expression profiles of genes were determined using DNA microarray and the level of transcription of genes of interest was further evaluated using quantitative real-time PCR (qRT-PCR). Gene clustering analysis of the microarray data from the FAS, LDS, and YDS subjects exhibited disease pattern-specific upregulation of expression of several genes in a particular cluster. Further analysis of transcription of selected genes using qRT-PCR led to identification of specific genes, including prostaglandin endoperoxide synthase 2, G0/G1 switch 2, carcinoembryonic antigen-related cell adhesion molecule 3, cystein-serine-rich nuclear protein 1, and interleukin 8 receptor, alpha which were highly expressed in LDS obesity constitution. Our current study can be considered as a valuable contribution to the understanding of possible explanation for obesity pattern differentiation in oriental medicine. Further studies can address a novel possibility that the genomic and oriental empirical approaches can be combined and implemented in systematic and synergistic development of personalized medicine. This clinical trial was registered in Clinical Research Information Service of Korea National Institute of Health ( https://cris.nih.go.kr/cris/index.jsp ). KCT0000387.

A METHOD TO REMOVE ENVIRONMENTAL INHIBITORS PRIOR TO THE DETECTION OF WATERBORNE ENTERIC VIRUSES BY REVERSE TRANSCRIPTION-POLYMERASE CHAIN REACTION

EPA Science Inventory

A method was developed to remove environmental inhibitors from sample concentrates prior to detection of human enteric viruses using the reverse transcription-polymerase chain reaction (RT-PCR).Environmental inhibitors, concentrated along with viruses during water sample processi...

SmartMal: a service-oriented behavioral malware detection framework for mobile devices.

PubMed

Wang, Chao; Wu, Zhizhong; Li, Xi; Zhou, Xuehai; Wang, Aili; Hung, Patrick C K

2014-01-01

This paper presents SmartMal--a novel service-oriented behavioral malware detection framework for vehicular and mobile devices. The highlight of SmartMal is to introduce service-oriented architecture (SOA) concepts and behavior analysis into the malware detection paradigms. The proposed framework relies on client-server architecture, the client continuously extracts various features and transfers them to the server, and the server's main task is to detect anomalies using state-of-art detection algorithms. Multiple distributed servers simultaneously analyze the feature vector using various detectors and information fusion is used to concatenate the results of detectors. We also propose a cycle-based statistical approach for mobile device anomaly detection. We accomplish this by analyzing the users' regular usage patterns. Empirical results suggest that the proposed framework and novel anomaly detection algorithm are highly effective in detecting malware on Android devices.

SmartMal: A Service-Oriented Behavioral Malware Detection Framework for Mobile Devices

PubMed Central

Wu, Zhizhong; Li, Xi; Zhou, Xuehai; Wang, Aili; Hung, Patrick C. K.

2014-01-01

This paper presents SmartMal—a novel service-oriented behavioral malware detection framework for vehicular and mobile devices. The highlight of SmartMal is to introduce service-oriented architecture (SOA) concepts and behavior analysis into the malware detection paradigms. The proposed framework relies on client-server architecture, the client continuously extracts various features and transfers them to the server, and the server's main task is to detect anomalies using state-of-art detection algorithms. Multiple distributed servers simultaneously analyze the feature vector using various detectors and information fusion is used to concatenate the results of detectors. We also propose a cycle-based statistical approach for mobile device anomaly detection. We accomplish this by analyzing the users' regular usage patterns. Empirical results suggest that the proposed framework and novel anomaly detection algorithm are highly effective in detecting malware on Android devices. PMID:25165729

Swinger RNAs with sharp switches between regular transcription and transcription systematically exchanging ribonucleotides: Case studies.

PubMed

Seligmann, Hervé

2015-09-01

During RNA transcription, DNA nucleotides A,C,G, T are usually matched by ribonucleotides A, C, G and U. However occasionally, this rule does not apply: transcript-DNA homologies are detectable only assuming systematic exchanges between ribonucleotides. Nine symmetric (X ↔ Y, e.g. A ↔ C) and fourteen asymmetric (X ↔ Y ↔ Z, e.g. A ↔ C ↔ G) exchanges exist, called swinger transcriptions. Putatively, polymerases occasionally stabilize in unspecified swinger conformations, possibly similar to transient conformations causing punctual misinsertions. This predicts chimeric transcripts, part regular, part swinger-transformed, reflecting polymerases switching to swinger polymerization conformation(s). Four chimeric Genbank transcripts (three from human mitochondrion and one murine cytosolic) are described here: (a) the 5' and 3' extremities reflect regular polymerization, the intervening sequence exchanges systematically between ribonucleotides (swinger rule G ↔ U, transcript (1), with sharp switches between regular and swinger sequences; (b) the 5' half is 'normal', the 3' half systematically exchanges ribonucleotides (swinger rule C ↔ G, transcript (2), with an intercalated sequence lacking homology; (c) the 3' extremity fits A ↔ G exchanges (10% of transcript length), the 5' half follows regular transcription; the intervening region seems a mix of regular and A ↔ G transcriptions (transcript 3); (d) murine cytosolic transcript 4 switches to A ↔ U + C ↔ G, and is fused with A ↔ U + C ↔ G swinger transformed precursor rRNA. In (c), each concomitant transcript 5' and 3' extremities match opposite genome strands. Transcripts 3 and 4 combine transcript fusions with partial swinger transcriptions. Occasional (usually sharp) switches between regular and swinger transcriptions reveal greater coding potential than detected until now, suggest stable polymerase swinger conformations. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Transcript Isoform Variation Associated with Cytosine Modification in Human Lymphoblastoid Cell Lines.

PubMed

Zhang, Xu; Zhang, Wei

2016-06-01

Cytosine modification on DNA is variable among individuals, which could correlate with gene expression variation. The effect of cytosine modification on interindividual transcript isoform variation (TIV), however, remains unclear. In this study, we assessed the extent of cytosine modification-specific TIV in lymphoblastoid cell lines (LCLs) derived from unrelated individuals of European and African descent. Our study detected cytosine modification-specific TIVs for 17% of the analyzed genes at a 5% false discovery rate. Forty-five percent of the TIV-associated cytosine modifications correlated with the overall gene expression levels as well, with the corresponding CpG sites overrepresented in transcript initiation sites, transcription factor binding sites, and distinct histone modification peaks, suggesting that alternative isoform transcription underlies the TIVs. Our analysis also revealed 33% of the TIV-associated cytosine modifications that affected specific exons, with the corresponding CpG sites overrepresented in exon/intron junctions, splicing branching points, and transcript termination sites, implying that the TIVs are attributable to alternative splicing or transcription termination. Genetic and epigenetic regulation of TIV shared target preference but exerted independent effects on 61% of the common exon targets. Cytosine modification-specific TIVs detected from LCLs were differentially enriched in those detected from various tissues in The Cancer Genome Atlas, indicating their developmental dependency. Genes containing cytosine modification-specific TIVs were enriched in pathways of cancers and metabolic disorders. Our study demonstrated a prominent effect of cytosine modification variation on the transcript isoform spectrum over gross transcript abundance and revealed epigenetic contributions to diseases that were mediated through cytosine modification-specific TIV. Copyright © 2016 by the Genetics Society of America.

Regulating RNA polymerase pausing and transcription elongation in embryonic stem cells

PubMed Central

Min, Irene M.; Waterfall, Joshua J.; Core, Leighton J.; Munroe, Robert J.; Schimenti, John; Lis, John T.

2011-01-01

Transitions between pluripotent stem cells and differentiated cells are executed by key transcription regulators. Comparative measurements of RNA polymerase distribution over the genome's primary transcription units in different cell states can identify the genes and steps in the transcription cycle that are regulated during such transitions. To identify the complete transcriptional profiles of RNA polymerases with high sensitivity and resolution, as well as the critical regulated steps upon which regulatory factors act, we used genome-wide nuclear run-on (GRO-seq) to map the density and orientation of transcriptionally engaged RNA polymerases in mouse embryonic stem cells (ESCs) and mouse embryonic fibroblasts (MEFs). In both cell types, progression of a promoter-proximal, paused RNA polymerase II (Pol II) into productive elongation is a rate-limiting step in transcription of ∼40% of mRNA-encoding genes. Importantly, quantitative comparisons between cell types reveal that transcription is controlled frequently at paused Pol II's entry into elongation. Furthermore, “bivalent” ESC genes (exhibiting both active and repressive histone modifications) bound by Polycomb group complexes PRC1 (Polycomb-repressive complex 1) and PRC2 show dramatically reduced levels of paused Pol II at promoters relative to an average gene. In contrast, bivalent promoters bound by only PRC2 allow Pol II pausing, but it is confined to extremely 5′ proximal regions. Altogether, these findings identify rate-limiting targets for transcription regulation during cell differentiation. PMID:21460038

Enhancer-promoter interference and its prevention in transgenic plants

USDA-ARS?s Scientific Manuscript database

Transcriptional enhancer elements have been shown to override the specificity of nearby promoters in a position- and orientation-independent manner. This is problematic when multiple enhancers/promoters co-exist within a single transgenic construct as it has the potential to cause the mis-expressio...

Reflection during Portfolio-Based Conversations

ERIC Educational Resources Information Center

Oosterbaan, Anne E.; van der Schaaf, Marieke F.; Baartman, Liesbeth K. J.; Stokking, Karel M.

2010-01-01

This study aims to explore the relationship between the occurrence of reflection (and non-reflection) and thinking activities (e.g., orientating, selecting, analysing) during portfolio-based conversations. Analysis of 21 transcripts of portfolio-based conversations revealed that 20% of the segments were made up of reflection (content reflection…

Characterization and Detection of a Widely Distributed Gene Cluster That Predicts Anaerobic Choline Utilization by Human Gut Bacteria

PubMed Central

Martínez-del Campo, Ana; Bodea, Smaranda; Hamer, Hilary A.; Marks, Jonathan A.; Haiser, Henry J.; Turnbaugh, Peter J.

2015-01-01

ABSTRACT Elucidation of the molecular mechanisms underlying the human gut microbiota’s effects on health and disease has been complicated by difficulties in linking metabolic functions associated with the gut community as a whole to individual microorganisms and activities. Anaerobic microbial choline metabolism, a disease-associated metabolic pathway, exemplifies this challenge, as the specific human gut microorganisms responsible for this transformation have not yet been clearly identified. In this study, we established the link between a bacterial gene cluster, the choline utilization (cut) cluster, and anaerobic choline metabolism in human gut isolates by combining transcriptional, biochemical, bioinformatic, and cultivation-based approaches. Quantitative reverse transcription-PCR analysis and in vitro biochemical characterization of two cut gene products linked the entire cluster to growth on choline and supported a model for this pathway. Analyses of sequenced bacterial genomes revealed that the cut cluster is present in many human gut bacteria, is predictive of choline utilization in sequenced isolates, and is widely but discontinuously distributed across multiple bacterial phyla. Given that bacterial phylogeny is a poor marker for choline utilization, we were prompted to develop a degenerate PCR-based method for detecting the key functional gene choline TMA-lyase (cutC) in genomic and metagenomic DNA. Using this tool, we found that new choline-metabolizing gut isolates universally possessed cutC. We also demonstrated that this gene is widespread in stool metagenomic data sets. Overall, this work represents a crucial step toward understanding anaerobic choline metabolism in the human gut microbiota and underscores the importance of examining this microbial community from a function-oriented perspective. PMID:25873372

Analysis of cellulose synthase genes from domesticated apple identifies collinear genes WDR53 and CesA8A: partial co-expression, bicistronic mRNA, and alternative splicing of CESA8A

PubMed Central

Guerriero, Gea; Spadiut, Oliver; Kerschbamer, Christine; Giorno, Filomena; Baric, Sanja; Ezcurra, Inés

2016-01-01

Cellulose synthase (CesA) genes constitute a complex multigene family with six major phylogenetic clades in angiosperms. The recently sequenced genome of domestic apple, Malus×domestica, was mined for CesA genes, by blasting full-length cellulose synthase protein (CESA) sequences annotated in the apple genome against protein databases from the plant models Arabidopsis thaliana and Populus trichocarpa. Thirteen genes belonging to the six angiosperm CesA clades and coding for proteins with conserved residues typical of processive glycosyltransferases from family 2 were detected. Based on their phylogenetic relationship to Arabidopsis CESAs, as well as expression patterns, a nomenclature is proposed to facilitate further studies. Examination of their genomic organization revealed that MdCesA8-A is closely linked and co-oriented with WDR53, a gene coding for a WD40 repeat protein. The WDR53 and CesA8 genes display conserved collinearity in dicots and are partially co-expressed in the apple xylem. Interestingly, the presence of a bicistronic WDR53–CesA8A transcript was detected in phytoplasma-infected phloem tissues of apple. The bicistronic transcript contains a spliced intergenic sequence that is predicted to fold into hairpin structures typical of internal ribosome entry sites, suggesting its potential cap-independent translation. Surprisingly, the CesA8A cistron is alternatively spliced and lacks the zinc-binding domain. The possible roles of WDR53 and the alternatively spliced CESA8 variant during cellulose biosynthesis in M.×domestica are discussed. PMID:23048131

Choice of Grating Orientation for Evaluation of Peripheral Vision

PubMed Central

Venkataraman, Abinaya Priya; Winter, Simon; Rosén, Robert; Lundström, Linda

2016-01-01

ABSTRACT Purpose Peripheral resolution acuity depends on the orientation of the stimuli. However, it is uncertain if such a meridional effect also exists for peripheral detection tasks because they are affected by optical errors. Knowledge of the quantitative differences in acuity for different grating orientations is crucial for choosing the appropriate stimuli for evaluations of peripheral resolution and detection tasks. We assessed resolution and detection thresholds for different grating orientations in the peripheral visual field. Methods Resolution and detection thresholds were evaluated for gratings of four different orientations in eight different visual field meridians in the 20-deg visual field in white light. Detection measurements in monochromatic light (543 nm; bandwidth, 10 nm) were also performed to evaluate the effects of chromatic aberration on the meridional effect. A combination of trial lenses and adaptive optics system was used to correct the monochromatic lower- and higher-order aberrations. Results For both resolution and detection tasks, gratings parallel to the visual field meridian had better threshold compared with the perpendicular gratings, whereas the two oblique gratings had similar thresholds. The parallel and perpendicular grating acuity differences for resolution and detection tasks were 0.16 logMAR and 0.11 logMAD, respectively. Elimination of chromatic errors did not affect the meridional preference in detection acuity. Conclusions Similar to peripheral resolution, detection also shows a meridional effect that appears to have a neural origin. The threshold difference seen for parallel and perpendicular gratings suggests the use of two oblique gratings as stimuli in alternative forced-choice procedures for peripheral vision evaluation to reduce measurement variation. PMID:26889822

Choice of Grating Orientation for Evaluation of Peripheral Vision.

PubMed

Venkataraman, Abinaya Priya; Winter, Simon; Rosén, Robert; Lundström, Linda

2016-06-01

Peripheral resolution acuity depends on the orientation of the stimuli. However, it is uncertain if such a meridional effect also exists for peripheral detection tasks because they are affected by optical errors. Knowledge of the quantitative differences in acuity for different grating orientations is crucial for choosing the appropriate stimuli for evaluations of peripheral resolution and detection tasks. We assessed resolution and detection thresholds for different grating orientations in the peripheral visual field. Resolution and detection thresholds were evaluated for gratings of four different orientations in eight different visual field meridians in the 20-deg visual field in white light. Detection measurements in monochromatic light (543 nm; bandwidth, 10 nm) were also performed to evaluate the effects of chromatic aberration on the meridional effect. A combination of trial lenses and adaptive optics system was used to correct the monochromatic lower- and higher-order aberrations. For both resolution and detection tasks, gratings parallel to the visual field meridian had better threshold compared with the perpendicular gratings, whereas the two oblique gratings had similar thresholds. The parallel and perpendicular grating acuity differences for resolution and detection tasks were 0.16 logMAR and 0.11 logMAD, respectively. Elimination of chromatic errors did not affect the meridional preference in detection acuity. Similar to peripheral resolution, detection also shows a meridional effect that appears to have a neural origin. The threshold difference seen for parallel and perpendicular gratings suggests the use of two oblique gratings as stimuli in alternative forced-choice procedures for peripheral vision evaluation to reduce measurement variation.

A Cell Culture Model of Latent and Lytic Herpes Simplex Virus Type 1 Infection in Spiral Ganglion.

PubMed

Liu, Yuehong; Li, Shufeng

2015-01-01

Reactivation of latent herpes simplex virus type 1 (HSV-1) in spiral ganglion neurons (SGNs) is supposed to be one of the causes of idiopathic sudden sensorineural hearing loss. This study aims to establish a cell culture model of latent and lytic HSV-1 infection in spiral ganglia. In the presence of acyclovir, primary cultures of SGNs were latently infected with HSV-1 expressing green fluorescent protein. Four days later, these cells were treated with trichostatin A (TSA), a known chemical reactivator of HSV-1. TCID50 was used to measure the titers of virus in cultures on Vero cells. RNA from cultures was detected for the presence of transcripts of ICP27 and latency-associated transcript (LAT) using reverse transcription polymerase chain reaction. There is no detectable infectious HSV-1 in latently infected cultures, whereas they could be observed in both lytically infected and latently infected/TSA-treated cultures. LAT was the only detectable transcript during latent infection, whereas lytic ICP27 transcript was detected in lytically infected and latently infected/TSA-treated cultures. Cultured SGNs can be both latently and lytically infected with HSV-1. Furthermore, latently infected SGNs can be reactivated using TSA, yielding infectious virus.

New Views on Strand Asymmetry in Insect Mitochondrial Genomes

PubMed Central

Wei, Shu-Jun; Shi, Min; Chen, Xue-Xin; Sharkey, Michael J.; van Achterberg, Cornelis; Ye, Gong-Yin; He, Jun-Hua

2010-01-01

Strand asymmetry in nucleotide composition is a remarkable feature of animal mitochondrial genomes. Understanding the mutation processes that shape strand asymmetry is essential for comprehensive knowledge of genome evolution, demographical population history and accurate phylogenetic inference. Previous studies found that the relative contributions of different substitution types to strand asymmetry are associated with replication alone or both replication and transcription. However, the relative contributions of replication and transcription to strand asymmetry remain unclear. Here we conducted a broad survey of strand asymmetry across 120 insect mitochondrial genomes, with special reference to the correlation between the signs of skew values and replication orientation/gene direction. The results show that the sign of GC skew on entire mitochondrial genomes is reversed in all species of three distantly related families of insects, Philopteridae (Phthiraptera), Aleyrodidae (Hemiptera) and Braconidae (Hymenoptera); the replication-related elements in the A+T-rich regions of these species are inverted, confirming that reversal of strand asymmetry (GC skew) was caused by inversion of replication origin; and finally, the sign of GC skew value is associated with replication orientation but not with gene direction, while that of AT skew value varies with gene direction, replication and codon positions used in analyses. These findings show that deaminations during replication and other mutations contribute more than selection on amino acid sequences to strand compositions of G and C, and that the replication process has a stronger affect on A and T content than does transcription. Our results may contribute to genome-wide studies of replication and transcription mechanisms. PMID:20856815

Regulation of transcription of the cell division gene ftsA during sporulation of Bacillus subtilis.

PubMed Central

Gholamhoseinian, A; Shen, Z; Wu, J J; Piggot, P

1992-01-01

Three distinct 5' ends of ftsA mRNA were identified by S1 mapping and by primer extension analysis. These are thought to represent three transcription start sites. The transcripts from the downstream and upstream sites were detected throughout growth. The transcript from the middle site was not detected during exponential growth but was detected within 30 min of the start of sporulation, when it was the predominant transcript. Insertion of a cat cassette in the middle promoter, ftsAp2 (p2), did not affect vegetative growth but prevented postexponential symmetrical division and spore formation. Transcription from p2 was dependent on RNA polymerase containing sigma H, and promoter p2 resembled the consensus sigma H promoter. Transcription from p2 did not require expression of the spo0A, spo0B, spo0E, spo0F, or spo0K loci. Northern (RNA) blot analysis indicated that ftsA is cotranscribed with the adjacent ftsZ gene. Multiple promoters provide a mechanism by which essential vegetative genes can be subjected to sporulation control independent of control during vegetative growth. In the case of ftsA,Z, the promoters provide a mechanism to permit septum formation in conditions of nutrient depletion that might be expected to shut down the vegetative division machinery. Images PMID:1624452

Transcriptional organization of the DNA region controlling expression of the K99 gene cluster.

PubMed

Roosendaal, B; Damoiseaux, J; Jordi, W; de Graaf, F K

1989-01-01

The transcriptional organization of the K99 gene cluster was investigated in two ways. First, the DNA region, containing the transcriptional signals was analyzed using a transcription vector system with Escherichia coli galactokinase (GalK) as assayable marker and second, an in vitro transcription system was employed. A detailed analysis of the transcription signals revealed that a strong promoter PA and a moderate promoter PB are located upstream of fanA and fanB, respectively. No promoter activity was detected in the intercistronic region between fanB and fanC. Factor-dependent terminators of transcription were detected and are probably located in the intercistronic region between fanA and fanB (T1), and between fanB and fanC (T2). A third terminator (T3) was observed between fanC and fanD and has an efficiency of 90%. Analysis of the regulatory region in an in vitro transcription system confirmed the location of the respective transcription signals. A model for the transcriptional organization of the K99 cluster is presented. Indications were obtained that the trans-acting regulatory polypeptides FanA and FanB both function as anti-terminators. A model for the regulation of expression of the K99 gene cluster is postulated.

Electrochemical branched-DNA assay for polymerase chain reaction-free detection and quantification of oncogenes in messenger RNA.

PubMed

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

2008-12-15

We describe a novel electrochemical branched-DNA (bDNA) assay for polymerase chain reaction (PCR)-free detection and quantification of p185 BCR-ABL leukemia fusion transcripts in the population of messenger ribonucleic acid (mRNA) extracted from cell lines. The bDNA amplifier carrying high loading of alkaline phosphatase (ALP) tracers was used to amplify the target 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-naphthyl phosphate. The voltammetric characteristics of substrate and enzymatic product as well as the parameters of SWV analysis were systematically optimized. A detection limit of 1 fM (1 x 10(-19) mol of target transcripts in 100 microL) and a 3-order-wide dynamic range of target concentration were achieved by the electrochemical bDNA assay. Such limit corresponded to approximately 17 fg of the p185 BCR-ABL fusion transcripts. The specificity and sensitivity of assay enabled direct detection of target transcripts in as little as 4.6 ng of mRNA population 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 transcripts in mRNA population. A mean transcript copy number of 62,900/ng of mRNA was determined, which was at least 50-fold higher than that of real-time quantitative PCR (qPCR). The finding was consistent with the underestimation of targets by qPCR reported earlier. In addition, the unique design based on bDNA technology increases the assay specificity as only the p185 BCR-ABL fusion transcripts will respond to the detection. The approach thus provides a simple, sensitive, accurate, and quantitative tool alternative to the qPCR for early disease diagnosis.

DiffSplice: the genome-wide detection of differential splicing events with RNA-seq

PubMed Central

Hu, Yin; Huang, Yan; Du, Ying; Orellana, Christian F.; Singh, Darshan; Johnson, Amy R.; Monroy, Anaïs; Kuan, Pei-Fen; Hammond, Scott M.; Makowski, Liza; Randell, Scott H.; Chiang, Derek Y.; Hayes, D. Neil; Jones, Corbin; Liu, Yufeng; Prins, Jan F.; Liu, Jinze

2013-01-01

The RNA transcriptome varies in response to cellular differentiation as well as environmental factors, and can be characterized by the diversity and abundance of transcript isoforms. Differential transcription analysis, the detection of differences between the transcriptomes of different cells, may improve understanding of cell differentiation and development and enable the identification of biomarkers that classify disease types. The availability of high-throughput short-read RNA sequencing technologies provides in-depth sampling of the transcriptome, making it possible to accurately detect the differences between transcriptomes. In this article, we present a new method for the detection and visualization of differential transcription. Our approach does not depend on transcript or gene annotations. It also circumvents the need for full transcript inference and quantification, which is a challenging problem because of short read lengths, as well as various sampling biases. Instead, our method takes a divide-and-conquer approach to localize the difference between transcriptomes in the form of alternative splicing modules (ASMs), where transcript isoforms diverge. Our approach starts with the identification of ASMs from the splice graph, constructed directly from the exons and introns predicted from RNA-seq read alignments. The abundance of alternative splicing isoforms residing in each ASM is estimated for each sample and is compared across sample groups. A non-parametric statistical test is applied to each ASM to detect significant differential transcription with a controlled false discovery rate. The sensitivity and specificity of the method have been assessed using simulated data sets and compared with other state-of-the-art approaches. Experimental validation using qRT-PCR confirmed a selected set of genes that are differentially expressed in a lung differentiation study and a breast cancer data set, demonstrating the utility of the approach applied on experimental biological data sets. The software of DiffSplice is available at http://www.netlab.uky.edu/p/bioinfo/DiffSplice. PMID:23155066

Widespread antisense transcription of Populus genome under drought.

PubMed

Yuan, Yinan; Chen, Su

2018-06-06

Antisense transcription is widespread in many genomes and plays important regulatory roles in gene expression. The objective of our study was to investigate the extent and functional relevance of antisense transcription in forest trees. We employed Populus, a model tree species, to probe the antisense transcriptional response of tree genome under drought, through stranded RNA-seq analysis. We detected nearly 48% of annotated Populus gene loci with antisense transcripts and 44% of them with co-transcription from both DNA strands. Global distribution of reads pattern across annotated gene regions uncovered that antisense transcription was enriched in untranslated regions while sense reads were predominantly mapped in coding exons. We further detected 1185 drought-responsive sense and antisense gene loci and identified a strong positive correlation between the expression of antisense and sense transcripts. Additionally, we assessed the antisense expression in introns and found a strong correlation between intronic expression and exonic expression, confirming antisense transcription of introns contributes to transcriptional activity of Populus genome under drought. Finally, we functionally characterized drought-responsive sense-antisense transcript pairs through gene ontology analysis and discovered that functional groups including transcription factors and histones were concordantly regulated at both sense and antisense transcriptional level. Overall, our study demonstrated the extensive occurrence of antisense transcripts of Populus genes under drought and provided insights into genome structure, regulation pattern and functional significance of drought-responsive antisense genes in forest trees. Datasets generated in this study serve as a foundation for future genetic analysis to improve our understanding of gene regulation by antisense transcription.

Transcription-induced DNA supercoiling: New roles of intranucleosomal DNA loops in DNA repair and transcription.

PubMed

Gerasimova, N S; Pestov, N A; Kulaeva, O I; Clark, D J; Studitsky, V M

2016-05-26

RNA polymerase II (Pol II) transcription through chromatin is accompanied by formation of small intranucleosomal DNA loops. Pol II captured within a small loop drives accumulation of DNA supercoiling, facilitating further transcription. DNA breaks relieve supercoiling and induce Pol II arrest, allowing detection of DNA damage hidden in chromatin structure.

Pervasive transcription: detecting functional RNAs in bacteria.

PubMed

Lybecker, Meghan; Bilusic, Ivana; Raghavan, Rahul

2014-01-01

Pervasive, or genome-wide, transcription has been reported in all domains of life. In bacteria, most pervasive transcription occurs antisense to protein-coding transcripts, although recently a new class of pervasive RNAs was identified that originates from within annotated genes. Initially considered to be non-functional transcriptional noise, pervasive transcription is increasingly being recognized as important in regulating gene expression. The function of pervasive transcription is an extensively debated question in the field of transcriptomics and regulatory RNA biology. Here, we highlight the most recent contributions addressing the purpose of pervasive transcription in bacteria and discuss their implications.

Text String Detection from Natural Scenes by Structure-based Partition and Grouping

PubMed Central

Yi, Chucai; Tian, YingLi

2012-01-01

Text information in natural scene images serves as important clues for many image-based applications such as scene understanding, content-based image retrieval, assistive navigation, and automatic geocoding. However, locating text from complex background with multiple colors is a challenging task. In this paper, we explore a new framework to detect text strings with arbitrary orientations in complex natural scene images. Our proposed framework of text string detection consists of two steps: 1) Image partition to find text character candidates based on local gradient features and color uniformity of character components. 2) Character candidate grouping to detect text strings based on joint structural features of text characters in each text string such as character size differences, distances between neighboring characters, and character alignment. By assuming that a text string has at least three characters, we propose two algorithms of text string detection: 1) adjacent character grouping method, and 2) text line grouping method. The adjacent character grouping method calculates the sibling groups of each character candidate as string segments and then merges the intersecting sibling groups into text string. The text line grouping method performs Hough transform to fit text line among the centroids of text candidates. Each fitted text line describes the orientation of a potential text string. The detected text string is presented by a rectangle region covering all characters whose centroids are cascaded in its text line. To improve efficiency and accuracy, our algorithms are carried out in multi-scales. The proposed methods outperform the state-of-the-art results on the public Robust Reading Dataset which contains text only in horizontal orientation. Furthermore, the effectiveness of our methods to detect text strings with arbitrary orientations is evaluated on the Oriented Scene Text Dataset collected by ourselves containing text strings in non-horizontal orientations. PMID:21411405

Text string detection from natural scenes by structure-based partition and grouping.

PubMed

Yi, Chucai; Tian, YingLi

2011-09-01

Text information in natural scene images serves as important clues for many image-based applications such as scene understanding, content-based image retrieval, assistive navigation, and automatic geocoding. However, locating text from a complex background with multiple colors is a challenging task. In this paper, we explore a new framework to detect text strings with arbitrary orientations in complex natural scene images. Our proposed framework of text string detection consists of two steps: 1) image partition to find text character candidates based on local gradient features and color uniformity of character components and 2) character candidate grouping to detect text strings based on joint structural features of text characters in each text string such as character size differences, distances between neighboring characters, and character alignment. By assuming that a text string has at least three characters, we propose two algorithms of text string detection: 1) adjacent character grouping method and 2) text line grouping method. The adjacent character grouping method calculates the sibling groups of each character candidate as string segments and then merges the intersecting sibling groups into text string. The text line grouping method performs Hough transform to fit text line among the centroids of text candidates. Each fitted text line describes the orientation of a potential text string. The detected text string is presented by a rectangle region covering all characters whose centroids are cascaded in its text line. To improve efficiency and accuracy, our algorithms are carried out in multi-scales. The proposed methods outperform the state-of-the-art results on the public Robust Reading Dataset, which contains text only in horizontal orientation. Furthermore, the effectiveness of our methods to detect text strings with arbitrary orientations is evaluated on the Oriented Scene Text Dataset collected by ourselves containing text strings in nonhorizontal orientations.

Rapid and reliable diagnostic method to detect Zika virus by real-time fluorescence reverse transcription loop-mediated isothermal amplification.

PubMed

Guo, Xu-Guang; Zhou, Yong-Zhuo; Li, Qin; Wang, Wei; Wen, Jin-Zhou; Zheng, Lei; Wang, Qian

2018-04-18

To detect Zika virus more rapidly and accurately, we developed a novel method that utilized a real-time fluorescence reverse transcription loop-mediated isothermal amplification (LAMP) technique. The NS5 gene was amplified by a set of six specific primers that recognized six distinct sequences. The amplification process, including 60 min of thermostatic reaction with Bst DNA polymerase following real-time fluorescence reverse transcriptase using genomic Zika virus standard strain (MR766), was conducted through fluorescent signaling. Among the six pairs of primers that we designate here, NS5 was the most efficient with a high sensitivity of up to 3.3 ng/μl and reproducible specificity on eight pathogen samples that were used as negative controls. The real-time fluorescence reverse transcription LAMP detection process can be completed within 35 min. Our study demonstrated that real-time fluorescence reverse transcription LAMP could be highly beneficial and convenient clinical application to detect Zika virus due to its high specificity and stability.

Host-location behavior of the tea green leafhopper Empoasca vitis Göthe (Hemiptera: Cicadellidae): olfactory and visual effects on their orientation.

PubMed

Zhang, X; Pengsakul, T; Tukayo, M; Yu, L; Fang, W; Luo, D

2017-09-25

The tea green leafhopper, Empoasca vitis Göthe, is one of the most serious pests in tea growing areas. This study investigated the roles played by olfaction and vision in host orientation behavior. The compound eye of E. vitis was found to be a photopic eye; few olfactory sensilla were found on the antennae, while abundant gustatory sensilla were recorded on the mouthparts. Three opsin genes (EV_LWop, EV_UVop, EV_Bop) were isolated and found to be mainly expressed in the compound eye compared with other parts of the body. Immunolocalization indicated that the opsins mainly located in the different regions of rhabdom. The transcription levels of EV_LWop, EV_Bop and EV_UVop were reduced by 77.3, 70.0 and 40.0%, respectively, by RNA interference induced by being fed a special RNA-rich diet for 6 days. The rate of tropism to host color was effectively impaired by 67.6 and 29.5% in the dsEV_LWop and dsEV_Bop treatment groups, but there was no significant change in the dsEV_UVop group. The determination of the cause of the tropism indicated that odors from the host over long distances were unable to attract E. vitis and were only detected when the insects were close to the host. The developed compound eye of E. vitis plays a leading role in host location, and the long-wavelength opsin significantly affects the tropism to host color; the lack of olfactory sensilla results in long-distance odors not being able to be detected until the insect is near to the host-plant. The understanding of these behavioral mechanisms, especially the importance of opsin genes is expected to be useful for pest management.

Effect of attention on the detection and identification of masked spatial patterns.

PubMed

Põder, Endel

2005-01-01

The effect of attention on the detection and identification of vertically and horizontally oriented Gabor patterns in the condition of simultaneous masking with obliquely oriented Gabors was studied. Attention was manipulated by varying the set size in a visual-search experiment. In the first experiment, small target Gabors were presented on the background of larger masking Gabors. In the detection task, the effect of set size was as predicted by unlimited-capacity signal detection theory. In the orientation identification task, increasing the set size from 1 to 8 resulted in a much larger decline in performance. The results of the additional experiments suggest that attention can reduce the crowding effect of maskers.

The notes from nature tool for unlocking biodiversity records from museum records through citizen science

PubMed Central

Hill, Andrew; Guralnick, Robert; Smith, Arfon; Sallans, Andrew; Rosemary Gillespie; Denslow, Michael; Gross, Joyce; Murrell, Zack; Tim Conyers; Oboyski, Peter; Ball, Joan; Thomer, Andrea; Prys-Jones, Robert; de Torre, Javier; Kociolek, Patrick; Fortson, Lucy

2012-01-01

Abstract Legacy data from natural history collections contain invaluable and irreplaceable information about biodiversity in the recent past, providing a baseline for detecting change and forecasting the future of biodiversity on a human-dominated planet. However, these data are often not available in formats that facilitate use and synthesis. New approaches are needed to enhance the rates of digitization and data quality improvement. Notes from Nature provides one such novel approach by asking citizen scientists to help with transcription tasks. The initial web-based prototype of Notes from Nature is soon widely available and was developed collaboratively by biodiversity scientists, natural history collections staff, and experts in citizen science project development, programming and visualization. This project brings together digital images representing different types of biodiversity records including ledgers , herbarium sheets and pinned insects from multiple projects and natural history collections. Experts in developing web-based citizen science applications then designed and built a platform for transcribing textual data and metadata from these images. The end product is a fully open source web transcription tool built using the latest web technologies. The platform keeps volunteers engaged by initially explaining the scientific importance of the work via a short orientation, and then providing transcription “missions” of well defined scope, along with dynamic feedback, interactivity and rewards. Transcribed records, along with record-level and process metadata, are provided back to the institutions.  While the tool is being developed with new users in mind, it can serve a broad range of needs from novice to trained museum specialist. Notes from Nature has the potential to speed the rate of biodiversity data being made available to a broad community of users. PMID:22859890

The notes from nature tool for unlocking biodiversity records from museum records through citizen science.

PubMed

Hill, Andrew; Guralnick, Robert; Smith, Arfon; Sallans, Andrew; Rosemary Gillespie; Denslow, Michael; Gross, Joyce; Murrell, Zack; Tim Conyers; Oboyski, Peter; Ball, Joan; Thomer, Andrea; Prys-Jones, Robert; de Torre, Javier; Kociolek, Patrick; Fortson, Lucy

2012-01-01

Legacy data from natural history collections contain invaluable and irreplaceable information about biodiversity in the recent past, providing a baseline for detecting change and forecasting the future of biodiversity on a human-dominated planet. However, these data are often not available in formats that facilitate use and synthesis. New approaches are needed to enhance the rates of digitization and data quality improvement. Notes from Nature provides one such novel approach by asking citizen scientists to help with transcription tasks. The initial web-based prototype of Notes from Nature is soon widely available and was developed collaboratively by biodiversity scientists, natural history collections staff, and experts in citizen science project development, programming and visualization. This project brings together digital images representing different types of biodiversity records including ledgers , herbarium sheets and pinned insects from multiple projects and natural history collections. Experts in developing web-based citizen science applications then designed and built a platform for transcribing textual data and metadata from these images. The end product is a fully open source web transcription tool built using the latest web technologies. The platform keeps volunteers engaged by initially explaining the scientific importance of the work via a short orientation, and then providing transcription "missions" of well defined scope, along with dynamic feedback, interactivity and rewards. Transcribed records, along with record-level and process metadata, are provided back to the institutions.  While the tool is being developed with new users in mind, it can serve a broad range of needs from novice to trained museum specialist. Notes from Nature has the potential to speed the rate of biodiversity data being made available to a broad community of users.

Beta-keratins of differentiating epidermis of snake comprise glycine-proline-serine-rich proteins with an avian-like gene organization.

PubMed

Dalla Valle, Luisa; Nardi, Alessia; Belvedere, Paola; Toni, Mattia; Alibardi, Lorenzo

2007-07-01

Beta-keratins of reptilian scales have been recently cloned and characterized in some lizards. Here we report for the first time the sequence of some beta-keratins from the snake Elaphe guttata. Five different cDNAs were obtained using 5'- and 3'-RACE analyses. Four sequences differ by only few nucleotides in the coding region, whereas the last cDNA shows, in this region, only 84% of identity. The gene corresponding to one of the cDNA sequences has a single intron present in the 5'-untranslated region. This genomic organization is similar to that of birds' beta-keratins. Cloning and Southern blotting analysis suggest that snake beta-keratins belong to a family of high-related genes as for geckos. PCR analysis suggests a head-to-tail orientation of genes in the same chromosome. In situ hybridization detected beta-keratin transcripts almost exclusively in differentiating oberhautchen and beta-cells of the snake epidermis in renewal phase. This is confirmed by Northern blotting that showed, in this phase, a high expression of two different transcripts whereas only the longer transcript is expressed at a much lower level in resting skin. The cDNA coding sequences encoded putative glycine-proline-serine rich proteins containing 137-139 amino acids, with apparent isoelectric point at 7.5 and 8.2. A central region, rich in proline, shows over 50% homology with avian scale, claw, and feather keratins. The prediction of secondary structure shows mainly a random coil conformation and few beta-strand regions in the central region, likely involved in the formation of a fibrous framework of beta-keratins. This region was possibly present in basic reptiles that originated reptiles and birds. Copyright 2007 Wiley-Liss, Inc.

GGRNA: an ultrafast, transcript-oriented search engine for genes and transcripts

PubMed Central

Naito, Yuki; Bono, Hidemasa

2012-01-01

GGRNA (http://GGRNA.dbcls.jp/) is a Google-like, ultrafast search engine for genes and transcripts. The web server accepts arbitrary words and phrases, such as gene names, IDs, gene descriptions, annotations of gene and even nucleotide/amino acid sequences through one simple search box, and quickly returns relevant RefSeq transcripts. A typical search takes just a few seconds, which dramatically enhances the usability of routine searching. In particular, GGRNA can search sequences as short as 10 nt or 4 amino acids, which cannot be handled easily by popular sequence analysis tools. Nucleotide sequences can be searched allowing up to three mismatches, or the query sequences may contain degenerate nucleotide codes (e.g. N, R, Y, S). Furthermore, Gene Ontology annotations, Enzyme Commission numbers and probe sequences of catalog microarrays are also incorporated into GGRNA, which may help users to conduct searches by various types of keywords. GGRNA web server will provide a simple and powerful interface for finding genes and transcripts for a wide range of users. All services at GGRNA are provided free of charge to all users. PMID:22641850

GGRNA: an ultrafast, transcript-oriented search engine for genes and transcripts.

PubMed

Naito, Yuki; Bono, Hidemasa

2012-07-01

GGRNA (http://GGRNA.dbcls.jp/) is a Google-like, ultrafast search engine for genes and transcripts. The web server accepts arbitrary words and phrases, such as gene names, IDs, gene descriptions, annotations of gene and even nucleotide/amino acid sequences through one simple search box, and quickly returns relevant RefSeq transcripts. A typical search takes just a few seconds, which dramatically enhances the usability of routine searching. In particular, GGRNA can search sequences as short as 10 nt or 4 amino acids, which cannot be handled easily by popular sequence analysis tools. Nucleotide sequences can be searched allowing up to three mismatches, or the query sequences may contain degenerate nucleotide codes (e.g. N, R, Y, S). Furthermore, Gene Ontology annotations, Enzyme Commission numbers and probe sequences of catalog microarrays are also incorporated into GGRNA, which may help users to conduct searches by various types of keywords. GGRNA web server will provide a simple and powerful interface for finding genes and transcripts for a wide range of users. All services at GGRNA are provided free of charge to all users.

Transcription-induced DNA supercoiling: New roles of intranucleosomal DNA loops in DNA repair and transcription

PubMed Central

Gerasimova, N. S.; Pestov, N. A.; Kulaeva, O. I.; Clark, D. J.; Studitsky, V. M.

2016-01-01

ABSTRACT RNA polymerase II (Pol II) transcription through chromatin is accompanied by formation of small intranucleosomal DNA loops. Pol II captured within a small loop drives accumulation of DNA supercoiling, facilitating further transcription. DNA breaks relieve supercoiling and induce Pol II arrest, allowing detection of DNA damage hidden in chromatin structure. PMID:27115204

Genome-wide differences in hepatitis C- vs alcoholism-associated hepatocellular carcinoma

PubMed Central

Derambure, Céline; Coulouarn, Cédric; Caillot, Frédérique; Daveau, Romain; Hiron, Martine; Scotte, Michel; François, Arnaud; Duclos, Celia; Goria, Odile; Gueudin, Marie; Cavard, Catherine; Terris, Benoit; Daveau, Maryvonne; Salier, Jean-Philippe

2008-01-01

AIM: To look at a comprehensive picture of etiology-dependent gene abnormalities in hepatocellular carcinoma in Western Europe. METHODS: With a liver-oriented microarray, transcript levels were compared in nodules and cirrhosis from a training set of patients with hepatocellular carcinoma (alcoholism, 12; hepatitis C, 10) and 5 controls. Loose or tight selection of informative transcripts with an abnormal abundance was statistically valid and the tightly selected transcripts were next quantified by qRTPCR in the nodules from our training set (12 + 10) and a test set (6 + 7). RESULTS: A selection of 475 transcripts pointed to significant gene over-representation on chromosome 8 (alcoholism) or -2 (hepatitis C) and ontology indicated a predominant inflammatory response (alcoholism) or changes in cell cycle regulation, transcription factors and interferon responsiveness (hepatitis C). A stringent selection of 23 transcripts whose differences between etiologies were significant in nodules but not in cirrhotic tissue indicated that the above dysregulations take place in tumor but not in the surrounding cirrhosis. These 23 transcripts separated our test set according to etiologies. The inflammation-associated transcripts pointed to limited alterations of free iron metabolism in alcoholic vs hepatitis C tumors. CONCLUSION: Etiology-specific abnormalities (chromosome preference; differences in transcriptomes and related functions) have been identified in hepatocellular carcinoma driven by alcoholism or hepatitis C. This may open novel avenues for differential therapies in this disease. PMID:18350606

Strong transcription blockage mediated by R-loop formation within a G-rich homopurine–homopyrimidine sequence localized in the vicinity of the promoter

PubMed Central

Soo Shin, Jane Hae

2017-01-01

Abstract Guanine-rich (G-rich) homopurine–homopyrimidine nucleotide sequences can block transcription with an efficiency that depends upon their orientation, composition and length, as well as the presence of negative supercoiling or breaks in the non-template DNA strand. We report that a G-rich sequence in the non-template strand reduces the yield of T7 RNA polymerase transcription by more than an order of magnitude when positioned close (9 bp) to the promoter, in comparison to that for a distal (∼250 bp) location of the same sequence. This transcription blockage is much less pronounced for a C-rich sequence, and is not significant for an A-rich sequence. Remarkably, the blockage is not pronounced if transcription is performed in the presence of RNase H, which specifically digests the RNA strands within RNA–DNA hybrids. The blockage also becomes less pronounced upon reduced RNA polymerase concentration. Based upon these observations and those from control experiments, we conclude that the blockage is primarily due to the formation of stable RNA–DNA hybrids (R-loops), which inhibit successive rounds of transcription. Our results could be relevant to transcription dynamics in vivo (e.g. transcription ‘bursting’) and may also have practical implications for the design of expression vectors. PMID:28498974

Strong transcription blockage mediated by R-loop formation within a G-rich homopurine-homopyrimidine sequence localized in the vicinity of the promoter.

PubMed

Belotserkovskii, Boris P; Soo Shin, Jane Hae; Hanawalt, Philip C

2017-06-20

Guanine-rich (G-rich) homopurine-homopyrimidine nucleotide sequences can block transcription with an efficiency that depends upon their orientation, composition and length, as well as the presence of negative supercoiling or breaks in the non-template DNA strand. We report that a G-rich sequence in the non-template strand reduces the yield of T7 RNA polymerase transcription by more than an order of magnitude when positioned close (9 bp) to the promoter, in comparison to that for a distal (∼250 bp) location of the same sequence. This transcription blockage is much less pronounced for a C-rich sequence, and is not significant for an A-rich sequence. Remarkably, the blockage is not pronounced if transcription is performed in the presence of RNase H, which specifically digests the RNA strands within RNA-DNA hybrids. The blockage also becomes less pronounced upon reduced RNA polymerase concentration. Based upon these observations and those from control experiments, we conclude that the blockage is primarily due to the formation of stable RNA-DNA hybrids (R-loops), which inhibit successive rounds of transcription. Our results could be relevant to transcription dynamics in vivo (e.g. transcription 'bursting') and may also have practical implications for the design of expression vectors. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Cell Wall Modifications in Maize Pulvini in Response to Gravitational Stress1[W][OA

PubMed Central

Zhang, Qisen; Pettolino, Filomena A.; Dhugga, Kanwarpal S.; Rafalski, J. Antoni; Tingey, Scott; Taylor, Jillian; Shirley, Neil J.; Hayes, Kevin; Beatty, Mary; Abrams, Suzanne R.; Zaharia, L. Irina; Burton, Rachel A.; Bacic, Antony; Fincher, Geoffrey B.

2011-01-01

Changes in cell wall polysaccharides, transcript abundance, metabolite profiles, and hormone concentrations were monitored in the upper and lower regions of maize (Zea mays) pulvini in response to gravistimulation, during which maize plants placed in a horizontal position returned to the vertical orientation. Heteroxylan levels increased in the lower regions of the pulvini, together with lignin, but xyloglucans and heteromannan contents decreased. The degree of substitution of heteroxylan with arabinofuranosyl residues decreased in the lower pulvini, which exhibited increased mechanical strength as the plants returned to the vertical position. Few or no changes in noncellulosic wall polysaccharides could be detected on the upper side of the pulvinus, and crystalline cellulose content remained essentially constant in both the upper and lower pulvinus. Microarray analyses showed that spatial and temporal changes in transcript profiles were consistent with the changes in wall composition that were observed in the lower regions of the pulvinus. In addition, the microarray analyses indicated that metabolic pathways leading to the biosynthesis of phytohormones were differentially activated in the upper and lower regions of the pulvinus in response to gravistimulation. Metabolite profiles and measured hormone concentrations were consistent with the microarray data, insofar as auxin, physiologically active gibberellic acid, and metabolites potentially involved in lignin biosynthesis increased in the elongating cells of the lower pulvinus. PMID:21697508

Inferring genome-wide interplay landscape between DNA methylation and transcriptional regulation.

PubMed

Tang, Binhua; Wang, Xin

2015-01-01

DNA methylation and transcriptional regulation play important roles in cancer cell development and differentiation processes. Based on the currently available cell line profiling information from the ENCODE Consortium, we propose a Bayesian inference model to infer and construct genome-wide interaction landscape between DNA methylation and transcriptional regulation, which sheds light on the underlying complex functional mechanisms important within the human cancer and disease context. For the first time, we select all the currently available cell lines (>=20) and transcription factors (>=80) profiling information from the ENCODE Consortium portal. Through the integration of those genome-wide profiling sources, our genome-wide analysis detects multiple functional loci of interest, and indicates that DNA methylation is cell- and region-specific, due to the interplay mechanisms with transcription regulatory activities. We validate our analysis results with the corresponding RNA-sequencing technique for those detected genomic loci. Our results provide novel and meaningful insights for the interplay mechanisms of transcriptional regulation and gene expression for the human cancer and disease studies.

Development of a rapid diagnostic assay for the detection of tomato chlorotic dwarf viroid based on isothermal reverse-transcription-recombinase polymerase amplification

USDA-ARS?s Scientific Manuscript database

A molecular diagnostic assay utilizing reverse transcription-recombinase polymerase amplification (RT-RPA) at an isothermal constant temperature of 39 °C and target-specific primers and probe were developed for the rapid, sensitive, and specific detection of tomato chlorotic dwarf viroid (TCDVd) in ...

One-step reverse transcription loop mediated isothermal amplification assay for detection of Apple chlorotic leaf spot virus

USDA-ARS?s Scientific Manuscript database

A reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the detection of Apple chlorotic leaf spot virus (ACLSV) was developed. In this method, a set of four primers was designed based on the conserved regions in the coat protein gene of ACLSV, and was synthesized for the ...

Real time coarse orientation detection in MR scans using multi-planar deep convolutional neural networks

NASA Astrophysics Data System (ADS)

Bhatia, Parmeet S.; Reda, Fitsum; Harder, Martin; Zhan, Yiqiang; Zhou, Xiang Sean

2017-02-01

Automatically detecting anatomy orientation is an important task in medical image analysis. Specifically, the ability to automatically detect coarse orientation of structures is useful to minimize the effort of fine/accurate orientation detection algorithms, to initialize non-rigid deformable registration algorithms or to align models to target structures in model-based segmentation algorithms. In this work, we present a deep convolution neural network (DCNN)-based method for fast and robust detection of the coarse structure orientation, i.e., the hemi-sphere where the principal axis of a structure lies. That is, our algorithm predicts whether the principal orientation of a structure is in the northern hemisphere or southern hemisphere, which we will refer to as UP and DOWN, respectively, in the remainder of this manuscript. The only assumption of our method is that the entire structure is located within the scan's field-of-view (FOV). To efficiently solve the problem in 3D space, we formulated it as a multi-planar 2D deep learning problem. In the training stage, a large number coronal-sagittal slice pairs are constructed as 2-channel images to train a DCNN to classify whether a scan is UP or DOWN. During testing, we randomly sample a small number of coronal-sagittal 2-channel images and pass them through our trained network. Finally, coarse structure orientation is determined using majority voting. We tested our method on 114 Elbow MR Scans. Experimental results suggest that only five 2-channel images are sufficient to achieve a high success rate of 97.39%. Our method is also extremely fast and takes approximately 50 milliseconds per 3D MR scan. Our method is insensitive to the location of the structure in the FOV.

Widespread promoter-mediated coordination of transcription and mRNA degradation

PubMed Central

2012-01-01

Background Previous work showed that mRNA degradation is coordinated with transcription in yeast, and in several genes the control of mRNA degradation was linked to promoter elements through two different mechanisms. Here we show at the genomic scale that the coordination of transcription and mRNA degradation is promoter-dependent in yeast and is also observed in humans. Results We first demonstrate that swapping upstream cis-regulatory sequences between two yeast species affects both transcription and mRNA degradation and suggest that while some cis-regulatory elements control either transcription or degradation, multiple other elements enhance both processes. Second, we show that adjacent yeast genes that share a promoter (through divergent orientation) have increased similarity in their patterns of mRNA degradation, providing independent evidence for the promoter-mediated coupling of transcription to mRNA degradation. Finally, analysis of the differences in mRNA degradation rates between mammalian cell types or mammalian species suggests a similar coordination between transcription and mRNA degradation in humans. Conclusions Our results extend previous studies and suggest a pervasive promoter-mediated coordination between transcription and mRNA degradation in yeast. The diverse genes and regulatory elements associated with this coordination suggest that it is generated by a global mechanism of gene regulation and modulated by gene-specific mechanisms. The observation of a similar coupling in mammals raises the possibility that coupling of transcription and mRNA degradation may reflect an evolutionarily conserved phenomenon in gene regulation. PMID:23237624

In vivo phosphorylation of WRKY transcription factor by MAPK.

PubMed

Ishihama, Nobuaki; Adachi, Hiroaki; Yoshioka, Miki; Yoshioka, Hirofumi

2014-01-01

Plants activate signaling networks in response to diverse pathogen-derived signals, facilitating transcriptional reprogramming through mitogen-activated protein kinase (MAPK) cascades. Identification of phosphorylation targets of MAPK and in vivo detection of the phosphorylated substrates are important processes to elucidate the signaling pathway in plant immune responses. We have identified a WRKY transcription factor, which is phosphorylated by defense-related MAPKs, SIPK and WIPK. Recent evidence demonstrated that some group I WRKY transcription factors, which contain a conserved motif in the N-terminal region, are activated by MAPK-dependent phosphorylation. In this chapter, we describe protocols for preparation of anti-phosphopeptide antibodies, detection of activated MAPKs using anti-phospho-MAPK antibody, and activated WRKY using anti-phospho-WRKY antibody, respectively.

Optimized Lentiviral Vector Design Improves Titer and Transgene Expression of Vectors Containing the Chicken β-Globin Locus HS4 Insulator Element

PubMed Central

Hanawa, Hideki; Yamamoto, Motoko; Zhao, Huifen; Shimada, Takashi; Persons, Derek A

2009-01-01

Hematopoietic cell gene therapy using retroviral vectors has achieved success in clinical trials. However, safety issues regarding vector insertional mutagenesis have emerged. In two different trials, vector insertion resulted in the transcriptional activation of proto-oncogenes. One strategy for potentially diminishing vector insertional mutagenesis is through the use of self-inactivating lentiviral vectors containing the 1.2-kb insulator element derived from the chicken β-globin locus. However, use of this element can dramatically decrease both vector titer and transgene expression, thereby compromising its practical use. Here, we studied lentiviral vectors containing either the full-length 1.2-kb insulator or the smaller 0.25-kb core element in both orientations in the partially deleted long-terminal repeat. We show that use of the 0.25-kb core insulator rescued vector titer by alleviating a postentry block to reverse transcription associated with the 1.2-kb element. In addition, in an orientation-dependent manner, the 0.25-kb core element significantly increased transgene expression from an internal promoter due to improved transcriptional termination. This element also demonstrated barrier activity, reducing variability of expression due to position effects. As it is known that the 0.25-kb core insulator has enhancer-blocking activity, this particular insulated lentiviral vector design may be useful for clinical application. PMID:19223867

Identification, occurrence, and validation of DRE and ABRE Cis-regulatory motifs in the promoter regions of genes of Arabidopsis thaliana.

PubMed

Mishra, Sonal; Shukla, Aparna; Upadhyay, Swati; Sanchita; Sharma, Pooja; Singh, Seema; Phukan, Ujjal J; Meena, Abha; Khan, Feroz; Tripathi, Vineeta; Shukla, Rakesh Kumar; Shrama, Ashok

2014-04-01

Plants posses a complex co-regulatory network which helps them to elicit a response under diverse adverse conditions. We used an in silico approach to identify the genes with both DRE and ABRE motifs in their promoter regions in Arabidopsis thaliana. Our results showed that Arabidopsis contains a set of 2,052 genes with ABRE and DRE motifs in their promoter regions. Approximately 72% or more of the total predicted 2,052 genes had a gap distance of less than 400 bp between DRE and ABRE motifs. For positional orientation of the DRE and ABRE motifs, we found that the DR form (one in direct and the other one in reverse orientation) was more prevalent than other forms. These predicted 2,052 genes include 155 transcription factors. Using microarray data from The Arabidopsis Information Resource (TAIR) database, we present 44 transcription factors out of 155 which are upregulated by more than twofold in response to osmotic stress and ABA treatment. Fifty-one transcripts from the one predicted above were validated using semiquantitative expression analysis to support the microarray data in TAIR. Taken together, we report a set of genes containing both DRE and ABRE motifs in their promoter regions in A. thaliana, which can be useful to understand the role of ABA under osmotic stress condition. © 2013 Institute of Botany, Chinese Academy of Sciences.

Computer-oriented emissions inventory procedure for urban and industrial sources

DOE Office of Scientific and Technical Information (OSTI.GOV)

Runca, E.; Zannetti, P.; Melli, P.

1978-06-01

A knowledge of the rate of emission of atmospheric pollutants is essential for the enforcement of air quality control policies. A computer-oriented emission inventory procedure has been developed and applied to Venice, Italy. By using optically readable forms this procedure avoids many of the errors inherent in the transcription and punching steps typical of approaches applied so far. Moreover, this procedure allows an easy updating of the inventory. Emission patterns of SO/sub 2/ in the area of Venice showed that the total urban emissions were about 6% of those emitted by industrial sources.

Chromogenic detection of yam mosaic virus by closed-tube reverse transcription loop-mediated isothermal amplification (CT-RT-LAMP).

PubMed

Nkere, Chukwuemeka K; Oyekanmi, Joshua O; Silva, Gonçalo; Bömer, Moritz; Atiri, Gabriel I; Onyeka, Joseph; Maroya, Norbert G; Seal, Susan E; Kumar, P Lava

2018-04-01

A closed-tube reverse transcription loop-mediated isothermal amplification (CT-RT-LAMP) assay was developed for the detection of yam mosaic virus (YMV, genus Potyvirus) infecting yam (Dioscorea spp.). The assay uses a set of six oligonucleotide primers targeting the YMV coat protein region, and the amplification products in YMV-positive samples are visualized by chromogenic detection with SYBR Green I dye. The CT-RT-LAMP assay detected YMV in leaf and tuber tissues of infected plants. The assay is 100 times more sensitive in detecting YMV than standard RT-PCR, while maintaining the same specificity.

Cellular cAMP uptake as trigger for electrotaxis

NASA Astrophysics Data System (ADS)

Guido, Isabella; Bodenschatz, Eberhard

Cells have the ability to detect continuous current electric fields and respond to them with a directed migratory movement. Dictyostelium discoideum cells, a key model organism for the study of eukaryotic chemotaxis, orient and migrate toward the cathode under the influence of an electric field. The underlying sensing mechanism and whether it is shared by the chemotactic response pathway remains unknown. By investigating the migration in the electric field of cell strains unable to migrate chemotactically (Amib-null) and with defective cAMP relay (ACA-null) we show that the starvation-induced transcription of a set of genes involved in the early developmental stage is not necessary for electrotaxis. However, the analysis of electrotaxis of vegetative cells as well as shortly starved cells shows that cells need to be stimulated with cAMP in order for them to migrate electrotactically. Indeed 30 minutes stimulation with cAMP pulses is enough to let cells orienting with the electric field although during this time the expression of receptors and the beginning of the development has not happened yet. We believe that the reason for this observed phenomenon lies on the endocytosis of the external cAMP which triggers electrotaxis as long as endocytosis and exocytosis are not balanced. This work is part of the MaxSynBio Consortium which is jointly funded by the Federal Ministry of Education and Research of Germany and the Max Planck Society.

Transcriptional regulation of intermediate progenitor cell generation during hippocampal development

PubMed Central

Harris, Lachlan; Zalucki, Oressia; Gobius, Ilan; McDonald, Hannah; Osinki, Jason; Harvey, Tracey J.; Essebier, Alexandra; Vidovic, Diana; Gladwyn-Ng, Ivan; Burne, Thomas H.; Heng, Julian I.; Richards, Linda J.; Gronostajski, Richard M.

2016-01-01

During forebrain development, radial glia generate neurons through the production of intermediate progenitor cells (IPCs). The production of IPCs is a central tenet underlying the generation of the appropriate number of cortical neurons, but the transcriptional logic underpinning this process remains poorly defined. Here, we examined IPC production using mice lacking the transcription factor nuclear factor I/X (Nfix). We show that Nfix deficiency delays IPC production and prolongs the neurogenic window, resulting in an increased number of neurons in the postnatal forebrain. Loss of additional Nfi alleles (Nfib) resulted in a severe delay in IPC generation while, conversely, overexpression of NFIX led to precocious IPC generation. Mechanistically, analyses of microarray and ChIP-seq datasets, coupled with the investigation of spindle orientation during radial glial cell division, revealed that NFIX promotes the generation of IPCs via the transcriptional upregulation of inscuteable (Insc). These data thereby provide novel insights into the mechanisms controlling the timely transition of radial glia into IPCs during forebrain development. PMID:27965439

Arabidopsis JACKDAW and MAGPIE zinc finger proteins delimit asymmetric cell division and stabilize tissue boundaries by restricting SHORT-ROOT action

PubMed Central

Welch, David; Hassan, Hala; Blilou, Ikram; Immink, Richard; Heidstra, Renze; Scheres, Ben

2007-01-01

In the Arabidopsis root, the SHORT-ROOT transcription factor moves outward to the ground tissue from its site of transcription in the stele and is required for the specification of the endodermis and the stem cell organizing quiescent center cells. In addition, SHORT-ROOT and the downstream transcription factor SCARECROW control an oriented cell division in ground tissue stem cell daughters. Here, we show that the JACKDAW and MAGPIE genes, which encode members of a plant-specific family of zinc finger proteins, act in a SHR-dependent feed-forward loop to regulate the range of action of SHORT-ROOT and SCARECROW. JACKDAW expression is initiated independent of SHORT-ROOT and regulates the SCARECROW expression domain outside the stele, while MAGPIE expression depends on SHORT-ROOT and SCARECROW. We provide evidence that JACKDAW and MAGPIE regulate tissue boundaries and asymmetric cell division and can control SHORT-ROOT and SCARECROW activity in a transcriptional and protein interaction network. PMID:17785527

High-confidence coding and noncoding transcriptome maps

PubMed Central

2017-01-01

The advent of high-throughput RNA sequencing (RNA-seq) has led to the discovery of unprecedentedly immense transcriptomes encoded by eukaryotic genomes. However, the transcriptome maps are still incomplete partly because they were mostly reconstructed based on RNA-seq reads that lack their orientations (known as unstranded reads) and certain boundary information. Methods to expand the usability of unstranded RNA-seq data by predetermining the orientation of the reads and precisely determining the boundaries of assembled transcripts could significantly benefit the quality of the resulting transcriptome maps. Here, we present a high-performing transcriptome assembly pipeline, called CAFE, that significantly improves the original assemblies, respectively assembled with stranded and/or unstranded RNA-seq data, by orienting unstranded reads using the maximum likelihood estimation and by integrating information about transcription start sites and cleavage and polyadenylation sites. Applying large-scale transcriptomic data comprising 230 billion RNA-seq reads from the ENCODE, Human BodyMap 2.0, The Cancer Genome Atlas, and GTEx projects, CAFE enabled us to predict the directions of about 220 billion unstranded reads, which led to the construction of more accurate transcriptome maps, comparable to the manually curated map, and a comprehensive lncRNA catalog that includes thousands of novel lncRNAs. Our pipeline should not only help to build comprehensive, precise transcriptome maps from complex genomes but also to expand the universe of noncoding genomes. PMID:28396519

1H NMR studies of the 5-(hydroxymethyl)-2'-deoxyuridine containing TF1 binding site.

PubMed

Pasternack, L B; Bramham, J; Mayol, L; Galeone, A; Jia, X; Kearns, D R

1996-07-15

The pyrimidine base 5-(hydroxymethyl)-2'-deoxyuridine (HmU) is a common nucleotide in SPO1 phage DNA. Numerous transcriptional proteins bind HmU-containing DNA preferentially implicating a regulatory function of HmU. We have investigated the conformation and dynamics of d-(5'-CHmUCHmUACACGHmUGHmUAGAG-OH-3')2 (HmU-DNA). This oligonucleotide mimics the consensus sequence of Transcription Factor 1 (TF1). The HmU-DNA was compared to the thymine-containing oligonucleotide. NOESY and DQF COSY spectroscopy provided resonance assignments of nonexchangeable and exchangeable protons, intranucleotide, internucleotide and intrastrand proton-proton distances, and dihedral angle constraints. Methylene protons of the hydroxymethyl group are nonequivalent protons and the hydroxymethyl group is not freely rotating. The hydroxymethyl group adopts a specific orientation with the OH group oriented on the 3' side of the plane of the base. Analysis of imino proton resonances and NOEs indicates additional end base pair fraying and a temperature-induced transition to a conformation in which the internal HmU-A base pairs are disrupted or have reduced lifetimes. Orientation of the hydroxymethyl group indicates the presence of internucleotide intrastrand hydrogen bonding between the HmU12C5 hydroxyl group and A13. All sugars in both DNAs show a C2'endo conformation (typical of B-DNA).

Micronuclei and other erythrocyte nuclear abnormalities in fishes from the Great Lakes Basin, USA

PubMed Central

Braham, Ryan P.; Shaw, Cassidy H.; Mazik, Patricia M.; Umbuzeiro, G.

2017-01-01

Biological markers (biomarkers) sensitive to genotoxic and mutagenic contamination in fishes are widely used to identify exposure effects in aquatic environments. The micronucleus assay was incorporated into a suite of indicators to assess exposure to genotoxic and mutagenic contamination at five Great Lakes Areas of Concern (AOCs), as well as one non‐AOC (reference) site. The assay allowed enumeration of micronuclei as well as other nuclear abnormalities for both site and species comparisons. Erythrocyte abnormality data was also compared to skin and liver tumor prevalence and hepatic transcript abundance. Erythrocyte abnormalities were observed at all sites with variable occurrence and severity among sites and species. Benthic‐oriented brown bullhead (Ameiurus nebulosus) and white sucker (Catostomus commersonii) expressed lower rates of erythrocyte abnormalities, but higher rates of skin and liver neoplasms, when compared to pelagic‐oriented largemouth bass (Micropterus salmoides) or smallmouth bass (Micropterus dolomieu) at the same site. The reduced erythrocyte abnormalities, increased transcript abundance associated with Phase I and II toxicant responsive pathways, and increased neoplastic lesions among benthic‐oriented taxa may indicate the development of contaminant resistance of these species to more acute effects. Environ. Mol. Mutagen. 58:570–581, 2017. © 2017 This article is a U.S. Government work and is in the public domain in the USA. Environmental and Molecular Mutagenesis published by Wiley Periodicals, Inc. on behalf of Environmental Mutagen Society PMID:28868735

Landscape of DNA Virus Associations across Human Malignant Cancers: Analysis of 3,775 Cases Using RNA-Seq

PubMed Central

Tannir, Nizar M.; Williams, Michelle D.; Chen, Yunxin; Yao, Hui; Zhang, Jianping; Thompson, Erika J.; Meric-Bernstam, Funda; Medeiros, L. Jeffrey; Weinstein, John N.

2013-01-01

Elucidation of tumor-DNA virus associations in many cancer types has enhanced our knowledge of fundamental oncogenesis mechanisms and provided a basis for cancer prevention initiatives. RNA-Seq is a novel tool to comprehensively assess such associations. We interrogated RNA-Seq data from 3,775 malignant neoplasms in The Cancer Genome Atlas database for the presence of viral sequences. Viral integration sites were also detected in expressed transcripts using a novel approach. The detection capacity of RNA-Seq was compared to available clinical laboratory data. Human papillomavirus (HPV) transcripts were detected using RNA-Seq analysis in head-and-neck squamous cell carcinoma, uterine endometrioid carcinoma, and squamous cell carcinoma of the lung. Detection of HPV by RNA-Seq correlated with detection by in situ hybridization and immunohistochemistry in squamous cell carcinoma tumors of the head and neck. Hepatitis B virus and Epstein-Barr virus (EBV) were detected using RNA-Seq in hepatocellular carcinoma and gastric carcinoma tumors, respectively. Integration sites of viral genes and oncogenes were detected in cancers harboring HPV or hepatitis B virus but not in EBV-positive gastric carcinoma. Integration sites of expressed viral transcripts frequently involved known coding areas of the host genome. No DNA virus transcripts were detected in acute myeloid leukemia, cutaneous melanoma, low- and high-grade gliomas of the brain, and adenocarcinomas of the breast, colon and rectum, lung, prostate, ovary, kidney, and thyroid. In conclusion, this study provides a large-scale overview of the landscape of DNA viruses in human malignant cancers. While further validation is necessary for specific cancer types, our findings highlight the utility of RNA-Seq in detecting tumor-associated DNA viruses and identifying viral integration sites that may unravel novel mechanisms of cancer pathogenesis. PMID:23740984

Transcriptional regulatory proteins as biosensing tools.

PubMed

Turner, Kendrick; Joel, Smita; Feliciano, Jessika; Feltus, Agatha; Pasini, Patrizia; Wynn, Daniel; Dau, Peter; Dikici, Emre; Deo, Sapna K; Daunert, Sylvia

2017-06-22

We have developed sensing systems employing different classes of transcriptional regulatory proteins genetically and chemically modified to incorporate a fluorescent reporter molecule for detection of arsenic, hydroxylated polychlorinated biphenyls (OH-PCBs), and cyclic AMP (cAMP). These are the first examples of optical sensing systems based on transcriptional regulatory proteins.

Subgroup 4 R2R3-MYBs in conifer trees: gene family expansion and contribution to the isoprenoid- and flavonoid-oriented responses

PubMed Central

Bedon, Frank; Bomal, Claude; Caron, Sébastien; Levasseur, Caroline; Boyle, Brian; Mansfield, Shawn D.; Schmidt, Axel; Gershenzon, Jonathan; Grima-Pettenati, Jacqueline; Séguin, Armand; MacKay, John

2010-01-01

Transcription factors play a fundamental role in plants by orchestrating temporal and spatial gene expression in response to environmental stimuli. Several R2R3-MYB genes of the Arabidopsis subgroup 4 (Sg4) share a C-terminal EAR motif signature recently linked to stress response in angiosperm plants. It is reported here that nearly all Sg4 MYB genes in the conifer trees Picea glauca (white spruce) and Pinus taeda (loblolly pine) form a monophyletic clade (Sg4C) that expanded following the split of gymnosperm and angiosperm lineages. Deeper sequencing in P. glauca identified 10 distinct Sg4C sequences, indicating over-represention of Sg4 sequences compared with angiosperms such as Arabidopsis, Oryza, Vitis, and Populus. The Sg4C MYBs share the EAR motif core. Many of them had stress-responsive transcript profiles after wounding, jasmonic acid (JA) treatment, or exposure to cold in P. glauca and P. taeda, with MYB14 transcripts accumulating most strongly and rapidly. Functional characterization was initiated by expressing the P. taeda MYB14 (PtMYB14) gene in transgenic P. glauca plantlets with a tissue-preferential promoter (cinnamyl alcohol dehydrogenase) and a ubiquitous gene promoter (ubiquitin). Histological, metabolite, and transcript (microarray and targeted quantitiative real-time PCR) analyses of PtMYB14 transgenics, coupled with mechanical wounding and JA application experiments on wild-type plantlets, allowed identification of PtMYB14 as a putative regulator of an isoprenoid-oriented response that leads to the accumulation of sesquiterpene in conifers. Data further suggested that PtMYB14 may contribute to a broad defence response implicating flavonoids. This study also addresses the potential involvement of closely related Sg4C sequences in stress responses and plant evolution. PMID:20732878

Impaired search for orientation but not color in hemi-spatial neglect.

PubMed

Wilkinson, David; Ko, Philip; Milberg, William; McGlinchey, Regina

2008-01-01

Patients with hemi-spatial neglect have trouble finding targets defined by a conjunction of visual features. The problem is widely believed to stem from a high-level deficit in attentional deployment, which in turn has led to disagreement over whether the detection of basic features is also disrupted. If one assumes that the detection of salient visual features can be based on the output of spared 'preattentive' processes (Treisman and Gelade, 1980), then feature detection should remain intact. However, if one assumes that all forms of detection require at least a modicum of focused attention (Duncan and Humphreys, 1992), then all forms of search will be disrupted to some degree. Here we measured the detection of feature targets that were defined by either a unique color or orientation. Comparable detection rates were observed in non-neglected space, which indicated that both forms of search placed similar demands on attention. For either of the above accounts to be true, the two targets should therefore be detected with equal efficiency in the neglected field. We found that while the detection rate for color was normal in four of our five patients, all showed an increased reaction time and/or error rate for orientation. This result points to a selective deficit in orientation discrimination, and implies that neglect disrupts specific feature representations. That is, the effects of neglect on visual search are not only attentional but also perceptual.

Comparison of propidium monoazide-quantitative PCR and reverse transcription quantitative PCR for viability detection of fresh Cryptosporidium oocysts following disinfection and after long-term storage in water samples

EPA Science Inventory

Purified oocysts of Cryptosporidium parvum were used to evaluate applicability of two quantitative PCR (qPCR) viability detection methods in raw surface water and disinfection treated water. Propidium monoazide-qPCR targeting hsp70 gene was compared to reverse transcription (RT)-...

Development of a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the detection of Sugarcane mosaic virus and Sorghum mosaic virus in sugarcane

USDA-ARS?s Scientific Manuscript database

A reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for detecting Sugarcane mosaic virus (SCMV) and Sorghum mosaic virus (SrMV) in sugarcane. Six sets of four primers corresponding to the conserved coat protein gene were designed for each virus and their succ...

Detection of the CLOCK/BMAL1 heterodimer using a nucleic acid probe with cycling probe technology.

PubMed

Nakagawa, Kazuhiro; Yamamoto, Takuro; Yasuda, Akio

2010-09-15

An isothermal signal amplification technique for specific DNA sequences, known as cycling probe technology (CPT), has enabled rapid acquisition of genomic information. Here we report an analogous technique for the detection of an activated transcription factor, a transcription element-binding assay with fluorescent amplification by apurinic/apyrimidinic (AP) site lysis cycle (TEFAL). This simple amplification assay can detect activated transcription factors by using a unique nucleic acid probe containing a consensus binding sequence and an AP site, which enables the CPT reaction with AP endonuclease. In this article, we demonstrate that this method detects the functional CLOCK/BMAL1 heterodimer via the TEFAL probe containing the E-box consensus sequence to which the CLOCK/BMAL1 heterodimer binds. Using TEFAL combined with immunoassays, we measured oscillations in the amount of CLOCK/BMAL1 heterodimer in serum-stimulated HeLa cells. Furthermore, we succeeded in measuring the circadian accumulation of the functional CLOCK/BMAL1 heterodimer in human buccal mucosa cells. TEFAL contributes greatly to the study of transcription factor activation in mammalian tissues and cell extracts and is a powerful tool for less invasive investigation of human circadian rhythms. 2010 Elsevier Inc. All rights reserved.

Carotenoid accumulation in orange-pigmented Capsicum annuum fruit, regulated at multiple levels

PubMed Central

Rodriguez-Uribe, Laura; Guzman, Ivette; Rajapakse, Wathsala; Richins, Richard D.; O’Connell, Mary A.

2012-01-01

The pericarp of Capsicum fruit is a rich dietary source of carotenoids. Accumulation of these compounds may be controlled, in part, by gene transcription of biosynthetic enzymes. The carotenoid composition in a number of orange-coloured C. annuum cultivars was determined using HPLC and compared with transcript abundances for four carotenogenic enzymes, Psy, LcyB, CrtZ-2, and Ccs determined by qRT-PCR. There were unique carotenoid profiles as well as distinct patterns of transcription of carotenogenic enzymes within the seven orange-coloured cultivars. In one cultivar, ‘Fogo’, carrying the mutant ccs-3 allele, transcripts were detected for this gene, but no CCS protein accumulated. The premature stop termination in ccs-3 prevented expression of the biosynthetic activity to synthesize the capsanthin and capsorubin forms of carotenoids. In two other orange-coloured cultivars, ‘Orange Grande’ and ‘Oriole’, both with wild-type versions of all four carotenogenic enzymes, no transcripts for Ccs were detected and no red pigments accumulated. Finally, in a third case, the orange-coloured cultivar, Canary, transcripts for all four of the wild-type carotenogenic enzymes were readily detected yet no CCS protein appeared to accumulate and no red carotenoids were synthesized. In the past, mutations in Psy and Ccs have been identified as the loci controlling colour in the fruit. Now there is evidence that a non-structural gene may control colour development in Capsicum. PMID:21948863

Carotenoid accumulation in orange-pigmented Capsicum annuum fruit, regulated at multiple levels.

PubMed

Rodriguez-Uribe, Laura; Guzman, Ivette; Rajapakse, Wathsala; Richins, Richard D; O'Connell, Mary A

2012-01-01

The pericarp of Capsicum fruit is a rich dietary source of carotenoids. Accumulation of these compounds may be controlled, in part, by gene transcription of biosynthetic enzymes. The carotenoid composition in a number of orange-coloured C. annuum cultivars was determined using HPLC and compared with transcript abundances for four carotenogenic enzymes, Psy, LcyB, CrtZ-2, and Ccs determined by qRT-PCR. There were unique carotenoid profiles as well as distinct patterns of transcription of carotenogenic enzymes within the seven orange-coloured cultivars. In one cultivar, 'Fogo', carrying the mutant ccs-3 allele, transcripts were detected for this gene, but no CCS protein accumulated. The premature stop termination in ccs-3 prevented expression of the biosynthetic activity to synthesize the capsanthin and capsorubin forms of carotenoids. In two other orange-coloured cultivars, 'Orange Grande' and 'Oriole', both with wild-type versions of all four carotenogenic enzymes, no transcripts for Ccs were detected and no red pigments accumulated. Finally, in a third case, the orange-coloured cultivar, Canary, transcripts for all four of the wild-type carotenogenic enzymes were readily detected yet no CCS protein appeared to accumulate and no red carotenoids were synthesized. In the past, mutations in Psy and Ccs have been identified as the loci controlling colour in the fruit. Now there is evidence that a non-structural gene may control colour development in Capsicum.

Molecular cloning and characterisation of banana fruit polyphenol oxidase.

PubMed

Gooding, P S; Bird, C; Robinson, S P

2001-09-01

Polyphenol oxidase (PPO; EC 1.10.3.2) is the enzyme thought to be responsible for browning in banana [Musa cavendishii (AAA group, Cavendish subgroup) cv. Williams] fruit. Banana flesh was high in PPO activity throughout growth and ripening. Peel showed high levels of activity early in development but activity declined until ripening started and then remained constant. PPO activity in fruit was not substantially induced after wounding or treatment with 5-methyl jasmonate. Banana flowers and unexpanded leaf roll had high PPO activities with lower activities observed in mature leaves, roots and stem. Four different PPO cDNA clones were amplified from banana fruit (BPO1, BPO11, BPO34 and BPO35). Full-length cDNA and genomic clones were isolated for the most abundant sequence (BPO1) and the genomic clone was found to contain an 85-bp intron. Introns have not been previously found in PPO genes. Northern analysis revealed the presence of BPO1 mRNA in banana flesh early in development but little BPO1 mRNA was detected at the same stage in banana peel. BPO11 transcript was only detected in very young flesh and there was no detectable expression of BPO34 or BPO35 in developing fruit samples. PPO transcripts were also low throughout ripening in both flesh and peel. BPO1 transcripts were readily detected in flowers, stem, roots and leaf roll samples but were not detected in mature leaves. BPO11 showed a similar pattern of expression to BPO1 in these tissues but transcript levels were much lower. BPO34 and BPO35 mRNAs were only detected at a low level in flowers and roots and BPO34 transcript was detected in mature leaves, the only clone to do so. The results suggest that browning of banana fruit during ripening results from release of pre-existing PPO enzyme, which is synthesised very early in fruit development.

Automated Texture Analysis and Determination of Fibre Orientation of Heart Tissue: A Morphometric Study.

PubMed

Zach, Bernhard; Hofer, Ernst; Asslaber, Martin; Ahammer, Helmut

2016-01-01

The human heart has a heterogeneous structure, which is characterized by different cell types and their spatial configurations. The physical structure, especially the fibre orientation and the interstitial fibrosis, determines the electrical excitation and in further consequence the contractility in macroscopic as well as in microscopic areas. Modern image processing methods and parameters could be used to describe the image content and image texture. In most cases the description of the texture is not satisfying because the fibre orientation, detected with common algorithms, is biased by elements such as fibrocytes or endothelial nuclei. The goal of this work is to figure out if cardiac tissue can be analysed and classified on a microscopic level by automated image processing methods with a focus on an accurate detection of the fibre orientation. Quantitative parameters for identification of textures of different complexity or pathological attributes inside the heart were determined. The focus was set on the detection of the fibre orientation, which was calculated on the basis of the cardiomyocytes' nuclei. It turned out that the orientation of these nuclei corresponded with a high precision to the fibre orientation in the image plane. Additionally, these nuclei also indicated very well the inclination of the fibre.

Corazonin Signaling Is Required in the Male for Sperm Transfer in the Oriental Fruit Fly Bactrocera dorsalis

PubMed Central

Hou, Qiu-Li; Chen, Er-Hu; Jiang, Hong-Bo; Yu, Shuai-Feng; Yang, Pei-Jin; Liu, Xiao-Qiang; Park, Yoonseong; Wang, Jin-Jun; Smagghe, Guy

2018-01-01

Corazonin (Crz) is a widely distributed neuropeptide (or neurohormone) in insects with diverse physiological functions. The present study aimed to reveal the functions of Crz and its receptor (CrzR) in the regulation of sexual behavior and fertility in male Bactrocera dorsalis. Tissue-specific expression analyses showed that the BdCrz transcript was most abundant in the central nervous system (CNS), and the BdCrzR transcript was most abundant in both the fat body and CNS. Immunochemical localization confirmed that three pairs of Crz-immunoreactive neurons are located in the dorsolateral protocerebrum region of male adult brain. Importantly, RNAi-mediated Crz knockdown lengthened mating duration in males, and knockdown of Crz or CrzR strongly decreased male fertility in the following 3 days, while the courtship behavior and mating efficiency were not affected. The reduced number of sperm in the reproductive organs of mated females indicated that Crz knockdown in males reduced sperm transfer. The findings of this study indicate that Crz contributes to the reproductive physiology of the oriental fruit fly B. dorsalis by regulating sperm transfer in male adults.

Corazonin Signaling Is Required in the Male for Sperm Transfer in the Oriental Fruit Fly Bactrocera dorsalis.

PubMed

Hou, Qiu-Li; Chen, Er-Hu; Jiang, Hong-Bo; Yu, Shuai-Feng; Yang, Pei-Jin; Liu, Xiao-Qiang; Park, Yoonseong; Wang, Jin-Jun; Smagghe, Guy

2018-01-01

Corazonin (Crz) is a widely distributed neuropeptide (or neurohormone) in insects with diverse physiological functions. The present study aimed to reveal the functions of Crz and its receptor (CrzR) in the regulation of sexual behavior and fertility in male Bactrocera dorsalis . Tissue-specific expression analyses showed that the BdCrz transcript was most abundant in the central nervous system (CNS), and the BdCrzR transcript was most abundant in both the fat body and CNS. Immunochemical localization confirmed that three pairs of Crz-immunoreactive neurons are located in the dorsolateral protocerebrum region of male adult brain. Importantly, RNAi-mediated Crz knockdown lengthened mating duration in males, and knockdown of Crz or CrzR strongly decreased male fertility in the following 3 days, while the courtship behavior and mating efficiency were not affected. The reduced number of sperm in the reproductive organs of mated females indicated that Crz knockdown in males reduced sperm transfer. The findings of this study indicate that Crz contributes to the reproductive physiology of the oriental fruit fly B. dorsalis by regulating sperm transfer in male adults.

Sorting Five Human Tumor Types Reveals Specific Biomarkers and Background Classification Genes.

PubMed

Roche, Kimberly E; Weinstein, Marvin; Dunwoodie, Leland J; Poehlman, William L; Feltus, Frank A

2018-05-25

We applied two state-of-the-art, knowledge independent data-mining methods - Dynamic Quantum Clustering (DQC) and t-Distributed Stochastic Neighbor Embedding (t-SNE) - to data from The Cancer Genome Atlas (TCGA). We showed that the RNA expression patterns for a mixture of 2,016 samples from five tumor types can sort the tumors into groups enriched for relevant annotations including tumor type, gender, tumor stage, and ethnicity. DQC feature selection analysis discovered 48 core biomarker transcripts that clustered tumors by tumor type. When these transcripts were removed, the geometry of tumor relationships changed, but it was still possible to classify the tumors using the RNA expression profiles of the remaining transcripts. We continued to remove the top biomarkers for several iterations and performed cluster analysis. Even though the most informative transcripts were removed from the cluster analysis, the sorting ability of remaining transcripts remained strong after each iteration. Further, in some iterations we detected a repeating pattern of biological function that wasn't detectable with the core biomarker transcripts present. This suggests the existence of a "background classification" potential in which the pattern of gene expression after continued removal of "biomarker" transcripts could still classify tumors in agreement with the tumor type.

CRTC1/MAML2 fusion transcript in central mucoepidermoid carcinoma of mandible--diagnostic and histogenetic implications.

PubMed

Bell, Diana; Holsinger, Christopher F; El-Naggar, Adel K

2010-12-01

Intraosseous salivary gland carcinomas are extremely rare, comprising only 2% to 3% of all mucoepidermoid carcinomas (MECs) reported. The t(11;19) translocation and its CRTC1/MAML1 fusion transcript have been identified in MEC at different sites and are believed to be associated with the development of a subset of these tumors. However, the status of the fusion transcript has not been reported in intraosseous MEC. Here, we report 3 examples of central MEC of the mandible, including a case with a history of primary retromolar MEC. Reverse transcriptase-polymerase chain reaction and DNA sequencing analyses of the microdissected components of these tumors were used for the detection and verification of the fusion transcript. We identified, for the first time, the t(11;19) fusion gene transcript in central MEC, including in the previous primary retromolar MEC. No fusion transcript was detected in the second primary noncentral MEC or in another central MEC. The results indicate that central MEC can manifest the fusion transcript. This finding may have diagnostic and histogenetic roles in the future analysis of this entity. Published by Elsevier Inc.

Expression of the genes for insecticidal crystal proteins in Bacillus thuringiensis: cryIVA, not cryIVB, is transcribed by RNA polymerase containing sigma H and that containing sigma E.

PubMed

Yoshisue, H; Ihara, K; Nishimoto, T; Sakai, H; Komano, T

1995-03-15

To investigate the mechanism of transcriptional regulation of cryIVA and cryIVB, encoding 130-kDa dipteran-active crystal proteins, in Bacillus thuringiensis subsp. israelensis, we introduced each gene into several sporulation mutants of Bacillus subtilis. A spoIIG mutation, the wild-type gene of which encodes sigma E precursor, completely blocked the cryIVB transcription. In contrast, low but detectable transcription of cryIVA was observed in the spoIIG mutant. In the wild-type B. subtilis, no transcription of cryIVB was detected before T2 (2 h after the onset of stationary phase), while the cryIVA transcription started at the late exponential phase at low levels. Furthermore, in a wild-type strain of B. thuringiensis subsp. israelensis, transcription of cryIVA began earlier than that of genes encoding other crystal components, cryIVB and cytA. A consensus sequence recognized by an RNA polymerase containing sigma H of B. subtilis was found upstream of the transcription start point of cryIVA, which overlapped with that recognized by sigma E.

ChimeRScope: a novel alignment-free algorithm for fusion transcript prediction using paired-end RNA-Seq data

PubMed Central

Li, You; Heavican, Tayla B.; Vellichirammal, Neetha N.; Iqbal, Javeed

2017-01-01

Abstract The RNA-Seq technology has revolutionized transcriptome characterization not only by accurately quantifying gene expression, but also by the identification of novel transcripts like chimeric fusion transcripts. The ‘fusion’ or ‘chimeric’ transcripts have improved the diagnosis and prognosis of several tumors, and have led to the development of novel therapeutic regimen. The fusion transcript detection is currently accomplished by several software packages, primarily relying on sequence alignment algorithms. The alignment of sequencing reads from fusion transcript loci in cancer genomes can be highly challenging due to the incorrect mapping induced by genomic alterations, thereby limiting the performance of alignment-based fusion transcript detection methods. Here, we developed a novel alignment-free method, ChimeRScope that accurately predicts fusion transcripts based on the gene fingerprint (as k-mers) profiles of the RNA-Seq paired-end reads. Results on published datasets and in-house cancer cell line datasets followed by experimental validations demonstrate that ChimeRScope consistently outperforms other popular methods irrespective of the read lengths and sequencing depth. More importantly, results on our in-house datasets show that ChimeRScope is a better tool that is capable of identifying novel fusion transcripts with potential oncogenic functions. ChimeRScope is accessible as a standalone software at (https://github.com/ChimeRScope/ChimeRScope/wiki) or via the Galaxy web-interface at (https://galaxy.unmc.edu/). PMID:28472320

Structural features of the rice chromosome 4 centromere.

PubMed

Zhang, Yu; Huang, Yuchen; Zhang, Lei; Li, Ying; Lu, Tingting; Lu, Yiqi; Feng, Qi; Zhao, Qiang; Cheng, Zhukuan; Xue, Yongbiao; Wing, Rod A; Han, Bin

2004-01-01

A complete sequence of a chromosome centromere is necessary for fully understanding centromere function. We reported the sequence structures of the first complete rice chromosome centromere through sequencing a large insert bacterial artificial chromosome clone-based contig, which covered the rice chromosome 4 centromere. Complete sequencing of the 124-kb rice chromosome 4 centromere revealed that it consisted of 18 tracts of 379 tandemly arrayed repeats known as CentO and a total of 19 centromeric retroelements (CRs) but no unique sequences were detected. Four tracts, composed of 65 CentO repeats, were located in the opposite orientation, and 18 CentO tracts were flanked by 19 retroelements. The CRs were classified into four types, and the type I retroelements appeared to be more specific to rice centromeres. The preferential insert of the CRs among CentO repeats indicated that the centromere-specific retroelements may contribute to centromere expansion during evolution. The presence of three intact retrotransposons in the centromere suggests that they may be responsible for functional centromere initiation through a transcription-mediated mechanism.

Interactions between the promoter and first intron are involved in transcriptional control of alpha 1(I) collagen gene expression.

PubMed Central

Bornstein, P; McKay, J; Liska, D J; Apone, S; Devarayalu, S

1988-01-01

The first intron of the human collagen alpha 1(I) gene contains several positively and negatively acting elements. We have studied the transcription of collagen-human growth hormone fusion genes, containing deletions and rearrangements of collagen intronic sequences, by transient transfection of chick tendon fibroblasts and NIH 3T3 cells. In chick tendon fibroblasts, but not in 3T3 cells, inversion of intronic sequences containing a previously studied 274-base-pair segment, A274, resulted in markedly reduced human growth hormone mRNA levels as determined by an RNase protection assay. This inhibitory effect was largely alleviated when deletions were introduced in the collagen promoter of plasmids containing negatively oriented intronic sequences. Evidence for interaction of the promoter with the intronic segment, A274, was obtained by gel mobility shift assays. We suggest that promoter-intron interactions, mediated by DNA-binding proteins, regulate collagen gene transcription. Inversion of intronic segments containing critical interactive elements might then lead to an altered geometry and reduced activity of a transcriptional complex in those cells with sufficiently high levels of appropriate transcription factors. We further suggest that the deleted promoter segment plays a key role in directing DNA interactions involved in transcriptional control. Images PMID:3211130

LocExpress: a web server for efficiently estimating expression of novel transcripts.

PubMed

Hou, Mei; Tian, Feng; Jiang, Shuai; Kong, Lei; Yang, Dechang; Gao, Ge

2016-12-22

The temporal and spatial-specific expression pattern of a transcript in multiple tissues and cell types can indicate key clues about its function. While several gene atlas available online as pre-computed databases for known gene models, it's still challenging to get expression profile for previously uncharacterized (i.e. novel) transcripts efficiently. Here we developed LocExpress, a web server for efficiently estimating expression of novel transcripts across multiple tissues and cell types in human (20 normal tissues/cells types and 14 cell lines) as well as in mouse (24 normal tissues/cell types and nine cell lines). As a wrapper to RNA-Seq quantification algorithm, LocExpress efficiently reduces the time cost by making abundance estimation calls increasingly within the minimum spanning bundle region of input transcripts. For a given novel gene model, such local context-oriented strategy allows LocExpress to estimate its FPKMs in hundreds of samples within minutes on a standard Linux box, making an online web server possible. To the best of our knowledge, LocExpress is the only web server to provide nearly real-time expression estimation for novel transcripts in common tissues and cell types. The server is publicly available at http://loc-express.cbi.pku.edu.cn .

Demonstration of interleukin-1 beta transcripts in acute myeloblastic leukemic cells by in situ hybridization.

PubMed

Nakamura, M; Kanakura, Y; Furukawa, Y; Ernst, T J; Griffin, J D

1990-07-01

The cells from some patients with acute myeloblastic leukemia will secrete autostimulatory cytokines in tissue culture without the addition of stimulators such as phorbol 12-myristate 13-acetate. Production of interleukin-1 beta (IL-1 beta), for example, has been observed in up to 50% of cases. In order to investigate the nature of the cell secreting IL-1 beta in AML, we used an antisense RNA probe to detect specific IL-1 beta transcripts in individual leukemic cells by in situ hybridization. In fresh, uncultured cells, IL-1 beta transcripts were observed in 1-40% of undifferentiated leukemic blast cells in 17 of 19 cases. In situ hybridization was at least as sensitive as Northern blot analysis in detecting IL-1 beta transcripts. No correlation of IL-1 beta transcript expression with FAB classification was observed. Normal blood and bone marrow mononuclear cells did not contain cells expressing IL-1 beta transcripts. These results support the concept that the regulation of cytokine genes in AML cells is aberrant.

Comprehensive Analysis of CBFβ-MYH11 Fusion Transcripts in Acute Myeloid Leukemia by RT-PCR Analysis

PubMed Central

Kadkol, ShriHari S.; Bruno, Annette; Dodge, Carol; Lindgren, Valerie; Ravandi, Farhad

2004-01-01

CBFβ-MYH11 fusion transcripts are expressed in acute myeloid leukemias of the M4Eo subtype. Patients who express CBFβ-MYH11 fusion transcripts respond favorably to high-dose chemotherapy and are generally spared allogeneic bone marrow transplantation. Hence it is important to identify this fusion in all patients with acute myeloid leukemia M4Eo leukemia. The fusion can be detected by cytogenetics, fluorescence in-situ hybridization (FISH), or by molecular analysis with RT-PCR. Multiple fusion transcripts arising as a result of various breakpoints in the CBFβ and MYH11 have been identified. In this report we describe a comprehensive RT-PCR assay to identify all known fusion transcripts and provide an algorithm for molecular analysis of CBFβ-MYH11 fusions from patient specimens. Further, identification of the fusion transcript by such an assay would help in the diagnosis and follow up of patients with cryptic inversion 16 translocations (such as patient 2 in this report) not detected by standard cytogenetics or FISH and for rational design of probes for quantitative analysis by real-time PCR. PMID:14736823

ORIENTATION REQUIREMENT TO DETECT MAGNETIC FIELD-INDUCTED ALTERATION OF GAP JUNCTION COMMUNICATION IN EPITHELIAL CELLS

EPA Science Inventory

ORIENTATION REQUIREMENT TO DETECT MAGNETIC FIELD-INDUCED ALTERATION OF GAP JUNCTION COMMUNICATION IN EPITHELIAL CELLS.
OBJECTIVE: We have shown that functional gap junction communication as measured by Lucifer yellow dye transfer (DT) in Clone-9 rat liver epithelial cells, c...

Consistency of influenza A virus detection test results across respiratory specimen collection methods using real-time reverse transcription-PCR.

PubMed

Spencer, Sarah; Gaglani, Manjusha; Naleway, Allison; Reynolds, Sue; Ball, Sarah; Bozeman, Sam; Henkle, Emily; Meece, Jennifer; Vandermause, Mary; Clipper, Lydia; Thompson, Mark

2013-11-01

In our prospective cohort study, we compared the performance of nasopharyngeal, oropharyngeal, and nasal swabs for the detection of influenza virus using real-time reverse transcription-PCR assay. Joint consideration of results from oropharyngeal and nasal swabs was as effective as consideration of results from nasopharyngeal swabs alone, as measured by sensitivity and noninferiority analysis.

Osteoclast Inhibitory Peptide-1 Therapy for Paget’s Disease

DTIC Science & Technology

2012-08-01

1 (SQSTM1/p62) gene have been widely identified in PDB patients. We previously detected expression of measles virus nucleocapsid (MVNP) transcripts...high bone turnover in PDB. 15. SUBJECT TERMS Paget’s Disease, measles virus nucleocapsid, sequestosome1 , osteoclast, osteoclast inhibitory peptide...detected expression of measles virus nucleocapsid (MVNP) transcripts in osteoclasts from patients with PDB. Also, we have shown that MVNP gene

Rapid and Quantitative Detection of Hepatitis A Virus from Green Onion and Strawberry Rinses by Use of Real-Time Reverse Transcription-PCR

PubMed Central

Shan, X. C.; Wolffs, P.; Griffiths, M. W.

2005-01-01

In this study, an immunomagnetic capture method and a real-time reverse transcription-PCR assay were used to quantify hepatitis A virus (HAV) in green onion and strawberry rinses. This combined protocol detected as low as 0.5 PFU HAV in produce rinses and concentrated HAV levels up to 20-fold. PMID:16151164

Properties of a U1 RNA enhancer-like sequence.

PubMed Central

Ciliberto, G; Palla, F; Tebb, G; Mattaj, I W; Philipson, L

1987-01-01

The properties of a X.laevis U1B snRNA gene enhancer have been studied by microinjection in Xenopus oocytes. The enhancer-like sequence, defined as a short DNA stretch that is able to activate transcription in an orientation independent manner, is interchangeable between different U snRNA genes. The enhancer sequence alone does not, however, efficiently activate transcription from an SV40 pol II promoter but regains its activity when combined with the U-gene specific proximal sequence element. DNase I protection experiments show that the X.laevis U1B enhancer can interact specifically with a nuclear factor present in mammalian cells. Images PMID:3031597

Detectable Changes in The Blood Transcriptome Are Present after Two Weeks of Antituberculosis Therapy

PubMed Central

Bloom, Chloe I.; Graham, Christine M.; Berry, Matthew P. R.; Wilkinson, Katalin A.; Oni, Tolu; Rozakeas, Fotini; Xu, Zhaohui; Rossello-Urgell, Jose; Chaussabel, Damien; Banchereau, Jacques; Pascual, Virginia; Lipman, Marc; Wilkinson, Robert J.; O’Garra, Anne

2012-01-01

Rationale Globally there are approximately 9 million new active tuberculosis cases and 1.4 million deaths annually. Effective antituberculosis treatment monitoring is difficult as there are no existing biomarkers of poor adherence or inadequate treatment earlier than 2 months after treatment initiation. Inadequate treatment leads to worsening disease, disease transmission and drug resistance. Objectives To determine if blood transcriptional signatures change in response to antituberculosis treatment and could act as early biomarkers of a successful response. Methods Blood transcriptional profiles of untreated active tuberculosis patients in South Africa were analysed before, during (2 weeks and 2 months), at the end of (6 months) and after (12 months) antituberculosis treatment, and compared to individuals with latent tuberculosis. An active-tuberculosis transcriptional signature and a specific treatment-response transcriptional signature were derived. The specific treatment response transcriptional signature was tested in two independent cohorts. Two quantitative scoring algorithms were applied to measure the changes in the transcriptional response. The most significantly represented pathways were determined using Ingenuity Pathway Analysis. Results An active tuberculosis 664-transcript signature and a treatment specific 320-transcript signature significantly diminished after 2 weeks of treatment in all cohorts, and continued to diminish until 6 months. The transcriptional response to treatment could be individually measured in each patient. Conclusions Significant changes in the transcriptional signatures measured by blood tests were readily detectable just 2 weeks after treatment initiation. These findings suggest that blood transcriptional signatures could be used as early surrogate biomarkers of successful treatment response. PMID:23056259

Rotation-invariant features for multi-oriented text detection in natural images.

PubMed

Yao, Cong; Zhang, Xin; Bai, Xiang; Liu, Wenyu; Ma, Yi; Tu, Zhuowen

2013-01-01

Texts in natural scenes carry rich semantic information, which can be used to assist a wide range of applications, such as object recognition, image/video retrieval, mapping/navigation, and human computer interaction. However, most existing systems are designed to detect and recognize horizontal (or near-horizontal) texts. Due to the increasing popularity of mobile-computing devices and applications, detecting texts of varying orientations from natural images under less controlled conditions has become an important but challenging task. In this paper, we propose a new algorithm to detect texts of varying orientations. Our algorithm is based on a two-level classification scheme and two sets of features specially designed for capturing the intrinsic characteristics of texts. To better evaluate the proposed method and compare it with the competing algorithms, we generate a comprehensive dataset with various types of texts in diverse real-world scenes. We also propose a new evaluation protocol, which is more suitable for benchmarking algorithms for detecting texts in varying orientations. Experiments on benchmark datasets demonstrate that our system compares favorably with the state-of-the-art algorithms when handling horizontal texts and achieves significantly enhanced performance on variant texts in complex natural scenes.

Leukemia patient-derived lymphoblastoid cell lines exhibit increased induction of leukemia-associated transcripts following high-dose irradiation.

PubMed

Spencer, A; Granter, N

1999-09-01

Improvement in diagnostic cytogenetic techniques has led to the recognition of an increasing number of leukemia-associated chromosomal translocations and inversions. These genetic lesions frequently are associated with the disruption of putative transcription factors and the production of hybrid transcripts that are implicated in leukemogenesis. Epidemiologic evidence suggests that some, but not all, individuals with a history of gamma-irradiation exposure are at increased risk of developing chronic myeloid leukemia (CML). CML is characterized by the Philadelphia chromosome and transcription of the resulting hybrid BCR-ABL gene. Utilizing the leukemia-associated BCR-ABL p210 transcript as a marker, we sought differences in the induction of illegitimate genetic recombination following high-dose gamma-irradiation of karyotypically normal lymphoblastoid cell lines (LCL) derived from individuals with and without a history of myeloid leukemias. Six LCL [4 leukemia patient derived [2 acute myeloid leukemia and 2 CML] and 2 from normal individuals were analyzed with reverse transcriptase polymerase chain reaction for BCR-ABL under stringent conditions following exposure to 0, 50, or 100 Gy of LET gamma-irradiation delivered via a Varian linear accelerator at 4 MV. Transcripts identical to disease-associated b2a2 and b3a2 transcripts were detected both spontaneously (background illegitimate genetic recombination) and following gamma-irradiation. Background BCR-ABL positivity was demonstrable in 4 of the 6 LCL, with no significant difference in detection between leukemic- and nonleukemic-derived LCL. Overall, increasing gamma-irradiation dose resulted in an increased frequency of BCR-ABL transcript detection (0 Gy vs 50 Gy vs 100 Gy,p = 0.0023, Chi-square test). Within the leukemic- but not the nonleukemic-derived LCL there was significantly greater BCR-ABL positivity after gamma-irradiation compared to unirradiated equivalents. Furthermore, the BCR-ABL positivity of both the AML- and CML-derived LCL after gamma-irradiation was significantly greater than that of the nonleukemic-derived LCL after gamma-irradiation. We speculate that this difference in the detection of illegitimate after gamma-irradiation recombination may be due to aberrant DNA double strand break repair mechanisms in individuals predisposed to the development of myeloid leukemias.

Multiplex nucleic acid sequence-based amplification for simultaneous detection of several enteric viruses in model ready-to-eat foods.

PubMed

Jean, Julie; D'Souza, Doris H; Jaykus, Lee-Ann

2004-11-01

Human enteric viruses are currently recognized as one of the most important causes of food-borne disease. Implication of enteric viruses in food-borne outbreaks can be difficult to confirm due to the inadequacy of the detection methods available. In this study, a nucleic acid sequence-based amplification (NASBA) method was developed in a multiplex format for the specific, simultaneous, and rapid detection of epidemiologically relevant human enteric viruses. Three previously reported primer sets were used in a single reaction for the amplification of RNA target fragments of 474, 371, and 165 nucleotides for the detection of hepatitis A virus and genogroup I and genogroup II noroviruses, respectively. Amplicons were detected by agarose gel electrophoresis and confirmed by electrochemiluminescence and Northern hybridization. Endpoint detection sensitivity for the multiplex NASBA assay was approximately 10(-1) reverse transcription-PCR-detectable units (or PFU, as appropriate) per reaction. When representative ready-to-eat foods (deli sliced turkey and lettuce) were inoculated with various concentrations of each virus and processed for virus detection with the multiplex NASBA method, all three human enteric viruses were simultaneously detected at initial inoculum levels of 10(0) to 10(2) reverse transcription-PCR-detectable units (or PFU)/9 cm2 in both food commodities. The multiplex NASBA system provides rapid and simultaneous detection of clinically relevant food-borne viruses in a single reaction tube and may be a promising alternative to reverse transcription-PCR for the detection of viral contamination of foods.

Contrast, size, and orientation-invariant target detection in infrared imagery

NASA Astrophysics Data System (ADS)

Zhou, Yi-Tong; Crawshaw, Richard D.

1991-08-01

Automatic target detection in IR imagery is a very difficult task due to variations in target brightness, shape, size, and orientation. In this paper, the authors present a contrast, size, and orientation invariant algorithm based on Gabor functions for detecting targets from a single IR image frame. The algorithms consists of three steps. First, it locates potential targets by using low-resolution Gabor functions which resist noise and background clutter effects, then, it removes false targets and eliminates redundant target points based on a similarity measure. These two steps mimic human vision processing but are different from Zeevi's Foveating Vision System. Finally, it uses both low- and high-resolution Gabor functions to verify target existence. This algorithm has been successfully tested on several IR images that contain multiple examples of military vehicles with different size and brightness in various background scenes and orientations.

Building Area Extraction from Polarimetric SAR Data via Stationarity Detection and Circular-Pol Correlation Coefficient

NASA Astrophysics Data System (ADS)

Xiang, Deliang; Su, Yi; Ban, Yifeng

2015-04-01

Since the buildings have complex geometries and may be misclassified as forests or mountains with volume scattering due to the significant cross-pol backscatter and lack reflection symmetry, especially the slant-oriented buildings, building area extraction is a challenging problem. In this paper, the time-frequency decomposition technique is adopted to acquire subaperture images, which correspond to the same scene responses under different azimuthal look angles. Stationarity detection approach with polarimetric G0 distribution is proposed to extract ortho-orientedbuildings and the circular polarization correlation coefficient is optimal in characterizing slant-oriented buildings. We test the aforementioned method using ESAR image with L-band. The results demonstrate that the proposed method can effectively extract both ortho-oriented and slant-oriented buildings and the overall detection accuracy as well as kappa value is 10%-20% higher than the compared methods.

Orientation selectivity sharpens motion detection in Drosophila

PubMed Central

Fisher, Yvette E.; Silies, Marion; Clandinin, Thomas R.

2015-01-01

SUMMARY Detecting the orientation and movement of edges in a scene is critical to visually guided behaviors of many animals. What are the circuit algorithms that allow the brain to extract such behaviorally vital visual cues? Using in vivo two-photon calcium imaging in Drosophila, we describe direction selective signals in the dendrites of T4 and T5 neurons, detectors of local motion. We demonstrate that this circuit performs selective amplification of local light inputs, an observation that constrains motion detection models and confirms a core prediction of the Hassenstein-Reichardt Correlator (HRC). These neurons are also orientation selective, responding strongly to static features that are orthogonal to their preferred axis of motion, a tuning property not predicted by the HRC. This coincident extraction of orientation and direction sharpens directional tuning through surround inhibition and reveals a striking parallel between visual processing in flies and vertebrate cortex, suggesting a universal strategy for motion processing. PMID:26456048

Establishing the behavioural limits for countershaded camouflage.

PubMed

Penacchio, Olivier; Harris, Julie M; Lovell, P George

2017-10-20

Countershading is a ubiquitous patterning of animals whereby the side that typically faces the highest illumination is darker. When tuned to specific lighting conditions and body orientation with respect to the light field, countershading minimizes the gradient of light the body reflects by counterbalancing shadowing due to illumination, and has therefore classically been thought of as an adaptation for visual camouflage. However, whether and how crypsis degrades when body orientation with respect to the light field is non-optimal has never been studied. We tested the behavioural limits on body orientation for countershading to deliver effective visual camouflage. We asked human participants to detect a countershaded target in a simulated three-dimensional environment. The target was optimally coloured for crypsis in a reference orientation and was displayed at different orientations. Search performance dramatically improved for deviations beyond 15 degrees. Detection time was significantly shorter and accuracy significantly higher than when the target orientation matched the countershading pattern. This work demonstrates the importance of maintaining body orientation appropriate for the displayed camouflage pattern, suggesting a possible selective pressure for animals to orient themselves appropriately to enhance crypsis.

Ontogenetic profile of innate immune related genes and their tissue-specific expression in brown trout, Salmo trutta (Linnaeus, 1758).

PubMed

Cecchini, Stefano; Paciolla, Mariateresa; Biffali, Elio; Borra, Marco; Ursini, Matilde V; Lioi, Maria B

2013-09-01

The innate immune system is a fundamental defense weapon of fish, especially during early stages of development when acquired immunity is still far from being completely developed. The present study aims at looking into ontogeny of innate immune system in the brown trout, Salmo trutta, using RT-PCR based approach. Total RNA extracted from unfertilized and fertilized eggs and hatchlings at 0, 1 h and 1, 2, 3, 4, 5, 6, 7 weeks post-fertilization was subjected to RT-PCR using self-designed primers to amplify some innate immune relevant genes (TNF-α, IL-1β, TGF-β and lysozyme c-type). The constitutive expression of β-actin was detected in all developmental stages. IL-1β and TNF-α transcripts were detected from 4 week post-fertilization onwards, whereas TGF-β transcript was detected only from 7 week post-fertilization onwards. Lysozyme c-type transcript was detected early from unfertilized egg stage onwards. Similarly, tissues such as muscle, ovary, heart, brain, gill, testis, liver, intestine, spleen, skin, posterior kidney, anterior kidney and blood collected from adult brown trout were subjected to detection of all selected genes by RT-PCR. TNF-α and lysozyme c-type transcripts were expressed in all tissues. IL-1β and TGF-β transcripts were expressed in all tissues except for the brain and liver, respectively. Taken together, our results show a spatial-temporal expression of some key innate immune-related genes, improving the basic knowledge of the function of innate immune system at early stage of brown trout. Copyright © 2013 Elsevier Ltd. All rights reserved.

Computerized evaluation of holographic interferograms for fatigue crack detection in riveted lap joints

NASA Astrophysics Data System (ADS)

Zhou, Xiang

Using an innovative portable holographic inspection and testing system (PHITS) developed at the Australian Defence Force Academy, fatigue cracks in riveted lap joints can be detected by visually inspecting the abnormal fringe changes recorded on holographic interferograms. In this thesis, for automatic crack detection, some modern digital image processing techniques are investigated and applied to holographic interferogram evaluation. Fringe analysis algorithms are developed for identification of the crack-induced fringe changes. Theoretical analysis of PHITS and riveted lap joints and two typical experiments demonstrate that the fatigue cracks in lightly-clamped joints induce two characteristic fringe changes: local fringe discontinuities at the cracking sites; and the global crescent fringe distribution near to the edge of the rivet hole. Both of the fringe features are used for crack detection in this thesis. As a basis of the fringe feature extraction, an algorithm for local fringe orientation calculation is proposed. For high orientation accuracy and computational efficiency, Gaussian gradient filtering and neighboring direction averaging are used to minimize the effects of image background variations and random noise. The neighboring direction averaging is also used to approximate the fringe directions in centerlines of bright and dark fringes. Experimental results indicate that for high orientation accuracy the scales of the Gaussian filter and neighboring direction averaging should be chosen according to the local fringe spacings. The orientation histogram technique is applied to detect the local fringe discontinuity due to the fatigue cracks. The Fourier descriptor technique is used to characterize the global fringe distribution change from a circular to a crescent distribution with the fatigue crack growth. Experiments and computer simulations are conducted to analyze the detectability and reliability of crack detection using the two techniques. Results demonstrate that the Fourier descriptor technique is more promising in the detection of the short cracks near the edge of the rivet head. However, it is not as reliable as the fringe orientation technique for detection of the long through cracks. For reliability, both techniques should be used in practical crack detection. Neither the Fourier descriptor technique nor the orientation histogram technique have been previously applied to holographic interferometry. While this work related primarily to interferograms of cracked rivets, the techniques would be readily applied to other areas of fringe pattern analysis.

Pseudouridines have context-dependent mutation and stop rates in high-throughput sequencing.

PubMed

Zhou, Katherine I; Clark, Wesley C; Pan, David W; Eckwahl, Matthew J; Dai, Qing; Pan, Tao

2018-05-11

The abundant RNA modification pseudouridine (Ψ) has been mapped transcriptome-wide by chemically modifying pseudouridines with carbodiimide and detecting the resulting reverse transcription stops in high-throughput sequencing. However, these methods have limited sensitivity and specificity, in part due to the use of reverse transcription stops. We sought to use mutations rather than just stops in sequencing data to identify pseudouridine sites. Here, we identify reverse transcription conditions that allow read-through of carbodiimide-modified pseudouridine (CMC-Ψ), and we show that pseudouridines in carbodiimide-treated human ribosomal RNA have context-dependent mutation and stop rates in high-throughput sequencing libraries prepared under these conditions. Furthermore, accounting for the context-dependence of mutation and stop rates can enhance the detection of pseudouridine sites. Similar approaches could contribute to the sequencing-based detection of many RNA modifications.

Transcriptional organization of the Escherichia coli pilus adhesin K99.

PubMed

Inoue, O J; Lee, J H; Isaacson, R E

1993-11-01

The production of the Escherichia coli pilus adhesin K99 requires the expression of eight unique proteins: FanA-H. The transcriptional organization of the K99 operon was investigated by Northern blot analysis. Four RNAs of 0.54, 1.4, 2.5 and 3.5 kb were detected. When a fanC probe was used all four RNAs were detected while the use of fanD, fanF and fanG probes detected two RNAs each. Using several deletion and TnphoA insertion mutants it was concluded that there were seven unique K99-specific transcripts, several of which were of the same approximate sizes (1.4 and 2.5 kb). It also was concluded that K99 was comprised of at least three complementation groups, two of which were regulated by catabolite repression.

Regulated expression of a novel TCP domain transcription factor indicates an involvement in the control of meristem activation processes in Solanum tuberosum.

PubMed

Faivre-Rampant, Odile; Bryan, Glenn J; Roberts, Alison G; Milbourne, Daniel; Viola, Roberto; Taylor, Mark A

2004-04-01

In this study, the aim was to determine whether TCP transcription factors are implicated in meristem activation in potato (Solanum tuberosum). By searching a database of potato EST sequences, with a sequence characteristically conserved in TCP domains, a potato tcp gene was identified. A BAC clone containing the tcp sequence was isolated and the genomic sequence was determined. Using a CAPS marker assay, the potato tcp gene (sttcp1) was mapped to chromosome 8. In dormant buds, relatively high levels of sttcp1-specific transcript were detected by in situ hybridization. By contrast, in sprouting buds, no expression of the sttcp1 could be detected. Furthermore, an inverse relationship between axillary bud size and the steady-state level of the sstcp1 transcript was demonstrated. In non-growing buds exhibiting correlative inhibition, sttcpI-specific transcript levels were also relatively high, but rapidly decreased when apical dominance was removed by excision of the apical bud.

Heterologous expression of the Phycomyces blakesleeanus phytoene dehydrogenase gene (carB) in Mucor circinelloides.

PubMed

Ruiz-Hidalgo, M J; Eslava, A P; Alvarez, M I; Benito, E P

1999-11-01

A phytoene dehydrogenase-deficient mutant of Mucor circinelloides accumulating only phytoene was transformed with the gene encoding the corresponding enzyme (carB gene) of Phycomyces blakesleeanus. Carotenoids derived from phytoene were detected in the transformants showing that the P. blakesleeanus carB gene complements the M. circinelloides carB mutation. These newly formed carotenoids accumulated in low quantities, indicating that functional complementation was poor. carB mRNA molecules correctly transcribed were detected in the transformants, but they represented a small proportion of the total population of carB-derived mRNAs, mostly constituted by truncated transcripts and by transcripts longer than the transcript that is functional in Phycomyces. These results showed that the P. blakesleeanus carB gene was expressed in M. circinelloides and suggested that the poor complementation observed was owing, at least in part, to the lack of specificity in the recognition of the transcription initiation and termination signals of the P. blakesleeanus carB gene by the M. circinelloides transcriptional machinery.

Viral DNA Replication Orientation and hnRNPs Regulate Transcription of the Human Papillomavirus 18 Late Promoter.

PubMed

Wang, Xiaohong; Liu, Haibin; Ge, Hui; Ajiro, Masahiko; Sharma, Nishi R; Meyers, Craig; Morozov, Pavel; Tuschl, Thomas; Klar, Amar; Court, Donald; Zheng, Zhi-Ming

2017-05-30

The life cycle of human papillomaviruses (HPVs) is tightly linked to keratinocyte differentiation. Although expression of viral early genes is initiated immediately upon virus infection of undifferentiated basal cells, viral DNA amplification and late gene expression occur only in the mid to upper strata of the keratinocytes undergoing terminal differentiation. In this report, we show that the relative activity of HPV18 TATA-less late promoter P 811 depends on its orientation relative to that of the origin (Ori) of viral DNA replication and is sensitive to the eukaryotic DNA polymerase inhibitor aphidicolin. Additionally, transfected 70-nucleotide (nt)-long single-strand DNA oligonucleotides that are homologous to the region near Ori induce late promoter activity. We also found that promoter activation in raft cultures leads to production of the late promoter-associated, sense-strand transcription initiation RNAs (tiRNAs) and splice-site small RNAs (spliRNAs). Finally, a cis -acting AAGTATGCA core element that functions as a repressor to the promoter was identified. This element interacts with hnRNP D0B and hnRNP A/B factors. Point mutations in the core prevented binding of hnRNPs and increased the promoter activity. Confirming this result, knocking down the expression of both hnRNPs in keratinocytes led to increased promoter activity. Taking the data together, our study revealed the mechanism of how the HPV18 late promoter is regulated by DNA replication and host factors. IMPORTANCE It has been known for decades that the activity of viral late promoters is associated with viral DNA replication among almost all DNA viruses. However, the mechanism of how DNA replication activates the viral late promoter and what components of the replication machinery are involved remain largely unknown. In this study, we characterized the P 811 promoter region of HPV18 and demonstrated that its activation depends on the orientation of DNA replication. Using single-stranded oligonucleotides targeting the replication fork on either leading or lagging strands, we showed that viral lagging-strand replication activates the promoter. We also identified a transcriptional repressor element located upstream of the promoter transcription start site which interacts with cellular proteins hnRNP D0B and hnRNP A/B and modulates the late promoter activity. This is the first report on how DNA replication activates a viral late promoter. Copyright © 2017 Wang et al.

Detection of phenols using engineered bacteria

DOEpatents

Wise, Arlene A.; Kuske, Cheryl R.; Terwilliger, Thomas C.

2007-12-04

Detection of phenols using engineered bacteria. A biosensor can be created by placing a reporter gene under control of an inducible promoter. The reporter gene produces a signal when a cognate transcriptional activator senses the inducing chemical. Creation of bacterial biosensors is currently restricted by limited knowledge of the genetic systems of bacteria that catabolize xenobiotics. By using mutagenic PCR to change the chemical specificity of the Pseudomonas species CF600 DmpR protein, the potential for engineering novel biosensors for detection of phenols has been demonstrated. DmpR, a well-characterized transcriptional activator of the P. CF600's dmp operon mediates growth on simple phenols. Transcription from Po, the promoter heading the dmp operon, is activated when the sensor domain of DmpR interacts with phenol and mono-substituted phenols. By altering the sensor domain of the DmpR, a group of DmpR derivatives that activate transcription of a Po-lacZ fusion in response to eight of the EPA's eleven priority pollutant phenols has been created. The assays and the sensor domain mutations that alter the chemical specificity of DmpR is described.

Detection of phenols using engineered bacteria

DOEpatents

Wise, Arlene A.; Kuske, Cheryl R.; Terwilliger, Thomas C.

2004-08-10

Detection of phenols using engineered bacteria. A biosensor can be created by placing a reporter gene under control of an inducible promoter. The reporter gene produces a signal when a cognate transcriptional activator senses the inducing chemical. Creation of bacterial biosensors is currently restricted by limited knowledge of the genetic systems of bacteria that catabolize xenobiotics. By using mutagenic PCR to change the chemical specificity of the Pseudomonas species CF600 DmpR protein, the potential for engineering novel biosensors for detection of phenols has been demonstrated. DmpR, a well-characterized transcriptional activator of the P. CF600's dmp operon mediates growth on simple phenols. Transcription from Po, the promoter heading the dmp operon, is activated when the sensor domain of DmpR interacts with phenol and mono-substituted phenols. By altering the sensor domain of the DmpR, a group of DmpR derivatives that activate transcription of a Po-lacZ fusion in response to eight of the EPA's eleven priority pollutant phenols has been created. The assays and the sensor domain mutations that alter the chemical specificity of DmpR is described.

Parameter selection with the Hotelling observer in linear iterative image reconstruction for breast tomosynthesis

NASA Astrophysics Data System (ADS)

Rose, Sean D.; Roth, Jacob; Zimmerman, Cole; Reiser, Ingrid; Sidky, Emil Y.; Pan, Xiaochuan

2018-03-01

In this work we investigate an efficient implementation of a region-of-interest (ROI) based Hotelling observer (HO) in the context of parameter optimization for detection of a rod signal at two orientations in linear iterative image reconstruction for DBT. Our preliminary results suggest that ROI-HO performance trends may be efficiently estimated by modeling only the 2D plane perpendicular to the detector and containing the X-ray source trajectory. In addition, the ROI-HO is seen to exhibit orientation dependent trends in detectability as a function of the regularization strength employed in reconstruction. To further investigate the ROI-HO performance in larger 3D system models, we present and validate an iterative methodology for calculating the ROI-HO. Lastly, we present a real data study investigating the correspondence between ROI-HO performance trends and signal conspicuity. Conspicuity of signals in real data reconstructions is seen to track well with trends in ROI-HO detectability. In particular, we observe orientation dependent conspicuity matching the orientation dependent detectability of the ROI-HO.

Transcriptome-wide effects of inverted SINEs on gene expression and their impact on RNA polymerase II activity.

PubMed

Tajaddod, Mansoureh; Tanzer, Andrea; Licht, Konstantin; Wolfinger, Michael T; Badelt, Stefan; Huber, Florian; Pusch, Oliver; Schopoff, Sandy; Janisiw, Michael; Hofacker, Ivo; Jantsch, Michael F

2016-10-25

Short interspersed elements (SINEs) represent the most abundant group of non-long-terminal repeat transposable elements in mammalian genomes. In primates, Alu elements are the most prominent and homogenous representatives of SINEs. Due to their frequent insertion within or close to coding regions, SINEs have been suggested to play a crucial role during genome evolution. Moreover, Alu elements within mRNAs have also been reported to control gene expression at different levels. Here, we undertake a genome-wide analysis of insertion patterns of human Alus within transcribed portions of the genome. Multiple, nearby insertions of SINEs within one transcript are more abundant in tandem orientation than in inverted orientation. Indeed, analysis of transcriptome-wide expression levels of 15 ENCODE cell lines suggests a cis-repressive effect of inverted Alu elements on gene expression. Using reporter assays, we show that the negative effect of inverted SINEs on gene expression is independent of known sensors of double-stranded RNAs. Instead, transcriptional elongation seems impaired, leading to reduced mRNA levels. Our study suggests that there is a bias against multiple SINE insertions that can promote intramolecular base pairing within a transcript. Moreover, at a genome-wide level, mRNAs harboring inverted SINEs are less expressed than mRNAs harboring single or tandemly arranged SINEs. Finally, we demonstrate a novel mechanism by which inverted SINEs can impact on gene expression by interfering with RNA polymerase II.

Consistency of Influenza A Virus Detection Test Results across Respiratory Specimen Collection Methods Using Real-Time Reverse Transcription-PCR

PubMed Central

Gaglani, Manjusha; Naleway, Allison; Reynolds, Sue; Ball, Sarah; Bozeman, Sam; Henkle, Emily; Meece, Jennifer; Vandermause, Mary; Clipper, Lydia; Thompson, Mark

2013-01-01

In our prospective cohort study, we compared the performance of nasopharyngeal, oropharyngeal, and nasal swabs for the detection of influenza virus using real-time reverse transcription-PCR assay. Joint consideration of results from oropharyngeal and nasal swabs was as effective as consideration of results from nasopharyngeal swabs alone, as measured by sensitivity and noninferiority analysis. PMID:24108606

Revealing stable processing products from ribosome-associated small RNAs by deep-sequencing data analysis.

PubMed

Zywicki, Marek; Bakowska-Zywicka, Kamilla; Polacek, Norbert

2012-05-01

The exploration of the non-protein-coding RNA (ncRNA) transcriptome is currently focused on profiling of microRNA expression and detection of novel ncRNA transcription units. However, recent studies suggest that RNA processing can be a multi-layer process leading to the generation of ncRNAs of diverse functions from a single primary transcript. Up to date no methodology has been presented to distinguish stable functional RNA species from rapidly degraded side products of nucleases. Thus the correct assessment of widespread RNA processing events is one of the major obstacles in transcriptome research. Here, we present a novel automated computational pipeline, named APART, providing a complete workflow for the reliable detection of RNA processing products from next-generation-sequencing data. The major features include efficient handling of non-unique reads, detection of novel stable ncRNA transcripts and processing products and annotation of known transcripts based on multiple sources of information. To disclose the potential of APART, we have analyzed a cDNA library derived from small ribosome-associated RNAs in Saccharomyces cerevisiae. By employing the APART pipeline, we were able to detect and confirm by independent experimental methods multiple novel stable RNA molecules differentially processed from well known ncRNAs, like rRNAs, tRNAs or snoRNAs, in a stress-dependent manner.

Position and Orientation Tracking in a Ubiquitous Monitoring System for Parkinson Disease Patients With Freezing of Gait Symptom

PubMed Central

Català, Andreu; Rodríguez Martín, Daniel; van der Aa, Nico; Chen, Wei; Rauterberg, Matthias

2013-01-01

Background Freezing of gait (FoG) is one of the most disturbing and least understood symptoms in Parkinson disease (PD). Although the majority of existing assistive systems assume accurate detections of FoG episodes, the detection itself is still an open problem. The specificity of FoG is its dependency on the context of a patient, such as the current location or activity. Knowing the patient's context might improve FoG detection. One of the main technical challenges that needs to be solved in order to start using contextual information for FoG detection is accurate estimation of the patient's position and orientation toward key elements of his or her indoor environment. Objective The objectives of this paper are to (1) present the concept of the monitoring system, based on wearable and ambient sensors, which is designed to detect FoG using the spatial context of the user, (2) establish a set of requirements for the application of position and orientation tracking in FoG detection, (3) evaluate the accuracy of the position estimation for the tracking system, and (4) evaluate two different methods for human orientation estimation. Methods We developed a prototype system to localize humans and track their orientation, as an important prerequisite for a context-based FoG monitoring system. To setup the system for experiments with real PD patients, the accuracy of the position and orientation tracking was assessed under laboratory conditions in 12 participants. To collect the data, the participants were asked to wear a smartphone, with and without known orientation around the waist, while walking over a predefined path in the marked area captured by two Kinect cameras with non-overlapping fields of view. Results We used the root mean square error (RMSE) as the main performance measure. The vision based position tracking algorithm achieved RMSE = 0.16 m in position estimation for upright standing people. The experimental results for the proposed human orientation estimation methods demonstrated the adaptivity and robustness to changes in the smartphone attachment position, when the fusion of both vision and inertial information was used. Conclusions The system achieves satisfactory accuracy on indoor position tracking for the use in the FoG detection application with spatial context. The combination of inertial and vision information has the potential for correct patient heading estimation even when the inertial wearable sensor device is put into an a priori unknown position. PMID:25098265

Position and orientation tracking in a ubiquitous monitoring system for Parkinson disease patients with freezing of gait symptom.

PubMed

Takač, Boris; Català, Andreu; Rodríguez Martín, Daniel; van der Aa, Nico; Chen, Wei; Rauterberg, Matthias

2013-07-15

Freezing of gait (FoG) is one of the most disturbing and least understood symptoms in Parkinson disease (PD). Although the majority of existing assistive systems assume accurate detections of FoG episodes, the detection itself is still an open problem. The specificity of FoG is its dependency on the context of a patient, such as the current location or activity. Knowing the patient's context might improve FoG detection. One of the main technical challenges that needs to be solved in order to start using contextual information for FoG detection is accurate estimation of the patient's position and orientation toward key elements of his or her indoor environment. The objectives of this paper are to (1) present the concept of the monitoring system, based on wearable and ambient sensors, which is designed to detect FoG using the spatial context of the user, (2) establish a set of requirements for the application of position and orientation tracking in FoG detection, (3) evaluate the accuracy of the position estimation for the tracking system, and (4) evaluate two different methods for human orientation estimation. We developed a prototype system to localize humans and track their orientation, as an important prerequisite for a context-based FoG monitoring system. To setup the system for experiments with real PD patients, the accuracy of the position and orientation tracking was assessed under laboratory conditions in 12 participants. To collect the data, the participants were asked to wear a smartphone, with and without known orientation around the waist, while walking over a predefined path in the marked area captured by two Kinect cameras with non-overlapping fields of view. We used the root mean square error (RMSE) as the main performance measure. The vision based position tracking algorithm achieved RMSE = 0.16 m in position estimation for upright standing people. The experimental results for the proposed human orientation estimation methods demonstrated the adaptivity and robustness to changes in the smartphone attachment position, when the fusion of both vision and inertial information was used. The system achieves satisfactory accuracy on indoor position tracking for the use in the FoG detection application with spatial context. The combination of inertial and vision information has the potential for correct patient heading estimation even when the inertial wearable sensor device is put into an a priori unknown position.

X-linked Alport syndrome caused by splicing mutations in COL4A5.

PubMed

Nozu, Kandai; Vorechovsky, Igor; Kaito, Hiroshi; Fu, Xue Jun; Nakanishi, Koichi; Hashimura, Yuya; Hashimoto, Fusako; Kamei, Koichi; Ito, Shuichi; Kaku, Yoshitsugu; Imasawa, Toshiyuki; Ushijima, Katsumi; Shimizu, Junya; Makita, Yoshio; Konomoto, Takao; Yoshikawa, Norishige; Iijima, Kazumoto

2014-11-07

X-linked Alport syndrome is caused by mutations in the COL4A5 gene. Although many COL4A5 mutations have been detected, the mutation detection rate has been unsatisfactory. Some men with X-linked Alport syndrome show a relatively mild phenotype, but molecular basis investigations have rarely been conducted to clarify the underlying mechanism. In total, 152 patients with X-linked Alport syndrome who were suspected of having Alport syndrome through clinical and pathologic investigations and referred to the hospital for mutational analysis between January of 2006 and January of 2013 were genetically diagnosed. Among those patients, 22 patients had suspected splice site mutations. Transcripts are routinely examined when suspected splice site mutations for abnormal transcripts are detected; 11 of them showed expected exon skipping, but others showed aberrant splicing patterns. The mutation detection strategy had two steps: (1) genomic DNA analysis using PCR and direct sequencing and (2) mRNA analysis using RT-PCR to detect RNA processing abnormalities. Six splicing consensus site mutations resulting in aberrant splicing patterns, one exonic mutation leading to exon skipping, and four deep intronic mutations producing cryptic splice site activation were identified. Interestingly, one case produced a cryptic splice site with a single nucleotide substitution in the deep intron that led to intronic exonization containing a stop codon; however, the patient showed a clearly milder phenotype for X-linked Alport syndrome in men with a truncating mutation. mRNA extracted from the kidney showed both normal and abnormal transcripts, with the normal transcript resulting in the milder phenotype. This novel mechanism leads to mild clinical characteristics. This report highlights the importance of analyzing transcripts to enhance the mutation detection rate and provides insight into genotype-phenotype correlations. This approach can clarify the cause of atypically mild phenotypes in X-linked Alport syndrome. Copyright © 2014 by the American Society of Nephrology.

Yeast-based biosensors: design and applications.

PubMed

Adeniran, Adebola; Sherer, Michael; Tyo, Keith E J

2015-02-01

Yeast-based biosensing (YBB) is an exciting research area, as many studies have demonstrated the use of yeasts to accurately detect specific molecules. Biosensors incorporating various yeasts have been reported to detect an incredibly large range of molecules including but not limited to odorants, metals, intracellular metabolites, carcinogens, lactate, alcohols, and sugars. We review the detection strategies available for different types of analytes, as well as the wide range of output methods that have been incorporated with yeast biosensors. We group biosensors into two categories: those that are dependent upon transcription of a gene to report the detection of a desired molecule and those that are independent of this reporting mechanism. Transcription-dependent biosensors frequently depend on heterologous expression of sensing elements from non-yeast organisms, a strategy that has greatly expanded the range of molecules available for detection by YBBs. Transcription-independent biosensors circumvent the problem of sensing difficult-to-detect analytes by instead relying on yeast metabolism to generate easily detected molecules when the analyte is present. The use of yeast as the sensing element in biosensors has proven to be successful and continues to hold great promise for a variety of applications. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permission@oup.com.

On Lying and Being Lied to: A Linguistic Analysis of Deception in Computer-Mediated Communication

ERIC Educational Resources Information Center

Hancock, Jeffrey T.; Curry, Lauren E.; Goorha, Saurabh; Woodworth, Michael

2008-01-01

This study investigated changes in both the liar's and the conversational partner's linguistic style across truthful and deceptive dyadic communication in a synchronous text-based setting. An analysis of 242 transcripts revealed that liars produced more words, more sense-based words (e.g., seeing, touching), and used fewer self-oriented but more…

Community-Based Individual Knowledge Construction in the Classroom: A Process-Oriented Account

ERIC Educational Resources Information Center

Looi, C.-K.; Chen, W.

2010-01-01

This paper explores the process of knowledge convergence and knowledge sharing in the context of classroom collaboration in which students do a group learning activity mediated by a generic representation tool. In analysing the transcript of the interactions of a group, we adapt the group cognition method of Stahl and the uptake analysis…

A rapid and efficient newly established method to detect COL1A1-PDGFB gene fusion in dermatofibrosarcoma protuberans

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yokoyama, Yoko; Shimizu, Akira; Okada, Etsuko

Highlights: Black-Right-Pointing-Pointer We developed new method to rapidly identify COL1A1-PDGFB fusion in DFSP. Black-Right-Pointing-Pointer New PCR method using a single primer pair detected COL1A1-PDGFB fusion in DFSP. Black-Right-Pointing-Pointer This is the first report of DFSP with a novel COL1A1 breakpoint in exon 5. -- Abstract: The detection of fusion transcripts of the collagen type 1{alpha}1 (COL1A1) and platelet-derived growth factor-BB (PDGFB) genes by genetic analysis has recognized as a reliable and valuable molecular tool for the diagnosis of dermatofibrosarcoma protuberans (DFSP). To detect the COL1A1-PDGFB fusion, almost previous reports performed reverse transcription polymerase chain reaction (RT-PCR) using multiplex forward primersmore » from COL1A1. However, it has possible technical difficulties with respect to the handling of multiple primers and reagents in the procedure. The objective of this study is to establish a rapid, easy, and efficient one-step method of PCR using only a single primer pair to detect the fusion transcripts of the COL1A1 and PDGFB in DFSP. To validate new method, we compared the results of RT-PCR in five patients of DFSP between the previous method using multiplex primers and our established one-step RT-PCR using a single primer pair. In all cases of DFSP, the COL1A1-PDGFB fusion was detected by both previous method and newly established one-step PCR. Importantly, we detected a novel COL1A1 breakpoint in exon 5. The newly developed method is valuable to rapidly identify COL1A1-PDGFB fusion transcripts in DFSP.« less

Microchip capillary electrophoresis with laser-induced fluorescence combined with one-step duplex reverse-transcription polymerase chain reaction for the rapid detection of Enterovirus 71 and Coxsackievirus A16 in throat swab specimens.

PubMed

Jia, Ruan; Chengjun, Sun; Heng, Chen; Chen, Zhou; Yuanqian, Li; Yongxin, Li

2015-07-01

Enterovirus 71 and Coxsackievirus A16 are the main pathogens causing hand-foot-mouth disease. In this paper, microchip capillary electrophoresis with laser-induced fluorescence combined with one-step duplex reverse transcript-polymerase chain reaction has been developed for the detection of Enterovirus 71 and Coxsackievirus A16 in throat swab specimens. The specific reverse transcription-polymerase chain reaction amplicons labeled with SYBR Orange were separated by microchip capillary electrophoresis and detected by laser induced fluorescence detector within 7 min. The intraday and interday relative standard deviation of migration time for DNA Marker was in the range of 1.36-2.94 and 2.78-3.96%, respectively. The detection limits were as low as 2.06 × 10(3) copies/mL for Enterovirus 71 and 5 × 10(3) copies/mL for Coxsackievirus A16. No cross-reactivity was observed with rotavirus, astrovirus, norovirus, and adenovirus, which showed good specificity of the method. This assay was validated using 100 throat swab specimens that were detected by real-time reverse-transcript polymerase chain reaction in parallel and the two methods produced the same results. This study provided a rapid, sensitive and specific method for the detection of Enterovirus 71 and Coxsackievirus A16, which make a contribution to significant time and cost saving for the identification and treatment of patients. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Both cell substratum regulation and hormonal regulation of milk protein gene expression are exerted primarily at the posttranscriptional level

DOE Office of Scientific and Technical Information (OSTI.GOV)

Eisenstein, R.S.; Rosen, J.M.

The mechanism by which individual peptide and steroid hormones and cell-substratum interactions regulate milk protein gene expression has been studied in the COMMA-D mammary epithelial cell line. In the presence of insulin, hydrocortisone, and prolactin, growth of COMMA-D cells on floating collagen gels in comparison with that on a plastic substratum resulted in a 2.5- to 3-fold increase in the relative rate of ..beta..-casein gene transcription but a 37-fold increase in ..beta..-casein mRNA accumulation. In contrast, whey acidic protein gene transcription was constitutive in COMMA-D cells grown on either substratum, but its mRNA was unstable and little intact mature mRNAmore » was detected. Culturing COMMA-D cells on collagen also promoted increased expression of other genes expressed in differentiated mammary epithelial cells, including those encoding ..cap alpha..- and ..gamma..-casein, transferrin, malic enzyme, and phosphoenolpyruvate carboxykinase but decreased the expression of actin and histone genes. Using COMMA-D cells, the authors defined further the role of individual hormones in influencing ..beta..-casein gene transcription. With insulin alone, a basal level of ..beta..-casein gene transcription was detected in COMMA-D cells grown on floating collagen gels. Addition of prolactin but not hydrocortisone resulted in a 2.5- to 3.0-fold increase in ..beta..-casein gene transcription, but both hormones were required to elicit the maximal 73-fold induction in mRNA accumulation. The posttranscriptional effect of hormones on casein mRNA accummulation preceded any detectable changes in the relative rate of transcription. Thus, regulation by both hormones and cell substratum of casein gene expression is exerted primarily at the post transcriptional level.« less

Detection of SYT-SSX mutant transcripts in formalin-fixed paraffin-embedded sarcoma tissues using one-step reverse transcriptase real-time PCR.

PubMed

Norlelawati, A T; Mohd Danial, G; Nora, H; Nadia, O; Zatur Rawihah, K; Nor Zamzila, A; Naznin, M

2016-04-01

Synovial sarcoma (SS) is a rare cancer and accounts for 5-10% of adult soft tissue sarcomas. Making an accurate diagnosis is difficult due to the overlapping histological features of SS with other types of sarcomas and the non-specific immunohistochemistry profile findings. Molecular testing is thus considered necessary to confirm the diagnosis since more than 90% of SS cases carry the transcript of t(X;18)(p11.2;q11.2). The purpose of this study is to diagnose SS at molecular level by testing for t(X;18) fusion-transcript expression through One-step reverse transcriptase real-time Polymerase Chain Reaction (PCR). Formalin-fixed paraffin-embedded tissue blocks of 23 cases of soft tissue sarcomas, which included 5 and 8 cases reported as SS as the primary diagnosis and differential diagnosis respectively, were retrieved from the Department of Pathology, Tengku Ampuan Afzan Hospital, Kuantan, Pahang. RNA was purified from the tissue block sections and then subjected to One-step reverse transcriptase real-time PCR using sequence specific hydrolysis probes for simultaneous detection of either SYT-SSX1 or SYT-SSX2 fusion transcript. Of the 23 cases, 4 cases were found to be positive for SYT-SSX fusion transcript in which 2 were diagnosed as SS whereas in the 2 other cases, SS was the differential diagnosis. Three cases were excluded due to failure of both amplification assays SYT-SSX and control β-2-microglobulin. The remaining 16 cases were negative for the fusion transcript. This study has shown that the application of One-Step reverse transcriptase real time PCR for the detection SYT-SSX transcript is feasible as an aid in confirming the diagnosis of synovial sarcoma.

Mitochondrial run-on transcription assay using biotin labeling.

PubMed

Kühn, Kristina

2015-01-01

RNA synthesis and different posttranscriptional processes shape the transcriptome of plant mitochondria. It is believed that mitochondrial transcription in plants is not stringently controlled, and that RNA degradation has a major impact on mitochondrial steady-state transcript levels. Nevertheless, the presence of two RNA polymerases with different gene specificities in mitochondria of dicotyledonous species indicates that transcriptional mechanisms may provide a means to control mitochondrial steady-state RNA pools and gene expression. To experimentally assess transcriptional activities in mitochondria, run-on transcription assays have been developed. These assays measure elongation rates for endogenous transcripts in freshly prepared mitochondrial extracts. The mitochondrial run-on transcription protocol described here has been optimized for the model plant Arabidopsis (Arabidopsis thaliana). It uses mitochondria prepared from soil-grown Arabidopsis plants and employs nonradioactive labeling for the subsequent detection of run-on transcripts.

Systematic analysis of transcribed loci in ENCODE regions using RACE sequencing reveals extensive transcription in the human genome.

PubMed

Wu, Jia Qian; Du, Jiang; Rozowsky, Joel; Zhang, Zhengdong; Urban, Alexander E; Euskirchen, Ghia; Weissman, Sherman; Gerstein, Mark; Snyder, Michael

2008-01-03

Recent studies of the mammalian transcriptome have revealed a large number of additional transcribed regions and extraordinary complexity in transcript diversity. However, there is still much uncertainty regarding precisely what portion of the genome is transcribed, the exact structures of these novel transcripts, and the levels of the transcripts produced. We have interrogated the transcribed loci in 420 selected ENCyclopedia Of DNA Elements (ENCODE) regions using rapid amplification of cDNA ends (RACE) sequencing. We analyzed annotated known gene regions, but primarily we focused on novel transcriptionally active regions (TARs), which were previously identified by high-density oligonucleotide tiling arrays and on random regions that were not believed to be transcribed. We found RACE sequencing to be very sensitive and were able to detect low levels of transcripts in specific cell types that were not detectable by microarrays. We also observed many instances of sense-antisense transcripts; further analysis suggests that many of the antisense transcripts (but not all) may be artifacts generated from the reverse transcription reaction. Our results show that the majority of the novel TARs analyzed (60%) are connected to other novel TARs or known exons. Of previously unannotated random regions, 17% were shown to produce overlapping transcripts. Furthermore, it is estimated that 9% of the novel transcripts encode proteins. We conclude that RACE sequencing is an efficient, sensitive, and highly accurate method for characterization of the transcriptome of specific cell/tissue types. Using this method, it appears that much of the genome is represented in polyA+ RNA. Moreover, a fraction of the novel RNAs can encode protein and are likely to be functional.

The hepatitis B virus large surface protein (LHBs) is a transcriptional activator.

PubMed

Hildt, E; Saher, G; Bruss, V; Hofschneider, P H

1996-11-01

It has been shown that a C-terminally truncated form of the middle-sized hepatitis B virus (HBV) surface protein (MHBst) functions as a transcriptional activator. This function is dependent on the cytosolic orientation of the N-terminal PreS2 domain of MHBst, but in the case of wild-type MHBs, the PreS2 domain is contranslationally translocated into the ER lumen. Recent reports demonstrated that the PreS2 domain of the large HBV surface protein (LHBs) initially remains on the cytosolic side of the ER membrane after translation. Therefore, the question arose as to whether the LHBs protein exhibits the same transcriptional activator function as MHBst. We show that LHBs, like MHBst, is indeed able to activate a variety of promoter elements. There is evidence for a PKC-dependent activation of AP-1 and NF-kappa B by LHBs. Downstream of the PKC the functionality of c-Raf-1 kinase is a prerequisite for LHBs-dependent activation of AP-1 and NF-kappa B since inhibition of c-Raf-1 kinase abolishes LHBs-dependent transcriptional activation of AP-1 and NF-kappa B.

An in vivo and in silico approach to study cis-antisense: a short cut to higher order response

NASA Astrophysics Data System (ADS)

Courtney, Colleen; Varanasi, Usha; Chatterjee, Anushree

2014-03-01

Antisense interactions are present in all domains of life. Typically sense, antisense RNA pairs originate from overlapping genes with convergent face to face promoters, and are speculated to be involved in gene regulation. Recent studies indicate the role of transcriptional interference (TI) in regulating expression of genes in convergent orientation. Modeling antisense, TI gene regulation mechanisms allows us to understand how organisms control gene expression. We present a modeling and experimental framework to understand convergent transcription that combines the effects of transcriptional interference and cis-antisense regulation. Our model shows that combining transcriptional interference and antisense RNA interaction adds multiple-levels of regulation which affords a highly tunable biological output, ranging from first order response to complex higher-order response. To study this system we created a library of experimental constructs with engineered TI and antisense interaction by using face-to-face inducible promoters separated by carefully tailored overlapping DNA sequences to control expression of a set of fluorescent reporter proteins. Studying this gene expression mechanism allows for an understanding of higher order behavior of gene expression networks.

Optimization of the elution buffer and concentration method for detecting hepatitis E virus in swine liver using a nested reverse transcription-polymerase chain reaction and real-time reverse transcription-polymerase chain reaction.

PubMed

Son, Na Ry; Seo, Dong Joo; Lee, Min Hwa; Seo, Sheungwoo; Wang, Xiaoyu; Lee, Bog-Hieu; Lee, Jeong-Su; Joo, In-Sun; Hwang, In-Gyun; Choi, Changsun

2014-09-01

The aim of this study was to develop an optimal technique for detecting hepatitis E virus (HEV) in swine livers. Here, three elution buffers and two concentration methods were compared with respect to enhancing recovery of HEV from swine liver samples. Real-time reverse transcription-polymerase chain reaction (RT-PCR) and nested RT-PCR were performed to detect HEV RNA. When phosphate-buffered saline (PBS, pH 7.4) was used to concentrate HEV in swine liver samples using ultrafiltration, real-time RT-PCR detected HEV in 6 of the 26 samples. When threonine buffer was used to concentrate HEV using polyethylene glycol (PEG) precipitation and ultrafiltration, real-time RT-PCR detected HEV in 1 and 3 of the 26 samples, respectively. When glycine buffer was used to concentrate HEV using ultrafiltration and PEG precipitation, real-time RT-PCR detected HEV in 1 and 3 samples of the 26 samples, respectively. When nested RT-PCR was used to detect HEV, all samples tested negative regardless of the type of elution buffer or concentration method used. Therefore, the combination of real-time RT-PCR and ultrafiltration with PBS buffer was the most sensitive and reliable method for detecting HEV in swine livers. Copyright © 2014 Elsevier B.V. All rights reserved.

The full transcription map of mouse papillomavirus type 1 (MmuPV1) in mouse wart tissues

PubMed Central

Kim, Bong-Hyun; Gotte, Deanna; Chen, Xiongfong; Cam, Maggie; Lambert, Paul F.

2017-01-01

Mouse papillomavirus type 1 (MmuPV1) provides, for the first time, the opportunity to study infection and pathogenesis of papillomaviruses in the context of laboratory mice. In this report, we define the transcriptome of MmuPV1 genome present in papillomas arising in experimentally infected mice using a combination of RNA-seq, PacBio Iso-seq, 5’ RACE, 3’ RACE, primer-walking RT-PCR, RNase protection, Northern blot and in situ hybridization analyses. We demonstrate that the MmuPV1 genome is transcribed unidirectionally from five major promoters (P) or transcription start sites (TSS) and polyadenylates its transcripts at two major polyadenylation (pA) sites. We designate the P7503, P360 and P859 as “early” promoters because they give rise to transcripts mostly utilizing the polyadenylation signal at nt 3844 and therefore can only encode early genes, and P7107 and P533 as “late” promoters because they give rise to transcripts utilizing polyadenylation signals at either nt 3844 or nt 7047, the latter being able to encode late, capsid proteins. MmuPV1 genome contains five splice donor sites and three acceptor sites that produce thirty-six RNA isoforms deduced to express seven predicted early gene products (E6, E7, E1, E1^M1, E1^M2, E2 and E8^E2) and three predicted late gene products (E1^E4, L2 and L1). The majority of the viral early transcripts are spliced once from nt 757 to 3139, while viral late transcripts, which are predicted to encode L1, are spliced twice, first from nt 7243 to either nt 3139 (P7107) or nt 757 to 3139 (P533) and second from nt 3431 to nt 5372. Thirteen of these viral transcripts were detectable by Northern blot analysis, with the P533-derived late E1^E4 transcripts being the most abundant. The late transcripts could be detected in highly differentiated keratinocytes of MmuPV1-infected tissues as early as ten days after MmuPV1 inoculation and correlated with detection of L1 protein and viral DNA amplification. In mature warts, detection of L1 was also found in more poorly differentiated cells, as previously reported. Subclinical infections were also observed. The comprehensive transcription map of MmuPV1 generated in this study provides further evidence that MmuPV1 is similar to high-risk cutaneous beta human papillomaviruses. The knowledge revealed will facilitate the use of MmuPV1 as an animal virus model for understanding of human papillomavirus gene expression, pathogenesis and immunology. PMID:29176795

Promoter-proximal rDNA terminator augments initiation by preventing disruption of the stable transcription complex caused by polymerase read-in

DOE Office of Scientific and Technical Information (OSTI.GOV)

Henderson, S.L.; Ryan, K.; Sollner-Webb, B.

1989-02-01

We have examined the mechanism by which transcriptional initiation at the mouse rDNA promoter is augmented by the RNA polymerase I terminator element that resides just upstream of it. Using templates in which terminator elements are instead positioned at the opposite side of the plasmid rather than proximal to the promoter, or conditions where transcription is terminated elsewhere in the plasmid by UV-induced lesions, we show that the terminator's stimulatory effect is not position dependent. Mouse terminator elements therefore do not stimulate via the previously postulated 'read-through enhancement' model in which terminated polymerases are handed off to an adjacent promotermore » in a concerted reaction. The position independence and orientation dependence of the terminator also makes it unlikely that the terminator functions as a promoter element or as an enhancer. Instead, terminators serve to augment initiation by preventing polymerases from reading completely around the plasmid and through the promoter from upstream, an event which we show interferes with subsequent rounds of initiation. Notably, this transcriptional interference arises because polymerase passage across a promoter disrupts the otherwise stable transcription complex, specifically releasing the bound transcription factor D. These liberated D molecules can then bind to other templates and activate their expression. The rDNA transcriptional interference is not due to a steric impediment to the binding of new polymerase molecules, and it does not similarly liberate the initiation-competent polymerase (factor C). These studies have also convincingly demonstrated that multiple rounds of transcription are obtained from rDNA template molecules in vitro.« less

Multiplex Nucleic Acid Sequence-Based Amplification for Simultaneous Detection of Several Enteric Viruses in Model Ready-To-Eat Foods†

PubMed Central

Jean, Julie; D'Souza, Doris H.; Jaykus, Lee-Ann

2004-01-01

Human enteric viruses are currently recognized as one of the most important causes of food-borne disease. Implication of enteric viruses in food-borne outbreaks can be difficult to confirm due to the inadequacy of the detection methods available. In this study, a nucleic acid sequence-based amplification (NASBA) method was developed in a multiplex format for the specific, simultaneous, and rapid detection of epidemiologically relevant human enteric viruses. Three previously reported primer sets were used in a single reaction for the amplification of RNA target fragments of 474, 371, and 165 nucleotides for the detection of hepatitis A virus and genogroup I and genogroup II noroviruses, respectively. Amplicons were detected by agarose gel electrophoresis and confirmed by electrochemiluminescence and Northern hybridization. Endpoint detection sensitivity for the multiplex NASBA assay was approximately 10−1 reverse transcription-PCR-detectable units (or PFU, as appropriate) per reaction. When representative ready-to-eat foods (deli sliced turkey and lettuce) were inoculated with various concentrations of each virus and processed for virus detection with the multiplex NASBA method, all three human enteric viruses were simultaneously detected at initial inoculum levels of 100 to 102 reverse transcription-PCR-detectable units (or PFU)/9 cm2 in both food commodities. The multiplex NASBA system provides rapid and simultaneous detection of clinically relevant food-borne viruses in a single reaction tube and may be a promising alternative to reverse transcription-PCR for the detection of viral contamination of foods. PMID:15528524

Implicit Change Identification: A Replication of Fernandez-Duque and Thornton (2003)

ERIC Educational Resources Information Center

Laloyaux, Cedric; Destrebecqz, Arnaud; Cleeremans, Axel

2006-01-01

Using a simple change detection task involving vertical and horizontal stimuli, I. M. Thornton and D. Fernandez-Duque (2000) showed that the implicit detection of a change in the orientation of an item influences performance in a subsequent orientation judgment task. However, S. R. Mitroff, D. J. Simons, and S. L. Franconeri (2002) were not able…

Rapid and Sensitive Detection of Norovirus Genomes in Oysters by a Two-Step Isothermal Amplification Assay System Combining Nucleic Acid Sequence-Based Amplification and Reverse Transcription-Loop-Mediated Isothermal Amplification Assays▿

PubMed Central

Fukuda, Shinji; Sasaki, Yukie; Seno, Masato

2008-01-01

We developed a two-step isothermal amplification assay system, which achieved the detection of norovirus (NoV) genomes in oysters with a sensitivity similar to that of reverse transcription-seminested PCR. The time taken for the amplification of NoV genomes from RNA extracts was shortened to about 3 h. PMID:18456857

A trans-acting Variant within the Transcription Factor RIM101 Interacts with Genetic Background to Determine its Regulatory Capacity.

PubMed

Read, Timothy; Richmond, Phillip A; Dowell, Robin D

2016-01-01

Most genetic variants associated with disease occur within regulatory regions of the genome, underscoring the importance of defining the mechanisms underlying differences in regulation of gene expression between individuals. We discovered a pair of co-regulated, divergently oriented transcripts, AQY2 and ncFRE6, that are expressed in one strain of Saccharomyces cerevisiae, ∑1278b, but not in another, S288c. By combining classical genetics techniques with high-throughput sequencing, we identified a trans-acting single nucleotide polymorphism within the transcription factor RIM101 that causes the background-dependent expression of both transcripts. Subsequent RNA-seq experiments revealed that RIM101 regulates many more targets in S288c than in ∑1278b and that deletion of RIM101 in both backgrounds abrogates the majority of differential expression between the strains. Strikingly, only three transcripts undergo a significant change in expression after swapping RIM101 alleles between backgrounds, implying that the differences in the RIM101 allele lead to a remarkably focused transcriptional response. However, hundreds of RIM101-dependent targets undergo a subtle but consistent shift in expression in the S288c RIM101-swapped strain, but not its ∑1278b counterpart. We conclude that ∑1278b may harbor a variant(s) that buffers against widespread transcriptional dysregulation upon introduction of a non-native RIM101 allele, emphasizing the importance of accounting for genetic background when assessing the impact of a regulatory variant.

The equivalent internal orientation and position noise for contour integration.

PubMed

Baldwin, Alex S; Fu, Minnie; Farivar, Reza; Hess, Robert F

2017-10-12

Contour integration is the joining-up of local responses to parts of a contour into a continuous percept. In typical studies observers detect contours formed of discrete wavelets, presented against a background of random wavelets. This measures performance for detecting contours in the limiting external noise that background provides. Our novel task measures contour integration without requiring any background noise. This allowed us to perform noise-masking experiments using orientation and position noise. From these we measure the equivalent internal noise for contour integration. We found an orientation noise of 6° and position noise of 3 arcmin. Orientation noise was 2.6x higher in contour integration compared to an orientation discrimination control task. Comparing against a position discrimination task found position noise in contours to be 2.4x lower. This suggests contour integration involves intermediate processing that enhances the quality of element position representation at the expense of element orientation. Efficiency relative to the ideal observer was lower for the contour tasks (36% in orientation noise, 21% in position noise) compared to the controls (54% and 57%).

Highly multiplexed simultaneous detection of RNAs and proteins in single cells.

PubMed

Frei, Andreas P; Bava, Felice-Alessio; Zunder, Eli R; Hsieh, Elena W Y; Chen, Shih-Yu; Nolan, Garry P; Gherardini, Pier Federico

2016-03-01

To enable the detection of expression signatures specific to individual cells, we developed PLAYR (proximity ligation assay for RNA), a method for highly multiplexed transcript quantification by flow and mass cytometry that is compatible with standard antibody staining. When used with mass cytometry, PLAYR allowed for the simultaneous quantification of more than 40 different mRNAs and proteins. In primary cells, we quantified multiple transcripts, with the identity and functional state of each analyzed cell defined on the basis of the expression of a separate set of transcripts or proteins. By expanding high-throughput deep phenotyping of cells beyond protein epitopes to include RNA expression, PLAYR opens a new avenue for the characterization of cellular metabolism.

A binary search approach to whole-genome data analysis.

PubMed

Brodsky, Leonid; Kogan, Simon; Benjacob, Eshel; Nevo, Eviatar

2010-09-28

A sequence analysis-oriented binary search-like algorithm was transformed to a sensitive and accurate analysis tool for processing whole-genome data. The advantage of the algorithm over previous methods is its ability to detect the margins of both short and long genome fragments, enriched by up-regulated signals, at equal accuracy. The score of an enriched genome fragment reflects the difference between the actual concentration of up-regulated signals in the fragment and the chromosome signal baseline. The "divide-and-conquer"-type algorithm detects a series of nonintersecting fragments of various lengths with locally optimal scores. The procedure is applied to detected fragments in a nested manner by recalculating the lower-than-baseline signals in the chromosome. The algorithm was applied to simulated whole-genome data, and its sensitivity/specificity were compared with those of several alternative algorithms. The algorithm was also tested with four biological tiling array datasets comprising Arabidopsis (i) expression and (ii) histone 3 lysine 27 trimethylation CHIP-on-chip datasets; Saccharomyces cerevisiae (iii) spliced intron data and (iv) chromatin remodeling factor binding sites. The analyses' results demonstrate the power of the algorithm in identifying both the short up-regulated fragments (such as exons and transcription factor binding sites) and the long--even moderately up-regulated zones--at their precise genome margins. The algorithm generates an accurate whole-genome landscape that could be used for cross-comparison of signals across the same genome in evolutionary and general genomic studies.

ENDOGENOUS RETROVIRUSES MOBILIZED DURING FRIEND MURINE LEUKEMIA VIRUS INFECTION

PubMed Central

Hansen, Ethan; Hendrick, Duncan; Malik, Frank; Evans, Leonard H.

2016-01-01

We have demonstrated in a mouse model that infection with a retrovirus can lead not only to the generation of recombinants between exogenous and endogenous gammaretrovirus, but also to the mobilization of endogenous proviruses by pseudotyping entire polytropic proviral transcripts and facilitating their infectious spread to new cells. However, the frequency of this occurrence, the kinetics, and the identity of mobilized endogenous proviruses was unclear. Here we find that these mobilized transcripts are detected after only one day of infection. They predominate over recombinant polytropic viruses early in infection, persist throughout the course of disease and are comprised of multiple different polytropic proviruses. Other endogenous retroviral elements such as intracisternal A particles (IAPs) were not detected. The integration of the endogenous transcripts into new cells could result in loss of transcriptional control and elevated expression which may facilitate pathogenesis, perhaps by contributing to the generation of polytropic recombinant viruses. PMID:27657834

Systematically frameshifting by deletion of every 4th or 4th and 5th nucleotides during mitochondrial transcription: RNA self-hybridization regulates delRNA expression.

PubMed

Seligmann, Hervé

2016-01-01

In mitochondria, secondary structures punctuate post-transcriptional RNA processing. Recently described transcripts match the human mitogenome after systematic deletions of every 4th, respectively every 4th and 5th nucleotides, called delRNAs. Here I explore predicted stem-loop hairpin formation by delRNAs, and their associations with delRNA transcription and detected peptides matching their translation. Despite missing 25, respectively 40% of the nucleotides in the original sequence, del-transformed sequences form significantly more secondary structures than corresponding randomly shuffled sequences, indicating biological function, independently of, and in combination with, previously detected delRNA and thereof translated peptides. Self-hybridization decreases delRNA abundances, indicating downregulation. Systematic deletions of the human mitogenome reveal new, unsuspected coding and structural informations. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

In Situ Detection of MicroRNA Expression with RNAscope Probes.

PubMed

Yin, Viravuth P

2018-01-01

Elucidating the spatial resolution of gene transcripts provides important insight into potential gene function. MicroRNAs are short, singled-stranded noncoding RNAs that control gene expression through base-pair complementarity with target mRNAs in the 3' untranslated region (UTR) and inhibiting protein expression. However, given their small size of ~22- to 24-nt and low expression levels, standard in situ hybridization detection methods are not amendable for microRNA spatial resolution. Here, I describe a technique that employs RNAscope probe design and propriety amplification technology that provides simultaneous single molecule detection of individual microRNA and its target gene. This method allows for rapid and sensitive detection of noncoding RNA transcripts in frozen tissue sections.

Interactive-predictive detection of handwritten text blocks

NASA Astrophysics Data System (ADS)

Ramos Terrades, O.; Serrano, N.; Gordó, A.; Valveny, E.; Juan, A.

2010-01-01

A method for text block detection is introduced for old handwritten documents. The proposed method takes advantage of sequential book structure, taking into account layout information from pages previously transcribed. This glance at the past is used to predict the position of text blocks in the current page with the help of conventional layout analysis methods. The method is integrated into the GIDOC prototype: a first attempt to provide integrated support for interactive-predictive page layout analysis, text line detection and handwritten text transcription. Results are given in a transcription task on a 764-page Spanish manuscript from 1891.

Mammalian synthetic biology: emerging medical applications

PubMed Central

Kis, Zoltán; Pereira, Hugo Sant'Ana; Homma, Takayuki; Pedrigi, Ryan M.; Krams, Rob

2015-01-01

In this review, we discuss new emerging medical applications of the rapidly evolving field of mammalian synthetic biology. We start with simple mammalian synthetic biological components and move towards more complex and therapy-oriented gene circuits. A comprehensive list of ON–OFF switches, categorized into transcriptional, post-transcriptional, translational and post-translational, is presented in the first sections. Subsequently, Boolean logic gates, synthetic mammalian oscillators and toggle switches will be described. Several synthetic gene networks are further reviewed in the medical applications section, including cancer therapy gene circuits, immuno-regulatory networks, among others. The final sections focus on the applicability of synthetic gene networks to drug discovery, drug delivery, receptor-activating gene circuits and mammalian biomanufacturing processes. PMID:25808341

Transition to adult-oriented health care: perspectives of youth and adults with complex physical disabilities.

PubMed

Young, Nancy L; Barden, Wendy S; Mills, Wendy A; Burke, Tricia A; Law, Mary; Boydell, Katherine

2009-01-01

The transition to adulthood is extremely difficult for individuals with disabilities. We sought to explore the specific issue of transition to adult-oriented health care in a Canadian context. We conducted semi-structured individual interviews with 15 youth and 15 adults with cerebral palsy, spina bifida, and acquired brain injuries of childhood, and their parents (n = 30). Respondents discussed their health care services, their experience with clinical transition, and contributing factors. We analyzed the transcripts using qualitative methods. All participants identified challenges in transition, including: lack of access to health care; lack of professionals' knowledge; lack of information and uncertainty regarding the transition process. Two solutions were identified: early provision of detailed information and more extensive support throughout the clinical transition process. The challenges of clinical transition were universal. More extensive information and support is needed during transition to ensure an efficient move to appropriate adult-oriented health care.

On the Binding Stress-Enhanced Sensitivity of (Pb(Mg1/3Nb2/3)O3)0.65-(PbTiO3) 0.35 (PMN-PT) Piezoelectric Plate Sensor (PEPS)

NASA Astrophysics Data System (ADS)

Wu, Wei

(Pb(Mg1/3Nb2/3)O3)0.65-(PbTiO 3)0.35 (PMN-PT) piezoelectric plate sensor (PEPS) showed enhanced sensitivity in chemical and biological sensing applications which has been attributed to binding-induced crystalline orientation switching in the PMN-PT layer. However, so far there has been no direct demonstration of PEPS crystalline orientation switching upon target-analyte binding. Using biotin and streptavidin binding as a model detection system and by direct X-Ray diffraction observations after analyte binding we have unambiguously demonstrated that switching of the crystalline orientations of the PMN-PT layer indeed occurred. In addition, we have shown that PEPS sensitivity enhancement increased with an increasing transverse electromechanical coupling constant, -k31, of the PMN-PT layer--which is known to correlate with the crystalline orientation switching capability--by increasing the grain size of the PMN-PT layer or by applying a DC bias electric field. Finally, unprecedented high sensitivity of PEPS with high -k31, (i.e., -k31 > 0.3) were illustrated by the aM (10-18 M) sensitivity of in situ DNA hybridization detection without amplification and by the 100 fg/ml (10-13 g/ml) sensitivity of rapid, in situ protein detection in biological fluids such as troponin I detection in serum for early sign of myocardial infarction (heart attack), Her2 detection in serum for cancer treatment and monitoring, Tn antigen and anti-Tn antibody detection in serum for early cancer detection, and Toxins detection in stool for Clostridium difficile infection detection.

Premature terminator analysis sheds light on a hidden world of bacterial transcriptional attenuation.

PubMed

Naville, Magali; Gautheret, Daniel

2010-01-01

Bacterial transcription attenuation occurs through a variety of cis-regulatory elements that control gene expression in response to a wide range of signals. The signal-sensing structures in attenuators are so diverse and rapidly evolving that only a small fraction have been properly annotated and characterized to date. Here we apply a broad-spectrum detection tool in order to achieve a more complete view of the transcriptional attenuation complement of key bacterial species. Our protocol seeks gene families with an unusual frequency of 5' terminators found across multiple species. Many of the detected attenuators are part of annotated elements, such as riboswitches or T-boxes, which often operate through transcriptional attenuation. However, a significant fraction of candidates were not previously characterized in spite of their unmistakable footprint. We further characterized some of these new elements using sequence and secondary structure analysis. We also present elements that may control the expression of several non-homologous genes, suggesting co-transcription and response to common signals. An important class of such elements, which we called mobile attenuators, is provided by 3' terminators of insertion sequences or prophages that may be exapted as 5' regulators when inserted directly upstream of a cellular gene. We show here that attenuators involve a complex landscape of signal-detection structures spanning the entire bacterial domain. We discuss possible scenarios through which these diverse 5' regulatory structures may arise or evolve.

Transcription Factors MYOCD, SRF, Mesp1 and SMARCD3 Enhance the Cardio-Inducing Effect of GATA4, TBX5, and MEF2C during Direct Cellular Reprogramming

PubMed Central

Christoforou, Nicolas; Chellappan, Malathi; Adler, Andrew F.; Kirkton, Robert D.; Wu, Tianyi; Addis, Russell C.; Bursac, Nenad; Leong, Kam W.

2013-01-01

Transient overexpression of defined combinations of master regulator genes can effectively induce cellular reprogramming: the acquisition of an alternative predicted phenotype from a differentiated cell lineage. This can be of particular importance in cardiac regenerative medicine wherein the heart lacks the capacity to heal itself, but simultaneously contains a large pool of fibroblasts. In this study we determined the cardio-inducing capacity of ten transcription factors to actuate cellular reprogramming of mouse embryonic fibroblasts into cardiomyocyte-like cells. Overexpression of transcription factors MYOCD and SRF alone or in conjunction with Mesp1 and SMARCD3 enhanced the basal but necessary cardio-inducing effect of the previously reported GATA4, TBX5, and MEF2C. In particular, combinations of five or seven transcription factors enhanced the activation of cardiac reporter vectors, and induced an upregulation of cardiac-specific genes. Global gene expression analysis also demonstrated a significantly greater cardio-inducing effect when the transcription factors MYOCD and SRF were used. Detection of cross-striated cells was highly dependent on the cell culture conditions and was enhanced by the addition of valproic acid and JAK inhibitor. Although we detected Ca2+ transient oscillations in the reprogrammed cells, we did not detect significant changes in resting membrane potential or spontaneously contracting cells. This study further elucidates the cardio-inducing effect of the transcriptional networks involved in cardiac cellular reprogramming, contributing to the ongoing rational design of a robust protocol required for cardiac regenerative therapies. PMID:23704920

A rapid assay for detection of Rose rosette virus using reverse transcription-recombinase polymerase amplification using multiple gene targets.

PubMed

Babu, Binoy; Washburn, Brian K; Miller, Steven H; Poduch, Kristina; Sarigul, Tulin; Knox, Gary W; Ochoa-Corona, Francisco M; Paret, Mathews L

2017-02-01

Rose rosette disease caused by Rose rosette virus (RRV; genus Emaravirus) is the most economically relevant disease of Knock Out ® series roses in the U.S. As there are no effective chemical control options for the disease, the most critical disease management strategies include the use of virus free clean plants for propagation and early detection and destruction of infected plants. The current diagnostic techniques for RRV including end-point reverse transcription-polymerase chain reaction (RT-PCR) and real-time PCR (RT-qPCR) are highly sensitive, but limited to diagnostic labs with the equipment and expertise; and is time consuming. To address this limitation, an isothermal reverse transcription-recombinase polymerase amplification (RT-RPA) assay based on multiple gene targets for specific detection of RRV was developed. The assay is highly specific and did not cross react with other viruses belonging to the inclusive and exclusive genus. Dilution assays using the in vitro transcripts showed that the primer sets designed (RPA-267, RPA-131, and RPA-321) are highly sensitive, consistently detecting RRV with a detection limit of 1fg/μL. Testing of the infected plants using the primer sets indicated that the virus could be detected from leaves, stems and petals of roses. The primer pair RPA-267 produced 100% positive detection of the virus from infected leaf tissues, while primer set RPA-131 produced 100% detection from stems and petals. The primer set RPA-321 produced 83%, 87.5% and 75% positive detection from leaves, petals and stem tissues, respectively. In addition, the assay has been efficiently used in the detection of RRV infecting Knock Out ® roses, collected from different states in the U.S. The assay can be completed in 20min as compared to the end-point RT-PCR assay (3-4h) and RT-qPCR (1.5h). The RT-RPA assay is reliable, rapid, highly sensitive, and can be easily used in diagnostic laboratories for detection of RRV with no need for any special equipment. Copyright © 2016 Elsevier B.V. All rights reserved.

Reverse transcription strand invasion based amplification (RT-SIBA): a method for rapid detection of influenza A and B.

PubMed

Eboigbodin, Kevin; Filén, Sanna; Ojalehto, Tuomas; Brummer, Mirko; Elf, Sonja; Pousi, Kirsi; Hoser, Mark

2016-06-01

Rapid and accurate diagnosis of influenza viruses plays an important role in infection control, as well as in preventing the misuse of antibiotics. Isothermal nucleic acid amplification methods offer significant advantages over the polymerase chain reaction (PCR), since they are more rapid and do not require the sophisticated instruments needed for thermal cycling. We previously described a novel isothermal nucleic acid amplification method, 'Strand Invasion Based Amplification' (SIBA®), with high analytical sensitivity and specificity, for the detection of DNA. In this study, we describe the development of a variant of the SIBA method, namely, reverse transcription SIBA (RT-SIBA), for the rapid detection of viral RNA targets. The RT-SIBA method includes a reverse transcriptase enzyme that allows one-step reverse transcription of RNA to complementary DNA (cDNA) and simultaneous amplification and detection of the cDNA by SIBA under isothermal reaction conditions. The RT-SIBA method was found to be more sensitive than PCR for the detection of influenza A and B and could detect 100 copies of influenza RNA within 15 min. The development of RT-SIBA will enable rapid and accurate diagnosis of viral RNA targets within point-of-care or central laboratory settings.

Direct ultrasensitive electrochemical biosensing of pathogenic DNA using homogeneous target-initiated transcription amplification

PubMed Central

Yan, Yurong; Ding, Shijia; Zhao, Dan; Yuan, Rui; Zhang, Yuhong; Cheng, Wei

2016-01-01

Sensitive and specific methodologies for detection of pathogenic gene at the point-of-care are still urgent demands in rapid diagnosis of infectious diseases. This work develops a simple and pragmatic electrochemical biosensing strategy for ultrasensitive and specific detection of pathogenic nucleic acids directly by integrating homogeneous target-initiated transcription amplification (HTITA) with interfacial sensing process in single analysis system. The homogeneous recognition and specific binding of target DNA with the designed hairpin probe triggered circular primer extension reaction to form DNA double-strands which contained T7 RNA polymerase promoter and served as templates for in vitro transcription amplification. The HTITA protocol resulted in numerous single-stranded RNA products which could synchronously hybridized with the detection probes and immobilized capture probes for enzyme-amplified electrochemical detection on the biosensor surface. The proposed electrochemical biosensing strategy showed very high sensitivity and selectivity for target DNA with a dynamic response range from 1 fM to 100 pM. Using salmonella as a model, the established strategy was successfully applied to directly detect invA gene from genomic DNA extract. This proposed strategy presented a simple, pragmatic platform toward ultrasensitive nucleic acids detection and would become a versatile and powerful tool for point-of-care pathogen identification. PMID:26729209

Rapid detection and quantification of Ebola Zaire virus by one-step real-time quantitative reverse transcription-polymerase chain reaction.

PubMed

Ro, Young-Tae; Ticer, Anysha; Carrion, Ricardo; Patterson, Jean L

2017-04-01

Given that Ebola virus causes severe hemorrhagic fever in humans with mortality rates as high as 90%, rapid and accurate detection of this virus is essential both for controlling infection and preventing further transmission. Here, a one-step qRT-PCR assay for rapid and quantitative detection of an Ebola Zaire strain using GP, VP24 or VP40 genes as a target is introduced. Routine assay conditions for hydrolysis probe detection were established from the manufacturer's protocol used in the assays. The analytical specificity and sensitivity of each assay was evaluated using in vitro synthesized viral RNA transcripts. The assays were highly specific for the RNA transcripts, no cross-reactivity being observed among them. The limits of detection of the assays ranged from 10 2 to 10 3 copies per reaction. The assays were also evaluated using viral RNAs extracted from cell culture-propagated viruses (Ebola Zaire, Sudan and Reston strains), confirming that they are gene- and strain-specific. The RT-PCR assays detected viral RNAs in blood samples from virus-infected animal, suggesting that they can be also a useful method for identifying Ebola virus in clinical samples. © 2017 The Societies and John Wiley & Sons Australia, Ltd.

Direct ultrasensitive electrochemical biosensing of pathogenic DNA using homogeneous target-initiated transcription amplification

NASA Astrophysics Data System (ADS)

Yan, Yurong; Ding, Shijia; Zhao, Dan; Yuan, Rui; Zhang, Yuhong; Cheng, Wei

2016-01-01

Sensitive and specific methodologies for detection of pathogenic gene at the point-of-care are still urgent demands in rapid diagnosis of infectious diseases. This work develops a simple and pragmatic electrochemical biosensing strategy for ultrasensitive and specific detection of pathogenic nucleic acids directly by integrating homogeneous target-initiated transcription amplification (HTITA) with interfacial sensing process in single analysis system. The homogeneous recognition and specific binding of target DNA with the designed hairpin probe triggered circular primer extension reaction to form DNA double-strands which contained T7 RNA polymerase promoter and served as templates for in vitro transcription amplification. The HTITA protocol resulted in numerous single-stranded RNA products which could synchronously hybridized with the detection probes and immobilized capture probes for enzyme-amplified electrochemical detection on the biosensor surface. The proposed electrochemical biosensing strategy showed very high sensitivity and selectivity for target DNA with a dynamic response range from 1 fM to 100 pM. Using salmonella as a model, the established strategy was successfully applied to directly detect invA gene from genomic DNA extract. This proposed strategy presented a simple, pragmatic platform toward ultrasensitive nucleic acids detection and would become a versatile and powerful tool for point-of-care pathogen identification.

Direct ultrasensitive electrochemical biosensing of pathogenic DNA using homogeneous target-initiated transcription amplification.

PubMed

Yan, Yurong; Ding, Shijia; Zhao, Dan; Yuan, Rui; Zhang, Yuhong; Cheng, Wei

2016-01-05

Sensitive and specific methodologies for detection of pathogenic gene at the point-of-care are still urgent demands in rapid diagnosis of infectious diseases. This work develops a simple and pragmatic electrochemical biosensing strategy for ultrasensitive and specific detection of pathogenic nucleic acids directly by integrating homogeneous target-initiated transcription amplification (HTITA) with interfacial sensing process in single analysis system. The homogeneous recognition and specific binding of target DNA with the designed hairpin probe triggered circular primer extension reaction to form DNA double-strands which contained T7 RNA polymerase promoter and served as templates for in vitro transcription amplification. The HTITA protocol resulted in numerous single-stranded RNA products which could synchronously hybridized with the detection probes and immobilized capture probes for enzyme-amplified electrochemical detection on the biosensor surface. The proposed electrochemical biosensing strategy showed very high sensitivity and selectivity for target DNA with a dynamic response range from 1 fM to 100 pM. Using salmonella as a model, the established strategy was successfully applied to directly detect invA gene from genomic DNA extract. This proposed strategy presented a simple, pragmatic platform toward ultrasensitive nucleic acids detection and would become a versatile and powerful tool for point-of-care pathogen identification.

A deep learning method for lincRNA detection using auto-encoder algorithm.

PubMed

Yu, Ning; Yu, Zeng; Pan, Yi

2017-12-06

RNA sequencing technique (RNA-seq) enables scientists to develop novel data-driven methods for discovering more unidentified lincRNAs. Meantime, knowledge-based technologies are experiencing a potential revolution ignited by the new deep learning methods. By scanning the newly found data set from RNA-seq, scientists have found that: (1) the expression of lincRNAs appears to be regulated, that is, the relevance exists along the DNA sequences; (2) lincRNAs contain some conversed patterns/motifs tethered together by non-conserved regions. The two evidences give the reasoning for adopting knowledge-based deep learning methods in lincRNA detection. Similar to coding region transcription, non-coding regions are split at transcriptional sites. However, regulatory RNAs rather than message RNAs are generated. That is, the transcribed RNAs participate the biological process as regulatory units instead of generating proteins. Identifying these transcriptional regions from non-coding regions is the first step towards lincRNA recognition. The auto-encoder method achieves 100% and 92.4% prediction accuracy on transcription sites over the putative data sets. The experimental results also show the excellent performance of predictive deep neural network on the lincRNA data sets compared with support vector machine and traditional neural network. In addition, it is validated through the newly discovered lincRNA data set and one unreported transcription site is found by feeding the whole annotated sequences through the deep learning machine, which indicates that deep learning method has the extensive ability for lincRNA prediction. The transcriptional sequences of lincRNAs are collected from the annotated human DNA genome data. Subsequently, a two-layer deep neural network is developed for the lincRNA detection, which adopts the auto-encoder algorithm and utilizes different encoding schemes to obtain the best performance over intergenic DNA sequence data. Driven by those newly annotated lincRNA data, deep learning methods based on auto-encoder algorithm can exert their capability in knowledge learning in order to capture the useful features and the information correlation along DNA genome sequences for lincRNA detection. As our knowledge, this is the first application to adopt the deep learning techniques for identifying lincRNA transcription sequences.

Carl Rogers' Responses in the 17th Session with Miss Mun: Comments from a Process-Experiential and Psychoanalytic Perspective.

ERIC Educational Resources Information Center

Gundrum, Monica; Lietaer, Germain; Van Hees-Matthijssen, Christiane

1999-01-01

Reproduces the transcript of one of Carl Rogers' filmed therapeutic sessions with Miss Mun, followed by an empirical and clinical-qualitative analysis. Five task oriented processes are examined in detail: the evocative impact of reflections of feeling; empathic affirmation as a marker of intense vulnerability; focusing reflections; working with…

Quantification of Yeast and Bacterial Gene Transcripts in Retail Cheeses by Reverse Transcription-Quantitative PCR

PubMed Central

Straub, Cécile; Castellote, Jessie; Onesime, Djamila; Bonnarme, Pascal; Irlinger, Françoise

2013-01-01

The cheese microbiota contributes to a large extent to the development of the typical color, flavor, and texture of the final product. Its composition is not well defined in most cases and varies from one cheese to another. The aim of the present study was to establish procedures for gene transcript quantification in cheeses by reverse transcription-quantitative PCR. Total RNA was extracted from five smear-ripened cheeses purchased on the retail market, using a method that does not involve prior separation of microbial cells. 16S rRNA and malate:quinone oxidoreductase gene transcripts of Corynebacterium casei, Brevibacterium aurantiacum, and Arthrobacter arilaitensis and 26S rRNA and beta tubulin gene transcripts of Geotrichum candidum and Debaryomyces hansenii could be detected and quantified in most of the samples. Three types of normalization were applied: against total RNA, against the amount of cheese, and against a reference gene. For the first two types of normalization, differences of reverse transcription efficiencies from one sample to another were taken into account by analysis of exogenous control mRNA. No good correlation was found between the abundances of target mRNA or rRNA transcripts and the viable cell concentration of the corresponding species. However, in most cases, no mRNA transcripts were detected for species that did not belong to the dominant species. The applications of gene expression measurement in cheeses containing an undefined microbiota, as well as issues concerning the strategy of normalization and the assessment of amplification specificity, are discussed. PMID:23124230

Recent behavioral history modifies coupling between cell activity and Arc gene transcription in hippocampal CA1 neurons.

PubMed

Guzowski, John F; Miyashita, Teiko; Chawla, Monica K; Sanderson, Jennifer; Maes, Levi I; Houston, Frank P; Lipa, Peter; McNaughton, Bruce L; Worley, Paul F; Barnes, Carol A

2006-01-24

The ability of neurons to alter their transcriptional programs in response to synaptic input is of fundamental importance to the neuroplastic mechanisms underlying learning and memory. Because of technical limitations of conventional gene detection methods, the current view of activity-dependent neural transcription derives from experiments in which neurons are assumed quiescent until a signaling stimulus is given. The present study was designed to move beyond this static model by examining how earlier episodes of neural activity influence transcription of the immediate-early gene Arc. Using a sensitive FISH method that detects primary transcript at genomic alleles, the proportion of hippocampal CA1 neurons that activate transcription of Arc RNA was constant at approximately 40% in response to both a single novel exploration session and daily sessions repeated over 9 days. This proportion is similar to the percentage of active neurons defined electrophysiologically. However, this close correspondence was disrupted in rats exposed briefly, but repeatedly, to the same environment within a single day. Arc transcription in CA1 neurons declined dramatically after as few as four 5-min sessions, despite stable electrophysiological activity during all sessions. Additional experiments indicate that the decrement in Arc transcription occurred at the cellular, rather than synaptic level, and was not simply linked to habituation to novelty. Thus, the neural genomic response is governed by recent, but not remote, cell firing history in the behaving animal. This state-dependence of neuronal transcriptional coupling provides a mechanism of metaplasticity and may regulate capacity for synaptic modification in neural networks.

Survey of protein–DNA interactions in Aspergillus oryzae on a genomic scale

PubMed Central

Wang, Chao; Lv, Yangyong; Wang, Bin; Yin, Chao; Lin, Ying; Pan, Li

2015-01-01

The genome-scale delineation of in vivo protein–DNA interactions is key to understanding genome function. Only ∼5% of transcription factors (TFs) in the Aspergillus genus have been identified using traditional methods. Although the Aspergillus oryzae genome contains >600 TFs, knowledge of the in vivo genome-wide TF-binding sites (TFBSs) in aspergilli remains limited because of the lack of high-quality antibodies. We investigated the landscape of in vivo protein–DNA interactions across the A. oryzae genome through coupling the DNase I digestion of intact nuclei with massively parallel sequencing and the analysis of cleavage patterns in protein–DNA interactions at single-nucleotide resolution. The resulting map identified overrepresented de novo TF-binding motifs from genomic footprints, and provided the detailed chromatin remodeling patterns and the distribution of digital footprints near transcription start sites. The TFBSs of 19 known Aspergillus TFs were also identified based on DNase I digestion data surrounding potential binding sites in conjunction with TF binding specificity information. We observed that the cleavage patterns of TFBSs were dependent on the orientation of TF motifs and independent of strand orientation, consistent with the DNA shape features of binding motifs with flanking sequences. PMID:25883143

CRTC1-MAML2 and CRTC3-MAML2 fusions were not detected in metaplastic Warthin tumor and metaplastic pleomorphic adenoma of salivary glands.

PubMed

Skálová, Alena; Vanecek, Tomas; Simpson, Roderick H W; Vazmitsel, Marina A; Majewska, Hanna; Mukensnabl, Petr; Hauer, Lukas; Andrle, Pavel; Hosticka, Lubor; Grossmann, Petr; Michal, Michal

2013-11-01

The recurrent translocations t(11;19) and t(11;15) resulting in CRTC1-MAML2 or CRTC3-MAML2 fusion oncogenes, respectively, are identified in a large proportion of mucoepidermoid carcinomas (MECs) of the salivary gland and have impact on prognosis. However, there are conflicting data on the specificity of this translocation, in particular, on its putative occurrence in Warthin tumor (WT) of the parotid gland as reported in few previous cases. It was speculated that extensive squamous metaplasia could explain the presence of t(11;19) translocation in a subset of WTs. We evaluated 76 salivary gland tumors, including 16 cases of metaplastic WT and 8 cases of pleomorphic adenoma (PA) with squamous and/or mucinous metaplasia, extensive enough morphologically to mimic MEC. Detection of CRTC1-MAML2 and CRTC3-MAML2 fusion transcripts and MAML2 gene break was performed using nested reverse transcription-polymerase chain reaction and fluorescence in situ hybridization (FISH), respectively. None of 16 analyzed metaplastic WTs showed positivity for fusion transcripts CRTC1-MAML2 or CRTC3-MAML2, and none showed rearrangement of the MAML2 gene by FISH. Similarly, we did not detect these transcripts or break of MAML2 gene in any case of PA with extensive squamous/mucinous metaplasia. For comparison, 40 cases of low-grade MEC were also evaluated. CRTC1-MAML2 and CRTC3-MAML2 fusion transcripts were detected in 17 and 5 cases, respectively. The FISH method using break-apart probe demonstrated the MAML2 gene rearrangement in 25 cases of low-grade MEC. In contrast to low-grade MEC, neither metaplastic WTs nor metaplastic PAs harbored translocations t(11;19) and anticipated t(11;15) resulting in CRTC1-MAML2 and CRTC3-MAML2 fusion transcripts, respectively, and/or MAML2 gene rearrangement.

From the viral perspective: infectious salmon anemia virus (ISAV) transcriptome during the infective process in Atlantic salmon (Salmo salar).

PubMed

Valenzuela-Miranda, Diego; Cabrejos, María Eugenia; Yañez, José Manuel; Gallardo-Escárate, Cristian

2015-04-01

The infectious salmon anemia virus (ISAV) is a severe disease that mainly affects the Atlantic salmon (Salmo salar) aquaculture industry. Although several transcriptional studies have aimed to understand Salmon-ISAV interaction through the evaluation of host-gene transcription, none of them has focused their attention upon the viral transcriptional dynamics. For this purpose, RNA-Seq and RT-qPCR analyses were conducted in gills, liver and head-kidney of S. salar challenged by cohabitation with ISAV. Results evidence the time and tissue transcript patterns involved in the viral expression and how the transcription levels of ISAV segments are directly linked with the protein abundance found in other virus of the Orthomyxoviridae family. In addition, RT-qPCR result evidenced that quantification of ISAV through amplification of segment 3 would result in a more sensitive approach for detection and quantification of ISAV. This study offers a more comprehensive approach regarding the ISAV infective process and gives novel knowledge for its molecular detection. Copyright © 2014 Elsevier B.V. All rights reserved.

Ring test evaluation of the detection of influenza A virus in swine oral fluids by real-time, reverse transcription polymerase chain reaction (rRT-PCR) and virus isolation

USDA-ARS?s Scientific Manuscript database

The probability of detecting influenza A virus (IAV) in oral fluid (OF) specimens was calculated for each of 13 real-time, reverse transcription polymerase chain reaction (rRT-PCR) and 7 virus isolation (VI) assays. To conduct the study, OF was inoculated with H1N1 or H3N2 IAV and serially 10-fold d...

Optimized approach for Ion Proton RNA sequencing reveals details of RNA splicing and editing features of the transcriptome.

PubMed

Brown, Roger B; Madrid, Nathaniel J; Suzuki, Hideaki; Ness, Scott A

2017-01-01

RNA-sequencing (RNA-seq) has become the standard method for unbiased analysis of gene expression but also provides access to more complex transcriptome features, including alternative RNA splicing, RNA editing, and even detection of fusion transcripts formed through chromosomal translocations. However, differences in library methods can adversely affect the ability to recover these different types of transcriptome data. For example, some methods have bias for one end of transcripts or rely on low-efficiency steps that limit the complexity of the resulting library, making detection of rare transcripts less likely. We tested several commonly used methods of RNA-seq library preparation and found vast differences in the detection of advanced transcriptome features, such as alternatively spliced isoforms and RNA editing sites. By comparing several different protocols available for the Ion Proton sequencer and by utilizing detailed bioinformatics analysis tools, we were able to develop an optimized random primer based RNA-seq technique that is reliable at uncovering rare transcript isoforms and RNA editing features, as well as fusion reads from oncogenic chromosome rearrangements. The combination of optimized libraries and rapid Ion Proton sequencing provides a powerful platform for the transcriptome analysis of research and clinical samples.

The pan-cancer pathological regulatory landscape

PubMed Central

Falco, Matias M.; Bleda, Marta; Carbonell-Caballero, José; Dopazo, Joaquín

2016-01-01

Dysregulation of the normal gene expression program is the cause of a broad range of diseases, including cancer. Detecting the specific perturbed regulators that have an effect on the generation and the development of the disease is crucial for understanding the disease mechanism and for taking decisions on efficient preventive and curative therapies. Moreover, detecting such perturbations at the patient level is even more important from the perspective of personalized medicine. We applied the Transcription Factor Target Enrichment Analysis, a method that detects the activity of transcription factors based on the quantification of the collective transcriptional activation of their targets, to a large collection of 5607 cancer samples covering eleven cancer types. We produced for the first time a comprehensive catalogue of altered transcription factor activities in cancer, a considerable number of them significantly associated to patient’s survival. Moreover, we described several interesting TFs whose activity do not change substantially in the cancer with respect to the normal tissue but ultimately play an important role in patient prognostic determination, which suggest they might be promising therapeutic targets. An additional advantage of this method is that it allows obtaining personalized TF activity estimations for individual patients. PMID:28000771

Gene-breaking: A new paradigm for human retrotransposon-mediated gene evolution

PubMed Central

Wheelan, Sarah J.; Aizawa, Yasunori; Han, Jeffrey S.; Boeke, Jef D.

2005-01-01

The L1 retrotransposon is the most highly successful autonomous retrotransposon in mammals. This prolific genome parasite may on occasion benefit its host through genome rearrangements or adjustments of host gene expression. In examining possible effects of L1 elements on host gene expression, we investigated whether a full-length L1 element inserted in the antisense orientation into an intron of a cellular gene may actually split the gene's transcript into two smaller transcripts: (1) a transcript containing the upstream exons and terminating in the major antisense polyadenylation site (MAPS) of the L1, and (2) a transcript derived from the L1 antisense promoter (ASP) that includes the downstream exons of the gene. Bioinformatic analysis and experimental follow-up provide evidence for this L1 “gene-breaking” hypothesis. We identified three human genes apparently “broken” by L1 elements, as well as 12 more candidate genes. Most of the inserted L1 elements in our 15 candidate genes predate the human/chimp divergence. If indeed split, the transcripts of these genes may in at least one case encode potentially interacting proteins, and in another case may encode novel proteins. Gene-breaking represents a new mechanism through which L1 elements remodel mammalian genomes. PMID:16024818

A dual promoter system regulating λ DNA replication initiation

PubMed Central

Olszewski, Paweł; Szambowska, Anna; Barańska, Sylwia; Narajczyk, Magdalena; Węgrzyn, Grzegorz; Glinkowska, Monika

2014-01-01

Transcription and DNA replication are tightly regulated to ensure coordination of gene expression with growth conditions and faithful transmission of genetic material to progeny. A large body of evidence has accumulated, indicating that encounters between protein machineries carrying out DNA and RNA synthesis occur in vivo and may have important regulatory consequences. This feature may be exacerbated in the case of compact genomes, like the one of bacteriophage λ, used in our study. Transcription that starts at the rightward pR promoter and proceeds through the λ origin of replication and downstream of it was proven to stimulate the initiation of λ DNA replication. Here, we demonstrate that the activity of a convergently oriented pO promoter decreases the efficiency of transcription starting from pR. Our results show, however, that a lack of the functional pO promoter negatively influences λ phage and λ-derived plasmid replication. We present data, suggesting that this effect is evoked by the enhanced level of the pR-driven transcription, occurring in the presence of the defective pO, which may result in the impeded formation of the replication initiation complex. Our data suggest that the cross talk between the two promoters regulates λ DNA replication and coordinates transcription and replication processes. PMID:24500197

Transcription blockage by homopurine DNA sequences: role of sequence composition and single-strand breaks

PubMed Central

Belotserkovskii, Boris P.; Neil, Alexander J.; Saleh, Syed Shayon; Shin, Jane Hae Soo; Mirkin, Sergei M.; Hanawalt, Philip C.

2013-01-01

The ability of DNA to adopt non-canonical structures can affect transcription and has broad implications for genome functioning. We have recently reported that guanine-rich (G-rich) homopurine-homopyrimidine sequences cause significant blockage of transcription in vitro in a strictly orientation-dependent manner: when the G-rich strand serves as the non-template strand [Belotserkovskii et al. (2010) Mechanisms and implications of transcription blockage by guanine-rich DNA sequences., Proc. Natl Acad. Sci. USA, 107, 12816–12821]. We have now systematically studied the effect of the sequence composition and single-stranded breaks on this blockage. Although substitution of guanine by any other base reduced the blockage, cytosine and thymine reduced the blockage more significantly than adenine substitutions, affirming the importance of both G-richness and the homopurine-homopyrimidine character of the sequence for this effect. A single-strand break in the non-template strand adjacent to the G-rich stretch dramatically increased the blockage. Breaks in the non-template strand result in much weaker blockage signals extending downstream from the break even in the absence of the G-rich stretch. Our combined data support the notion that transcription blockage at homopurine-homopyrimidine sequences is caused by R-loop formation. PMID:23275544

Clinorotation influences rDNA and NopA100 localization in nucleoli

NASA Astrophysics Data System (ADS)

Sobol, M. A.; González-Camacho, F.; Rodríguez-Vilariño, V.; Kordyum, E. L.; Medina, F. J.

The nucleolus is the transcription site of rRNA genes as well as the site of processing and initial packaging of their transcripts. The plant nucleolin homologue NopA100 is involved in the regulation of r-chromatin condensation/expansion and rDNA transcription as well as in rRNA processing. We have investigated with immunogold electron microscopy the location of nucleolar DNA and NopA100 in cress root meristematic cells grown under slow horizontal clinorotation, reproducing an important feature of microgravity, namely the absence of an orienting action of a gravity vector, compared to control conditions. We demonstrate redistribution of both rDNA and NopA100 in nucleolar subcomponents induced by clinorotation. Ribosomal DNA concentrated predominantly in fibrillar centers in the form of condensed r-chromatin inclusions and internal non condensed fibrils, redistributing from the dense fibrillar component and the transition zone between fibrillar centers and the dense fibrillar component, recognized as the loci of rDNA transcription. The content of NopA100 was much higher in the inner space of fibrillar centers and reduced in the dense fibrillar component as compared to the control. Based on these data, an effect of slow horizontal clinorotation in lowering the level of rDNA transcription as well as rRNA processing is suggested.

Task-oriented situation recognition

NASA Astrophysics Data System (ADS)

Bauer, Alexander; Fischer, Yvonne

2010-04-01

From the advances in computer vision methods for the detection, tracking and recognition of objects in video streams, new opportunities for video surveillance arise: In the future, automated video surveillance systems will be able to detect critical situations early enough to enable an operator to take preventive actions, instead of using video material merely for forensic investigations. However, problems such as limited computational resources, privacy regulations and a constant change in potential threads have to be addressed by a practical automated video surveillance system. In this paper, we show how these problems can be addressed using a task-oriented approach. The system architecture of the task-oriented video surveillance system NEST and an algorithm for the detection of abnormal behavior as part of the system are presented and illustrated for the surveillance of guests inside a video-monitored building.

Direct observation of binding stress-induced crystalline orientation change in piezoelectric plate sensors

NASA Astrophysics Data System (ADS)

Wu, Wei; Shih, Wei-Heng; Shih, Wan Y.

2016-03-01

We have examined the mechanism of the detection resonance frequency shift, Δf/f, of a 1370 μm long and 537 μm wide [Pb(Mg1/3Nb2/3)O3]0.65[PbTiO3]0.35 (PMN-PT) piezoelectric plate sensor (PEPS) made of a 8-μm thick PMN-PT freestanding film. The Δf/f of the PEPS was monitored in a three-step binding model detections of (1) binding of maleimide-activated biotin to the sulfhydryl on the PEPS surface followed by (2) binding of streptavidin to the bound biotin and (3) subsequent binding of biotinylated probe deoxyribonucleic acid to the bound streptavidin. We used a PMN-PT surrogate made of the same 8-μm thick PMN-PT freestanding film that the PEPS was made of but was about 1 cm in length and width to carry out crystalline orientation study using X-ray diffraction (XRD) scan around the (002)/(200) peaks after each of the binding steps. The result of the XRD studies indicated that each binding step caused the crystalline orientation of the PMN-PT thin layer to switch from the vertical (002) orientation to the horizontal (200) orientation, and most of the PEPS detection Δf/f was due to the change in the lateral Young's modulus of the PMN-PT thin layer as a result of the crystalline orientation change.

Effects of the adenovirus 2 late promoter on simian virus 40 transcription and replication.

PubMed Central

Grass, D S; Manley, J L

1986-01-01

A 100-base-pair fragment of adenovirus 2 (Ad2) DNA encompassing the major late transcriptional promoter was inserted into the simian virus 40 (SV40) late promoter region at SV40 nucleotide 294 to study the effects of a strong TATA box-containing promoter on SV40 late transcription. pSVAdE contains the insert in an orientation such that it would promote transcription towards the origin and early region of SV40, while the insert is in the opposite orientation in pSVAdL. Nuclease S1 analysis with 5'-end-labeled probes showed that in cells transfected with pSVAdE, the late mRNA initiation sites are essentially the same as in wild type, demonstrating that an insert of 100 base pairs can have no effect on utilization of the SV40 late start sites. In pSVAdL-transfected cells, however, the major late viral initiation site is now in the insert at +1 with respect to the Ad2 major late cap site. However, all of the SV40 initiation sites are still utilized and with the same efficiency relative to each other as in wild type. Thus, it appears that the Ad2 late promoter and the SV40 late promoter can function independently on the same DNA molecule, even when one promoter is embedded within the other. By using cytosine arabinoside to block DNA replication and thereby inhibit the onset of late expression, it has been shown that both the Ad2 late promoter and the SV40 late promoter have similar requirement for DNA replication in this context. In addition, pSVAdL showed dramatically diminished virus viability and VPI expression compared with both wildtype and pSVAdE. Possible explanations for this unexpected finding are discussed. Images PMID:3001338

Transcriptional regulation of PCFT by KLF4, HNF4α, CDX2 and C/EBPα: Implication in its site-specific expression in the small intestine

DOE Office of Scientific and Technical Information (OSTI.GOV)

Furumiya, Mai; Department of Biopharmaceutics, College of Pharmacy, Kinjo Gakuin University, 2-1723 Omori, Moriyama-ku, Nagoya 463-8521; Inoue, Katsuhisa

2013-02-08

Highlights: ► We examined transcription factors that may regulate PCFT expression in the intestine. ► PCFT promoter activity is basically induced by KLF4. ► KLF4-induced PCFT promoter activity is enhanced by HNF4α synergistically. ► CDX2 and C/EBPα suppress PCFT promoter activity induced by KLF4 and HNF4α. -- Abstract: Proton-coupled folate transporter (PCFT), which is responsible for the intestinal uptake of folates and analogs, is expressed only in the proximal region in the small intestine. The present study was to examine its transcriptional regulation, which may be involved in such a unique expression profile and potentially in its alteration, using dual-luciferasemore » reporter assays in human embryonic kidney (HEK) 293 cells. The luciferase activity derived from the reporter construct containing the 5′-flanking sequence of −1695/+96 of the human PCFT gene was enhanced most extensively by the introduction of Krüppel-like factor 4 (KLF4). The KLF4-induced luciferase activity was further enhanced by hepatocyte nuclear factor 4α (HNF4α) synergistically. To the contrary, caudal-type homeobox transcription factor 2 (CDX2) and CCAAT/enhancer-binding protein α (C/EBPα) extensively suppressed the luciferase activity induced by KLF4 alone and also that induced by KLF4 and HNF4α. Western blot analysis using the rat small intestine indicated uniform expression of KLF4 along the intestinal tract, proximal-oriented expression of HNF4α, distal-oriented expression of CDX2 and C/EBPα. These results suggest that the activity of PCFT promoter is basically induced by KLF4 and the gradiented expression profile of PCFT may be at least in part accounted for by those of HNF4α, CDX2 and C/EBPα.« less

Contrast limited adaptive histogram equalization image processing to improve the detection of simulated spiculations in dense mammograms.

PubMed

Pisano, E D; Zong, S; Hemminger, B M; DeLuca, M; Johnston, R E; Muller, K; Braeuning, M P; Pizer, S M

1998-11-01

The purpose of this project was to determine whether Contrast Limited Adaptive Histogram Equalization (CLAHE) improves detection of simulated spiculations in dense mammograms. Lines simulating the appearance of spiculations, a common marker of malignancy when visualized with masses, were embedded in dense mammograms digitized at 50 micron pixels, 12 bits deep. Film images with no CLAHE applied were compared to film images with nine different combinations of clip levels and region sizes applied. A simulated spiculation was embedded in a background of dense breast tissue, with the orientation of the spiculation varied. The key variables involved in each trial included the orientation of the spiculation, contrast level of the spiculation and the CLAHE settings applied to the image. Combining the 10 CLAHE conditions, 4 contrast levels and 4 orientations gave 160 combinations. The trials were constructed by pairing 160 combinations of key variables with 40 backgrounds. Twenty student observers were asked to detect the orientation of the spiculation in the image. There was a statistically significant improvement in detection performance for spiculations with CLAHE over unenhanced images when the region size was set at 32 with a clip level of 2, and when the region size was set at 32 with a clip level of 4. The selected CLAHE settings should be tested in the clinic with digital mammograms to determine whether detection of spiculations associated with masses detected at mammography can be improved.

Attention to baseline: does orienting visuospatial attention really facilitate target detection?

PubMed

Albares, Marion; Criaud, Marion; Wardak, Claire; Nguyen, Song Chi Trung; Ben Hamed, Suliann; Boulinguez, Philippe

2011-08-01

Standard protocols testing the orientation of visuospatial attention usually present spatial cues before targets and compare valid-cue trials with invalid-cue trials. The valid/invalid contrast results in a relative behavioral or physiological difference that is generally interpreted as a benefit of attention orientation. However, growing evidence suggests that inhibitory control of response is closely involved in this kind of protocol that requires the subjects to withhold automatic responses to cues, probably biasing behavioral and physiological baselines. Here, we used two experiments to disentangle the inhibitory control of automatic responses from orienting of visuospatial attention in a saccadic reaction time task in humans, a variant of the classical cue-target detection task and a sustained visuospatial attentional task. Surprisingly, when referring to a simple target detection task in which there is no need to refrain from reacting to avoid inappropriate responses, we found no consistent evidence of facilitation of target detection at the attended location. Instead, we observed a cost at the unattended location. Departing from the classical view, our results suggest that reaction time measures of visuospatial attention probably relie on the attenuation of elementary processes involved in visual target detection and saccade initiation away from the attended location rather than on facilitation at the attended location. This highlights the need to use proper control conditions in experimental designs to disambiguate relative from absolute cueing benefits on target detection reaction times, both in psychophysical and neurophysiological studies.

Widespread evidence of cooperative DNA binding by transcription factors in Drosophila development

PubMed Central

Kazemian, Majid; Pham, Hannah; Wolfe, Scot A.; Brodsky, Michael H.; Sinha, Saurabh

2013-01-01

Regulation of eukaryotic gene transcription is often combinatorial in nature, with multiple transcription factors (TFs) regulating common target genes, often through direct or indirect mutual interactions. Many individual examples of cooperative binding by directly interacting TFs have been identified, but it remains unclear how pervasive this mechanism is during animal development. Cooperative TF binding should be manifest in genomic sequences as biased arrangements of TF-binding sites. Here, we explore the extent and diversity of such arrangements related to gene regulation during Drosophila embryogenesis. We used the DNA-binding specificities of 322 TFs along with chromatin accessibility information to identify enriched spacing and orientation patterns of TF-binding site pairs. We developed a new statistical approach for this task, specifically designed to accurately assess inter-site spacing biases while accounting for the phenomenon of homotypic site clustering commonly observed in developmental regulatory regions. We observed a large number of short-range distance preferences between TF-binding site pairs, including examples where the preference depends on the relative orientation of the binding sites. To test whether these binding site patterns reflect physical interactions between the corresponding TFs, we analyzed 27 TF pairs whose binding sites exhibited short distance preferences. In vitro protein–protein binding experiments revealed that >65% of these TF pairs can directly interact with each other. For five pairs, we further demonstrate that they bind cooperatively to DNA if both sites are present with the preferred spacing. This study demonstrates how DNA-binding motifs can be used to produce a comprehensive map of sequence signatures for different mechanisms of combinatorial TF action. PMID:23847101

Widespread evidence of cooperative DNA binding by transcription factors in Drosophila development.

PubMed

Kazemian, Majid; Pham, Hannah; Wolfe, Scot A; Brodsky, Michael H; Sinha, Saurabh

2013-09-01

Regulation of eukaryotic gene transcription is often combinatorial in nature, with multiple transcription factors (TFs) regulating common target genes, often through direct or indirect mutual interactions. Many individual examples of cooperative binding by directly interacting TFs have been identified, but it remains unclear how pervasive this mechanism is during animal development. Cooperative TF binding should be manifest in genomic sequences as biased arrangements of TF-binding sites. Here, we explore the extent and diversity of such arrangements related to gene regulation during Drosophila embryogenesis. We used the DNA-binding specificities of 322 TFs along with chromatin accessibility information to identify enriched spacing and orientation patterns of TF-binding site pairs. We developed a new statistical approach for this task, specifically designed to accurately assess inter-site spacing biases while accounting for the phenomenon of homotypic site clustering commonly observed in developmental regulatory regions. We observed a large number of short-range distance preferences between TF-binding site pairs, including examples where the preference depends on the relative orientation of the binding sites. To test whether these binding site patterns reflect physical interactions between the corresponding TFs, we analyzed 27 TF pairs whose binding sites exhibited short distance preferences. In vitro protein-protein binding experiments revealed that >65% of these TF pairs can directly interact with each other. For five pairs, we further demonstrate that they bind cooperatively to DNA if both sites are present with the preferred spacing. This study demonstrates how DNA-binding motifs can be used to produce a comprehensive map of sequence signatures for different mechanisms of combinatorial TF action.

Genomic Heat Shock Element Sequences Drive Cooperative Human Heat Shock Factor 1 DNA Binding and Selectivity*

PubMed Central

Jaeger, Alex M.; Makley, Leah N.; Gestwicki, Jason E.; Thiele, Dennis J.

2014-01-01

The heat shock transcription factor 1 (HSF1) activates expression of a variety of genes involved in cell survival, including protein chaperones, the protein degradation machinery, anti-apoptotic proteins, and transcription factors. Although HSF1 activation has been linked to amelioration of neurodegenerative disease, cancer cells exhibit a dependence on HSF1 for survival. Indeed, HSF1 drives a program of gene expression in cancer cells that is distinct from that activated in response to proteotoxic stress, and HSF1 DNA binding activity is elevated in cycling cells as compared with arrested cells. Active HSF1 homotrimerizes and binds to a DNA sequence consisting of inverted repeats of the pentameric sequence nGAAn, known as heat shock elements (HSEs). Recent comprehensive ChIP-seq experiments demonstrated that the architecture of HSEs is very diverse in the human genome, with deviations from the consensus sequence in the spacing, orientation, and extent of HSE repeats that could influence HSF1 DNA binding efficacy and the kinetics and magnitude of target gene expression. To understand the mechanisms that dictate binding specificity, HSF1 was purified as either a monomer or trimer and used to evaluate DNA-binding site preferences in vitro using fluorescence polarization and thermal denaturation profiling. These results were compared with quantitative chromatin immunoprecipitation assays in vivo. We demonstrate a role for specific orientations of extended HSE sequences in driving preferential HSF1 DNA binding to target loci in vivo. These studies provide a biochemical basis for understanding differential HSF1 target gene recognition and transcription in neurodegenerative disease and in cancer. PMID:25204655

Detection of Abnormal Events via Optical Flow Feature Analysis

PubMed Central

Wang, Tian; Snoussi, Hichem

2015-01-01

In this paper, a novel algorithm is proposed to detect abnormal events in video streams. The algorithm is based on the histogram of the optical flow orientation descriptor and the classification method. The details of the histogram of the optical flow orientation descriptor are illustrated for describing movement information of the global video frame or foreground frame. By combining one-class support vector machine and kernel principal component analysis methods, the abnormal events in the current frame can be detected after a learning period characterizing normal behaviors. The difference abnormal detection results are analyzed and explained. The proposed detection method is tested on benchmark datasets, then the experimental results show the effectiveness of the algorithm. PMID:25811227

Interactive web-based identification and visualization of transcript shared sequences.

PubMed

Azhir, Alaleh; Merino, Louis-Henri; Nauen, David W

2018-05-12

We have developed TraC (Transcript Consensus), a web-based tool for detecting and visualizing shared sequences among two or more mRNA transcripts such as splice variants. Results including exon-exon boundaries are returned in a highly intuitive, data-rich, interactive plot that permits users to explore the similarities and differences of multiple transcript sequences. The online tool (http://labs.pathology.jhu.edu/nauen/trac/) is free to use. The source code is freely available for download (https://github.com/nauenlab/TraC). Copyright © 2018 Elsevier Inc. All rights reserved.

Detection and differentiation of wild-type and vaccine strains of canine distemper virus by a duplex reverse transcription polymerase chain reaction

PubMed Central

Dong, X. Y.; Li, W. H.; Zhu, J. L.; Liu, W. J.; Zhao, M. Q.; Luo, Y. W.; Chen, J. D.

2015-01-01

Canine distemper virus (CDV) is the cause of canine distemper (CD) which is a severe and highly contagious disease in dogs. In the present study, a duplex reverse transcription polymerase chain reaction (RT-PCR) method was developed for the detection and differentiation of wild-type and vaccine strains of CDV. Four primers were designed to detect and discriminate the two viruses by generating 638- and 781-bp cDNA products, respectively. Furthermore, the duplex RT-PCR method was used to detect 67 field samples suspected of CD from Guangdong province in China. Results showed that, 33 samples were to be wild-type-like. The duplex RT-PCR method exhibited high specificity and sensitivity which could be used to effectively detect and differentiate wild-type and vaccine CDV, indicating its use for clinical detection and epidemiological surveillance. PMID:27175171

Optical Strategies for Studying Metastatic Mechanisms, Tumor Cell Detection and Treatment of Prostate Cancer

DTIC Science & Technology

2005-10-01

increase in VEGF- A levels following PDT treatment (Figure 7) of orthotopic prostate tumors. Task 2: Design of optical monitoring tools to detect circulating...in intracellular VEGF- A (Figure 5, B) at 0,5 J/cm 2 (1.6 fold). Surprisingly we did not measure any significant increase in intracellular VEGF- A ... levels at the lower dose (0,25 J/cm 2). VEGF-A is known to be regulated at the transcriptional and post-transcriptional levels. We therefore used primers

Structural dissection of an interaction between transcription initiation and termination factors implicated in promoter-terminator cross-talk.

PubMed

Bratkowski, Matthew; Unarta, Ilona Christy; Zhu, Lizhe; Shubbar, Murtada; Huang, Xuhui; Liu, Xin

2018-02-02

Functional cross-talk between the promoter and terminator of a gene has long been noted. Promoters and terminators are juxtaposed to form gene loops in several organisms, and gene looping is thought to be involved in transcriptional regulation. The general transcription factor IIB (TFIIB) and the C-terminal domain phosphatase Ssu72, essential factors of the transcription preinitiation complex and the mRNA processing and polyadenylation complex, respectively, are important for gene loop formation. TFIIB and Ssu72 interact both genetically and physically, but the molecular basis of this interaction is not known. Here we present a crystal structure of the core domain of TFIIB in two new conformations that differ in the relative distance and orientation of the two cyclin-like domains. The observed extraordinary conformational plasticity may underlie the binding of TFIIB to multiple transcription factors and promoter DNAs that occurs in distinct stages of transcription, including initiation, reinitiation, and gene looping. We mapped the binding interface of the TFIIB-Ssu72 complex using a series of systematic, structure-guided in vitro binding and site-specific photocross-linking assays. Our results indicate that Ssu72 competes with acidic activators for TFIIB binding and that Ssu72 disrupts an intramolecular TFIIB complex known to impede transcription initiation. We also show that the TFIIB-binding site on Ssu72 overlaps with the binding site of symplekin, a component of the mRNA processing and polyadenylation complex. We propose a hand-off model in which Ssu72 mediates a conformational transition in TFIIB, accounting for the role of Ssu72 in transcription reinitiation, gene looping, and promoter-terminator cross-talk. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Automated Detection of Solar Loops by the Oriented Connectivity Method

NASA Technical Reports Server (NTRS)

Lee, Jong Kwan; Newman, Timothy S.; Gary, G. Allen

2004-01-01

An automated technique to segment solar coronal loops from intensity images of the Sun s corona is introduced. It exploits physical characteristics of the solar magnetic field to enable robust extraction from noisy images. The technique is a constructive curve detection approach, constrained by collections of estimates of the magnetic fields orientation. Its effectiveness is evaluated through experiments on synthetic and real coronal images.

The equine herpesvirus-1 IR3 gene that lies antisense to the sole immediate-early (IE) gene is trans-activated by the IE protein, and is poorly expressed to a protein

PubMed Central

Ahn, Byung Chul; Breitenbach, Jonathan E.; Kim, Seong K.; O’Callaghan, Dennis J.

2007-01-01

The unique IR3 gene of equine herpesvirus 1 (EHV-1) is expressed as a late 1.0-kb transcript. Previous studies confirmed the IR3 transcription initiation site and tentatively identified other cis-acting elements specific to IR3 such as a TATA box, a 443 base pair 5′untranslated region (UTR), a 285 base pair open reading frame (ORF) and a poly adenylation (A) signal (Holden et al., 1992 DNA Seq 3, 143-52). Transient transfection assays revealed that the IR3 promoter is strongly trans-activated by the IE protein (IEP) and that coexpression of the IEP with the early EICP0 and IR4 regulatory proteins results in maximal trans-activation of the IR3 promoter. Gel shift assays revealed that the IEP directly binds to the IR3 promoter region. Western blot analysis showed that the IR3 protein produced in E. coli was detected by antibodies to IR3 synthetic peptides; however, the IR3 protein was not detected in EHV-1 infected cell extracts by these same anti-IR3 antibodies, even though the IR3 transcript was detected by northern blot. These findings suggest that the IR3 may not be expressed to a protein. Expression of an IR3/GFP fusion gene was not observed, but expression of a GFP/IR3 fusion gene was detected by fluorescent microscopy. In further attempts to detect the IR3/GFP fusion protein using anti-GFP antibody, western blot analysis showed that the IR3/GFP fusion protein was not detected in vivo. Interestingly, a truncated form of the GFP/IR3 protein was synthesized from the GFP/IR3 fusion gene. However, GFP/IR3 and IR3/GFP fusion proteins of the predicted sizes were synthesized by in vitro coupled transcription and translation of the fusion genes, suggesting poor expression of the IR3 protein in vivo. The possible role of the IR3 transcript in EHV-1 infection is discussed. PMID:17306852

A low-temperature-responsive element involved in the regulation of the Arabidopsis thaliana At1g71850/At1g71860 divergent gene pair.

PubMed

Liu, Shijuan; Chen, Huiqing; Li, Xiulan; Zhang, Wei

2016-08-01

The bidirectional promoter of the Arabidopsis thaliana gene pair At1g71850/At1g71860 harbors low-temperature-responsive elements, which participate in anti-correlated transcription regulation of the driving genes in response to environmental low temperature. A divergent gene pair is defined as two adjacent genes organized head to head in opposite orientation, sharing a common promoter region. Divergent gene pairs are mainly coexpressed, but some display opposite regulation. The mechanistic basis of such anti-correlated regulation is not well understood. Here, the regulation of the Arabidopsis thaliana gene pair At1g71850/At1g71860 was investigated. Semi-quantitative RT-PCR and Genevestigator analyses showed that while one of the pair was upregulated by exposure to low temperature, the same treatment downregulated the other. Promoter::GUS fusion transgenes were used to show that this behavior was driven by a bidirectional promoter, which harbored an as-1 motif, associated with the low-temperature response; mutation of this sequence produced a significant decrease in cold-responsive expression. With regard to the as-1 motif in the native orientation repressing the promoter's low-temperature responsiveness, the same as-1 motif introduced in the reverse direction showed a slight enhancement in the promoter's responsiveness to low-temperature exposure, indicating that the orientation of the motif was important for the promoter's activity. These findings provide new insights into the complex transcriptional regulation of bidirectional gene pairs as well as plant stress response.

Micronuclei and other erythrocyte nuclear abnormalities in fishes from the Great Lakes Basin, USA.

PubMed

Braham, Ryan P; Blazer, Vicki S; Shaw, Cassidy H; Mazik, Patricia M

2017-10-01

Biological markers (biomarkers) sensitive to genotoxic and mutagenic contamination in fishes are widely used to identify exposure effects in aquatic environments. The micronucleus assay was incorporated into a suite of indicators to assess exposure to genotoxic and mutagenic contamination at five Great Lakes Areas of Concern (AOCs), as well as one non-AOC (reference) site. The assay allowed enumeration of micronuclei as well as other nuclear abnormalities for both site and species comparisons. Erythrocyte abnormality data was also compared to skin and liver tumor prevalence and hepatic transcript abundance. Erythrocyte abnormalities were observed at all sites with variable occurrence and severity among sites and species. Benthic-oriented brown bullhead (Ameiurus nebulosus) and white sucker (Catostomus commersonii) expressed lower rates of erythrocyte abnormalities, but higher rates of skin and liver neoplasms, when compared to pelagic-oriented largemouth bass (Micropterus salmoides) or smallmouth bass (Micropterus dolomieu) at the same site. The reduced erythrocyte abnormalities, increased transcript abundance associated with Phase I and II toxicant responsive pathways, and increased neoplastic lesions among benthic-oriented taxa may indicate the development of contaminant resistance of these species to more acute effects. Environ. Mol. Mutagen. 58:570-581, 2017. © 2017 This article is a U.S. Government work and is in the public domain in the USA. Environmental and Molecular Mutagenesis published by Wiley Periodicals, Inc. on behalf of Environmental Mutagen Society. © 2017 This article is a U.S. Government work and is in the public domain in the USA. Environmental and Molecular Mutagenesis published by Wiley Periodicals, Inc. on behalf of Environmental Mutagen Society.

Sensemaking in the formation of basic life support teams - a proof-of-concept, qualitative study of simulated in-hospital cardiac arrests.

PubMed

Hallas, Peter; Lauridsen, Johnny; Brabrand, Mikkel

2018-01-29

The formation of critical care teams is a complex process where team members need to get a shared understanding of a serious situation. No previous studies have focused on how this shared understanding is achieved during the formation of cardiac arrest teams. "Sensemaking" is a concept well known in organizational studies. It refers to the collaborative effort among members in a dialogue to create meaning in an ambiguous situation, often by using subtle variations in the sentences in the dialogue. Sentences with high degrees of "sensemaking" activity can be thematized as "co-orientation", "re-presentation" and/or "subordination" (among others). We sought to establish if elements of "sensemaking" occur in the formation of in-hospital cardiac arrest teams. Videos of ten simulations of unannounced in-hospital cardiac arrests treated by basic life support (BLS) providers. We transcribed all verbal communication from the moment the first responder stepped into the room until the moment external chest compression were initiated (verbatim transcription). Transcriptions were then analyzed with a focus on identifying three elements of sensemaking: Co-orientation, Re-presentation and Sub-ordination. Sensemaking elements could be identified in seven of ten scenarios as part of team formation. Co-orientation was the element that was used most consistently, occurring in all of the eight scenarios that included sensemaking efforts. Sensemaking is an element in the communication in some cardiac arrest teams. It is possible that the active moderation of sensemaking should be considered a non-technical skill in cardiac arrest teams.

Fabrication of antibody microarrays by light-induced covalent and oriented immobilization.

PubMed

Adak, Avijit K; Li, Ben-Yuan; Huang, Li-De; Lin, Ting-Wei; Chang, Tsung-Che; Hwang, Kuo Chu; Lin, Chun-Cheng

2014-07-09

Antibody microarrays have important applications for the sensitive detection of biologically important target molecules and as biosensors for clinical applications. Microarrays produced by oriented immobilization of antibodies generally have higher antigen-binding capacities than those in which antibodies are immobilized with random orientations. Here, we present a UV photo-cross-linking approach that utilizes boronic acid to achieve oriented immobilization of an antibody on a surface while retaining the antigen-binding activity of the immobilized antibody. A photoactive boronic acid probe was designed and synthesized in which boronic acid provided good affinity and specificity for the recognition of glycan chains on the Fc region of the antibody, enabling covalent tethering to the antibody upon exposure to UV light. Once irradiated with optimal UV exposure (16 mW/cm(2)), significant antibody immobilization on a boronic acid-presenting surface with maximal antigen detection sensitivity in a single step was achieved, thus obviating the necessity of prior antibody modifications. The developed approach is highly modular, as demonstrated by its implementation in sensitive sandwich immunoassays for the protein analytes Ricinus communis agglutinin 120, human prostate-specific antigen, and interleukin-6 with limits of detection of 7.4, 29, and 16 pM, respectively. Furthermore, the present system enabled the detection of multiple analytes in samples without any noticeable cross-reactivities. Antibody coupling via the use of boronic acid and UV light represents a practical, oriented immobilization method with significant implications for the construction of a large array of immunosensors for diagnostic applications.

Perceptual Learning Selectively Refines Orientation Representations in Early Visual Cortex

PubMed Central

Jehee, Janneke F.M.; Ling, Sam; Swisher, Jascha D.; van Bergen, Ruben S.; Tong, Frank

2013-01-01

Although practice has long been known to improve perceptual performance, the neural basis of this improvement in humans remains unclear. Using fMRI in conjunction with a novel signal detection-based analysis, we show that extensive practice selectively enhances the neural representation of trained orientations in the human visual cortex. Twelve observers practiced discriminating small changes in the orientation of a laterally presented grating over 20 or more daily one-hour training sessions. Training on average led to a two-fold improvement in discrimination sensitivity, specific to the trained orientation and the trained location, with minimal improvement found for untrained orthogonal orientations or for orientations presented in the untrained hemifield. We measured the strength of orientation-selective responses in individual voxels in early visual areas (V1–V4) using signal detection measures, both pre- and post-training. Although the overall amplitude of the BOLD response was no greater after training, practice nonetheless specifically enhanced the neural representation of the trained orientation at the trained location. This training-specific enhancement of orientation-selective responses was observed in the primary visual cortex (V1) as well as higher extrastriate visual areas V2–V4, and moreover, reliably predicted individual differences in the behavioral effects of perceptual learning. These results demonstrate that extensive training can lead to targeted functional reorganization of the human visual cortex, refining the cortical representation of behaviorally relevant information. PMID:23175828

Perceptual learning selectively refines orientation representations in early visual cortex.

PubMed

Jehee, Janneke F M; Ling, Sam; Swisher, Jascha D; van Bergen, Ruben S; Tong, Frank

2012-11-21

Although practice has long been known to improve perceptual performance, the neural basis of this improvement in humans remains unclear. Using fMRI in conjunction with a novel signal detection-based analysis, we show that extensive practice selectively enhances the neural representation of trained orientations in the human visual cortex. Twelve observers practiced discriminating small changes in the orientation of a laterally presented grating over 20 or more daily 1 h training sessions. Training on average led to a twofold improvement in discrimination sensitivity, specific to the trained orientation and the trained location, with minimal improvement found for untrained orthogonal orientations or for orientations presented in the untrained hemifield. We measured the strength of orientation-selective responses in individual voxels in early visual areas (V1-V4) using signal detection measures, both before and after training. Although the overall amplitude of the BOLD response was no greater after training, practice nonetheless specifically enhanced the neural representation of the trained orientation at the trained location. This training-specific enhancement of orientation-selective responses was observed in the primary visual cortex (V1) as well as higher extrastriate visual areas V2-V4, and moreover, reliably predicted individual differences in the behavioral effects of perceptual learning. These results demonstrate that extensive training can lead to targeted functional reorganization of the human visual cortex, refining the cortical representation of behaviorally relevant information.

The Acoustic Correlates of Perceived Masculinity, Perceived Femininity, and Perceived Sexual Orientation

ERIC Educational Resources Information Center

Munson, Benjamin

2007-01-01

Previous studies have shown that a subset of gay, lesbian, and bisexual (GLB) and heterosexual adults produce distinctive patterns of phonetic variation that allow listeners to detect their sexual orientation from audio-only samples of read speech. The current investigation examined the extent to which judgments of sexual orientation from speech…

The Distribution of Catalase Activity, Isozyme Protein, and Transcript in the Tissues of the Developing Maize Seedling 1

PubMed Central

Redinbaugh, Margaret G.; Sabre, Mara; Scandalios, John G.

1990-01-01

The catalase activity, CAT-2 and CAT-3 isozyme protein levels, and the steady-state mRNA levels for each of the three catalase genes were determined in the scutellum, root, epicotyl, and leaf of the developing maize (Zea mays L.) seedling. Catalase activity was highest in the scutellum, with 10-fold lower enzyme activity in the leaf and epicotyl. Very low levels of catalase activity were found in the root. The highest levels of CAT-2 protein were found in the scutellum, with about 10-fold lower levels in the green leaf. CAT-2 protein was present in trace amounts early in root development and no CAT-2 protein was detected in the epicotyl. Shortly after germination, CAT-3 protein was present at high levels in both the epicotyl and green leaf. With development, the amount of CAT-3 protein decreased slowly in the epicotyl and rapidly in the green leaf. Low levels of this isozyme were detected in the scutellum and root. The Cat1 transcript accumulated to low levels in all four tissues during the 14 day developmental period. High levels of the Cat2 transcript were found in the scutellum, with moderate levels of the mRNA in the green leaf. The Cat2 transcript levels were very low in the root and epicotyl. While the Cat3 mRNA level in the scutellum was low, high levels of the Cat3 transcript were detected in the root, epicotyl, and leaf. There was a positive correlation between the accumulation of a catalase isozyme and its transcript, indicating that the tissue specificity of maize catalase gene expression was regulated pretranslationally. Images Figure 3 Figure 4 PMID:16667285

ASPIC: a novel method to predict the exon-intron structure of a gene that is optimally compatible to a set of transcript sequences.

PubMed

Bonizzoni, Paola; Rizzi, Raffaella; Pesole, Graziano

2005-10-05

Currently available methods to predict splice sites are mainly based on the independent and progressive alignment of transcript data (mostly ESTs) to the genomic sequence. Apart from often being computationally expensive, this approach is vulnerable to several problems--hence the need to develop novel strategies. We propose a method, based on a novel multiple genome-EST alignment algorithm, for the detection of splice sites. To avoid limitations of splice sites prediction (mainly, over-predictions) due to independent single EST alignments to the genomic sequence our approach performs a multiple alignment of transcript data to the genomic sequence based on the combined analysis of all available data. We recast the problem of predicting constitutive and alternative splicing as an optimization problem, where the optimal multiple transcript alignment minimizes the number of exons and hence of splice site observations. We have implemented a splice site predictor based on this algorithm in the software tool ASPIC (Alternative Splicing PredICtion). It is distinguished from other methods based on BLAST-like tools by the incorporation of entirely new ad hoc procedures for accurate and computationally efficient transcript alignment and adopts dynamic programming for the refinement of intron boundaries. ASPIC also provides the minimal set of non-mergeable transcript isoforms compatible with the detected splicing events. The ASPIC web resource is dynamically interconnected with the Ensembl and Unigene databases and also implements an upload facility. Extensive bench marking shows that ASPIC outperforms other existing methods in the detection of novel splicing isoforms and in the minimization of over-predictions. ASPIC also requires a lower computation time for processing a single gene and an EST cluster. The ASPIC web resource is available at http://aspic.algo.disco.unimib.it/aspic-devel/.

In situ hybridization evidence for the coexistence of ASIC and TRPV1 within rat single sensory neurons.

PubMed

Ugawa, Shinya; Ueda, Takashi; Yamamura, Hisao; Shimada, Shoichi

2005-05-20

The activation of nociceptors by protons plays a crucial role in the initiation and maintenance of acidosis-linked pain. Acid-sensing ion channel (ASIC) and transient receptor potential/vanilloid receptor subtype-1 (TRPV1) encode proton-activated cation channels expressed by nociceptors and the opening of these channels results in nociceptor excitation. Histological relations among ASIC clones and the colocalization of each ASIC subunit and TRPV1 within single sensory neurons were examined on serial sections of rat dorsal root ganglia (DRG) using in situ hybridization histochemistry. ASIC1a transcripts were expressed in 20-25% of the DRG neurons, and most of the neurons had small (<30 microm)-diameter cell bodies. ASIC1b transcripts and ASIC3 transcripts were expressed in approximately 10% and 30-35% of the DRG neurons, respectively, and the greater part of each population was located in small-to-medium (30-50 microm)-diameter cells. The ASIC1a transcripts and ASIC1b transcripts were basically localized in the distinct populations of the DRG neurons, while approximately 20% of the ASIC1a-positive neurons and approximately 10% of the ASIC1b-positive neurons expressed ASIC3 transcripts. TRPV1 transcripts were expressed in 35-40% of the DRG neurons, and most of the TRPV1-positive neurons had small-diameter cell bodies. Intense expression signals for ASIC1a transcripts were detected in 40-45% of the TRPV1-positive neurons. Neurons expressing both ASIC1b and TRPV1 transcripts were barely detected in the DRG. Approximately 30% of the TRPV1-positive neurons expressed ASIC3 transcripts, and the double-labeled neurons were comprised of both small-diameter and medium-diameter cells. Approximately 13% of the TRPV1-positive neurons expressed both ASIC1a and ASIC3 transcripts.

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

PubMed Central

Bojanovič, Klara; D'Arrigo, Isotta

2017-01-01

ABSTRACT Bacteria cope with and adapt to stress by modulating gene expression in response to specific environmental cues. In this study, the transcriptional response of Pseudomonas putida KT2440 to osmotic, oxidative, and imipenem stress conditions at two time points was investigated via identification of differentially expressed mRNAs and small RNAs (sRNAs). A total of 440 sRNA transcripts were detected, of which 10% correspond to previously annotated sRNAs, 40% to novel intergenic transcripts, and 50% to novel transcripts antisense to annotated genes. Each stress elicits a unique response as far as the extent and dynamics of the transcriptional changes. Nearly 200 protein-encoding genes exhibited significant changes in all stress types, implicating their participation in a general stress response. Almost half of the sRNA transcripts were differentially expressed under at least one condition, suggesting possible functional roles in the cellular response to stress conditions. The data show a larger fraction of differentially expressed sRNAs than of mRNAs with >5-fold expression changes. The work provides detailed insights into the mechanisms through which P. putida responds to different stress conditions and increases understanding of bacterial adaptation in natural and industrial settings. IMPORTANCE This study maps the complete transcriptional response of P. putida KT2440 to osmotic, oxidative, and imipenem stress conditions at short and long exposure times. Over 400 sRNA transcripts, consisting of both intergenic and antisense transcripts, were detected, increasing the number of identified sRNA transcripts in the strain by a factor of 10. Unique responses to each type of stress are documented, including both the extent and dynamics of the gene expression changes. The work adds rich detail to previous knowledge of stress response mechanisms due to the depth of the RNA sequencing data. Almost half of the sRNAs exhibit significant expression changes under at least one condition, suggesting their involvement in adaptation to stress conditions and identifying interesting candidates for further functional characterization. PMID:28130298

In vitro comparative study of vibro-acoustography versus pulse-echo ultrasound in imaging permanent prostate brachytherapy seeds

PubMed Central

Mitri, F.G.; Davis, B.J.; Greenleaf, J.F.; Fatemi, M.

2010-01-01

Background Permanent prostate brachytherapy (PPB) is a common treatment for early stage prostate cancer. While the modern approach using trans-rectal ultrasound guidance has demonstrated excellent outcome, the efficacy of PPB depends on achieving complete radiation dose coverage of the prostate by obtaining a proper radiation source (seed) distribution. Currently, brachytherapy seed placement is guided by trans-rectal ultrasound imaging and fluoroscopy. A significant percentage of seeds are not detected by trans-rectal ultrasound because certain seed orientations are invisible making accurate intra-operative feedback of radiation dosimetry very difficult, if not impossible. Therefore, intra-operative correction of suboptimal seed distributions cannot easily be done with current methods. Vibro-acoustography (VA) is an imaging modality that is capable of imaging solids at any orientation, and the resulting images are speckle free. Objective and methods The purpose of this study is to compare the capabilities of VA and pulse-echo ultrasound in imaging PPB seeds at various angles and show the sensitivity of detection to seed orientation. In the VA experiment, two intersecting ultrasound beams driven at f1 = 3.00 MHz and f2 = 3.020 MHz respectively were focused on the seeds attached to a latex membrane while the amplitude of the acoustic emission produced at the difference frequency 20 kHz was detected by a low frequency hydrophone. Results Finite element simulations and results of experiments conducted under well-controlled conditions in a water tank on a series of seeds indicate that the seeds can be detected at any orientation with VA, whereas pulse-echo ultrasound is very sensitive to the seed orientation. Conclusion It is concluded that vibro-acoustography is superior to pulse-echo ultrasound for detection of PPB seeds. PMID:18538365

Depletion of polycistronic transcripts using short interfering RNAs: cDNA synthesis method affects levels of non-targeted genes determined by quantitative PCR.

PubMed

Hanning, Jennifer E; Groves, Ian J; Pett, Mark R; Coleman, Nicholas

2013-05-21

Short interfering RNAs (siRNAs) are often used to deplete viral polycistronic transcripts, such as those encoded by human papillomavirus (HPV). There are conflicting data in the literature concerning how siRNAs targeting one HPV gene can affect levels of other genes in the polycistronic transcripts. We hypothesised that the conflict might be partly explained by the method of cDNA synthesis used prior to transcript quantification. We treated HPV16-positive cervical keratinocytes with siRNAs targeting the HPV16 E7 gene and used quantitative PCR to compare transcript levels of E7 with those of E6 and E2, viral genes located upstream and downstream of the target site respectively. We compared our findings from cDNA generated using oligo-dT primers alone with those from cDNA generated using a combination of random hexamer and oligo-dT primers. Our data show that when polycistronic transcripts are targeted by siRNAs, there is a period when untranslatable cleaved mRNA upstream of the siRNA binding site remains detectable by PCR, if cDNA is generated using random hexamer primers. Such false indications of mRNA abundance are avoided using oligo-dT primers. The period corresponds to the time taken for siRNA activity and degradation of the cleaved transcripts. Genes downstream of the siRNA binding site are detectable during this interval, regardless of how the cDNA is generated. These data emphasise the importance of the cDNA synthesis method used when measuring transcript abundance following siRNA depletion of polycistronic transcripts. They provide a partial explanation for erroneous reports suggesting that siRNAs targeting HPV E7 can have gene-specific effects.

Depletion of polycistronic transcripts using short interfering RNAs: cDNA synthesis method affects levels of non-targeted genes determined by quantitative PCR

PubMed Central

2013-01-01

Background Short interfering RNAs (siRNAs) are often used to deplete viral polycistronic transcripts, such as those encoded by human papillomavirus (HPV). There are conflicting data in the literature concerning how siRNAs targeting one HPV gene can affect levels of other genes in the polycistronic transcripts. We hypothesised that the conflict might be partly explained by the method of cDNA synthesis used prior to transcript quantification. Findings We treated HPV16-positive cervical keratinocytes with siRNAs targeting the HPV16 E7 gene and used quantitative PCR to compare transcript levels of E7 with those of E6 and E2, viral genes located upstream and downstream of the target site respectively. We compared our findings from cDNA generated using oligo-dT primers alone with those from cDNA generated using a combination of random hexamer and oligo-dT primers. Our data show that when polycistronic transcripts are targeted by siRNAs, there is a period when untranslatable cleaved mRNA upstream of the siRNA binding site remains detectable by PCR, if cDNA is generated using random hexamer primers. Such false indications of mRNA abundance are avoided using oligo-dT primers. The period corresponds to the time taken for siRNA activity and degradation of the cleaved transcripts. Genes downstream of the siRNA binding site are detectable during this interval, regardless of how the cDNA is generated. Conclusions These data emphasise the importance of the cDNA synthesis method used when measuring transcript abundance following siRNA depletion of polycistronic transcripts. They provide a partial explanation for erroneous reports suggesting that siRNAs targeting HPV E7 can have gene-specific effects. PMID:23693071

Transcriptional and translational adaptation to aerobic nitrate anabolism in the denitrifier Paracoccus denitrificans

PubMed Central

Luque-Almagro, Victor M.; Manso, Isabel; Sullivan, Matthew J.; Rowley, Gary; Ferguson, Stuart J.; Moreno-Vivián, Conrado; Richardson, David J.; Gates, Andrew J.

2017-01-01

Transcriptional adaptation to nitrate-dependent anabolism by Paracoccus denitrificans PD1222 was studied. A total of 74 genes were induced in cells grown with nitrate as N-source compared with ammonium, including nasTSABGHC and ntrBC genes. The nasT and nasS genes were cotranscribed, although nasT was more strongly induced by nitrate than nasS. The nasABGHC genes constituted a transcriptional unit, which is preceded by a non-coding region containing hairpin structures involved in transcription termination. The nasTS and nasABGHC transcripts were detected at similar levels with nitrate or glutamate as N-source, but nasABGHC transcript was undetectable in ammonium-grown cells. The nitrite reductase NasG subunit was detected by two-dimensional polyacrylamide gel electrophoresis in cytoplasmic fractions from nitrate-grown cells, but it was not observed when either ammonium or glutamate was used as the N-source. The nasT mutant lacked both nasABGHC transcript and nicotinamide adenine dinucleotide (NADH)-dependent nitrate reductase activity. On the contrary, the nasS mutant showed similar levels of the nasABGHC transcript to the wild-type strain and displayed NasG protein and NADH–nitrate reductase activity with all N-sources tested, except with ammonium. Ammonium repression of nasABGHC was dependent on the Ntr system. The ntrBC and ntrYX genes were expressed at low levels regardless of the nitrogen source supporting growth. Mutational analysis of the ntrBCYX genes indicated that while ntrBC genes are required for nitrate assimilation, ntrYX genes can only partially restore growth on nitrate in the absence of ntrBC genes. The existence of a regulation mechanism for nitrate assimilation in P. denitrificans, by which nitrate induction operates at both transcriptional and translational levels, is proposed. PMID:28385879

Zebrafish fin immune responses during high mortality infections with viral haemorrhagic septicemia rhabdovirus. A proteomic and transcriptomic approach.

PubMed

Encinas, Paloma; Rodriguez-Milla, Miguel A; Novoa, Beatriz; Estepa, Amparo; Figueras, Antonio; Coll, Julio

2010-09-27

Despite rhabdoviral infections being one of the best known fish diseases, the gene expression changes induced at the surface tissues after the natural route of infection (infection-by-immersion) have not been described yet. This work describes the differential infected versus non-infected expression of proteins and immune-related transcripts in fins and organs of zebrafish Danio rerio shortly after infection-by-immersion with viral haemorrhagic septicemia virus (VHSV). Two-dimensional differential gel electrophoresis detected variations on the protein levels of the enzymes of the glycolytic pathway and cytoskeleton components but it detected very few immune-related proteins. Differential expression of immune-related gene transcripts estimated by quantitative polymerase chain reaction arrays and hybridization to oligo microarrays showed that while more transcripts increased in fins than in organs (spleen, head kidney and liver), more transcripts decreased in organs than in fins. Increased differential transcript levels in fins detected by both arrays corresponded to previously described infection-related genes such as complement components (c3b, c8 and c9) or class I histocompatibility antigens (mhc1) and to newly described genes such as secreted immunoglobulin domain (sid4), macrophage stimulating factor (mst1) and a cluster differentiation antigen (cd36). The genes described would contribute to the knowledge of the earliest molecular events occurring in the fish surfaces at the beginning of natural rhabdoviral infections and/or might be new candidates to be tested as adjuvants for fish vaccines.

Regulated expression of homeobox genes Msx-1 and Msx-2 in mouse mammary gland development suggests a role in hormone action and epithelial-stromal interactions.

PubMed

Friedmann, Y; Daniel, C W

1996-07-10

The murine homeobox genes Msx-1 and Msx-2 are related to the Drosophila msh gene and are expressed in a variety of tissues during mouse embryogenesis. We now report the developmentally regulated expression of Msx-1 and Msx-2 in the mouse mammary gland and show that their expression patterns point toward significant functional roles. Msx-1 and Msx-2 transcripts were present in glands of virgin mice and in glands of mice in early pregnancy, but transcripts decreased dramatically during late pregnancy. Low levels of Msx-1 transcripts were detected in glands from lactating animals and during the first days of involution, whereas Msx-2 expression was not detected during lactation or early involution. Expression of both genes increased gradually as involution progressed. Msx-2 but not Msx-1 expression was decreased following ovariectomy or following exposure to anti-estrogen implanted directly into the gland. Hormonal regulation of Msx-2 expression was confirmed when transcripts returned to normal levels after estrogen was administered to ovariectomized animals. In situ molecular hybridization for Msx-1 showed transcripts localized to the mammary epithelium, whereas Msx-2 expression was confined to the periductal stroma. Mammary stroma from which mammary epithelium had been removed did not transcribe detectable amounts of Msx-2, showing that expression is regulated by contiguous mammary epithelium, and indicating a role for these homeobox genes in mesenchymal-epithelial interactions during mammary development.

Detection and inpainting of facial wrinkles using texture orientation fields and Markov random field modeling.

PubMed

Batool, Nazre; Chellappa, Rama

2014-09-01

Facial retouching is widely used in media and entertainment industry. Professional software usually require a minimum level of user expertise to achieve the desirable results. In this paper, we present an algorithm to detect facial wrinkles/imperfection. We believe that any such algorithm would be amenable to facial retouching applications. The detection of wrinkles/imperfections can allow these skin features to be processed differently than the surrounding skin without much user interaction. For detection, Gabor filter responses along with texture orientation field are used as image features. A bimodal Gaussian mixture model (GMM) represents distributions of Gabor features of normal skin versus skin imperfections. Then, a Markov random field model is used to incorporate the spatial relationships among neighboring pixels for their GMM distributions and texture orientations. An expectation-maximization algorithm then classifies skin versus skin wrinkles/imperfections. Once detected automatically, wrinkles/imperfections are removed completely instead of being blended or blurred. We propose an exemplar-based constrained texture synthesis algorithm to inpaint irregularly shaped gaps left by the removal of detected wrinkles/imperfections. We present results conducted on images downloaded from the Internet to show the efficacy of our algorithms.

When transcription goes on Holliday: Double Holliday junctions block RNA polymerase II transcription in vitro.

PubMed

Pipathsouk, Anne; Belotserkovskii, Boris P; Hanawalt, Philip C

2017-02-01

Non-canonical DNA structures can obstruct transcription. This transcription blockage could have various biological consequences, including genomic instability and gratuitous transcription-coupled repair. Among potential structures causing transcription blockage are Holliday junctions (HJs), which can be generated as intermediates in homologous recombination or during processing of stalled replication forks. Of particular interest is the double Holliday junction (DHJ), which contains two HJs. Topological considerations impose the constraint that the total number of helical turns in the DNA duplexes between the junctions cannot be altered as long as the flanking DNA duplexes are intact. Thus, the DHJ structure should strongly resist transient unwinding during transcription; consequently, it is predicted to cause significantly stronger blockage than single HJ structures. The patterns of transcription blockage obtained for RNA polymerase II transcription in HeLa cell nuclear extracts were in accordance with this prediction. However, we did not detect transcription blockage with purified T7 phage RNA polymerase; we discuss a possible explanation for this difference. In general, our findings implicate naturally occurring Holliday junctions in transcription arrest. Copyright © 2016 Elsevier B.V. All rights reserved.

Designing deep sequencing experiments: detecting structural variation and estimating transcript abundance.

PubMed

Bashir, Ali; Bansal, Vikas; Bafna, Vineet

2010-06-18

Massively parallel DNA sequencing technologies have enabled the sequencing of several individual human genomes. These technologies are also being used in novel ways for mRNA expression profiling, genome-wide discovery of transcription-factor binding sites, small RNA discovery, etc. The multitude of sequencing platforms, each with their unique characteristics, pose a number of design challenges, regarding the technology to be used and the depth of sequencing required for a particular sequencing application. Here we describe a number of analytical and empirical results to address design questions for two applications: detection of structural variations from paired-end sequencing and estimating mRNA transcript abundance. For structural variation, our results provide explicit trade-offs between the detection and resolution of rearrangement breakpoints, and the optimal mix of paired-read insert lengths. Specifically, we prove that optimal detection and resolution of breakpoints is achieved using a mix of exactly two insert library lengths. Furthermore, we derive explicit formulae to determine these insert length combinations, enabling a 15% improvement in breakpoint detection at the same experimental cost. On empirical short read data, these predictions show good concordance with Illumina 200 bp and 2 Kbp insert length libraries. For transcriptome sequencing, we determine the sequencing depth needed to detect rare transcripts from a small pilot study. With only 1 Million reads, we derive corrections that enable almost perfect prediction of the underlying expression probability distribution, and use this to predict the sequencing depth required to detect low expressed genes with greater than 95% probability. Together, our results form a generic framework for many design considerations related to high-throughput sequencing. We provide software tools http://bix.ucsd.edu/projects/NGS-DesignTools to derive platform independent guidelines for designing sequencing experiments (amount of sequencing, choice of insert length, mix of libraries) for novel applications of next generation sequencing.

[Application of transcription mediated amplification and real-time reverse transcription polymerase chain reaction in detection of human immunodeficiency virus RNA].

PubMed

Wu, Daxian; Tao, Shuhui; Liu, Shuiping; Zhou, Jiebin; Tan, Deming; Hou, Zhouhua

2017-07-28

To observe the sensitivity of transcription mediated amplification (TMA), and to compare its performance with real-time reverse transcription polymerase chain reaction (real-time RT-PCR) in detecting human immunodeficiency virus RNA (HIV RNA).
 Methods: TMA system was established with TaqMan probes, specific primers, moloney murine leukemia virus (MMLV) reverse transcriptase, T7 RNA polymerase, and reaction substrates. The sensitivity of TMA was evaluated by amplifying a group of 10-fold diluted HIV RNA standards which were transcribed in vitro. A total of 60 plasma of HIV infected patients were measured by TMA and Cobas Amplicor HIV-1 Monitor test to observe the positive rate. The correlation and concordance of the above two technologies were investigated by linear regression and Bland-Altman analysis.
 Results: TMA system was established successfully and HIV RNA transcribed standards at concentration of equal or more than 10 copies/mL could be detected by TMA technology. Among 60 samples of plasma from HIV infected patients, 46 were positively detected and 12 were negatively amplified by both TMA and Cobas reagents; 2 samples were positively tested by Cobas reagent but negatively tested by TMA system. The concordance rate of the two methods was 97.1% and the difference of positive detection rate between the two methods was not statistically significant (P>0.05). Linear regression was used for 46 samples which were positively detected by both TMA and Cobas reagents and showed an excellent correlation between the two reagents (r=0.997, P<0.001). Bland-Altma analysis revealed that the mean different value of HIV RNA levels for denary logarithm was 0.02. Forty-four samples were included in 95% of credibility interval of concordance.
 Conclusion: TMA system has the potential of high sensitivity. TMA and real-time RT-PCR keep an excellent correlation and consistency in detecting HIV RNA.

Structure of transcribed chromatin is a sensor of DNA damage

PubMed Central

Pestov, Nikolay A.; Gerasimova, Nadezhda S.; Kulaeva, Olga I.; Studitsky, Vasily M.

2015-01-01

Early detection and repair of damaged DNA is essential for cell functioning and survival. Although multiple cellular systems are involved in the repair of single-strand DNA breaks (SSBs), it remains unknown how SSBs present in the nontemplate strand (NT-SSBs) of DNA organized in chromatin are detected. The effect of NT-SSBs on transcription through chromatin by RNA polymerase II was studied. NT-SSBs localized in the promoter-proximal region of nucleosomal DNA and hidden in the nucleosome structure can induce a nearly quantitative arrest of RNA polymerase downstream of the break, whereas more promoter-distal SSBs moderately facilitate transcription. The location of the arrest sites on nucleosomal DNA suggests that formation of small intranucleosomal DNA loops causes the arrest. This mechanism likely involves relief of unconstrained DNA supercoiling accumulated during transcription through chromatin by NT-SSBs. These data suggest the existence of a novel chromatin-specific mechanism that allows the detection of NT-SSBs by the transcribing enzyme. PMID:26601207

Quantitative Detection of Low-Abundance Transcripts at Single-Cell Level in Human Epidermal Keratinocytes by Digital Droplet Reverse Transcription-Polymerase Chain Reaction.

PubMed

Auvré, Frédéric; Coutier, Julien; Martin, Michèle T; Fortunel, Nicolas O

2018-05-08

Genetic and epigenetic characterization of the large cellular diversity observed within tissues is essential to understanding the molecular networks that ensure the regulation of homeostasis, repair, and regeneration, but also pathophysiological processes. Skin is composed of multiple cell lineages and is therefore fully concerned by this complexity. Even within one particular lineage, such as epidermal keratinocytes, different immaturity statuses or differentiation stages are represented, which are still incompletely characterized. Accordingly, there is presently great demand for methods and technologies enabling molecular investigation at single-cell level. Also, most current methods used to analyze gene expression at RNA level, such as RT-qPCR, do not directly provide quantitative data, but rather comparative ratios between two conditions. A second important need in skin biology is thus to determine the number of RNA molecules in a given cell sample. Here, we describe a workflow that we have set up to meet these specific needs, by means of transcript quantification in cellular micro-samples using flow cytometry sorting and reverse transcription-digital droplet polymerase chain reaction. As a proof-of-principle, the workflow was tested for the detection of transcription factor transcripts expressed at low levels in keratinocyte precursor cells. A linear correlation was found between quantification values and keratinocyte input numbers in a low quantity range from 40 cells to 1 cell. Interpretable signals were repeatedly obtained from single-cell samples corresponding to estimated expression levels as low as 10-20 transcript copies per keratinocyte or less. The present workflow may have broad applications for the detection and quantification of low-abundance nucleic acid species in single cells, opening up perspectives for the study of cell-to-cell genetic and molecular heterogeneity. Interestingly, the process described here does not require internal references such as house-keeping gene expression, as it is initiated with defined cell numbers, precisely sorted by flow cytometry.

Object-Oriented Query Language For Events Detection From Images Sequences

NASA Astrophysics Data System (ADS)

Ganea, Ion Eugen

2015-09-01

In this paper is presented a method to represent the events extracted from images sequences and the query language used for events detection. Using an object oriented model the spatial and temporal relationships between salient objects and also between events are stored and queried. This works aims to unify the storing and querying phases for video events processing. The object oriented language syntax used for events processing allow the instantiation of the indexes classes in order to improve the accuracy of the query results. The experiments were performed on images sequences provided from sport domain and it shows the reliability and the robustness of the proposed language. To extend the language will be added a specific syntax for constructing the templates for abnormal events and for detection of the incidents as the final goal of the research.

CRX ChIP-seq reveals the cis-regulatory architecture of mouse photoreceptors

PubMed Central

Corbo, Joseph C.; Lawrence, Karen A.; Karlstetter, Marcus; Myers, Connie A.; Abdelaziz, Musa; Dirkes, William; Weigelt, Karin; Seifert, Martin; Benes, Vladimir; Fritsche, Lars G.; Weber, Bernhard H.F.; Langmann, Thomas

2010-01-01

Approximately 98% of mammalian DNA is noncoding, yet we understand relatively little about the function of this enigmatic portion of the genome. The cis-regulatory elements that control gene expression reside in noncoding regions and can be identified by mapping the binding sites of tissue-specific transcription factors. Cone-rod homeobox (CRX) is a key transcription factor in photoreceptor differentiation and survival, but its in vivo targets are largely unknown. Here, we used chromatin immunoprecipitation with massively parallel sequencing (ChIP-seq) on CRX to identify thousands of cis-regulatory regions around photoreceptor genes in adult mouse retina. CRX directly regulates downstream photoreceptor transcription factors and their target genes via a network of spatially distributed regulatory elements around each locus. CRX-bound regions act in a synergistic fashion to activate transcription and contain multiple CRX binding sites which interact in a spacing- and orientation-dependent manner to fine-tune transcript levels. CRX ChIP-seq was also performed on Nrl−/− retinas, which represent an enriched source of cone photoreceptors. Comparison with the wild-type ChIP-seq data set identified numerous rod- and cone-specific CRX-bound regions as well as many shared elements. Thus, CRX combinatorially orchestrates the transcriptional networks of both rods and cones by coordinating the expression of photoreceptor genes including most retinal disease genes. In addition, this study pinpoints thousands of noncoding regions of relevance to both Mendelian and complex retinal disease. PMID:20693478

Defining the Status of RNA Polymerase at Promoters

PubMed Central

Core, Leighton J.; Waterfall, Joshua J.; Gilchrist, Daniel A.; Fargo, David C.; Kwak, Hojoong; Adelman, Karen; Lis, John T.

2012-01-01

Summary Recent genome-wide studies in metazoans have shown that RNA Polymerase II (Pol II) accumulates to high densities on many promoters at a rate-limited step in transcription. However, the status of this Pol II remains an area of debate. Here, we compare quantitative outputs of GRO-seq and ChIP-seq assays and demonstrate the majority of the Pol II on Drosophila promoters is transcriptionally-engaged - very little exists in a preinitiation or arrested complex. These promoter-proximal polymerases are inhibited from further elongation by detergent sensitive factors, and knockdown of negative elongation factor, NELF, reduces their levels. These results not only solidify that pausing occurs at most promoters, but demonstrate that it is the major rate-limiting step in early transcription at these promoters. Finally, the divergent elongation complexes seen at mammalian promoters are far less prevalent in Drosophila, and this specificity in orientation correlates with directional core promoter elements, which are abundant in Drosophila. PMID:23062713

TIPMaP: a web server to establish transcript isoform profiles from reliable microarray probes.

PubMed

Chitturi, Neelima; Balagannavar, Govindkumar; Chandrashekar, Darshan S; Abinaya, Sadashivam; Srini, Vasan S; Acharya, Kshitish K

2013-12-27

Standard 3' Affymetrix gene expression arrays have contributed a significantly higher volume of existing gene expression data than other microarray platforms. These arrays were designed to identify differentially expressed genes, but not their alternatively spliced transcript forms. No resource can currently identify expression pattern of specific mRNA forms using these microarray data, even though it is possible to do this. We report a web server for expression profiling of alternatively spliced transcripts using microarray data sets from 31 standard 3' Affymetrix arrays for human, mouse and rat species. The tool has been experimentally validated for mRNAs transcribed or not-detected in a human disease condition (non-obstructive azoospermia, a male infertility condition). About 4000 gene expression datasets were downloaded from a public repository. 'Good probes' with complete coverage and identity to latest reference transcript sequences were first identified. Using them, 'Transcript specific probe-clusters' were derived for each platform and used to identify expression status of possible transcripts. The web server can lead the user to datasets corresponding to specific tissues, conditions via identifiers of the microarray studies or hybridizations, keywords, official gene symbols or reference transcript identifiers. It can identify, in the tissues and conditions of interest, about 40% of known transcripts as 'transcribed', 'not-detected' or 'differentially regulated'. Corresponding additional information for probes, genes, transcripts and proteins can be viewed too. We identified the expression of transcripts in a specific clinical condition and validated a few of these transcripts by experiments (using reverse transcription followed by polymerase chain reaction). The experimental observations indicated higher agreements with the web server results, than contradictions. The tool is accessible at http://resource.ibab.ac.in/TIPMaP. The newly developed online tool forms a reliable means for identification of alternatively spliced transcript-isoforms that may be differentially expressed in various tissues, cell types or physiological conditions. Thus, by making better use of existing data, TIPMaP avoids the dependence on precious tissue-samples, in experiments with a goal to establish expression profiles of alternative splice forms--at least in some cases.

A Portable Reverse Transcription Recombinase Polymerase Amplification Assay for Rapid Detection of Foot-and-Mouth Disease Virus

PubMed Central

Abd El Wahed, Ahmed; El-Deeb, Ayman; El-Tholoth, Mohamed; Abd El Kader, Hanaa; Ahmed, Abeer; Hassan, Sayed; Hoffmann, Bernd; Haas, Bernd; Shalaby, Mohamed A.; Hufert, Frank T.; Weidmann, Manfred

2013-01-01

Foot-and-mouth disease (FMD) is a trans-boundary viral disease of livestock, which causes huge economic losses and constitutes a serious infectious threat for livestock farming worldwide. Early diagnosis of FMD helps to diminish its impact by adequate outbreak management. In this study, we describe the development of a real-time reverse transcription recombinase polymerase amplification (RT-RPA) assay for the detection of FMD virus (FMDV). The FMDV RT-RPA design targeted the 3D gene of FMDV and a 260 nt molecular RNA standard was used for assay validation. The RT-RPA assay was fast (4–10 minutes) and the analytical sensitivity was determined at 1436 RNA molecules detected by probit regression analysis. The FMDV RT-RPA assay detected RNA prepared from all seven FMDV serotypes but did not detect classical swine fever virus or swine vesicular disease virus. The FMDV RT-RPA assay was used in the field during the recent FMD outbreak in Egypt. In clinical samples, reverse transcription polymerase chain reaction (RT-PCR) and RT-RPA showed a diagnostic sensitivity of 100% and 98%, respectively. In conclusion, FMDV RT-RPA was quicker and much easier to handle in the field than real-time RT-PCR. Thus RT-RPA could be easily implemented to perform diagnostics at quarantine stations or farms for rapid spot-of-infection detection. PMID:23977101

A portable reverse transcription recombinase polymerase amplification assay for rapid detection of foot-and-mouth disease virus.

PubMed

Abd El Wahed, Ahmed; El-Deeb, Ayman; El-Tholoth, Mohamed; Abd El Kader, Hanaa; Ahmed, Abeer; Hassan, Sayed; Hoffmann, Bernd; Haas, Bernd; Shalaby, Mohamed A; Hufert, Frank T; Weidmann, Manfred

2013-01-01

Foot-and-mouth disease (FMD) is a trans-boundary viral disease of livestock, which causes huge economic losses and constitutes a serious infectious threat for livestock farming worldwide. Early diagnosis of FMD helps to diminish its impact by adequate outbreak management. In this study, we describe the development of a real-time reverse transcription recombinase polymerase amplification (RT-RPA) assay for the detection of FMD virus (FMDV). The FMDV RT-RPA design targeted the 3D gene of FMDV and a 260 nt molecular RNA standard was used for assay validation. The RT-RPA assay was fast (4-10 minutes) and the analytical sensitivity was determined at 1436 RNA molecules detected by probit regression analysis. The FMDV RT-RPA assay detected RNA prepared from all seven FMDV serotypes but did not detect classical swine fever virus or swine vesicular disease virus. The FMDV RT-RPA assay was used in the field during the recent FMD outbreak in Egypt. In clinical samples, reverse transcription polymerase chain reaction (RT-PCR) and RT-RPA showed a diagnostic sensitivity of 100% and 98%, respectively. In conclusion, FMDV RT-RPA was quicker and much easier to handle in the field than real-time RT-PCR. Thus RT-RPA could be easily implemented to perform diagnostics at quarantine stations or farms for rapid spot-of-infection detection.

Time-gated detection of protein-protein interactions with transcriptional readout

PubMed Central

Sanchez, Mateo I; Coukos, Robert; von Zastrow, Mark

2017-01-01

Transcriptional assays, such as yeast two-hybrid and TANGO, that convert transient protein-protein interactions (PPIs) into stable expression of transgenes are powerful tools for PPI discovery, screens, and analysis of cell populations. However, such assays often have high background and lose information about PPI dynamics. We have developed SPARK (Specific Protein Association tool giving transcriptional Readout with rapid Kinetics), in which proteolytic release of a membrane-tethered transcription factor (TF) requires both a PPI to deliver a protease proximal to its cleavage peptide and blue light to uncage the cleavage site. SPARK was used to detect 12 different PPIs in mammalian cells, with 5 min temporal resolution and signal ratios up to 37. By shifting the light window, we could reconstruct PPI time-courses. Combined with FACS, SPARK enabled 51 fold enrichment of PPI-positive over PPI-negative cells. Due to its high specificity and sensitivity, SPARK has the potential to advance PPI analysis and discovery. PMID:29189201

Rapid detection of biothreat agents based on cellular machinery.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lane, Todd W.; Gantt, Richard W.

This research addresses rapid and sensitive identification of biological agents in a complex background. We attempted to devise a method by which the specificity of the cellular transcriptional machinery could be used to detect and identify bacterial bio-terror agents in a background of other organisms. Bacterial cells contain RNA polymerases and transcription factors that transcribe genes into mRNA for translation into proteins. RNA polymerases in conjunction with transcription factors recognize regulatory elements (promoters) upstream of the gene. These promoters are, in many cases, recognized by the polymerase and transcription factor combinations of one species only. We have engineered a plasmid,more » for Escherichia coli, containing the virA promoter from the target species Shigella flexneri. This promoter was fused to a reporter gene Green Fluorescent Protein (GFP). In theory the indicator strain (carrying the plasmid) is mixed with the target strain and the two are lysed. The cellular machinery from both cells mixes and the GFP is produced. This report details the results of testing this system.« less

Rapid and specific detection of Yam mosaic virus by reverse-transcription recombinase polymerase amplification.

PubMed

Silva, Gonçalo; Bömer, Moritz; Nkere, Chukwuemeka; Kumar, P Lava; Seal, Susan E

2015-09-15

Yam mosaic virus (YMV; genus Potyvirus) is considered to cause the most economically important viral disease of yams (Dioscorea spp.) in West Africa which is the dominant region for yam production globally. Yams are a vegetatively propagated crop and the use of virus-free planting material forms an essential component of disease control. Current serological and PCR-based diagnostic methods for YMV are time consuming involving a succession of target detection steps. In this study, a novel assay for specific YMV detection is described that is based on isothermal reverse transcription-recombinase polymerase amplification (RT-exoRPA). This test has been shown to be reproducible and able to detect as little as 14 pg/μl of purified RNA obtained from an YMV-infected plant, a sensitivity equivalent to that obtained with the reverse transcription-polymerase chain reaction (RT-PCR) in current general use. The RT-exoRPA assay has, however, several advantages over the RT-PCR; positive samples can be detected in less than 30 min, and amplification only requires a single incubation temperature (optimum 37°C). These features make the RT-exoRPA assay a promising candidate for adapting into a field test format to be used by yam breeding programmes or certification laboratories. Copyright © 2015 Elsevier B.V. All rights reserved.

The role of repressor kinetics in relief of transcriptional interference between convergent promoters

PubMed Central

Hao, Nan; Palmer, Adam C.; Ahlgren-Berg, Alexandra; Shearwin, Keith E.; Dodd, Ian B.

2016-01-01

Transcriptional interference (TI), where transcription from a promoter is inhibited by the activity of other promoters in its vicinity on the same DNA, enables transcription factors to regulate a target promoter indirectly, inducing or relieving TI by controlling the interfering promoter. For convergent promoters, stochastic simulations indicate that relief of TI can be inhibited if the repressor at the interfering promoter has slow binding kinetics, making it either sensitive to frequent dislodgement by elongating RNA polymerases (RNAPs) from the target promoter, or able to be a strong roadblock to these RNAPs. In vivo measurements of relief of TI by CI or Cro repressors in the bacteriophage λ PR–PRE system show strong relief of TI and a lack of dislodgement and roadblocking effects, indicative of rapid CI and Cro binding kinetics. However, repression of the same λ promoter by a catalytically dead CRISPR Cas9 protein gave either compromised or no relief of TI depending on the orientation at which it binds DNA, consistent with dCas9 being a slow kinetics repressor. This analysis shows how the intrinsic properties of a repressor can be evolutionarily tuned to set the magnitude of relief of TI. PMID:27378773

DLEU2 encodes an antisense RNA for the putative bicistronic RFP2/LEU5 gene in humans and mouse.

PubMed

Corcoran, Martin M; Hammarsund, Marianne; Zhu, Chaoyong; Lerner, Mikael; Kapanadze, Bagrat; Wilson, Bill; Larsson, Catharina; Forsberg, Lars; Ibbotson, Rachel E; Einhorn, Stefan; Oscier, David G; Grandér, Dan; Sangfelt, Olle

2004-08-01

Our group previously identified two novel genes, RFP2/LEU5 and DLEU2, within a 13q14.3 genomic region of loss seen in various malignancies. However, no specific inactivating mutations were found in these or other genes in the vicinity of the deletion, suggesting that a nonclassical tumor-suppressor mechanism may be involved. Here, we present data showing that the DLEU2 gene encodes a putative noncoding antisense RNA, with one exon directly overlapping the first exon of the RFP2/LEU5 gene in the opposite orientation. In addition, the RFP2/LEU5 transcript can be alternatively spliced to produce either several monocistronic transcripts or a putative bicistronic transcript encoding two separate open-reading frames, adding to the complexity of the locus. The finding that these gene structures are conserved in the mouse, including the putative bicistronic RFP2/LEU5 transcript as well as the antisense relationship with DLEU2, further underlines the significance of this unusual organization and suggests a biological function for DLEU2 in the regulation of RFP2/LEU5. Copyright 2004 Wiley-Liss, Inc.

Detection of preferential particle orientation in the atmosphere. Development of an alternative polarization lidar system

DOE PAGES

Geier, Manfred; Arienti, Marco

2014-07-19

Increasing interest in polarimetric characterization of atmospheric aerosols has led to the development of complete sample-measuring (Mueller) polarimeters that are capable of measuring the entire backscattering phase matrix of a probed volume. The Mueller polarimeters consist of several moving parts, which limit measurement rates and complicate data analysis. In this paper, we present the concept of a less complex polarization lidar setup for detection of preferential orientation of atmospheric particulates. On the basis of theoretical considerations of data inversion stability and propagation of measurement uncertainties, an optimum optical configuration is established for two modes of operation (with either a linearmore » or a circular polarized incident laser beam). We discovered that the conceptualized setup falls in the category of incomplete sample-measuring polarimeters and uses four detection channels for simultaneous measurement of the backscattered light. Likewise, the expected performance characteristics are discussed through an example of a typical aerosol with a small fraction of particles oriented in a preferred direction. As a result, the theoretical analysis suggests that achievable accuracies in backscatter cross-sections and depolarization ratios are similar to those with conventional two-channel configurations, while in addition preferential orientation can be detected with the proposed four-channel system for a wide range of conditions.« less

An Intergenic Region Shared by At4g35985 and At4g35987 in Arabidopsis thaliana Is a Tissue Specific and Stress Inducible Bidirectional Promoter Analyzed in Transgenic Arabidopsis and Tobacco Plants

PubMed Central

Banerjee, Joydeep; Sahoo, Dipak Kumar; Dey, Nrisingha; Houtz, Robert L.; Maiti, Indu Bhushan

2013-01-01

On chromosome 4 in the Arabidopsis genome, two neighboring genes (calmodulin methyl transferase At4g35987 and senescence associated gene At4g35985) are located in a head-to-head divergent orientation sharing a putative bidirectional promoter. This 1258 bp intergenic region contains a number of environmental stress responsive and tissue specific cis-regulatory elements. Transcript analysis of At4g35985 and At4g35987 genes by quantitative real time PCR showed tissue specific and stress inducible expression profiles. We tested the bidirectional promoter-function of the intergenic region shared by the divergent genes At4g35985 and At4g35987 using two reporter genes (GFP and GUS) in both orientations in transient tobacco protoplast and Agro-infiltration assays, as well as in stably transformed transgenic Arabidopsis and tobacco plants. In transient assays with GFP and GUS reporter genes the At4g35985 promoter (P85) showed stronger expression (about 3.5 fold) compared to the At4g35987 promoter (P87). The tissue specific as well as stress responsive functional nature of the bidirectional promoter was evaluated in independent transgenic Arabidopsis and tobacco lines. Expression of P85 activity was detected in the midrib of leaves, leaf trichomes, apical meristemic regions, throughout the root, lateral roots and flowers. The expression of P87 was observed in leaf-tip, hydathodes, apical meristem, root tips, emerging lateral root tips, root stele region and in floral tissues. The bidirectional promoter in both orientations shows differential up-regulation (2.5 to 3 fold) under salt stress. Use of such regulatory elements of bidirectional promoters showing spatial and stress inducible promoter-functions in heterologous system might be an important tool for plant biotechnology and gene stacking applications. PMID:24260266

[Effect of Leonurus Heterophyllus Sweet on tissue factor transcription and expression in human umbilical vein endothelial cells in vitro].

PubMed

Zheng, Lian; Fang, Chi-hua

2007-06-01

To investigate the effect of Leonurus Heterophyllus Sweet, (LHS) on tissue factor (TF) transcription and expression induced by thrombin in human umbilical vein endothelial cells (HUVECs). HUVECs were incubated with different concentrations of LHS and the TF mRNA expression was detected by reverse transcript-polymerase chain reaction (RT-PCR). LHS treatment of HUVECs at different concentrations and for different times resulted in significant differences in TF expression (Plt;0.01). The transcription of TF in LHS-treated cells was significantly different from that of the blank control group (Plt;0.01). LHS can decrease the expression of TF and intervene with TF transcription in HUVECs in vitro.

Transcriptome profiling of Kentucky bluegrass (Poa pratensis L.) accessions in response to salt stress.

PubMed

Bushman, B Shaun; Amundsen, Keenan L; Warnke, Scott E; Robins, Joseph G; Johnson, Paul G

2016-01-13

Kentucky bluegrass (Poa pratensis L.) is a prominent turfgrass in the cool-season regions, but it is sensitive to salt stress. Previously, a relatively salt tolerant Kentucky bluegrass accession was identified that maintained green colour under consistent salt applications. In this study, a transcriptome study between the tolerant (PI 372742) accession and a salt susceptible (PI 368233) accession was conducted, under control and salt treatments, and in shoot and root tissues. Sample replicates grouped tightly by tissue and treatment, and fewer differentially expressed transcripts were detected in the tolerant PI 372742 samples compared to the susceptible PI 368233 samples, and in root tissues compared to shoot tissues. A de novo assembly resulted in 388,764 transcripts, with 36,587 detected as differentially expressed. Approximately 75 % of transcripts had homology based annotations, with several differences in GO terms enriched between the PI 368233 and PI 372742 samples. Gene expression profiling identified salt-responsive gene families that were consistently down-regulated in PI 372742 and unlikely to contribute to salt tolerance in Kentucky bluegrass. Gene expression profiling also identified sets of transcripts relating to transcription factors, ion and water transport genes, and oxidation-reduction process genes with likely roles in salt tolerance. The transcript assembly represents the first such assembly in the highly polyploidy, facultative apomictic Kentucky bluegrass. The transcripts identified provide genetic information on how this plant responds to and tolerates salt stress in both shoot and root tissues, and can be used for further genetic testing and introgression.

Combined in vitro transcription and reverse transcription to amplify and label complex synthetic oligonucleotide probe libraries.

PubMed

Murgha, Yusuf; Beliveau, Brian; Semrau, Kassandra; Schwartz, Donald; Wu, Chao-Ting; Gulari, Erdogan; Rouillard, Jean-Marie

2015-06-01

Oligonucleotide microarrays allow the production of complex custom oligonucleotide libraries for nucleic acid detection-based applications such as fluorescence in situ hybridization (FISH). We have developed a PCR-free method to make single-stranded DNA (ssDNA) fluorescent probes through an intermediate RNA library. A double-stranded oligonucleotide library is amplified by transcription to create an RNA library. Next, dye- or hapten-conjugate primers are used to reverse transcribe the RNA to produce a dye-labeled cDNA library. Finally the RNA is hydrolyzed under alkaline conditions to obtain the single-stranded fluorescent probes library. Starting from unique oligonucleotide library constructs, we present two methods to produce single-stranded probe libraries. The two methods differ in the type of reverse transcription (RT) primer, the incorporation of fluorescent dye, and the purification of fluorescent probes. The first method employs dye-labeled reverse transcription primers to produce multiple differentially single-labeled probe subsets from one microarray library. The fluorescent probes are purified from excess primers by oligonucleotide-bead capture. The second method uses an RNA:DNA chimeric primer and amino-modified nucleotides to produce amino-allyl probes. The excess primers and RNA are hydrolyzed under alkaline conditions, followed by probe purification and labeling with amino-reactive dyes. The fluorescent probes created by the combination of transcription and reverse transcription can be used for FISH and to detect any RNA and DNA targets via hybridization.

The chimeric transcript RUNX1-GLRX5: a biomarker for good postoperative prognosis in Stage IA non-small-cell lung cancer.

PubMed

Ishikawa, Rie; Amano, Yosuke; Kawakami, Masanori; Sunohara, Mitsuhiro; Watanabe, Kousuke; Kage, Hidenori; Ohishi, Nobuya; Yatomi, Yutaka; Nakajima, Jun; Fukayama, Masashi; Nagase, Takahide; Takai, Daiya

2016-02-01

Stage IA non-small-cell lung cancer cases have been recognized as having a low risk of relapse; however, occasionally, relapse may occur. To predict clinical outcome in Stage IA non-small-cell lung cancer patients, we searched for chimeric transcripts that can be used as biomarkers and identified a novel chimeric transcript, RUNX1-GLRX5, comprising RUNX1, a transcription factor, and GLRX5. This chimera was detected in approximately half of the investigated Stage IA non-small-cell lung cancer patients (44/104 cases, 42.3%). Although there was no significant difference in the overall survival rate between RUNX1-GLRX5-positive and -negative cases (P = 0.088), a significantly lower relapse rate was observed in the RUNX1-GLRX5-positive cases (P = 0.039), indicating that this chimera can be used as a biomarker for good prognosis in Stage IA patients. Detection of the RUNX1-GLRX5 chimeric transcript may therefore be useful for the determination of a postoperative treatment plan for Stage IA non-small-cell lung cancer patients. © The Author 2015. Published by Oxford University Press.

Integrating alternative splicing detection into gene prediction.

PubMed

Foissac, Sylvain; Schiex, Thomas

2005-02-10

Alternative splicing (AS) is now considered as a major actor in transcriptome/proteome diversity and it cannot be neglected in the annotation process of a new genome. Despite considerable progresses in term of accuracy in computational gene prediction, the ability to reliably predict AS variants when there is local experimental evidence of it remains an open challenge for gene finders. We have used a new integrative approach that allows to incorporate AS detection into ab initio gene prediction. This method relies on the analysis of genomically aligned transcript sequences (ESTs and/or cDNAs), and has been implemented in the dynamic programming algorithm of the graph-based gene finder EuGENE. Given a genomic sequence and a set of aligned transcripts, this new version identifies the set of transcripts carrying evidence of alternative splicing events, and provides, in addition to the classical optimal gene prediction, alternative optimal predictions (among those which are consistent with the AS events detected). This allows for multiple annotations of a single gene in a way such that each predicted variant is supported by a transcript evidence (but not necessarily with a full-length coverage). This automatic combination of experimental data analysis and ab initio gene finding offers an ideal integration of alternatively spliced gene prediction inside a single annotation pipeline.

Differential expression of diacylglycerol acyltransferase (DGAT) genes in olive tissues.

PubMed

Giannoulia, K; Haralampidis, K; Poghosyan, Z; Murphy, D J; Hatzopoulos, P

2000-12-01

Fatty acids are accumulated in triacylglycerols (TAGs), in specialized organelles of seeds named oil bodies. The major site of TAG accumulation is detected in developing seed and mesocarp of certain species. We have isolated two cDNAs encoding DGAT enzymes from olives. The deduced polypeptides differ by 26 amino acids in size. However, they have high homology and almost identical hydropathy profiles. The DGAT gene is expressed in all tissues that synthesize TAGs. However, higher levels of DGAT transcripts have been detected in seed tissues of developing olive drupe. DGAT expression and mRNA accumulation in drupe tissues is developmentally regulated. Each DGAT transcript shows a distinct profile of accumulation. The existence of two different DGAT transcripts might reflect two different enzymes with discrete function and/or localization.

Annotation and sequence diversity of transposable elements in common bean (Phaseolus vulgaris).

PubMed

Gao, Dongying; Abernathy, Brian; Rohksar, Daniel; Schmutz, Jeremy; Jackson, Scott A

2014-01-01

Common bean (Phaseolus vulgaris) is an important legume crop grown and consumed worldwide. With the availability of the common bean genome sequence, the next challenge is to annotate the genome and characterize functional DNA elements. Transposable elements (TEs) are the most abundant component of plant genomes and can dramatically affect genome evolution and genetic variation. Thus, it is pivotal to identify TEs in the common bean genome. In this study, we performed a genome-wide transposon annotation in common bean using a combination of homology and sequence structure-based methods. We developed a 2.12-Mb transposon database which includes 791 representative transposon sequences and is available upon request or from www.phytozome.org. Of note, nearly all transposons in the database are previously unrecognized TEs. More than 5,000 transposon-related expressed sequence tags (ESTs) were detected which indicates that some transposons may be transcriptionally active. Two Ty1-copia retrotransposon families were found to encode the envelope-like protein which has rarely been identified in plant genomes. Also, we identified an extra open reading frame (ORF) termed ORF2 from 15 Ty3-gypsy families that was located between the ORF encoding the retrotransposase and the 3'LTR. The ORF2 was in opposite transcriptional orientation to retrotransposase. Sequence homology searches and phylogenetic analysis suggested that the ORF2 may have an ancient origin, but its function is not clear. These transposon data provide a useful resource for understanding the genome organization and evolution and may be used to identify active TEs for developing transposon-tagging system in common bean and other related genomes.

Natural variation in expression of genes associated with carotenoid biosynthesis and accumulation in cassava (Manihot esculenta Crantz) storage root.

PubMed

Carvalho, Luiz Jcb; Agustini, Marco Av; Anderson, James V; Vieira, Eduardo A; de Souza, Claudia Rb; Chen, Songbi; Schaal, Barbara A; Silva, Joseane P

2016-06-10

Cassava (Manihot esculenta Crantz) storage root provides a staple food source for millions of people worldwide. Increasing the carotenoid content in storage root of cassava could provide improved nutritional and health benefits. Because carotenoid accumulation has been associated with storage root color, this study characterized carotenoid profiles, and abundance of key transcripts associated with carotenoid biosynthesis, from 23 landraces of cassava storage root ranging in color from white-to-yellow-to-pink. This study provides important information to plant breeding programs aimed at improving cassava storage root nutritional quality. Among the 23 landraces, five carotenoid types were detected in storage root with white color, while carotenoid types ranged from 1 to 21 in storage root with pink and yellow color. The majority of storage root in these landraces ranged in color from pale-to-intense yellow. In this color group, total β-carotene, containing all-E-, 9-Z-, and 13-Z-β-carotene isomers, was the major carotenoid type detected, varying from 26.13 to 76.72 %. Although no α-carotene was observed, variable amounts of a α-ring derived xanthophyll, lutein, was detected; with greater accumulation of α-ring xanthophylls than of β-ring xanthophyll. Lycopene was detected in a landrace (Cas51) with pink color storage root, but it was not detected in storage root with yellow color. Based on microarray and qRT-PCR analyses, abundance of transcripts coding for enzymes involved in carotenoid biosynthesis were consistent with carotenoid composition determined by contrasting HPLC-Diode Array profiles from storage root of landraces IAC12, Cas64, and Cas51. Abundance of transcripts encoding for proteins regulating plastid division were also consistent with the observed differences in total β-carotene accumulation. Among the 23 cassava landraces with varying storage root color and diverse carotenoid types and profiles, landrace Cas51 (pink color storage root) had low LYCb transcript abundance, whereas landrace Cas64 (intense yellow storage root) had decreased HYb transcript abundance. These results may explain the increased amounts of lycopene and total β-carotene observed in landraces Cas51 and Cas64, respectively. Overall, total carotenoid content in cassava storage root of color class representatives were associated with spatial patterns of secondary growth, color, and abundance of transcripts linked to plastid division. Finally, a partial carotenoid biosynthesis pathway is proposed.

Effects of Orientation and Weatherproofing on the Detection of Bat Echolocation Calls

Treesearch

E. Britzke; B. Slack; M Armstrong; S. Loeb

2010-01-01

Ultrasonic detectors are powerful tools for the study of bat ecology. Many options are available for deploying acoustic detectors including various weatherproofing designs and microphone orientations, but the impacts of these options on the quantity and quality of the bat calls that are recorded are unknown. We compared the impacts of three microphone orientations (...

Transcription profile of boar spermatozoa as revealed by RNA-sequencing

USDA-ARS?s Scientific Manuscript database

High-throughput RNA sequencing (RNA-Seq) overcomes the limitations of the current hybridization-based techniques to detect the actual pool of RNA transcripts in spermatozoa. The application of this technology in livestock can speed the discovery of potential predictors of male fertility. As a first ...

Microarray Detection Call Methodology as a Means to Identify and Compare Transcripts Expressed within Syncytial Cells from Soybean (Glycine max) Roots Undergoing Resistant and Susceptible Reactions to the Soybean Cyst Nematode (Heterodera glycines)

PubMed Central

Klink, Vincent P.; Overall, Christopher C.; Alkharouf, Nadim W.; MacDonald, Margaret H.; Matthews, Benjamin F.

2010-01-01

Background. A comparative microarray investigation was done using detection call methodology (DCM) and differential expression analyses. The goal was to identify genes found in specific cell populations that were eliminated by differential expression analysis due to the nature of differential expression methods. Laser capture microdissection (LCM) was used to isolate nearly homogeneous populations of plant root cells. Results. The analyses identified the presence of 13,291 transcripts between the 4 different sample types. The transcripts filtered down into a total of 6,267 that were detected as being present in one or more sample types. A comparative analysis of DCM and differential expression methods showed a group of genes that were not differentially expressed, but were expressed at detectable amounts within specific cell types. Conclusion. The DCM has identified patterns of gene expression not shown by differential expression analyses. DCM has identified genes that are possibly cell-type specific and/or involved in important aspects of plant nematode interactions during the resistance response, revealing the uniqueness of a particular cell population at a particular point during its differentiation process. PMID:20508855

Detection and characterization of latent HSV RNA by in situ and northern blot hybridization in guinea pigs.

PubMed

Burke, R L; Hartog, K; Croen, K D; Ostrove, J M

1991-04-01

Following intravaginal infection of guinea pigs, herpes simplex virus establishes a latent infection in the sensory lumbosacral ganglia. Using the techniques of in situ and Northern blot hybridization, we have characterized this latent HSV-2 virus and compared it to latent HSV-1 at the same anatomical site. For HSV-2, a single 1.8-kb latency-associated transcript (LAT) was detected. In contrast, as described for latent HSV-1 in the trigeminal ganglia of rabbits and mice, two HSV-1 LAT species were detected in the lumbosacral ganglia, an abundant transcript of 1.8 kb and a less abundant transcript of 1.55 kb. Despite these differences in LAT expression, the clinical course of the acute and recurrent genital disease was similar for both viruses. LAT was detected in 0.3-6.0% of the sensory neurons of sacral but not in lumbar ganglia. The abundance of LAT correlated with the severity of the initial infection, but not with the frequency of recurrent disease. Thus, vaccination strategies that substantially reduced or eliminated symptomatic disease following challenge infection appeared to block the establishment of a latent infection.

Isolation of genes negatively or positively co-expressed with human recombination activating gene 1 (RAG1) by differential display PCR (DD RT-PCR).

PubMed

Verkoczy, L K; Berinstein, N L

1998-10-01

Differential display PCR (DD RT-PCR) has been extensively used for analysis of differential gene expression, but continues to be hampered by technical limitations that impair its effectiveness. In order to isolate novel genes co-expressing with human RAG1, we have developed an effective, multi-tiered screening/purification approach which effectively complements the standard DD RT-PCR methodology. In 'primary' screens, standard DD RT-PCR was used, detecting 22 reproducible differentially expressed amplicons between clonally related cell variants with differential constitutive expression of RAG mRNAs. 'Secondary' screens used differential display (DD) amplicons as probes in low and high stringency northern blotting. Eight of 22 independent DD amplicons detected nine independent differentially expressed transcripts. 'Tertiary' screens used reconfirmed amplicons as probes in northern analysis of multiple RAG-and RAG+sources. Reconfirmed DD amplicons detected six independent RAG co-expressing transcripts. All DD amplicons reconfirmed by northern blot were a heterogeneous mixture of cDNAs, necessitating further purification to isolate single cDNAs prior to subcloning and sequencing. To effectively select the appropriate cDNAs from DD amplicons, we excised and eluted the cDNA(s) directly from regions of prior northern blots in which differentially expressed transcripts were detected. Sequences of six purified cDNA clones specifically detecting RAG co-expressing transcripts included matches to portions of the human RAG2 and BSAP regions and to four novel partial cDNAs (three with homologies to human ESTs). Overall, our results also suggest that even when using clonally related variants from the same cell line in addition to all appropriate internal controls previously reported, further screening and purification steps are still required in order to efficiently and specifically isolate differentially expressed genes by DD RT-PCR.

Molecular detection of a novel paramyxovirus in fruit bats from Indonesia

PubMed Central

2012-01-01

Background Fruit bats are known to harbor zoonotic paramyxoviruses including Nipah, Hendra, and Menangle viruses. The aim of this study was to detect the presence of paramyxovirus RNA in fruit bats from Indonesia. Methods RNA samples were obtained from the spleens of 110 fruit bats collected from four locations in Indonesia. All samples were screened by semi-nested broad spectrum reverse transcription PCR targeting the paramyxovirus polymerase (L) genes. Results Semi-nested reverse transcription PCR detected five previously unidentified paramyxoviruses from six fruit bats. Phylogenetic analysis showed that these virus sequences were related to henipavirus or rubulavirus. Conclusions This study indicates the presence of novel paramyxoviruses among fruit bat populations in Indonesia. PMID:23082748

Activation of both acfA and acfD transcription by Vibrio cholerae ToxT requires binding to two centrally located DNA sites in an inverted repeat conformation.

PubMed

Withey, Jeffrey H; DiRita, Victor J

2005-05-01

The Gram-negative bacterium Vibrio cholerae is the infectious agent responsible for the disease Asiatic cholera. The genes required for V. cholerae virulence, such as those encoding the cholera toxin (CT) and toxin-coregulated pilus (TCP), are controlled by a cascade of transcriptional activators. Ultimately, the direct transcriptional activator of the majority of V. cholerae virulence genes is the AraC/XylS family member ToxT protein, the expression of which is activated by the ToxR and TcpP proteins. Previous studies have identified the DNA sites to which ToxT binds upstream of the ctx operon, encoding CT, and the tcpA operon, encoding, among other products, the major subunit of the TCP. These known ToxT binding sites are seemingly dissimilar in sequence other than being A/T rich. Further results suggested that ctx and tcpA each has a pair of ToxT binding sites arranged in a direct repeat orientation upstream of the core promoter elements. In this work, using both transcriptional lacZ fusions and in vitro copper-phenanthroline footprinting experiments, we have identified the ToxT binding sites between the divergently transcribed acfA and acfD genes, which encode components of the accessory colonization factor required for efficient intestinal colonization by V. cholerae. Our results indicate that ToxT binds to a pair of DNA sites between acfA and acfD in an inverted repeat orientation. Moreover, a mutational analysis of the ToxT binding sites indicates that both binding sites are required by ToxT for transcriptional activation of both acfA and acfD. Using copper-phenanthroline footprinting to assess the occupancy of ToxT on DNA having mutations in one of these binding sites, we found that protection by ToxT of the unaltered binding site was not affected, whereas protection by ToxT of the mutant binding site was significantly reduced in the region of the mutations. The results of further footprinting experiments using DNA templates having +5 bp and +10 bp insertions between the two ToxT binding sites indicate that both binding sites are occupied by ToxT regardless of their positions relative to each other. Based on these results, we propose that ToxT binds independently to two DNA sites between acfA and acfD to activate transcription of both genes.

Rapid and sensitive detection of Lily symptomless virus by reverse transcription loop-mediated isothermal amplification.

PubMed

He, Xiangfeng; Xue, Fei; Xu, Shufa; Wang, Wenhe

2016-12-01

Lily symptomless virus (LSV) is one of the most prevalent viruses that infect lily plants worldwide. A rapid and sensitive reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for detection of LSV, using two primer pairs that specifically amplified the conserved sequence of LSV coat protein. The optimum reaction conditions were as follows: 4mM MgCl 2 and 0.8M betaine with incubation at 64°C for 30min. The limit of detection of LSV from infected lily leaves was 10-fold higher for RT-LAMP than for conventional RT-PCR. Moreover, RT-LAMP detected LSV in not only symptomatic, but also in symptomless tissues of infected plants. These findings indicate that our RT-LAMP method for LSV can serve as a low-cost, simple, and rapid alternative to conventional detection assays. Copyright © 2016 Elsevier B.V. All rights reserved.

A Dictyostelium mutant deficient in severin, an F-actin fragmenting protein, shows normal motility and chemotaxis

PubMed Central

1989-01-01

A severin deficient mutant of Dictyostelium discoideum has been isolated by the use of colony immunoblotting after chemical mutagenesis. In homogenates of wild-type cells, severin is easily detected as a very active F-actin fragmenting protein. Tests for severin in the mutant, HG1132, included viscometry for the assay of F- actin fragmentation in fractions from DEAE-cellulose columns, labeling of blots with monoclonal and polyclonal antibodies, and immunofluorescent-labeling of cryosections. Severin could not be detected in the mutant using these methods. The mutation in HG1132 is recessive and has been mapped to linkage group VII. The mutant failed to produce the normal severin mRNA, but small amounts of a transcript that was approximately 100 bases larger than the wild-type mRNA were detected in the mutant throughout all stages of development. On the DNA level a new Mbo II restriction site was found in the mutant within the coding region of the severin gene. The severin deficient mutant cells grew at an approximately normal rate, aggregated and formed fruiting bodies with viable spores. By the use of an image processing system, speed of cell movement, turning rates, and precision of chemotactic orientation in a stable gradient of cyclic AMP were quantitated, and no significant differences between wild-type and mutant cells were found. Thus, under the culture conditions used, severin proved to be neither essential for growth of D. discoideum nor for any cell function that is important for aggregation or later development. PMID:2537840

Variations in the non-coding transcriptome as a driver of inter-strain divergence and physiological adaptation in bacteria.

PubMed

Kopf, Matthias; Klähn, Stephan; Scholz, Ingeborg; Hess, Wolfgang R; Voß, Björn

2015-04-22

In all studied organisms, a substantial portion of the transcriptome consists of non-coding RNAs that frequently execute regulatory functions. Here, we have compared the primary transcriptomes of the cyanobacteria Synechocystis sp. PCC 6714 and PCC 6803 under 10 different conditions. These strains share 2854 protein-coding genes and a 16S rRNA identity of 99.4%, indicating their close relatedness. Conserved major transcriptional start sites (TSSs) give rise to non-coding transcripts within the sigB gene, from the 5'UTRs of cmpA and isiA, and 168 loci in antisense orientation. Distinct differences include single nucleotide polymorphisms rendering promoters inactive in one of the strains, e.g., for cmpR and for the asRNA PsbA2R. Based on the genome-wide mapped location, regulation and classification of TSSs, non-coding transcripts were identified as the most dynamic component of the transcriptome. We identified a class of mRNAs that originate by read-through from an sRNA that accumulates as a discrete and abundant transcript while also serving as the 5'UTR. Such an sRNA/mRNA structure, which we name 'actuaton', represents another way for bacteria to remodel their transcriptional network. Our findings support the hypothesis that variations in the non-coding transcriptome constitute a major evolutionary element of inter-strain divergence and capability for physiological adaptation.

Transcriptional interference networks coordinate the expression of functionally related genes clustered in the same genomic loci

PubMed Central

Boldogköi, Zsolt

2012-01-01

The regulation of gene expression is essential for normal functioning of biological systems in every form of life. Gene expression is primarily controlled at the level of transcription, especially at the phase of initiation. Non-coding RNAs are one of the major players at every level of genetic regulation, including the control of chromatin organization, transcription, various post-transcriptional processes, and translation. In this study, the Transcriptional Interference Network (TIN) hypothesis was put forward in an attempt to explain the global expression of antisense RNAs and the overall occurrence of tandem gene clusters in the genomes of various biological systems ranging from viruses to mammalian cells. The TIN hypothesis suggests the existence of a novel layer of genetic regulation, based on the interactions between the transcriptional machineries of neighboring genes at their overlapping regions, which are assumed to play a fundamental role in coordinating gene expression within a cluster of functionally linked genes. It is claimed that the transcriptional overlaps between adjacent genes are much more widespread in genomes than is thought today. The Waterfall model of the TIN hypothesis postulates a unidirectional effect of upstream genes on the transcription of downstream genes within a cluster of tandemly arrayed genes, while the Seesaw model proposes a mutual interdependence of gene expression between the oppositely oriented genes. The TIN represents an auto-regulatory system with an exquisitely timed and highly synchronized cascade of gene expression in functionally linked genes located in close physical proximity to each other. In this study, we focused on herpesviruses. The reason for this lies in the compressed nature of viral genes, which allows a tight regulation and an easier investigation of the transcriptional interactions between genes. However, I believe that the same or similar principles can be applied to cellular organisms too. PMID:22783276

Transcriptional interference networks coordinate the expression of functionally related genes clustered in the same genomic loci.

PubMed

Boldogköi, Zsolt

2012-01-01

The regulation of gene expression is essential for normal functioning of biological systems in every form of life. Gene expression is primarily controlled at the level of transcription, especially at the phase of initiation. Non-coding RNAs are one of the major players at every level of genetic regulation, including the control of chromatin organization, transcription, various post-transcriptional processes, and translation. In this study, the Transcriptional Interference Network (TIN) hypothesis was put forward in an attempt to explain the global expression of antisense RNAs and the overall occurrence of tandem gene clusters in the genomes of various biological systems ranging from viruses to mammalian cells. The TIN hypothesis suggests the existence of a novel layer of genetic regulation, based on the interactions between the transcriptional machineries of neighboring genes at their overlapping regions, which are assumed to play a fundamental role in coordinating gene expression within a cluster of functionally linked genes. It is claimed that the transcriptional overlaps between adjacent genes are much more widespread in genomes than is thought today. The Waterfall model of the TIN hypothesis postulates a unidirectional effect of upstream genes on the transcription of downstream genes within a cluster of tandemly arrayed genes, while the Seesaw model proposes a mutual interdependence of gene expression between the oppositely oriented genes. The TIN represents an auto-regulatory system with an exquisitely timed and highly synchronized cascade of gene expression in functionally linked genes located in close physical proximity to each other. In this study, we focused on herpesviruses. The reason for this lies in the compressed nature of viral genes, which allows a tight regulation and an easier investigation of the transcriptional interactions between genes. However, I believe that the same or similar principles can be applied to cellular organisms too.

Optimization of oligonucleotide arrays and RNA amplification protocols for analysis of transcript structure and alternative splicing.

PubMed

Castle, John; Garrett-Engele, Phil; Armour, Christopher D; Duenwald, Sven J; Loerch, Patrick M; Meyer, Michael R; Schadt, Eric E; Stoughton, Roland; Parrish, Mark L; Shoemaker, Daniel D; Johnson, Jason M

2003-01-01

Microarrays offer a high-resolution means for monitoring pre-mRNA splicing on a genomic scale. We have developed a novel, unbiased amplification protocol that permits labeling of entire transcripts. Also, hybridization conditions, probe characteristics, and analysis algorithms were optimized for detection of exons, exon-intron edges, and exon junctions. These optimized protocols can be used to detect small variations and isoform mixtures, map the tissue specificity of known human alternative isoforms, and provide a robust, scalable platform for high-throughput discovery of alternative splicing.

Optimization of oligonucleotide arrays and RNA amplification protocols for analysis of transcript structure and alternative splicing

PubMed Central

Castle, John; Garrett-Engele, Phil; Armour, Christopher D; Duenwald, Sven J; Loerch, Patrick M; Meyer, Michael R; Schadt, Eric E; Stoughton, Roland; Parrish, Mark L; Shoemaker, Daniel D; Johnson, Jason M

2003-01-01

Microarrays offer a high-resolution means for monitoring pre-mRNA splicing on a genomic scale. We have developed a novel, unbiased amplification protocol that permits labeling of entire transcripts. Also, hybridization conditions, probe characteristics, and analysis algorithms were optimized for detection of exons, exon-intron edges, and exon junctions. These optimized protocols can be used to detect small variations and isoform mixtures, map the tissue specificity of known human alternative isoforms, and provide a robust, scalable platform for high-throughput discovery of alternative splicing. PMID:14519201

Improved Sectional Image Analysis Technique for Evaluating Fiber Orientations in Fiber-Reinforced Cement-Based Materials.

PubMed

Lee, Bang Yeon; Kang, Su-Tae; Yun, Hae-Bum; Kim, Yun Yong

2016-01-12

The distribution of fiber orientation is an important factor in determining the mechanical properties of fiber-reinforced concrete. This study proposes a new image analysis technique for improving the evaluation accuracy of fiber orientation distribution in the sectional image of fiber-reinforced concrete. A series of tests on the accuracy of fiber detection and the estimation performance of fiber orientation was performed on artificial fiber images to assess the validity of the proposed technique. The validation test results showed that the proposed technique estimates the distribution of fiber orientation more accurately than the direct measurement of fiber orientation by image analysis.

Improved Sectional Image Analysis Technique for Evaluating Fiber Orientations in Fiber-Reinforced Cement-Based Materials

PubMed Central

Lee, Bang Yeon; Kang, Su-Tae; Yun, Hae-Bum; Kim, Yun Yong

2016-01-01

The distribution of fiber orientation is an important factor in determining the mechanical properties of fiber-reinforced concrete. This study proposes a new image analysis technique for improving the evaluation accuracy of fiber orientation distribution in the sectional image of fiber-reinforced concrete. A series of tests on the accuracy of fiber detection and the estimation performance of fiber orientation was performed on artificial fiber images to assess the validity of the proposed technique. The validation test results showed that the proposed technique estimates the distribution of fiber orientation more accurately than the direct measurement of fiber orientation by image analysis. PMID:28787839

SP10 Infectivity Is Aborted after Bacteriophage SP10 Infection Induces nonA Transcription on the Prophage SPβ Region of the Bacillus subtilis Genome

PubMed Central

Yamamoto, Tatsuya; Obana, Nozomu; Yee, Lii Mien; Asai, Kei; Nomura, Nobuhiko

2014-01-01

Bacteria have developed various strategies for phage resistance. Infection with phage induces the transcription of part of the phage resistance gene, but the regulatory mechanisms of such transcription remain largely unknown. The phage resistance gene nonA is located on the SPβ prophage region of the Bacillus subtilis Marburg strain genome. The nonA transcript was detected at the late stage of SP10 infection but is undetectable in noninfected cells. The nonA transcript was detected after the induction of the sigma factor Orf199-Orf200 (σOrf199-200), when sigma factors encoded in the SP10 genome were expressed from a xylose-inducible plasmid. Thus, the SP10 sigma factor is an activator of a set of SP10 genes and nonA. The nonA gene encodes a 72-amino-acid protein with a transmembrane motif and has no significant homology with any protein in any database. NonA overexpression halted cell growth and reduced the efficiency of B. subtilis colony formation and respiration activity. In addition, SP10 virion protein synthesis was inhibited in the nonA+ strain, and SP10 virion particles were scarce in it. These results indicate that NonA is a novel protein that can abort SP10 infection, and its transcription was regulated by SP10 sigma factor. PMID:24272782

Patterns of Positive Selection of the Myogenic Regulatory Factor Gene Family in Vertebrates

PubMed Central

Zhao, Xiao; Yu, Qi; Huang, Ling; Liu, Qing-Xin

2014-01-01

The functional divergence of transcriptional factors is critical in the evolution of transcriptional regulation. However, the mechanism of functional divergence among these factors remains unclear. Here, we performed an evolutionary analysis for positive selection in members of the myogenic regulatory factor (MRF) gene family of vertebrates. We selected 153 complete vertebrate MRF nucleotide sequences from our analyses, which revealed substantial evidence of positive selection. Here, we show that sites under positive selection were more frequently detected and identified from the genes encoding the myogenic differentiation factors (MyoG and Myf6) than the genes encoding myogenic determination factors (Myf5 and MyoD). Additionally, the functional divergence within the myogenic determination factors or differentiation factors was also under positive selection pressure. The positive selection sites were more frequently detected from MyoG and MyoD than Myf6 and Myf5, respectively. Amino acid residues under positive selection were identified mainly in their transcription activation domains and on the surface of protein three-dimensional structures. These data suggest that the functional gain and divergence of myogenic regulatory factors were driven by distinct positive selection of their transcription activation domains, whereas the function of the DNA binding domains was conserved in evolution. Our study evaluated the mechanism of functional divergence of the transcriptional regulation factors within a family, whereby the functions of their transcription activation domains diverged under positive selection during evolution. PMID:24651579

Examination of soil effect upon GPR detectability of landmine with different orientations

NASA Astrophysics Data System (ADS)

Ebrahim, Shereen M.; Medhat, N. I.; Mansour, Khamis K.; Gaber, A.

2018-06-01

Landmines represent a serious environmental problem for several countries as it causes severe injured and many victims. In this paper, the response of GPR from different parameters of the landmine targets has been shown and the data is correlated with observed field experiment made in 2012 at Miami Crandon Park test site. The ability of GPR for detecting non-metallic mines with different orientations was revealed and soil effect upon the GPR signal was examined putting into consideration the soil parameters in different locations in Egypt such as in Sinai and El Alamein. The simulation results showed that PMN-2 landmine was detected at 5 cm and 15 cm depths, even at the minimum radar cross section vertical orientation. The B-Scan (2D GPR profiles) of PMN-2 target at 15 cm depth figured out high reflectivity for Wadi deposits due to large contrast between PMN-2 landmine material and soil of sand dunes.

Epistolary and Expository Interaction Patterns in a Computer Conference Transcript.

ERIC Educational Resources Information Center

Fahy, Patrick J.

2002-01-01

Discusses the relationship of gender and discourse types, including epistolary and expository, in computer-mediated communication such as listservs. Describes a study that used transcript analysis to determine whether gender patterns could be detected in an online graduate course and considers the strategic value of discourse styles in group…

Evidence for the Involvement of Xenobiotic-responsive Nuclear Receptors in Transcriptional Effects Upon Perfluoroalkyl Acid Exposure in Diverse Species.

EPA Science Inventory

Humans and other species have detectable body burdens of a number of perfluorinated alkyl acids (PFAA) including perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS). In mouse and rat liver these compounds elicit transcriptional and phenotypic effects similar to pe...

Evidence for the Involvement of Xenobiotic-response Nuclear Receptors in Transcriptional Effects Upon Perfluroroalkyl Acid Exposure in Diverse Species

EPA Science Inventory

Humans and ecological species have been found to have detectable body burdens of a number of perfluorinated alkyl acids (PFAA) including perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS). In mouse and rat liver these compounds elicit transcriptional and phenotyp...

A Novel PCR Assay for Listeria welshimeri Targeting Transcriptional Regulator Gene lwe1801

USDA-ARS?s Scientific Manuscript database

Transcriptional regulator genes encode a group of specialized molecules that play essential roles in microbial responses to changing external conditions. These genes have been shown to possess species or group specificity and are useful as detection targets for diagnostic application. The present st...

Role of Setbp1 in Myeloid Leukemia Development

DTIC Science & Technology

2014-09-05

CTC ACT GTG GAG ACG ATT C 3’ 5’ TTC TTA TCC AGC ACA CCA AGC TT 3’ Hoxa9 5’ TGT CTC CTC TCC CCC AAA CC 3’ 5’ GAG ATG AGG CCT GGG ATTTAG A 3’ Hoxa10...highlighted in yellow and noncoding exons in gray . Arrows indicate transcription start sites. The location and orientation of viral integrations are

Structures of ω repressors bound to direct and inverted DNA repeats explain modulation of transcription

PubMed Central

Weihofen, Wilhelm Andreas; Cicek, Aslan; Pratto, Florencia; Alonso, Juan Carlos; Saenger, Wolfram

2006-01-01

Repressor ω regulates transcription of genes required for copy number control, accurate segregation and stable maintenance of inc18 plasmids hosted by Gram-positive bacteria. ω belongs to homodimeric ribbon-helix-helix (RHH2) repressors typified by a central, antiparallel β-sheet for DNA major groove binding. Homodimeric ω2 binds cooperatively to promotors with 7 to 10 consecutive non-palindromic DNA heptad repeats (5′-A/TATCACA/T-3′, symbolized by →) in palindromic inverted, converging (→←) or diverging (←→) orientation and also, unique to ω2 and contrasting other RHH2 repressors, to non-palindromic direct (→→) repeats. Here we investigate with crystal structures how ω2 binds specifically to heptads in minimal operators with (→→) and (→←) repeats. Since the pseudo-2-fold axis relating the monomers in ω2 passes the central C–G base pair of each heptad with ∼0.3 Å downstream offset, the separation between the pseudo-2-fold axes is exactly 7 bp in (→→), ∼0.6 Å shorter in (→←) but would be ∼0.6 Å longer in (←→). These variations grade interactions between adjacent ω2 and explain modulations in cooperative binding affinity of ω2 to operators with different heptad orientations. PMID:16528102

Dual Regulation of Bacillus subtilis kinB Gene Encoding a Sporulation Trigger by SinR through Transcription Repression and Positive Stringent Transcription Control.

PubMed

Fujita, Yasutaro; Ogura, Mitsuo; Nii, Satomi; Hirooka, Kazutake

2017-01-01

It is known that transcription of kinB encoding a trigger for Bacillus subtilis sporulation is under repression by SinR, a master repressor of biofilm formation, and under positive stringent transcription control depending on the adenine species at the transcription initiation nucleotide (nt). Deletion and base substitution analyses of the kinB promoter (P kinB ) region using lacZ fusions indicated that either a 5-nt deletion (Δ5, nt -61/-57, +1 is the transcription initiation nt) or the substitution of G at nt -45 with A (G-45A) relieved kinB repression. Thus, we found a pair of SinR-binding consensus sequences (GTTCTYT; Y is T or C) in an inverted orientation (SinR-1) between nt -57/-42, which is most likely a SinR-binding site for kinB repression. This relief from SinR repression likely requires SinI, an antagonist of SinR. Surprisingly, we found that SinR is essential for positive stringent transcription control of P kinB . Electrophoretic mobility shift assay (EMSA) analysis indicated that SinR bound not only to SinR-1 but also to SinR-2 (nt -29/-8) consisting of another pair of SinR consensus sequences in a tandem repeat arrangement; the two sequences partially overlap the '-35' and '-10' regions of P kinB . Introduction of base substitutions (T-27C C-26T) in the upstream consensus sequence of SinR-2 affected positive stringent transcription control of P kinB , suggesting that SinR binding to SinR-2 likely causes this positive control. EMSA also implied that RNA polymerase and SinR are possibly bound together to SinR-2 to form a transcription initiation complex for kinB transcription. Thus, it was suggested in this work that derepression of kinB from SinR repression by SinI induced by Spo0A∼P and occurrence of SinR-dependent positive stringent transcription control of kinB might induce effective sporulation cooperatively, implying an intimate interplay by stringent response, sporulation, and biofilm formation.

Robust control of mitotic spindle orientation in the developing epidermis

PubMed Central

Poulson, Nicholas D.

2010-01-01

Progenitor cells must balance self-amplification and production of differentiated progeny during development and homeostasis. In the epidermis, progenitors divide symmetrically to increase surface area and asymmetrically to promote stratification. In this study, we show that individual epidermal cells can undergo both types of division, and therefore, the balance is provided by the sum of individual cells’ choices. In addition, we define two control points for determining a cell’s mode of division. First is the expression of the mouse Inscuteable gene, which is sufficient to drive asymmetric cell division (ACD). However, there is robust control of division orientation as excessive ACDs are prevented by a change in the localization of NuMA, an effector of spindle orientation. Finally, we show that p63, a transcriptional regulator of stratification, does not control either of these processes. These data have uncovered two important regulatory points controlling ACD in the epidermis and allow a framework for analysis of how external cues control this important choice. PMID:21098114

CRISPR-spacer integration reporter plasmids reveal distinct genuine acquisition specificities among CRISPR-Cas I-E variants of Escherichia coli

PubMed Central

Díez-Villaseñor, César; Guzmán, Noemí M.; Almendros, Cristóbal; García-Martínez, Jesús; Mojica, Francisco J.M.

2013-01-01

Prokaryotes immunize themselves against transmissible genetic elements by the integration (acquisition) in clustered regularly interspaced short palindromic repeats (CRISPR) loci of spacers homologous to invader nucleic acids, defined as protospacers. Following acquisition, mono-spacer CRISPR RNAs (termed crRNAs) guide CRISPR-associated (Cas) proteins to degrade (interference) protospacers flanked by an adjacent motif in extrachomosomal DNA. During acquisition, selection of spacer-precursors adjoining the protospacer motif and proper orientation of the integrated fragment with respect to the leader (sequence leading transcription of the flanking CRISPR array) grant efficient interference by at least some CRISPR-Cas systems. This adaptive stage of the CRISPR action is poorly characterized, mainly due to the lack of appropriate genetic strategies to address its study and, at least in Escherichia coli, the need of Cas overproduction for insertion detection. In this work, we describe the development and application in Escherichia coli strains of an interference-independent assay based on engineered selectable CRISPR-spacer integration reporter plasmids. By using this tool without the constraint of interference or cas overexpression, we confirmed fundamental aspects of this process such as the critical requirement of Cas1 and Cas2 and the identity of the CTT protospacer motif for the E. coli K12 system. In addition, we defined the CWT motif for a non-K12 CRISPR-Cas variant, and obtained data supporting the implication of the leader in spacer orientation, the preferred acquisition from plasmids harboring cas genes and the occurrence of a sequential cleavage at the insertion site by a ruler mechanism. PMID:23445770

CRISPR-spacer integration reporter plasmids reveal distinct genuine acquisition specificities among CRISPR-Cas I-E variants of Escherichia coli.

PubMed

Díez-Villaseñor, César; Guzmán, Noemí M; Almendros, Cristóbal; García-Martínez, Jesús; Mojica, Francisco J M

2013-05-01

Prokaryotes immunize themselves against transmissible genetic elements by the integration (acquisition) in clustered regularly interspaced short palindromic repeats (CRISPR) loci of spacers homologous to invader nucleic acids, defined as protospacers. Following acquisition, mono-spacer CRISPR RNAs (termed crRNAs) guide CRISPR-associated (Cas) proteins to degrade (interference) protospacers flanked by an adjacent motif in extrachomosomal DNA. During acquisition, selection of spacer-precursors adjoining the protospacer motif and proper orientation of the integrated fragment with respect to the leader (sequence leading transcription of the flanking CRISPR array) grant efficient interference by at least some CRISPR-Cas systems. This adaptive stage of the CRISPR action is poorly characterized, mainly due to the lack of appropriate genetic strategies to address its study and, at least in Escherichia coli, the need of Cas overproduction for insertion detection. In this work, we describe the development and application in Escherichia coli strains of an interference-independent assay based on engineered selectable CRISPR-spacer integration reporter plasmids. By using this tool without the constraint of interference or cas overexpression, we confirmed fundamental aspects of this process such as the critical requirement of Cas1 and Cas2 and the identity of the CTT protospacer motif for the E. coli K12 system. In addition, we defined the CWT motif for a non-K12 CRISPR-Cas variant, and obtained data supporting the implication of the leader in spacer orientation, the preferred acquisition from plasmids harboring cas genes and the occurrence of a sequential cleavage at the insertion site by a ruler mechanism.

Occupancy of RNA Polymerase II Phosphorylated on Serine 5 (RNAP S5P) and RNAP S2P on Varicella-Zoster Virus Genes 9, 51, and 66 Is Independent of Transcript Abundance and Polymerase Location within the Gene.

PubMed

Henderson, Heather H; Timberlake, Kensey B; Austin, Zoe A; Badani, Hussain; Sanford, Bridget; Tremblay, Keriann; Baird, Nicholas L; Jones, Kenneth; Rovnak, Joel; Frietze, Seth; Gilden, Don; Cohrs, Randall J

2016-02-01

Regulation of gene transcription in varicella-zoster virus (VZV), a ubiquitous human neurotropic alphaherpesvirus, requires coordinated binding of multiple host and virus proteins onto specific regions of the virus genome. Chromatin immunoprecipitation (ChIP) is widely used to determine the location of specific proteins along a genomic region. Since the size range of sheared virus DNA fragments governs the limit of accurate protein localization, particularly for compact herpesvirus genomes, we used a quantitative PCR (qPCR)-based assay to determine the efficiency of VZV DNA shearing before ChIP, after which the assay was used to determine the relationship between transcript abundance and the occupancy of phosphorylated RNA polymerase II (RNAP) on the gene promoter, body, and terminus of VZV genes 9, 51, and 66. The abundance of VZV gene 9, 51, and 66 transcripts in VZV-infected human fetal lung fibroblasts was determined by reverse transcription-linked quantitative PCR. Our results showed that the C-terminal domain of RNAP is hyperphosphorylated at serine 5 (S5(P)) on VZV genes 9, 51, and 66 independently of transcript abundance and the location within the virus gene at both 1 and 3 days postinfection (dpi). In contrast, phosphorylated serine 2 (S2(P))-modified RNAP was not detected at any virus gene location at 3 dpi and was detected at levels only slightly above background levels at 1 dpi. Regulation of herpesvirus gene transcription is an elaborate choreography between proteins and DNA that is revealed by chromatin immunoprecipitation (ChIP). We used a quantitative PCR-based assay to determine fragment size after DNA shearing, a critical parameter in ChIP assays, and exposed a basic difference in the mechanism of transcription between mammalian cells and VZV. We found that hyperphosphorylation at serine 5 of the C-terminal domain of RNAP along the lengths of VZV genes (the promoter, body, and transcription termination site) was independent of mRNA abundance. In contrast, little to no enrichment of serine 3 phosphorylation of RNAP was detected at these virus gene regions. This is distinct from the findings for RNAP at highly regulated host genes, where RNAP S5(P) occupancy decreased and S2(P) levels increased as the polymerase transited through the gene. Overall, these results suggest that RNAP associates with human and virus transcriptional units through different mechanisms. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Visual detection of Ebola virus using reverse transcription loop-mediated isothermal amplification combined with nucleic acid strip detection.

PubMed

Xu, Changping; Wang, Hualei; Jin, Hongli; Feng, Na; Zheng, Xuexing; Cao, Zengguo; Li, Ling; Wang, Jianzhong; Yan, Feihu; Wang, Lina; Chi, Hang; Gai, Weiwei; Wang, Chong; Zhao, Yongkun; Feng, Yan; Wang, Tiecheng; Gao, Yuwei; Lu, Yiyu; Yang, Songtao; Xia, Xianzhu

2016-05-01

Ebola virus (species Zaire ebolavirus) (EBOV) is highly virulent in humans. The largest recorded outbreak of Ebola hemorrhagic fever in West Africa to date was caused by EBOV. Therefore, it is necessary to develop a detection method for this virus that can be easily distributed and implemented. In the current study, we developed a visual assay that can detect EBOV-associated nucleic acids. This assay combines reverse transcription loop-mediated isothermal amplification and nucleic acid strip detection (RT-LAMP-NAD). Nucleic acid amplification can be achieved in a one-step process at a constant temperature (58 °C, 35 min), and the amplified products can be visualized within 2-5 min using a nucleic acid strip detection device. The assay is capable of detecting 30 copies of artificial EBOV glycoprotein (GP) RNA and RNA encoding EBOV GP from 10(2) TCID50 recombinant viral particles per ml with high specificity. Overall, the RT-LAMP-NAD method is simple and has high sensitivity and specificity; therefore, it is especially suitable for the rapid detection of EBOV in African regions.

Internally Controlled, Multiplex Real-Time Reverse Transcription PCR for Dengue Virus and Yellow Fever Virus Detection.

PubMed

Rojas, Alejandra; Diagne, Cheikh T; Stittleburg, Victoria D; Mohamed-Hadley, Alisha; de Guillén, Yvalena Arévalo; Balmaseda, Angel; Faye, Oumar; Faye, Ousmane; Sall, Amadou A; Harris, Eva; Pinsky, Benjamin A; Waggoner, Jesse J

2018-04-02

The differential diagnosis of dengue virus (DENV) and yellow fever virus (YFV) infections in endemic areas is complicated by nonspecific early clinical manifestations. In this study, we describe an internally controlled, multiplex real-time reverse transcription PCR (rRT-PCR) for the detection of DENV and YFV. The DENV-YFV assay demonstrated specific detection and had a dynamic range of 2.0-8.0 log 10 copies/μL of eluate for each DENV serotype and YFV. Clinical performance was similar to a published pan-DENV assay: 48/48 acute-phase samples from dengue cases were detected in both assays. For YFV detection, mock samples were prepared with nine geographically diverse YFV isolates over a range of concentrations. The DENV-YFV assay detected 62/65 replicates, whereas 54/65 were detected using a reference YFV rRT-PCR. Given the reemergence of DENV and YFV in areas around the world, the DENV-YFV assay should be a useful tool to narrow the differential diagnosis and provide early case detection.

Comparison of nested competitive RT-PCR and real-time RT-PCR for the detection and quantification of AML1/MTG8 fusion transcripts in t(8;21) positive acute myelogenous leukemia.

PubMed

Wattjes, M P; Krauter, J; Nagel, S; Heidenreich, O; Ganser, A; Heil, G

2000-02-01

The chromosomal translocation t(8;21)(q22;q22) is one of the most frequent karyotypic aberrations in acute myeloid leukemia (AML) and results in a chimeric fusion transcript AML1/MTG8. Since AML1/MTG8 fusion transcripts remain detectable by RT-PCR in t(8;21) AML patients in long-term hematological remission, quantitative assessment of AML1/MTG8 transcripts is necessary for the monitoring of minimal residual disease (MRD) in these patients. Competitive RT-PCR and recently real-time RT-PCR are increasingly used for detection and quantification of leukemia specific fusion transcripts. For the direct comparison of both methods we cloned a 42 bp DNA fragment into the original AML1/MTG8 sequence. The resulting molecule was used as an internal competitor for our novel competitive nested RT-PCR for AML1/MTG8 and as an external standard for the generation of AML1/MTG8 standard curves in a real-time PCR assay. Using this standard molecule for both PCR techniques, we compared their sensitivity, linearity and reproducibility. Both methods were comparable with regard to all parameters tested irrespective of analyzing serial dilutions of plasmids, cell lines or samples from t(8;21) positive AML patients at different stages of the disease. Therefore, both techniques can be recommended for the monitoring of MRD in these particular AML patients. However, the automatization of the real-time PCR technique offers some technical advantages.

Oriented antibody immobilization on self-assembled monolayers applied as impedance biosensors

NASA Astrophysics Data System (ADS)

Tsugimura, Kaiki; Ohnuki, Hitoshi; Wu, Haiyun; Endo, Hideaki; Tsuya, Daiju; Izumi, Mitsuru

2017-11-01

Oriented immobilization of antibodies on a sensor chip is crucial for enhancing both the sensitivity and antigen-binding capacity of immunosensors. Here, we report a comparative study of the effect of oriented and random antibody immobilization on the binding efficiency by electrochemical impedance spectroscopy (EIS). Oriented immobilization of anti-myoglobin immunoglobulin G (anti-Myo IgG) was achieved by bonding to an Fc receptor of protein G (PrG) on a self-assembled monolayer (SAM), which results in the myoglobin (Myo) binding sites being exposed outside the sensing surface. Random immobilization of anti-Myo IgG was achieved by direct covalent attachment to the SAM surface. Both immobilizations were applied to interdigitated electrodes to enhance the electrochemical signal, and the Myo biosensor performance was then evaluated by a series of EIS measurements. We found that (i) the rate of the normalized charge transfer resistance for the oriented sample was 3 times higher than that for the random sample and (ii) the detection limit was 0.001 ng/mL, which is the lowest recorded detection limit among Myo immunosensors based on EIS. These findings indicate that oriented antibody immobilization is crucial for preparing highly sensitive EIS-based biosensors.

Diversity of transcripts and transcript processing forms in plastids of the dinoflagellate alga Karenia mikimotoi.

PubMed

Dorrell, Richard G; Hinksman, George A; Howe, Christopher J

2016-02-01

Plastids produce a vast diversity of transcripts. These include mature transcripts containing coding sequences, and their processing precursors, as well as transcripts that lack direct coding functions, such as antisense transcripts. Although plastid transcriptomes have been characterised for many plant species, less is known about the transcripts produced in other plastid lineages. We characterised the transcripts produced in the fucoxanthin-containing plastids of the dinoflagellate alga Karenia mikimotoi. This plastid lineage, acquired through tertiary endosymbiosis, utilises transcript processing pathways that are very different from those found in plants and green algae, including 3' poly(U) tail addition, and extensive substitutional editing of transcript sequences. We have sequenced the plastid transcriptome of K. mikimotoi, and have detected evidence for divergent evolution of fucoxanthin plastid genomes. We have additionally characterised polycistronic and monocistronic transcripts from two plastid loci, psbD-tRNA (Met)-ycf4 and rpl36-rps13-rps11. We find evidence for a range of transcripts produced from each locus that differ in terms of editing state, 5' end cleavage position, and poly(U) tail addition. Finally, we identify antisense transcripts in K. mikimotoi, which appear to undergo different processing events from the corresponding sense transcripts. Overall, our study provides insights into the diversity of transcripts and processing intermediates found in plastid lineages across the eukaryotes.

In Situ Detection and Quantification of AR-V7, AR-FL, PSA, and KRAS Point Mutations in Circulating Tumor Cells.

PubMed

El-Heliebi, Amin; Hille, Claudia; Laxman, Navya; Svedlund, Jessica; Haudum, Christoph; Ercan, Erkan; Kroneis, Thomas; Chen, Shukun; Smolle, Maria; Rossmann, Christopher; Krzywkowski, Tomasz; Ahlford, Annika; Darai, Evangelia; von Amsberg, Gunhild; Alsdorf, Winfried; König, Frank; Löhr, Matthias; de Kruijff, Inge; Riethdorf, Sabine; Gorges, Tobias M; Pantel, Klaus; Bauernhofer, Thomas; Nilsson, Mats; Sedlmayr, Peter

2018-03-01

Liquid biopsies can be used in castration-resistant prostate cancer (CRPC) to detect androgen receptor splice variant 7 (AR-V7), a splicing product of the androgen receptor. Patients with AR-V7-positive circulating tumor cells (CTCs) have greater benefit of taxane chemotherapy compared with novel hormonal therapies, indicating a treatment-selection biomarker. Likewise, in those with pancreatic cancer (PaCa), KRAS mutations act as prognostic biomarkers. Thus, there is an urgent need for technology investigating the expression and mutation status of CTCs. Here, we report an approach that adds AR-V7 or KRAS status to CTC enumeration, compatible with multiple CTC-isolation platforms. We studied 3 independent CTC-isolation devices (CellCollector, Parsortix, CellSearch) for the evaluation of AR-V7 or KRAS status of CTCs with in situ padlock probe technology. Padlock probes allow highly specific detection and visualization of transcripts on a cellular level. We applied padlock probes for detecting AR-V7, androgen receptor full length (AR-FL), and prostate-specific antigen (PSA) in CRPC and KRAS wild-type (wt) and mutant (mut) transcripts in PaCa in CTCs from 46 patients. In situ analysis showed that 71% (22 of 31) of CRPC patients had detectable AR-V7 expression ranging from low to high expression [1-76 rolling circle products (RCPs)/CTC]. In PaCa patients, 40% (6 of 15) had KRAS mut expressing CTCs with 1 to 8 RCPs/CTC. In situ padlock probe analysis revealed CTCs with no detectable cytokeratin expression but positivity for AR-V7 or KRAS mut transcripts. Padlock probe technology enables quantification of AR-V7, AR-FL, PSA, and KRAS mut/wt transcripts in CTCs. The technology is easily applicable in routine laboratories and compatible with multiple CTC-isolation devices. © 2017 American Association for Clinical Chemistry.

Rapid and sensitive detection of human astrovirus in water samples by loop-mediated isothermal amplification with hydroxynaphthol blue dye.

PubMed

Yang, Bo-Yun; Liu, Xiao-Lu; Wei, Yu-Mei; Wang, Jing-Qi; He, Xiao-Qing; Jin, Yi; Wang, Zi-Jian

2014-02-14

The aim of this paper was to develop a reverse transcription loop-mediated isothermal amplification (RT-LAMP) method for rapid, sensitive and inexpensive detection of astrovirus. The detection limit of LAMP using in vitro RNA transcripts was 3.6 × 10 copies·μL⁻¹, which is as sensitive as the presently used PCR assays. However, the LAMP products could be identified as different colors with the naked eye following staining with hydroxynaphthol blue dye (HNB). No cross-reactivity with other gastroenteric viruses (rotavirus and norovirus) was observed, indicating the relatively high specificity of LAMP. The RT-LAMP method with HNB was used to effectively detect astrovirus in reclaimed water samples. The LAMP technique described in this study is a cheap, sensitive, specific and rapid method for the detection of astrovirus. The RT-LAMP method can be simply applied for the specific detection of astrovirus and has the potential to be utilized in the field as a screening test.

Probability, not linear summation, mediates the detection of concentric orientation-defined textures.

PubMed

Schmidtmann, Gunnar; Jennings, Ben J; Bell, Jason; Kingdom, Frederick A A

2015-01-01

Previous studies investigating signal integration in circular Glass patterns have concluded that the information in these patterns is linearly summed across the entire display for detection. Here we test whether an alternative form of summation, probability summation (PS), modeled under the assumptions of Signal Detection Theory (SDT), can be rejected as a model of Glass pattern detection. PS under SDT alone predicts that the exponent β of the Quick- (or Weibull-) fitted psychometric function should decrease with increasing signal area. We measured spatial integration in circular, radial, spiral, and parallel Glass patterns, as well as comparable patterns composed of Gabors instead of dot pairs. We measured the signal-to-noise ratio required for detection as a function of the size of the area containing signal, with the remaining area containing dot-pair or Gabor-orientation noise. Contrary to some previous studies, we found that the strength of summation never reached values close to linear summation for any stimuli. More importantly, the exponent β systematically decreased with signal area, as predicted by PS under SDT. We applied a model for PS under SDT and found that it gave a good account of the data. We conclude that probability summation is the most likely basis for the detection of circular, radial, spiral, and parallel orientation-defined textures.

Vehicle detection and orientation estimation using the radon transform

NASA Astrophysics Data System (ADS)

Pelapur, Rengarajan; Bunyak, Filiz; Palaniappan, Kannappan; Seetharaman, Gunasekaran

2013-05-01

Determining the location and orientation of vehicles in satellite and airborne imagery is a challenging task given the density of cars and other vehicles and complexity of the environment in urban scenes almost anywhere in the world. We have developed a robust and accurate method for detecting vehicles using a template-based directional chamfer matching, combined with vehicle orientation estimation based on a refined segmentation, followed by a Radon transform based profile variance peak analysis approach. The same algorithm was applied to both high resolution satellite imagery and wide area aerial imagery and initial results show robustness to illumination changes and geometric appearance distortions. Nearly 80% of the orientation angle estimates for 1585 vehicles across both satellite and aerial imagery were accurate to within 15? of the ground truth. In the case of satellite imagery alone, nearly 90% of the objects have an estimated error within +/-1.0° of the ground truth.

Detection of License Plate using Sliding Window, Histogram of Oriented Gradient, and Support Vector Machines Method

NASA Astrophysics Data System (ADS)

Astawa, INGA; Gusti Ngurah Bagus Caturbawa, I.; Made Sajayasa, I.; Dwi Suta Atmaja, I. Made Ari

2018-01-01

The license plate recognition usually used as part of system such as parking system. License plate detection considered as the most important step in the license plate recognition system. We propose methods that can be used to detect the vehicle plate on mobile phone. In this paper, we used Sliding Window, Histogram of Oriented Gradient (HOG), and Support Vector Machines (SVM) method to license plate detection so it will increase the detection level even though the image is not in a good quality. The image proceed by Sliding Window method in order to find plate position. Feature extraction in every window movement had been done by HOG and SVM method. Good result had shown in this research, which is 96% of accuracy.

Portable nuclear material detector and process

DOEpatents

Hofstetter, Kenneth J [Aiken, SC; Fulghum, Charles K [Aiken, SC; Harpring, Lawrence J [North Augusta, SC; Huffman, Russell K [Augusta, GA; Varble, Donald L [Evans, GA

2008-04-01

A portable, hand held, multi-sensor radiation detector is disclosed. The detection apparatus has a plurality of spaced sensor locations which are contained within a flexible housing. The detection apparatus, when suspended from an elevation, will readily assume a substantially straight, vertical orientation and may be used to monitor radiation levels from shipping containers. The flexible detection array can also assume a variety of other orientations to facilitate any unique container shapes or to conform to various physical requirements with respect to deployment of the detection array. The output of each sensor within the array is processed by at least one CPU which provides information in a usable form to a user interface. The user interface is used to provide the power requirements and operating instructions to the operational components within the detection array.

Linguistic Indicators of Wives’ Attachment Security and Communal Orientation During Military Deployment

PubMed Central

Borelli, Jessica L.; Sbarra, David A.; Randall, Ashley K.; Snavely, Jonathan E.; St. John, Heather K.; Ruiz, Sarah K.

2013-01-01

Military deployment affects thousands of families each year, yet little is known about its impact on non-deployed spouses (NDSs) and romantic relationships. This report examines two factors–attachment security and a communal orientation with respect to the deployment– that may be crucial to successful dyadic adjustment by the NDS. Thirty-seven female NDSs reported on their relationship satisfaction before and during their partner’s deployment, and 20 also did so two weeks following their partner’s return. Participants provided a stream-of-conscious speech sample regarding their relationship during the deployment; linguistic coding of sample transcripts provided measures of each participant’s (a) narrative coherence, hypothesized to reflect attachment security with respect to their deployed spouse; and, (b) frequency of first person plural pronoun use (we-talk), hypothesized to reflect a communal orientation to coping. More frequent first person plural pronouns— we-talk— was uniquely associated with higher relationship satisfaction during the deployment, and greater narrative coherence was uniquely associated with higher relationship satisfaction post-deployment. Discussion centers on the value of relationship security and communal orientations in predicting how couples cope with deployment and other types of relationship stressors. PMID:24033247

Performance characteristics of a reverse transcriptase-polymerase chain reaction assay for the detection of tumor-specific fusion transcripts from archival tissue.

PubMed

Fritsch, Michael K; Bridge, Julia A; Schuster, Amy E; Perlman, Elizabeth J; Argani, Pedram

2003-01-01

Pediatric small round cell tumors still pose tremendous diagnostic problems. In difficult cases, the ability to detect tumor-specific gene fusion transcripts for several of these neoplasms, including Ewing sarcoma/peripheral primitive neuroectodermal tumor (ES/PNET), synovial sarcoma (SS), alveolar rhabdomyosarcoma (ARMS), and desmoplastic small round cell tumor (DSRCT) using reverse transcriptase-polymerase chain reaction (RT-PCR), can be extremely helpful. Few studies to date, however, have systematically examined several different tumor types for the presence of multiple different fusion transcripts in order to determine the specificity and sensitivity of the RT-PCR method, and no study has addressed this issue for formalin-fixed material. The objectives of this study were to address the specificity, sensitivity, and practicality of such an assay applied strictly to formalin-fixed tissue blocks. Our results demonstrate that, for these tumors, the overall sensitivity for detecting each fusion transcript is similar to that reported in the literature for RT-PCR on fresh or formalin-fixed tissues. The specificity of the assay is very high, being essentially 100% for each primer pair when interpreting the results from visual inspection of agarose gels. However, when these same agarose gels were examined using Southern blotting, a small number of tumors also yielded reproducibly detectable weak signals for unexpected fusion products, in addition to a strong signal for the expected fusion product. Fluorescence in situ hybridization (FISH) studies in one such case indicated that a rearrangement that would account for the unexpected fusion was not present, while another case was equivocal. The overall specificity for each primer pair used in this assay ranged from 94 to 100%. Therefore, RT-PCR using formalin-fixed paraffin-embedded tissue sections can be used to detect chimeric transcripts as a reliable, highly sensitive, and highly specific diagnostic assay. However, we strongly suggest that the final interpretation of the results from this assay be viewed in light of the other features of the case, including clinical history, histology, and immunohistochemistry, by the diagnostic pathologist. Additional studies such as FISH may be useful in clarifying the nature of equivocal or unexpected results.

Retinal ganglion cell dendritic fields in old-world monkeys are oriented radially.

PubMed

Schall, J D; Perry, V H; Leventhal, A G

1986-03-12

We analyzed the dendritic field morphology of 297 ganglion cells from peripheral regions of monkey retina. Most of the dendritic fields were elongated, and there was a significant tendency for the dendritic fields to be oriented radially, i.e., like the spokes of a wheel with the fovea at the hub. An overrepresentation of radial orientations in the peripheral retina of primates might explain why humans are best able to detect stimuli which are oriented radially using peripheral vision.

Uncovering the Repertoire of Endogenous Flaviviral Elements in Aedes Mosquito Genomes

PubMed Central

Suzuki, Yasutsugu; Frangeul, Lionel; Dickson, Laura B.; Blanc, Hervé; Verdier, Yann; Vinh, Joelle

2017-01-01

ABSTRACT Endogenous viral elements derived from nonretroviral RNA viruses have been described in various animal genomes. Whether they have a biological function, such as host immune protection against related viruses, is a field of intense study. Here, we investigated the repertoire of endogenous flaviviral elements (EFVEs) in Aedes mosquitoes, the vectors of arboviruses such as dengue and chikungunya viruses. Previous studies identified three EFVEs from Aedes albopictus cell lines and one from Aedes aegypti cell lines. However, an in-depth characterization of EFVEs in wild-type mosquito populations and individual mosquitoes in vivo has not been performed. We detected the full-length DNA sequence of the previously described EFVEs and their respective transcripts in several A. albopictus and A. aegypti populations from geographically distinct areas. However, EFVE-derived proteins were not detected by mass spectrometry. Using deep sequencing, we detected the production of PIWI-interacting RNA-like small RNAs, in an antisense orientation, targeting the EFVEs and their flanking regions in vivo. The EFVEs were integrated in repetitive regions of the mosquito genomes, and their flanking sequences varied among mosquito populations. We bioinformatically predicted several new EFVEs from a Vietnamese A. albopictus population and observed variation in the occurrence of those elements among mosquitoes. Phylogenetic analysis of an A. aegypti EFVE suggested that it integrated prior to the global expansion of the species and subsequently diverged among and within populations. The findings of this study together reveal the substantial structural and nucleotide diversity of flaviviral integrations in Aedes genomes. Unraveling this diversity will help to elucidate the potential biological function of these EFVEs. IMPORTANCE Endogenous viral elements (EVEs) are whole or partial viral sequences integrated in host genomes. Interestingly, some EVEs have important functions for host fitness and antiviral defense. Because mosquitoes also have EVEs in their genomes, characterizing these EVEs is a prerequisite for their potential use to manipulate the mosquito antiviral response. In the study described here, we focused on EVEs related to the Flavivirus genus, to which dengue and Zika viruses belong, in individual Aedes mosquitoes from geographically distinct areas. We show the existence in vivo of flaviviral EVEs previously identified in mosquito cell lines, and we detected new ones. We show that EVEs have evolved differently in each mosquito population. They produce transcripts and small RNAs but not proteins, suggesting a function at the RNA level. Our study uncovers the diverse repertoire of flaviviral EVEs in Aedes mosquito populations and contributes to an understanding of their role in the host antiviral system. PMID:28539440

Uncovering the Repertoire of Endogenous Flaviviral Elements in Aedes Mosquito Genomes.

PubMed

Suzuki, Yasutsugu; Frangeul, Lionel; Dickson, Laura B; Blanc, Hervé; Verdier, Yann; Vinh, Joelle; Lambrechts, Louis; Saleh, Maria-Carla

2017-08-01

Endogenous viral elements derived from nonretroviral RNA viruses have been described in various animal genomes. Whether they have a biological function, such as host immune protection against related viruses, is a field of intense study. Here, we investigated the repertoire of endogenous flaviviral elements (EFVEs) in Aedes mosquitoes, the vectors of arboviruses such as dengue and chikungunya viruses. Previous studies identified three EFVEs from Aedes albopictus cell lines and one from Aedes aegypti cell lines. However, an in-depth characterization of EFVEs in wild-type mosquito populations and individual mosquitoes in vivo has not been performed. We detected the full-length DNA sequence of the previously described EFVEs and their respective transcripts in several A. albopictus and A. aegypti populations from geographically distinct areas. However, EFVE-derived proteins were not detected by mass spectrometry. Using deep sequencing, we detected the production of PIWI-interacting RNA-like small RNAs, in an antisense orientation, targeting the EFVEs and their flanking regions in vivo The EFVEs were integrated in repetitive regions of the mosquito genomes, and their flanking sequences varied among mosquito populations. We bioinformatically predicted several new EFVEs from a Vietnamese A. albopictus population and observed variation in the occurrence of those elements among mosquitoes. Phylogenetic analysis of an A. aegypti EFVE suggested that it integrated prior to the global expansion of the species and subsequently diverged among and within populations. The findings of this study together reveal the substantial structural and nucleotide diversity of flaviviral integrations in Aedes genomes. Unraveling this diversity will help to elucidate the potential biological function of these EFVEs. IMPORTANCE Endogenous viral elements (EVEs) are whole or partial viral sequences integrated in host genomes. Interestingly, some EVEs have important functions for host fitness and antiviral defense. Because mosquitoes also have EVEs in their genomes, characterizing these EVEs is a prerequisite for their potential use to manipulate the mosquito antiviral response. In the study described here, we focused on EVEs related to the Flavivirus genus, to which dengue and Zika viruses belong, in individual Aedes mosquitoes from geographically distinct areas. We show the existence in vivo of flaviviral EVEs previously identified in mosquito cell lines, and we detected new ones. We show that EVEs have evolved differently in each mosquito population. They produce transcripts and small RNAs but not proteins, suggesting a function at the RNA level. Our study uncovers the diverse repertoire of flaviviral EVEs in Aedes mosquito populations and contributes to an understanding of their role in the host antiviral system. Copyright © 2017 Suzuki et al.

Tissue contaminants and associated transcriptional response in trout liver from high elevation lakes of Washington

USGS Publications Warehouse

Moran, P.W.; Aluru, N.; Black, R.W.; Vijayan, M.M.

2007-01-01

The consistent cold temperatures and large amount of precipitation in the Olympic and Cascade ranges of Washington State are thought to enhance atmospheric deposition of contaminants. However, little is known about contaminant levels in organisms residing in these remote high elevation lakes. We measured total mercury and 28 organochlorine compounds in trout collected from 14 remote lakes in the Olympic, Mt. Rainer, and North Cascades National Parks. Mercury was detected in trout from all lakes sampled (15 to 262 ??g/kg ww), while two organochlorines, total polychlorinated biphenyls (tPCB) and dichlorodiphenyldichloroethylene (DDE), were also detected in these fish tissues (<25 ??g/kg ww). In sediments, organochlorine levels were below detection, while median total and methyl mercury were 30.4 and 0.34 ??g/ kg dry weight (ww), respectively. Using fish from two lakes, representing different contaminant loading levels (Wilcox lake: high; Skymo lake: low), we examined transcriptional response in the liver using a custom-made low-density targeted rainbow trout cDNA microarray. We detected significant differences in liver transcriptional response, including significant changes in metabolic, endocrine, and immune-related genes, in fish collected from Wilcox Lake compared to Skymo Lake. Overall, our results suggest that local urban areas contribute to the observed contaminant patterns in these high elevation lakes, while the transcriptional changes point to a biological response associated with exposure to these contaminants in fish. Specifically, the gene expression pattern leads us to hypothesize a role for mercury in disrupting the metabolic and reproductive pathways in fish from high elevation lakes in western Washington. ?? 2007 American Chemical Society.

Mammalian synthetic biology: emerging medical applications.

PubMed

Kis, Zoltán; Pereira, Hugo Sant'Ana; Homma, Takayuki; Pedrigi, Ryan M; Krams, Rob

2015-05-06

In this review, we discuss new emerging medical applications of the rapidly evolving field of mammalian synthetic biology. We start with simple mammalian synthetic biological components and move towards more complex and therapy-oriented gene circuits. A comprehensive list of ON-OFF switches, categorized into transcriptional, post-transcriptional, translational and post-translational, is presented in the first sections. Subsequently, Boolean logic gates, synthetic mammalian oscillators and toggle switches will be described. Several synthetic gene networks are further reviewed in the medical applications section, including cancer therapy gene circuits, immuno-regulatory networks, among others. The final sections focus on the applicability of synthetic gene networks to drug discovery, drug delivery, receptor-activating gene circuits and mammalian biomanufacturing processes. © 2015 The Author(s) Published by the Royal Society. All rights reserved.

Continuous-flow, microfluidic, qRT-PCR system for RNA virus detection.

PubMed

Fernández-Carballo, B Leticia; McBeth, Christine; McGuiness, Ian; Kalashnikov, Maxim; Baum, Christoph; Borrós, Salvador; Sharon, Andre; Sauer-Budge, Alexis F

2018-01-01

One of the main challenges in the diagnosis of infectious diseases is the need for rapid and accurate detection of the causative pathogen in any setting. Rapid diagnosis is key to avoiding the spread of the disease, to allow proper clinical decisions to be made in terms of patient treatment, and to mitigate the rise of drug-resistant pathogens. In the last decade, significant interest has been devoted to the development of point-of-care reverse transcription polymerase chain reaction (PCR) platforms for the detection of RNA-based viral pathogens. We present the development of a microfluidic, real-time, fluorescence-based, continuous-flow reverse transcription PCR system. The system incorporates a disposable microfluidic chip designed to be produced industrially with cost-effective roll-to-roll embossing methods. The chip has a long microfluidic channel that directs the PCR solution through areas heated to different temperatures. The solution first travels through a reverse transcription zone where RNA is converted to complementary DNA, which is later amplified and detected in real time as it travels through the thermal cycling area. As a proof of concept, the system was tested for Ebola virus detection. Two different master mixes were tested, and the limit of detection of the system was determined, as was the maximum speed at which amplification occurred. Our results and the versatility of our system suggest its promise for the detection of other RNA-based viruses such as Zika virus or chikungunya virus, which constitute global health threats worldwide. Graphical abstract Photograph of the RT-PCR thermoplastic chip.

Identification of a novel herpes simplex virus type 1 transcript and protein (AL3) expressed during latency.

PubMed

Jaber, Tareq; Henderson, Gail; Li, Sumin; Perng, Guey-Chuen; Carpenter, Dale; Wechsler, Steven L; Jones, Clinton

2009-10-01

The herpes simplex virus type 1 (HSV-1) latency-associated transcript (LAT) is abundantly expressed in latently infected sensory neurons. In small animal models of infection, expression of the first 1.5 kb of LAT coding sequences is necessary and sufficient for wild-type reactivation from latency. The ability of LAT to inhibit apoptosis is important for reactivation from latency. Within the first 1.5 kb of LAT coding sequences and LAT promoter sequences, additional transcripts have been identified. For example, the anti-sense to LAT transcript (AL) is expressed in the opposite direction to LAT from the 5' end of LAT and LAT promoter sequences. In addition, the upstream of LAT (UOL) transcript is expressed in the LAT direction from sequences in the LAT promoter. Further examination of the first 1.5 kb of LAT coding sequences revealed two small ORFs that are anti-sense with respect to LAT (AL2 and AL3). A transcript spanning AL3 was detected in productively infected cells, mouse neuroblastoma cells stably expressing LAT and trigeminal ganglia (TG) of latently infected mice. Peptide-specific IgG directed against AL3 specifically recognized a protein migrating near 15 kDa in cells stably transfected with LAT, mouse neuroblastoma cells transfected with a plasmid containing the AL3 ORF and TG of latently infected mice. The inability to detect the AL3 protein during productive infection may have been because the 5' terminus of the AL3 transcript was downstream of the first in-frame methionine of the AL3 ORF during productive infection.

Quantitative expression analysis of selected transcription factors in pavement, basal and trichome cells of mature leaves from Arabidopsis thaliana

PubMed Central

Schliep, Martin; Ebert, Berit; Simon-Rosin, Ulrike; Zoeller, Daniela

2010-01-01

Gene expression levels of several transcription factors from Arabidopsis thaliana that were described previously to be involved in leaf development and trichome formation were analysed in trichome, basal and pavement cells of mature leaves. Single cell samples of these three cells types were collected by glass micro-capillaries. Real-time reverse transcription (RT)-PCR was used to analyse expression patterns of the following transcription factors: MYB23, MYB55, AtHB1, FILAMENTOUS FLOWER (FIL)/YABBY1 (YAB1), TRIPTYCHON (TRY) and CAPRICE (CPC). A difference in the expression patterns of TRY and CPC was revealed. Contrary to the CPC expression pattern, no transcripts of TRY could be detected in pavement cells. FIL/YAB1 was exclusively expressed in trichome cells. AtHB1 was highly expressed throughout all three cell types. MYB55 was higher expressed in basal cells than in trichome and pavement cells. MYB23 showed a pattern of low expression in pavement cells, medium in basal cells and high expression in trichomes. Expression patterns obtained by single cell sampling and real-time RT-PCR were compared to promoter GUS fusions of the selected transcription factors. Therefore, we regenerated two transgenic Arabidopsis lines that expressed the GUS reporter gene under control of the promoters of MYB55 and YAB1. In conclusion, despite their function in leaf morphogenesis, all six transcription factors were detected in mature leaves. Furthermore, single cell sampling and promoter GUS staining patterns demonstrated the predominant presence of MYB55 in basal cells as compared to pavement cells and trichomes. PMID:20101514

Quantitative expression analysis of selected transcription factors in pavement, basal and trichome cells of mature leaves from Arabidopsis thaliana.

PubMed

Schliep, Martin; Ebert, Berit; Simon-Rosin, Ulrike; Zoeller, Daniela; Fisahn, Joachim

2010-05-01

Gene expression levels of several transcription factors from Arabidopsis thaliana that were described previously to be involved in leaf development and trichome formation were analysed in trichome, basal and pavement cells of mature leaves. Single cell samples of these three cells types were collected by glass micro-capillaries. Real-time reverse transcription (RT)-PCR was used to analyse expression patterns of the following transcription factors: MYB23, MYB55, AtHB1, FILAMENTOUS FLOWER (FIL)/YABBY1 (YAB1), TRIPTYCHON (TRY) and CAPRICE (CPC). A difference in the expression patterns of TRY and CPC was revealed. Contrary to the CPC expression pattern, no transcripts of TRY could be detected in pavement cells. FIL/YAB1 was exclusively expressed in trichome cells. AtHB1 was highly expressed throughout all three cell types. MYB55 was higher expressed in basal cells than in trichome and pavement cells. MYB23 showed a pattern of low expression in pavement cells, medium in basal cells and high expression in trichomes. Expression patterns obtained by single cell sampling and real-time RT-PCR were compared to promoter GUS fusions of the selected transcription factors. Therefore, we regenerated two transgenic Arabidopsis lines that expressed the GUS reporter gene under control of the promoters of MYB55 and YAB1. In conclusion, despite their function in leaf morphogenesis, all six transcription factors were detected in mature leaves. Furthermore, single cell sampling and promoter GUS staining patterns demonstrated the predominant presence of MYB55 in basal cells as compared to pavement cells and trichomes.

Identical genomic footprints of the adenovirus EIIa promoter are detected before and after EIa induction.

PubMed Central

Devaux, B; Albrecht, G; Kedinger, C

1987-01-01

Genomic DNase I footprinting was used to compare specific protein binding to the adenovirus type 5 early, EIa-inducible, EIIa promoter. Identical protection patterns of the promoter region were observed whether EIIa transcription was undetectable or fully induced. These results suggest that EIa-mediated transcriptional induction does not increase binding of limiting transcription factors to the promoter but rather that transactivation results from the proper interactions between factors already bound to their cognate sequences. Images PMID:2963956

Transcript copy number estimation using a mouse whole-genome oligonucleotide microarray

PubMed Central

Carter, Mark G; Sharov, Alexei A; VanBuren, Vincent; Dudekula, Dawood B; Carmack, Condie E; Nelson, Charlie; Ko, Minoru SH

2005-01-01

The ability to quantitatively measure the expression of all genes in a given tissue or cell with a single assay is an exciting promise of gene-expression profiling technology. An in situ-synthesized 60-mer oligonucleotide microarray designed to detect transcripts from all mouse genes was validated, as well as a set of exogenous RNA controls derived from the yeast genome (made freely available without restriction), which allow quantitative estimation of absolute endogenous transcript abundance. PMID:15998450

Detection of Citrus leprosis virus C using specific primers and TaqMan probe in one-step real-time reverse-transcription polymerase chain reaction assays.

PubMed

Choudhary, Nandlal; Wei, G; Govindarajulu, A; Roy, Avijit; Li, Wenbin; Picton, Deric D; Nakhla, M K; Levy, L; Brlansky, R H

2015-11-01

Citrus leprosis virus C (CiLV-C), a causal agent of the leprosis disease in citrus, is mostly present in the South and Central America and spreading toward the North America. To enable better diagnosis and inhibit the further spread of this re-emerging virus a quantitative (q) real-time reverse transcription polymerase chain reaction (qRT-PCR) assay is needed for early detection of CiLV-C when the virus is present in low titer in citrus leprosis samples. Using the genomic sequence of CiLV-C, specific primers and probe were designed and synthesized to amplify a 73 nt amplicon from the movement protein (MP) gene. A standard curve of the 73 nt amplicon MP gene was developed using known 10(10)-10(1) copies of in vitro synthesized RNA transcript to estimate the copy number of RNA transcript in the citrus leprosis samples. The one-step qRT-PCR detection assays for CiLV-C were determined to be 1000 times more sensitive when compared to the one-step conventional reverse transcription polymerase chain reaction (RT-PCR) CiLV-C detection method. To evaluate the quality of the total RNA extracts, NADH dehydrogenase gene specific primers (nad5) and probe were included in reactions as an internal control. The one-step qRT-PCR specificity was successfully validated by testing for the presence of CiLV-C in the total RNA extracts of the citrus leprosis samples collected from Belize, Costa Rica, Mexico and Panama. Implementation of the one-step qRT-PCR assays for CiLV-C diagnosis should assist regulatory agencies in surveillance activities to monitor the distribution pattern of CiLV-C in countries where it is present and to prevent further dissemination into citrus growing countries where there is no report of CiLV-C presence. Published by Elsevier B.V.

Development of a reverse transcription quantitative polymerase chain reaction-based assay for broad coverage detection of African and Asian Zika virus lineages.

PubMed

Yang, Yang; Wong, Gary; Ye, Baoguo; Li, Shihua; Li, Shanqin; Zheng, Haixia; Wang, Qiang; Liang, Mifang; Gao, George F; Liu, Lei; Liu, Yingxia; Bi, Yuhai

2017-06-01

The Zika virus (ZIKV) is an arbovirus that has spread rapidly worldwide within recent times. There is accumulating evidence that associates ZIKV infections with Guillain-Barré Syndrome (GBS) and microcephaly in humans. The ZIKV is genetically diverse and can be separated into Asian and African lineages. A rapid, sensitive, and specific assay is needed for the detection of ZIKV across various pandemic regions. So far, the available primers and probes do not cover the genetic diversity and geographic distribution of all ZIKV strains. To this end, we have developed a one-step quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay based on conserved sequences in the ZIKV envelope (E) gene. The detection limit of the assay was determined to be five RNA transcript copies and 2.94 × 10 -3 50% tissue culture infectious doses (TCID 50 ) of live ZIKV per reaction. The assay was highly specific and able to detect five different ZIKV strains covering the Asian and African lineages without nonspecific amplification, when tested against other flaviviruses. The assay was also successful in testing for ZIKV in clinical samples. Our assay represents an improvement over the current methods available for the detection ZIKV and would be valuable as a diagnostic tool in various pandemic regions.

[Detection and classification of medication errors at Joan XXIII University Hospital].

PubMed

Jornet Montaña, S; Canadell Vilarrasa, L; Calabuig Mũoz, M; Riera Sendra, G; Vuelta Arce, M; Bardají Ruiz, A; Gallart Mora, M J

2004-01-01

Medication errors are multifactorial and multidisciplinary, and may originate in processes such as drug prescription, transcription, dispensation, preparation and administration. The goal of this work was to measure the incidence of detectable medication errors that arise within a unit dose drug distribution and control system, from drug prescription to drug administration, by means of an observational method confined to the Pharmacy Department, as well as a voluntary, anonymous report system. The acceptance of this voluntary report system's implementation was also assessed. A prospective descriptive study was conducted. Data collection was performed at the Pharmacy Department from a review of prescribed medical orders, a review of pharmaceutical transcriptions, a review of dispensed medication and a review of medication returned in unit dose medication carts. A voluntary, anonymous report system centralized in the Pharmacy Department was also set up to detect medication errors. Prescription errors were the most frequent (1.12%), closely followed by dispensation errors (1.04%). Transcription errors (0.42%) and administration errors (0.69%) had the lowest overall incidence. Voluntary report involved only 4.25% of all detected errors, whereas unit dose medication cart review contributed the most to error detection. Recognizing the incidence and types of medication errors that occur in a health-care setting allows us to analyze their causes and effect changes in different stages of the process in order to ensure maximal patient safety.

Probabilistic Surface Characterization for Safe Landing Hazard Detection and Avoidance (HDA)

NASA Technical Reports Server (NTRS)

Johnson, Andrew E. (Inventor); Ivanov, Tonislav I. (Inventor); Huertas, Andres (Inventor)

2015-01-01

Apparatuses, systems, computer programs and methods for performing hazard detection and avoidance for landing vehicles are provided. Hazard assessment takes into consideration the geometry of the lander. Safety probabilities are computed for a plurality of pixels in a digital elevation map. The safety probabilities are combined for pixels associated with one or more aim points and orientations. A worst case probability value is assigned to each of the one or more aim points and orientations.

Transcriptional "silencer" element in rat repetitive sequences associated with the rat insulin 1 gene locus.

PubMed Central

Laimins, L; Holmgren-König, M; Khoury, G

1986-01-01

The enhancer elements from either simian virus 40 or murine sarcoma virus activate the expression of a transfected rat insulin 1 (rI1) gene when placed within 2.0 kilobases or less of the rI1 gene cap site. Inclusion of 4.0 kilobases of upstream rI1 sequence, however, results in a substantial reduction in the enhancer-dependent insulin gene expression. These observations suggested that a negative transcriptional regulatory element was present between 2.0 and 4.0 kilobases of the rI1 sequence. To test this notion, we employed a heterologous enhancer-dependent transcription assay in which the simian virus 40 72-base-pair repeat is linked to a human beta-globin gene. Addition of the upstream rI1 element to this system decreased the level of enhancer-dependent beta-globin transcription by a factor of 5 to 15. This rI1 "silencer" element functions in a manner relatively independent of position and orientation and requires a cis-dependent relationship to the transcription unit on which it acts. Thus, the silencer sequence seems to have a number of the characteristics of enhancer elements, and we suggest that it may function by the converse of the enhancer mechanism. The rI1 silencer sequence was identified as a member of a long interspersed rat repetitive family. Thus, a potential role for certain repetitive sequences interspersed throughout the eukaryotic genome may be to regulate gene expression by retaining transcriptional activity within defined domains. Images PMID:3010279

Cloning of a methanol-inducible moxF promoter and its analysis in moxB mutants of Methylobacterium extorquens AM1rif.

PubMed Central

Morris, C J; Lidstrom, M E

1992-01-01

In Methylobacterium extorquens AM1, gene encoding methanol dehydrogenase polypeptides are transcriptionally regulated in response to C1 compounds, including methanol (M. E. Lidstrom and D. I. Stirling, Annu. Rev. Microbiol. 44:27-57, 1990). In order to study this regulation, a transcriptional fusion has been constructed between a beta-galactosidase reporter gene and a 1.55-kb XhoI-SalI fragment of M. extorquens AM1rif DNA encoding the N terminus of the methanol dehydrogenase large subunit (moxF) and 1,289 bp of upstream DNA. The fusion exhibited orientation-specific promoter activity in M. extorquens AM1rif but was expressed constitutively when the transcriptional fusion was located on the plasmid. However, correct regulation was restored when the construction was inserted in the M. extorquens AM1rif chromosome. This DNA fragment was shown to contain both the moxFJGI promoter and the sequences necessary in cis for its transcriptional regulation by methanol. Transcription from this promoter was studied in the M. extorquens AM1rif moxB mutant strains UV4rif and UV25rif, which have a pleiotropic phenotype with regard to the components of methanol oxidation. In these mutants, beta-galactosidase activity from the fusion was reduced to a level equal to that of the vector background when the fusion was present in both plasmid and chromosomal locations. Since both constitutive and methanol-inducible promoter activities were lost in the mutants, moxB appears to be required for transcription of the genes encoding the methanol dehydrogenase polypeptides. Images PMID:1624436

Requirement of multiple cis-acting elements in the human cytomegalovirus major immediate-early distal enhancer for viral gene expression and replication.

PubMed

Meier, Jeffery L; Keller, Michael J; McCoy, James J

2002-01-01

We have shown previously that the human cytomegalovirus (HCMV) major immediate-early (MIE) distal enhancer is needed for MIE promoter-dependent transcription and viral replication at low multiplicities of infection (MOI). To understand how this region works, we constructed and analyzed a series of HCMVs with various distal enhancer mutations. We show that the distal enhancer is composed of at least two parts that function independently to coordinately activate MIE promoter-dependent transcription and viral replication. One such part is contained in a 47-bp segment that has consensus binding sites for CREB/ATF, SP1, and YY1. At low MOI, these working parts likely function in cis to directly activate MIE gene expression, thus allowing viral replication to ensue. Three findings support the view that these working parts are likely cis-acting elements. (i) Deletion of either part of a bisegmented distal enhancer only slightly alters MIE gene transcription and viral replication. (ii) Reversing the distal enhancer's orientation largely preserves MIE gene transcription and viral replication. (iii) Placement of stop codons at -300 or -345 in all reading frames does not impair MIE gene transcription and viral replication. Lastly, we show that these working parts are dispensable at high MOI, partly because of compensatory stimulation of MIE promoter activity and viral replication that is induced by a virion-associated component(s) present at a high viral particle/cell ratio. We conclude that the distal enhancer is a complex multicomponent cis-acting region that is required to augment both MIE promoter-dependent transcription and HCMV replication.

Comparison of Remote Sensing Image Processing Techniques to Identify Tornado Damage Areas from Landsat TM Data

PubMed Central

Myint, Soe W.; Yuan, May; Cerveny, Randall S.; Giri, Chandra P.

2008-01-01

Remote sensing techniques have been shown effective for large-scale damage surveys after a hazardous event in both near real-time or post-event analyses. The paper aims to compare accuracy of common imaging processing techniques to detect tornado damage tracks from Landsat TM data. We employed the direct change detection approach using two sets of images acquired before and after the tornado event to produce a principal component composite images and a set of image difference bands. Techniques in the comparison include supervised classification, unsupervised classification, and object-oriented classification approach with a nearest neighbor classifier. Accuracy assessment is based on Kappa coefficient calculated from error matrices which cross tabulate correctly identified cells on the TM image and commission and omission errors in the result. Overall, the Object-oriented Approach exhibits the highest degree of accuracy in tornado damage detection. PCA and Image Differencing methods show comparable outcomes. While selected PCs can improve detection accuracy 5 to 10%, the Object-oriented Approach performs significantly better with 15-20% higher accuracy than the other two techniques. PMID:27879757

Novel kinase fusion transcripts found in endometrial cancer

PubMed Central

Tamura, Ryo; Yoshihara, Kosuke; Yamawaki, Kaoru; Suda, Kazuaki; Ishiguro, Tatsuya; Adachi, Sosuke; Okuda, Shujiro; Inoue, Ituro; Verhaak, Roel G. W.; Enomoto, Takayuki

2015-01-01

Recent advances in RNA-sequencing technology have enabled the discovery of gene fusion transcripts in the transcriptome of cancer cells. However, it remains difficult to differentiate the therapeutically targetable fusions from passenger events. We have analyzed RNA-sequencing data and DNA copy number data from 25 endometrial cancer cell lines to identify potential therapeutically targetable fusion transcripts, and have identified 124 high-confidence fusion transcripts, of which 69% are associated with gene amplifications. As targetable fusion candidates, we focused on three in-frame kinase fusion transcripts that retain a kinase domain (CPQ-PRKDC, CAPZA2-MET, and VGLL4-PRKG1). We detected only CPQ-PRKDC fusion transcript in three of 122 primary endometrial cancer tissues. Cell proliferation of the fusion-positive cell line was inhibited by knocking down the expression of wild-type PRKDC but not by blocking the CPQ-PRKDC fusion transcript expression. Quantitative real-time RT-PCR demonstrated that the expression of the CPQ-PRKDC fusion transcript was significantly lower than that of wild-type PRKDC, corresponding to a low transcript allele fraction of this fusion, based on RNA-sequencing read counts. In endometrial cancers, the CPQ-PRKDC fusion transcript may be a passenger aberration related to gene amplification. Our findings suggest that transcript allele fraction is a useful predictor to find bona-fide therapeutic-targetable fusion transcripts. PMID:26689674

Novel kinase fusion transcripts found in endometrial cancer.

PubMed

Tamura, Ryo; Yoshihara, Kosuke; Yamawaki, Kaoru; Suda, Kazuaki; Ishiguro, Tatsuya; Adachi, Sosuke; Okuda, Shujiro; Inoue, Ituro; Verhaak, Roel G W; Enomoto, Takayuki

2015-12-22

Recent advances in RNA-sequencing technology have enabled the discovery of gene fusion transcripts in the transcriptome of cancer cells. However, it remains difficult to differentiate the therapeutically targetable fusions from passenger events. We have analyzed RNA-sequencing data and DNA copy number data from 25 endometrial cancer cell lines to identify potential therapeutically targetable fusion transcripts, and have identified 124 high-confidence fusion transcripts, of which 69% are associated with gene amplifications. As targetable fusion candidates, we focused on three in-frame kinase fusion transcripts that retain a kinase domain (CPQ-PRKDC, CAPZA2-MET, and VGLL4-PRKG1). We detected only CPQ-PRKDC fusion transcript in three of 122 primary endometrial cancer tissues. Cell proliferation of the fusion-positive cell line was inhibited by knocking down the expression of wild-type PRKDC but not by blocking the CPQ-PRKDC fusion transcript expression. Quantitative real-time RT-PCR demonstrated that the expression of the CPQ-PRKDC fusion transcript was significantly lower than that of wild-type PRKDC, corresponding to a low transcript allele fraction of this fusion, based on RNA-sequencing read counts. In endometrial cancers, the CPQ-PRKDC fusion transcript may be a passenger aberration related to gene amplification. Our findings suggest that transcript allele fraction is a useful predictor to find bona-fide therapeutic-targetable fusion transcripts.

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

PubMed

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

2017-04-01

Bacteria cope with and adapt to stress by modulating gene expression in response to specific environmental cues. In this study, the transcriptional response of Pseudomonas putida KT2440 to osmotic, oxidative, and imipenem stress conditions at two time points was investigated via identification of differentially expressed mRNAs and small RNAs (sRNAs). A total of 440 sRNA transcripts were detected, of which 10% correspond to previously annotated sRNAs, 40% to novel intergenic transcripts, and 50% to novel transcripts antisense to annotated genes. Each stress elicits a unique response as far as the extent and dynamics of the transcriptional changes. Nearly 200 protein-encoding genes exhibited significant changes in all stress types, implicating their participation in a general stress response. Almost half of the sRNA transcripts were differentially expressed under at least one condition, suggesting possible functional roles in the cellular response to stress conditions. The data show a larger fraction of differentially expressed sRNAs than of mRNAs with >5-fold expression changes. The work provides detailed insights into the mechanisms through which P. putida responds to different stress conditions and increases understanding of bacterial adaptation in natural and industrial settings. IMPORTANCE This study maps the complete transcriptional response of P. putida KT2440 to osmotic, oxidative, and imipenem stress conditions at short and long exposure times. Over 400 sRNA transcripts, consisting of both intergenic and antisense transcripts, were detected, increasing the number of identified sRNA transcripts in the strain by a factor of 10. Unique responses to each type of stress are documented, including both the extent and dynamics of the gene expression changes. The work adds rich detail to previous knowledge of stress response mechanisms due to the depth of the RNA sequencing data. Almost half of the sRNAs exhibit significant expression changes under at least one condition, suggesting their involvement in adaptation to stress conditions and identifying interesting candidates for further functional characterization. Copyright © 2017 American Society for Microbiology.

Strain history of ice shells of the Galilean satellites from radar detection of crystal orientation fabric

NASA Astrophysics Data System (ADS)

Barr, Amy C.; Stillman, David E.

2011-03-01

Orbital radar sounding has been suggested as a means of determining the subsurface thermal and physical structure of the outer ice I shells of the Galilean satellites. At radar frequencies, the dielectric permittivity of single- and polycrystalline water ice I is anisotropic. Crystal orientation fabric (COF), which is indicative of strain history, can be unambiguously detected by comparing the received power of dual co-polarization (linear polarization parallel and perpendicular to the orbit) radar data. Regions with crystal orientations dictated by the local strain field (“fabric”) form in terrestrial ice masses where accumulated strain and temperature are high, similar to conditions expected in a convecting outer ice I shell on Europa, Ganymede, or Callisto. We use simulations of solid-state ice shell convection to show that crystal orientation fabric can form in the warm convecting sublayer of the ice shells for plausible grain sizes. Changes in received power from parallel and perpendicular polarizations in the ice shells due to fabric could be detected if multi-polarization data is collected. With proper instrument design, radar sounding could be used to shed light on the strain history of the satellites' ice shells in addition to their present day internal structures.

Activated but not resting T cells or thymocytes express colony-stimulating factor 1 mRNA without co-expressing c-fms mRNA.

PubMed

Cerdan, C; Courcoul, M; Razanajaona, D; Pierrès, A; Maroc, N; Lopez, M; Mannoni, P; Mawas, C; Olive, D; Birg, F

1990-02-01

Following the observation that, besides acute myeloid leukemia cells, acute lymphoid leukemia cells of either B or T phenotype could express the transcript for the colony-stimulating factor 1 (CSF-1), a growth factor known to be restricted to the monocytic-macrophage lineage, various sources of resting and/or activated T cells and thymocytes were screened for expression of this hemopoietic growth factor. We report here that the CSF-1 transcript was rapidly (7 h) induced in T cells by a variety of stimuli, but was not detectable in either resting T cells or thymocytes. In addition, secretion of CSF-1 was detectable in the supernatants of activated T cells by 72 h, with a peak around 92-120 h. In contrast to activated monocytes, the transcript of the c-fms proto-oncogene, the product of which is the receptor for CSF-1, was not detectable in either resting or activated T cells. This observation could be relevant to the intimate relationships between T cells and antigen-presenting cells during immune responses.

Inventory of high-abundance mRNAs in skeletal muscle of normal men.

PubMed

Welle, S; Bhatt, K; Thornton, C A

1999-05-01

G42875rial analysis of gene expression (SAGE) method was used to generate a catalog of 53,875 short (14 base) expressed sequence tags from polyadenylated RNA obtained from vastus lateralis muscle of healthy young men. Over 12,000 unique tags were detected. The frequency of occurrence of each tag reflects the relative abundance of the corresponding mRNA. The mRNA species that were detected 10 or more times, each comprising >/=0.02% of the mRNA population, accounted for 64% of the mRNA mass but <10% of the total number of mRNA species detected. Almost all of the abundant tags matched mRNA or EST sequences cataloged in GenBank. Mitochondrial transcripts accounted for approximately 20% of the polyadenylated RNA. Transcripts encoding proteins of the myofibrils were the most abundant nuclear-encoded mRNAs. Transcripts encoding ribosomal proteins, and those encoding proteins involved in energy metabolism, also were very abundant. The database can be used as a reference for investigations of alterations in gene expression associated with conditions that influence muscle function, such as muscular dystrophies, aging, and exercise.

Early Detection of Dengue Virus by Use of Reverse Transcription-Recombinase Polymerase Amplification

PubMed Central

Teoh, Boon-Teong; Sam, Sing-Sin; Tan, Kim-Kee; Danlami, Mohammed Bashar; Shu, Meng-Hooi; Johari, Jefree; Hooi, Poh-Sim; Brooks, David; Piepenburg, Olaf; Nentwich, Oliver; Wilder-Smith, Annelies; Franco, Leticia; Tenorio, Antonio

2015-01-01

A method for the rapid diagnosis of early dengue virus (DENV) infection is highly needed. Here, a prototype reverse transcription-recombinase polymerase amplification (RT-RPA) assay was developed. The assay detected DENV RNA in <20 min without the need for thermocycling amplification. The assay enabled the detection of as few as 10 copies of DENV RNA. The designed RT-RPA primers and exo probe detected the DENV genome of at least 12 genotypes of DENV circulating globally without cross-reacting with other arboviruses. We assessed the diagnostic performance of the RT-RPA assay for the detection of DENV RNA in 203 serum samples of patients with clinically suspected dengue. The sera were simultaneously tested for DENV using a reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay, quantitative RT-PCR (qRT-PCR), and IgM- and IgG-capture enzyme-linked immunosorbent assays (ELISA). Acute DENV infection was confirmed in 130 samples and 61 of the samples (46.9%) were classified as viremic with qRT-PCR. The RT-RPA assay showed good concordance (κ of ≥0.723) with the RT-LAMP and qRT-PCR assays in detecting the dengue viremic samples. When used in combination with ELISA, both the RT-RPA and RT-LAMP assays increased the detection of acute DENV infection to ≥95.7% (≥45/47) in samples obtained within 5 days of illness. The results from the study suggest that the RT-RPA assay is the most rapid molecular diagnostic tool available for the detection of DENV. Hence, it is possible to use the RT-RPA assay in a laboratory to complement routine serology testing for dengue. PMID:25568438

Rapid and sensitive detection of Zika virus by reverse transcription loop-mediated isothermal amplification.

PubMed

Wang, Xuan; Yin, Fenggui; Bi, Yuhai; Cheng, Gong; Li, Jing; Hou, Lidan; Li, Yunlong; Yang, Baozhi; Liu, Wenjun; Yang, Limin

2016-12-01

Zika virus (ZIKV) is an arbovirus that recently emerged and has expanded worldwide, causing a global threat and raising international concerns. Current molecular diagnostics, e.g., real-time PCR and reverse transcription PCR (RT-PCR), are time consuming, expensive, and can only be deployed in a laboratory instead of for field diagnostics. This study aimed to develop a one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) platform showing sensitivity, specificity, and more convenience than previous methods, being easily distributed and implemented. Specific primers were designed and screened to target the entire ZIKV genome. The analytical sensitivity and specificity of the assay were evaluated and compared with traditional PCR and quantitative real-time PCR. Three different simulated clinical sample quick preparation protocols were evaluated to establish a rapid and straightforward treatment procedure for clinical specimens in open field detection. The RT-LAMP assay for detection of ZIKV demonstrated superior specificity and sensitivity compared to traditional PCR at the optimum reaction temperature. For the ZIKV RNA standard, the limit of detection was 20 copies/test. For the simulated ZIKV clinical samples, the limit of detection was 0.02 pfu/test, which was one order of magnitude higher than RT-PCR and similar to real-time PCR. The detection limit of simulated ZIKV specimens prepared using a protease quick processing method was consistent with that of samples prepared using commercial nucleic acid extraction kits, indicating that our ZIKV detection method could be used in point-of-care testing. The RT-LAMP assay had excellent sensitivity and specificity for detecting ZIKV and can be deployed together with a rapid specimen processing method, offering the possibility for ZIKV diagnosis outside of the laboratory. Copyright © 2016 Elsevier B.V. All rights reserved.

Acoustic Evidence for Phonologically Mismatched Speech Errors

ERIC Educational Resources Information Center

Gormley, Andrea

2015-01-01

Speech errors are generally said to accommodate to their new phonological context. This accommodation has been validated by several transcription studies. The transcription methodology is not the best choice for detecting errors at this level, however, as this type of error can be difficult to perceive. This paper presents an acoustic analysis of…

Brainstem Transcription of Speech Is Disrupted in Children with Autism Spectrum Disorders

ERIC Educational Resources Information Center

Russo, Nicole; Nicol, Trent; Trommer, Barbara; Zecker, Steve; Kraus, Nina

2009-01-01

Language impairment is a hallmark of autism spectrum disorders (ASD). The origin of the deficit is poorly understood although deficiencies in auditory processing have been detected in both perception and cortical encoding of speech sounds. Little is known about the processing and transcription of speech sounds at earlier (brainstem) levels or…

Isolation and Characterization of a TERMINAL FLOWER Homolog and Its Correlation with Juvenility in Citrus

PubMed Central

Pillitteri, Lynn Jo; Lovatt, Carol J.; Walling, Linda L.

2004-01-01

TERMINAL FLOWER is a key regulator of floral timing in Arabidopsis and other herbaceous species. A homolog of this gene, CsTFL, was isolated from the hybrid perennial tree crop Washington navel orange (Citrus sinensis L. Osbeck). The deduced amino acid sequence of CsTFL was 65% identical to the Arabidopsis TFL1 protein. Wild-type Arabidopsis plants ectopically expressing CsTFL showed late-flowering phenotypes similar to those described for overexpression of Arabidopsis TFL1. In addition, the 35S:CsTFL transgene complemented the tfl1-2 mutant. The severity of the overexpression phenotypes correlated with the amount of CsTFL transcript that accumulated. Unlike many model systems that have been studied, C. sinensis maintains two distinguishable CsTFL alleles. CsTFL transcripts from either allele were not detected in adult vegetative tissues using reverse transcription-PCR, but CsTFL RNAs were detected in all floral organs. In addition, real-time PCR determined that juvenility in citrus was positively correlated with CsTFL transcript accumulation and negatively correlated with the floral-regulatory genes, LEAFY and APETALA1, RNA levels. PMID:15235113

Transcriptional response of peripheral lymphocytes to early fibrosarcoma: a model system for cancer detection based on hybridization signatures.

PubMed

Marques, Márcia M C; Junta, Cristina M; Zárate-Blades, Carlos R; Sakamoto-Hojo, Elza Tiemi; Donadi, Eduardo A; Passos, Geraldo A S

2009-07-01

Since circulating leukocytes, mainly B and T cells, continuously maintain vigilant and comprehensive immune surveillance, these cells could be used as reporters for signs of infection or other pathologies, including cancer. Activated lymphocyte clones trigger a sensitive transcriptional response, which could be identified by gene expression profiling. To assess this hypothesis, we conducted microarray analysis of the gene expression profile of lymphocytes isolated from immunocompetent BALB/c mice subcutaneously injected with different numbers of tumorigenic B61 fibrosarcoma cells. Flow cytometry demonstrated that the number of circulating T (CD3(+)CD4(+) or CD3(+)CD8(+)) or B (CD19(+)) cells did not change. However, the lymphocytes isolated from tumor cell-injected animals expressed a unique transcriptional profile that was identifiable before the development of a palpable tumor mass. This finding demonstrates that the transcriptional response appears before alterations in the main lymphocyte subsets and that the gene expression profile of peripheral lymphocytes can serve as a sensitive and accurate method for the early detection of cancer.

Strand-Specific RNA-Seq Analyses of Fruiting Body Development in Coprinopsis cinerea

DOE PAGES

Muraguchi, Hajime; Umezawa, Kiwamu; Niikura, Mai; ...

2015-10-28

We report that the basidiomycete fungus Coprinopsis cinerea is an important model system for multicellular development. Fruiting bodies of C. cinerea are typical mushrooms, which can be produced synchronously on defined media in the laboratory. To investigate the transcriptome in detail during fruiting body development, high-throughput sequencing (RNA-seq) was performed using cDNA libraries strand-specifically constructed from 13 points (stages/tissues) with two biological replicates. The reads were aligned to 14,245 predicted transcripts, and counted for forward and reverse transcripts. Differentially expressed genes (DEGs) between two adjacent points and between vegetative mycelium and each point were detected by Tag Count Comparison (TCC).more » To validate RNA-seq data, expression levels of selected genes were compared using RPKM values in RNA-seq data and qRT-PCR data, and DEGs detected in microarray data were examined in MA plots of RNA-seq data by TCC. We discuss events deduced from GO analysis of DEGs. In addition, we uncovered both transcription factor candidates and antisense transcripts that are likely to be involved in developmental regulation for fruiting.« less

Strand-Specific RNA-Seq Analyses of Fruiting Body Development in Coprinopsis cinerea

DOE Office of Scientific and Technical Information (OSTI.GOV)

Muraguchi, Hajime; Umezawa, Kiwamu; Niikura, Mai

We report that the basidiomycete fungus Coprinopsis cinerea is an important model system for multicellular development. Fruiting bodies of C. cinerea are typical mushrooms, which can be produced synchronously on defined media in the laboratory. To investigate the transcriptome in detail during fruiting body development, high-throughput sequencing (RNA-seq) was performed using cDNA libraries strand-specifically constructed from 13 points (stages/tissues) with two biological replicates. The reads were aligned to 14,245 predicted transcripts, and counted for forward and reverse transcripts. Differentially expressed genes (DEGs) between two adjacent points and between vegetative mycelium and each point were detected by Tag Count Comparison (TCC).more » To validate RNA-seq data, expression levels of selected genes were compared using RPKM values in RNA-seq data and qRT-PCR data, and DEGs detected in microarray data were examined in MA plots of RNA-seq data by TCC. We discuss events deduced from GO analysis of DEGs. In addition, we uncovered both transcription factor candidates and antisense transcripts that are likely to be involved in developmental regulation for fruiting.« less

Coupled Transcriptome and Proteome Analysis of Human Lymphotropic Tumor Viruses: Insights on the Detection and Discovery of Viral Genes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dresang, Lindsay R.; Teuton, Jeremy R.; Feng, Huichen

Kaposi's sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV) are related human tumor viruses that cause primary effusion lymphomas (PEL) and Burkitt's lymphomas (BL), respectively. Viral genes expressed in naturally-infected cancer cells contribute to disease pathogenesis; knowing which viral genes are expressed is critical in understanding how these viruses cause cancer. To evaluate the expression of viral genes, we used high-resolution separation and mass spectrometry coupled with custom tiling arrays to align the viral proteomes and transcriptomes of three PEL and two BL cell lines under latent and lytic culture conditions. Results The majority of viral genes were efficiently detected atmore » the transcript and/or protein level on manipulating the viral life cycle. Overall the correlation of expressed viral proteins and transcripts was highly complementary in both validating and providing orthogonal data with latent/lytic viral gene expression. Our approach also identified novel viral genes in both KSHV and EBV, and extends viral genome annotation. Several previously uncharacterized genes were validated at both transcript and protein levels. Conclusions This systems biology approach coupling proteome and transcriptome measurements provides a comprehensive view of viral gene expression that could not have been attained using each methodology independently. Detection of viral proteins in combination with viral transcripts is a potentially powerful method for establishing virus-disease relationships.« less

Expression and Stress-Dependent Induction of Potassium Channel Transcripts in the Common Ice Plant1

PubMed Central

Su, Hua; Golldack, Dortje; Katsuhara, Maki; Zhao, Chengsong; Bohnert, Hans J.

2001-01-01

We have characterized transcripts for three potassium channel homologs in the AKT/KAT subfamily (Shaker type) from the common ice plant (Mesembryanthemum crystallinum), with a focus on their expression during salt stress (up to 500 mm NaCl). Mkt1 and 2, Arabidopsis AKT homologs, and Kmt1, a KAT homolog, are members of small gene families with two to three isoforms each. Mkt1 is root specific; Mkt2 is found in leaves, flowers, and seed capsules; and Kmt1 is expressed in leaves and seed capsules. Mkt1 is present in all cells of the root, and in leaves a highly conserved isoform is detected present in all cells with highest abundance in the vasculature. MKT1 for which antibodies were made is localized to the plasma membrane. Following salt stress, MKT1 (transcripts and protein) is drastically down-regulated, Mkt2 transcripts do not change significantly, and Kmt1 is strongly and transiently (maximum at 6 h) up-regulated in leaves and stems. The detection and stress-dependent behavior of abundant transcripts representing subfamilies of potassium channels provides information about tissue specificity and the complex regulation of genes encoding potassium uptake systems in a halophytic plant. PMID:11161018

Splice Site Mutations in the ATP7A Gene

PubMed Central

Møller, Lisbeth Birk

2011-01-01

Menkes disease (MD) is caused by mutations in the ATP7A gene. We describe 33 novel splice site mutations detected in patients with MD or the milder phenotypic form, Occipital Horn Syndrome. We review these 33 mutations together with 28 previously published splice site mutations. We investigate 12 mutations for their effect on the mRNA transcript in vivo. Transcriptional data from another 16 mutations were collected from the literature. The theoretical consequences of splice site mutations, predicted with the bioinformatics tool Human Splice Finder, were investigated and evaluated in relation to in vivo results. Ninety-six percent of the mutations identified in 45 patients with classical MD were predicted to have a significant effect on splicing, which concurs with the absence of any detectable wild-type transcript in all 19 patients investigated in vivo. Sixty-seven percent of the mutations identified in 12 patients with milder phenotypes were predicted to have no significant effect on splicing, which concurs with the presence of wild-type transcript in 7 out of 9 patients investigated in vivo. Both the in silico predictions and the in vivo results support the hypothesis previously suggested by us and others, that the presence of some wild-type transcript is correlated to a milder phenotype. PMID:21494555

Comparison of the Transcription and Replication Strategies of Marburg Virus and Ebola Virus by Using Artificial Replication Systems

PubMed Central

Mühlberger, Elke; Weik, Michael; Volchkov, Viktor E.; Klenk, Hans-Dieter; Becker, Stephan

1999-01-01

The members of the family Filoviridae, Marburg virus (MBGV) and Ebola virus (EBOV), are very similar in terms of morphology, genome organization, and protein composition. To compare the replication and transcription strategies of both viruses, an artificial replication system based on the vaccinia virus T7 expression system was established for EBOV. Specific transcription and replication of an artificial monocistronic minireplicon was demonstrated by reporter gene expression and detection of the transcribed and replicated RNA species. As it was shown previously for MBGV, three of the four EBOV nucleocapsid proteins, NP, VP35, and L, were essential and sufficient for replication. In contrast to MBGV, EBOV-specific transcription was dependent on the presence of the fourth nucleocapsid protein, VP30. When EBOV VP30 was replaced by MBGV VP30, EBOV-specific transcription was observed but with lower efficiency. Exchange of NP, VP35, and L between the two replication systems did not lead to detectable reporter gene expression. It was further observed that neither MBGV nor EBOV were able to replicate the heterologous minigenomes. A chimeric minigenome, however, containing the EBOV leader and the MBGV trailer was encapsidated, replicated, transcribed, and packaged by both viruses. PMID:9971816

Absence of 60-Hz, 0.1-mT magnetic field-induced changes in oncogene transcription rates or levels in CEM-CM3 cells.

PubMed

Jahreis, G P; Johnson, P G; Zhao, Y L; Hui, S W

1998-12-22

Our objective was to assess the reproducibility of the 60-Hz magnetic field-induced, time-dependent transcription changes of c-fos, c-jun and c-myc oncogenes in CEM-CM3 cells reported by Phillips et al. (Biochim. Biophys. Acta, 1132 (1992) 140-144). Cells were exposed to a 60-Hz magnetic field (MF) at 0.1 mT (rms), generated by a pair of Helmholtz coils energized in a reinforcing (MF) mode, or to a null magnetic field when the coils were energized in a bucking (sham) mode. After MF or sham exposure for 15, 30, 60 or 120 min, nuclei and cytoplasmic RNA were extracted. Transcription rates were measured by a nuclear run-on assay, and values were normalized against either their zero-time exposure values, or against those of the c-G3PDH (housekeeping) gene at the same time points. There was no significant difference, at P=0.05, detected between MF and either sham-exposed or control cells at any time point. Transcript levels of the oncogenes were measured by Northern analysis and normalized as above. No significant difference (P=0.05) in transcript levels between MF and either sham-exposed or control cells was detected.

Global survey of genomic imprinting by transcriptome sequencing.

PubMed

Babak, Tomas; Deveale, Brian; Armour, Christopher; Raymond, Christopher; Cleary, Michele A; van der Kooy, Derek; Johnson, Jason M; Lim, Lee P

2008-11-25

Genomic imprinting restricts gene expression to a paternal or maternal allele. To date, approximately 90 imprinted transcripts have been identified in mouse, of which the majority were detected after intense interrogation of clusters of imprinted genes identified by phenotype-driven assays in mice with uniparental disomies [1]. Here we use selective priming and parallel sequencing to measure allelic bias in whole transcriptomes. By distinguishing parent-of-origin bias from strain-specific bias in embryos derived from a reciprocal cross of mice, we constructed a genome-wide map of imprinted transcription. This map was able to objectively locate over 80% of known imprinted loci and allowed the detection and confirmation of six novel imprinted genes. Even in the intensely studied embryonic day 9.5 developmental stage that we analyzed, more than half of all imprinted single-nucleotide polymorphisms did not overlap previously discovered imprinted transcripts; a large fraction of these represent novel noncoding RNAs within known imprinted loci. For example, a previously unnoticed, maternally expressed antisense transcript was mapped within the Grb10 locus. This study demonstrates the feasibility of using transcriptome sequencing for mapping of imprinted gene expression in physiologically normal animals. Such an approach will allow researchers to study imprinting without restricting themselves to individual loci or specific transcripts.

TAFII-independent activation mediated by human TBP in the presence of the positive cofactor PC4.

PubMed Central

Wu, S Y; Kershnar, E; Chiang, C M

1998-01-01

TFIID is a multiprotein complex comprised of the TATA-binding protein (TBP) and an array of TBP-associated factors (TAFIIs). Whereas TBP is sufficient for basal transcription in conjunction with other general transcription factors and RNA polymerase II, TAFIIs are additionally required for activator-dependent transcription in mammalian cell-free transcription systems. However, recent in vivo studies carried out in yeast suggest that TAFIIs are not globally required for activator function. The discrepancy between in vivo yeast studies and in vitro mammalian cell-free systems remains to be resolved. In this study, we describe a mammalian cell-free transcription system reconstituted with only recombinant proteins and epitope-tagged multiprotein complexes. Transcriptional activation can be recapitulated in this highly purified in vitro transcription system in the absence of TAFIIs. This TBP-mediated activation is not induced by human mediator, another transcriptional coactivator complex potentially implicated in activator response. In contrast, general transcription factors TFIIH and TFIIA play a significant role in TBP-mediated activation, which can be detected in vitro with Gal4 fusion proteins containing various transcriptional activation domains. Our data, therefore, suggest that TFIIH and TFIIA can mediate activator function in the absence of TAFIIs. PMID:9687514

In Vivo Orientation of Single Myosin Lever Arms in Zebrafish Skeletal Muscle

PubMed Central

Sun, Xiaojing; Ekker, Stephen C.; Shelden, Eric A.; Takubo, Naoko; Wang, Yihua; Burghardt, Thomas P.

2014-01-01

Cardiac and skeletal myosin assembled in the muscle lattice power contraction by transducing ATP free energy into the mechanical work of moving actin. Myosin catalytic/lever-arm domains comprise the transduction/mechanical coupling machinery that move actin by lever-arm rotation. In vivo, myosin is crowded and constrained by the fiber lattice as side chains are mutated and otherwise modified under normal, diseased, or aging conditions that collectively define the native myosin environment. Single-myosin detection uniquely defines bottom-up characterization of myosin functionality. The marriage of in vivo and single-myosin detection to study zebrafish embryo models of human muscle disease is a multiscaled technology that allows one-to-one registration of a selected myosin molecular alteration with muscle filament-sarcomere-cell-fiber-tissue-organ- and organism level phenotypes. In vivo single-myosin lever-arm orientation was observed at superresolution using a photoactivatable-green-fluorescent-protein (PAGFP)-tagged myosin light chain expressed in zebrafish skeletal muscle. By simultaneous observation of multiphoton excitation fluorescence emission and second harmonic generation from myosin, we demonstrated tag specificity for the lever arm. Single-molecule detection used highly inclined parallel beam illumination and was verified by quantized photoactivation and photobleaching. Single-molecule emission patterns from relaxed muscle in vivo provided extensive superresolved dipole orientation constraints that were modeled using docking scenarios generated for the myosin (S1) and GFP crystal structures. The dipole orientation data provided sufficient constraints to estimate S1/GFP coordination. The S1/GFP coordination in vivo is rigid and the lever-arm orientation distribution is well-ordered in relaxed muscle. For comparison, single myosins in relaxed permeabilized porcine papillary muscle fibers indicated slightly differently oriented lever arms and rigid S1/GFP coordination. Lever arms in both muscles indicated one preferred spherical polar orientation and widely distributed azimuthal orientations relative to the fiber symmetry axis. Cardiac myosin is more radially displaced from the fiber axis. Probe rigidity implies the PAGFP tag reliably indicates cross-bridge orientation in situ and in vivo. PMID:25229148

Expression of growth hormone and its transcription factor, Pit-1, in early bovine development.

PubMed

Joudrey, E M; Lechniak, D; Petrik, J; King, W A

2003-03-01

During bovine embryogenesis, bovine growth hormone (bGH) contributes to proliferation, differentiation, and modulation of embryo metabolism. Pituitary-specific transcription factor-1 (Pit-1) is a transcription factor that binds to promoters of GH, prolactin (PRL), and thyroid-stimulating hormone-beta (TSHbeta) encoding genes. A polymorphism in the fifth exon of the bGH gene resulting in a leucine (Leu) to valine (Val) substitution provides an Alu I restriction site when the Leu allele is present. To determine the onset of embryonic expression of the bGH gene, oocytes derived from ovaries homozygous for Leu alleles were fertilized in vitro with spermatozoa obtained from a Val homozygote. For each developmental stage examined, three separate pools of embryos composed of approximately 100 cell samples underwent RNA isolation, reverse transcription to cDNA, and amplification by nested PCR (nPCR). Bovine GH gene transcripts were identified at 2- to 4-cell (n = 162), 8- to 16-cell (n = 73), morulae (n = 51), and blastocyst (n = 15) stages. Likewise, transcripts for Pit-1 were detected at 2-cell (n = 125), 4-cell (n = 114), 8-cell (n = 56), 12-to-32-cell (n = 32), morulae (n = 68), and blastocyst (n = 14) stages. After digestion with Alu1, bGH cDNA was genotyped by restriction fragment length polymorphism (RFLP) analysis. Bovine GH mRNA was present in all pools of stages examined. Both Leu and Val alleles (maternal and paternal) were only detected in pools of embryos that had reached 8- to 16-cell stage. Results suggest that transcription of the bGH gene begins at the 8- to 16-cell stage in bovine embryos, possibly under control of the transcription factor, Pit-1, and that RFLP analysis of the bGH gene can be used to determine parental origin of transcripts in early embryonic development. Copyright 2003 Wiley-Liss, Inc.

Transcriptional and translational adaptation to aerobic nitrate anabolism in the denitrifier Paracoccus denitrificans.

PubMed

Luque-Almagro, Victor M; Manso, Isabel; Sullivan, Matthew J; Rowley, Gary; Ferguson, Stuart J; Moreno-Vivián, Conrado; Richardson, David J; Gates, Andrew J; Roldán, M Dolores

2017-05-10

Transcriptional adaptation to nitrate-dependent anabolism by Paracoccus denitrificans PD1222 was studied. A total of 74 genes were induced in cells grown with nitrate as N-source compared with ammonium, including nasTSABGHC and ntrBC genes. The nasT and nasS genes were cotranscribed, although nasT was more strongly induced by nitrate than nasS The nasABGHC genes constituted a transcriptional unit, which is preceded by a non-coding region containing hairpin structures involved in transcription termination. The nasTS and nasABGHC transcripts were detected at similar levels with nitrate or glutamate as N-source, but nasABGHC transcript was undetectable in ammonium-grown cells. The nitrite reductase NasG subunit was detected by two-dimensional polyacrylamide gel electrophoresis in cytoplasmic fractions from nitrate-grown cells, but it was not observed when either ammonium or glutamate was used as the N-source. The nasT mutant lacked both nasABGHC transcript and nicotinamide adenine dinucleotide (NADH)-dependent nitrate reductase activity. On the contrary, the nasS mutant showed similar levels of the nasABGHC transcript to the wild-type strain and displayed NasG protein and NADH-nitrate reductase activity with all N-sources tested, except with ammonium. Ammonium repression of nasABGHC was dependent on the Ntr system. The ntrBC and ntrYX genes were expressed at low levels regardless of the nitrogen source supporting growth. Mutational analysis of the ntrBCYX genes indicated that while ntrBC genes are required for nitrate assimilation, ntrYX genes can only partially restore growth on nitrate in the absence of ntrBC genes. The existence of a regulation mechanism for nitrate assimilation in P. denitrificans , by which nitrate induction operates at both transcriptional and translational levels, is proposed. © 2017 The Author(s).

Responses to Orientation Discontinuities in V1 and V2: Physiological Dissociations and Functional Implications

PubMed Central

Purpura, Keith P.; Victor, Jonathan D.

2014-01-01

Segmenting the visual image into objects is a crucial stage of visual processing. Object boundaries are typically associated with differences in luminance, but discontinuities in texture also play an important role. We showed previously that a subpopulation of neurons in V2 in anesthetized macaques responds to orientation discontinuities parallel to their receptive field orientation. Such single-cell responses could be a neurophysiological correlate of texture boundary detection. Neurons in V1, on the other hand, are known to have contextual response modulations such as iso-orientation surround suppression, which also produce responses to orientation discontinuities. Here, we use pseudorandom multiregion grating stimuli of two frame durations (20 and 40 ms) to probe and compare texture boundary responses in V1 and V2 in anesthetized macaque monkeys. In V1, responses to texture boundaries were observed for only the 40 ms frame duration and were independent of the orientation of the texture boundary. However, in transient V2 neurons, responses to such texture boundaries were robust for both frame durations and were stronger for boundaries parallel to the neuron's preferred orientation. The dependence of these processes on stimulus duration and orientation indicates that responses to texture boundaries in V2 arise independently of contextual modulations in V1. In addition, because the responses in transient V2 neurons are sensitive to the orientation of the texture boundary but those of V1 neurons are not, we suggest that V2 responses are the correlate of texture boundary detection, whereas contextual modulation in V1 serves other purposes, possibly related to orientation “pop-out.” PMID:24599456

Regulation of expression of two LY-6 family genes by intron retention and transcription induced chimerism

PubMed Central

Calvanese, Vincenzo; Mallya, Meera; Campbell, R Duncan; Aguado, Begoña

2008-01-01

Background Regulation of the expression of particular genes can rely on mechanisms that are different from classical transcriptional and translational control. The LY6G5B and LY6G6D genes encode LY-6 domain proteins, whose expression seems to be regulated in an original fashion, consisting of an intron retention event which generates, through an early premature stop codon, a non-coding transcript, preventing expression in most cell lines and tissues. Results The MHC LY-6 non-coding transcripts have shown to be stable and very abundant in the cell, and not subject to Nonsense Mediated Decay (NMD). This retention event appears not to be solely dependent on intron features, because in the case of LY6G5B, when the intron is inserted in the artificial context of a luciferase expression plasmid, it is fully spliced but strongly stabilises the resulting luciferase transcript. In addition, by quantitative PCR we found that the retained and spliced forms are differentially expressed in tissues indicating an active regulation of the non-coding transcript. EST database analysis revealed that these genes have an alternative expression pathway with the formation of Transcription Induced Chimeras (TIC). This data was confirmed by RT-PCR, revealing the presence of different transcripts that would encode the chimeric proteins CSNKβ-LY6G5B and G6F-LY6G6D, in which the LY-6 domain would join to a kinase domain and an Ig-like domain, respectively. Conclusion In conclusion, the LY6G5B and LY6G6D intron-retained transcripts are not subjected to NMD and are more abundant than the properly spliced forms. In addition, these genes form chimeric transcripts with their neighbouring same orientation 5' genes. Of interest is the fact that the 5' genes (CSNKβ or G6F) undergo differential splicing only in the context of the chimera (CSNKβ-LY6G5B or G6F-LY6G6C) and not on their own. PMID:18817541

Knowing where is different from knowing what: Distinct response time profiles and accuracy effects for target location, orientation, and color probability.

PubMed

Jabar, Syaheed B; Filipowicz, Alex; Anderson, Britt

2017-11-01

When a location is cued, targets appearing at that location are detected more quickly. When a target feature is cued, targets bearing that feature are detected more quickly. These attentional cueing effects are only superficially similar. More detailed analyses find distinct temporal and accuracy profiles for the two different types of cues. This pattern parallels work with probability manipulations, where both feature and spatial probability are known to affect detection accuracy and reaction times. However, little has been done by way of comparing these effects. Are probability manipulations on space and features distinct? In a series of five experiments, we systematically varied spatial probability and feature probability along two dimensions (orientation or color). In addition, we decomposed response times into initiation and movement components. Targets appearing at the probable location were reported more quickly and more accurately regardless of whether the report was based on orientation or color. On the other hand, when either color probability or orientation probability was manipulated, response time and accuracy improvements were specific for that probable feature dimension. Decomposition of the response time benefits demonstrated that spatial probability only affected initiation times, whereas manipulations of feature probability affected both initiation and movement times. As detection was made more difficult, the two effects further diverged, with spatial probability disproportionally affecting initiation times and feature probability disproportionately affecting accuracy. In conclusion, all manipulations of probability, whether spatial or featural, affect detection. However, only feature probability affects perceptual precision, and precision effects are specific to the probable attribute.

The lizard celestial compass detects linearly polarized light in the blue.

PubMed

Beltrami, Giulia; Parretta, Antonio; Petrucci, Ferruccio; Buttini, Paola; Bertolucci, Cristiano; Foà, Augusto

2012-09-15

The present study first examined whether ruin lizards, Podarcis sicula, are able to orientate using plane-polarized light produced by an LCD screen. Ruin lizards were trained and tested indoors, inside a hexagonal Morris water maze positioned under an LCD screen producing white polarized light with a single E-vector, which provided an axial cue. White polarized light did not include wavelengths in the UV. Lizards orientated correctly either when tested with E-vector parallel to the training axis or after 90 deg rotation of the E-vector direction, thus validating the apparatus. Further experiments examined whether there is a preferential region of the light spectrum to perceive the E-vector direction of polarized light. For this purpose, lizards reaching learning criteria under white polarized light were subdivided into four experimental groups. Each group was tested for orientation under a different spectrum of plane-polarized light (red, green, cyan and blue) with equalized photon flux density. Lizards tested under blue polarized light orientated correctly, whereas lizards tested under red polarized light were completely disoriented. Green polarized light was barely discernible by lizards, and thus insufficient for a correct functioning of their compass. When exposed to cyan polarized light, lizard orientation performances were optimal, indistinguishable from lizards detecting blue polarized light. Overall, the present results demonstrate that perception of linear polarization in the blue is necessary - and sufficient - for a proper functioning of the sky polarization compass of ruin lizards. This may be adaptively important, as detection of polarized light in the blue improves functioning of the polarization compass under cloudy skies, i.e. when the alternative celestial compass based on detection of the sun disk is rendered useless because the sun is obscured by clouds.

Individual differences in attention strategies during detection, fine discrimination, and coarse discrimination

PubMed Central

Hecker, Elizabeth A.; Serences, John T.; Srinivasan, Ramesh

2013-01-01

Interacting with the environment requires the ability to flexibly direct attention to relevant features. We examined the degree to which individuals attend to visual features within and across Detection, Fine Discrimination, and Coarse Discrimination tasks. Electroencephalographic (EEG) responses were measured to an unattended peripheral flickering (4 or 6 Hz) grating while individuals (n = 33) attended to orientations that were offset by 0°, 10°, 20°, 30°, 40°, and 90° from the orientation of the unattended flicker. These unattended responses may be sensitive to attentional gain at the attended spatial location, since attention to features enhances early visual responses throughout the visual field. We found no significant differences in tuning curves across the three tasks in part due to individual differences in strategies. We sought to characterize individual attention strategies using hierarchical Bayesian modeling, which grouped individuals into families of curves that reflect attention to the physical target orientation (“on-channel”) or away from the target orientation (“off-channel”) or a uniform distribution of attention. The different curves were related to behavioral performance; individuals with “on-channel” curves had lower thresholds than individuals with uniform curves. Individuals with “off-channel” curves during Fine Discrimination additionally had lower thresholds than those assigned to uniform curves, highlighting the perceptual benefits of attending away from the physical target orientation during fine discriminations. Finally, we showed that a subset of individuals with optimal curves (“on-channel”) during Detection also demonstrated optimal curves (“off-channel”) during Fine Discrimination, indicating that a subset of individuals can modulate tuning optimally for detection and discrimination. PMID:23678013

Expressed sequence tag analysis of human RPE/choroid for the NEIBank Project: over 6000 non-redundant transcripts, novel genes and splice variants.

PubMed

Wistow, Graeme; Bernstein, Steven L; Wyatt, M Keith; Fariss, Robert N; Behal, Amita; Touchman, Jeffrey W; Bouffard, Gerald; Smith, Don; Peterson, Katherine

2002-06-15

The retinal pigment epithelium (RPE) and choroid comprise a functional unit of the eye that is essential to normal retinal health and function. Here we describe expressed sequence tag (EST) analysis of human RPE/choroid as part of a project for ocular bioinformatics. A cDNA library (cs) was made from human RPE/choroid and sequenced. Data were analyzed and assembled using the program GRIST (GRouping and Identification of Sequence Tags). Complete sequencing, Northern and Western blots, RH mapping, peptide antibody synthesis and immunofluorescence (IF) have been used to examine expression patterns and genome location for selected transcripts and proteins. Ten thousand individual sequence reads yield over 6300 unique gene clusters of which almost half have no matches with named genes. One of the most abundant transcripts is from a gene (named "alpha") that maps to the BBS1 region of chromosome 11. A number of tissue preferred transcripts are common to both RPE/choroid and iris. These include oculoglycan/opticin, for which an alternative splice form is detected in RPE/choroid, and "oculospanin" (Ocsp), a novel tetraspanin that maps to chromosome 17q. Antiserum to Ocsp detects expression in RPE, iris, ciliary body, and retinal ganglion cells by IF. A newly identified gene for a zinc-finger protein (TIRC) maps to 19q13.4. Variant transcripts of several genes were also detected. Most notably, the predominant form of Bestrophin represented in cs contains a longer open reading frame as a result of splice junction skipping. The unamplified cs library gives a view of the transcriptional repertoire of the adult RPE/choroid. A large number of potentially novel genes and splice forms and candidates for genetic diseases are revealed. Clones from this collection are being included in a large, nonredundant set for cDNA microarray construction.

Identification and characterization of transcripts from the biotin biosynthetic operon of Bacillus subtilis.

PubMed Central

Perkins, J B; Bower, S; Howitt, C L; Yocum, R R; Pero, J

1996-01-01

Northern (RNA) blot analysis of the Bacillus subtilis biotin operon, bioWAFDBIorf2, detected at least two steady-state polycistronic transcripts initiated from a putative vegetative (Pbio) promoter that precedes the operon, i.e., a full-length 7.2-kb transcript covering the entire operon and a more abundant 5.1-kb transcript covering just the first five genes of the operon. Biotin and the B. subtilis birA gene product regulated synthesis of the transcripts. Moreover, replacing the putative Pbio promoter and regulatory sequence with a constitutive SP01 phage promoter resulted in higher-level constitutive synthesis. Removal of a rho-independent terminator-like sequence located between the fifth (bioB) and sixth (bioI) genes prevented accumulation of the 5.1-kb transcript, suggesting that the putative terminator functions to limit expression of bioI, which is thought to be involved in an early step in biotin synthesis. PMID:8892842

Identification and characterization of transcripts from the biotin biosynthetic operon of Bacillus subtilis.

PubMed

Perkins, J B; Bower, S; Howitt, C L; Yocum, R R; Pero, J

1996-11-01

Northern (RNA) blot analysis of the Bacillus subtilis biotin operon, bioWAFDBIorf2, detected at least two steady-state polycistronic transcripts initiated from a putative vegetative (Pbio) promoter that precedes the operon, i.e., a full-length 7.2-kb transcript covering the entire operon and a more abundant 5.1-kb transcript covering just the first five genes of the operon. Biotin and the B. subtilis birA gene product regulated synthesis of the transcripts. Moreover, replacing the putative Pbio promoter and regulatory sequence with a constitutive SP01 phage promoter resulted in higher-level constitutive synthesis. Removal of a rho-independent terminator-like sequence located between the fifth (bioB) and sixth (bioI) genes prevented accumulation of the 5.1-kb transcript, suggesting that the putative terminator functions to limit expression of bioI, which is thought to be involved in an early step in biotin synthesis.

Growth-dependent regulation of rRNA synthesis is mediated by a transcription initiation factor (TIF-IA).

PubMed

Buttgereit, D; Pflugfelder, G; Grummt, I

1985-11-25

Mouse RNA polymerase I requires at least two chromatographically distinct transcription factors (designated TIF-IA and TIF-IB) to initiate transcription accurately and efficiently in vitro. In this paper we describe the partial purification of TIF-IA by a four-step fractionation procedure. The amount or activity of TIF-IA fluctuates in response to the physiological state of the cells. Extracts from quiescent cells are incapable of specific transcription and do not contain detectable levels of TIF-IA. Transcriptionally inactive extracts can be restored by the addition of TIF-IA preparations that have been highly purified from exponentially growing cells. During the fractionating procedure TIF-IA co-purifies with RNA polymerase I, suggesting that it is functionally associated with the transcribing enzyme. We suggest that only those enzyme molecules that are associated with TIF-IA are capable to interact with TIF-IB and to initiate transcription.

TALE-mediated modulation of transcriptional enhancers in vivo.

PubMed

Crocker, Justin; Stern, David L

2013-08-01

We tested whether transcription activator-like effectors (TALEs) could mediate repression and activation of endogenous enhancers in the Drosophila genome. TALE repressors (TALERs) targeting each of the five even-skipped (eve) stripe enhancers generated repression specifically of the focal stripes. TALE activators (TALEAs) targeting the eve promoter or enhancers caused increased expression primarily in cells normally activated by the promoter or targeted enhancer, respectively. This effect supports the view that repression acts in a dominant fashion on transcriptional activators and that the activity state of an enhancer influences TALE binding or the ability of the VP16 domain to enhance transcription. In these assays, the Hairy repression domain did not exhibit previously described long-range transcriptional repression activity. The phenotypic effects of TALER and TALEA expression in larvae and adults are consistent with the observed modulations of eve expression. TALEs thus provide a novel tool for detection and functional modulation of transcriptional enhancers in their native genomic context.

High-Throughput Protein Expression Using a Combination of Ligation-Independent Cloning (LIC) and Infrared Fluorescent Protein (IFP) Detection

PubMed Central

Dortay, Hakan; Akula, Usha Madhuri; Westphal, Christin; Sittig, Marie; Mueller-Roeber, Bernd

2011-01-01

Protein expression in heterologous hosts for functional studies is a cumbersome effort. Here, we report a superior platform for parallel protein expression in vivo and in vitro. The platform combines highly efficient ligation-independent cloning (LIC) with instantaneous detection of expressed proteins through N- or C-terminal fusions to infrared fluorescent protein (IFP). For each open reading frame, only two PCR fragments are generated (with three PCR primers) and inserted by LIC into ten expression vectors suitable for protein expression in microbial hosts, including Escherichia coli, Kluyveromyces lactis, Pichia pastoris, the protozoon Leishmania tarentolae, and an in vitro transcription/translation system. Accumulation of IFP-fusion proteins is detected by infrared imaging of living cells or crude protein extracts directly after SDS-PAGE without additional processing. We successfully employed the LIC-IFP platform for in vivo and in vitro expression of ten plant and fungal proteins, including transcription factors and enzymes. Using the IFP reporter, we additionally established facile methods for the visualisation of protein-protein interactions and the detection of DNA-transcription factor interactions in microtiter and gel-free format. We conclude that IFP represents an excellent reporter for high-throughput protein expression and analysis, which can be easily extended to numerous other expression hosts using the setup reported here. PMID:21541323

Experimental infection of European flat oyster Ostrea edulis with ostreid herpesvirus 1 microvar (OsHV-1μvar): Mortality, viral load and detection of viral transcripts by in situ hybridization.

PubMed

López Sanmartín, Monserrat; Power, Deborah M; de la Herrán, Roberto; Navas, José I; Batista, Frederico M

2016-06-02

Ostreid herpesvirus 1 (OsHV-1) infections have been reported in several bivalve species. Mortality of Pacific oyster Crassostrea gigas spat has increased considerably in Europe since 2008 linked to the spread of a variant of OsHV-1 called μvar. In the present study we demonstrated that O. edulis juveniles can be infected by OsHV-1μvar when administered as an intramuscular injection. Mortality in the oysters injected with OsHV-1μvar was first detected 4 days after injection and reached 25% mortality at day 10. Moreover, the high viral load observed and the detection of viral transcripts by in situ hybridization in several tissues of dying oysters suggested that OsHV-1μvar was the cause of mortality in the O. edulis juveniles. This is therefore the first study to provide evidence about the pathogenicity of OsHV-1μvar in a species that does not belong to the Crassostrea genus. Additionally, we present a novel method to detect OsHV-1 transcripts in infected individuals' using in situ hybridization. Copyright © 2016 Elsevier B.V. All rights reserved.

Activity-dependent gene expression in honey bee mushroom bodies in response to orientation flight.

PubMed

Lutz, Claudia C; Robinson, Gene E

2013-06-01

The natural history of adult worker honey bees (Apis mellifera) provides an opportunity to study the molecular basis of learning in an ecological context. Foragers must learn to navigate between the hive and floral locations that may be up to miles away. Young pre-foragers prepare for this task by performing orientation flights near the hive, during which they begin to learn navigational cues such as the appearance of the hive, the position of landmarks, and the movement of the sun. Despite well-described spatial learning and navigation behavior, there is currently limited information on the neural basis of insect spatial learning. We found that Egr, an insect homolog of Egr-1, is rapidly and transiently upregulated in the mushroom bodies in response to orientation. This result is the first example of an Egr-1 homolog acting as a learning-related immediate-early gene in an insect and also demonstrates that honey bee orientation uses a molecular mechanism that is known to be involved in many other forms of learning. This transcriptional response occurred both in naïve bees and in foragers induced to re-orient. Further experiments suggest that visual environmental novelty, rather than exercise or memorization of specific visual cues, acts as the stimulus for Egr upregulation. Our results implicate the mushroom bodies in spatial learning and emphasize the deep conservation of Egr-related pathways in experience-dependent plasticity.

Reality of working in a community-based, recovery-oriented mental health rehabilitation unit: A pragmatic grounded theory analysis.

PubMed

Parker, Stephen; Dark, Frances; Newman, Ellie; Korman, Nicole; Rasmussen, Zoe; Meurk, Carla

2017-08-01

In the present study, we explored the experiences of staff working at a recovery-oriented, community-based residential mental health rehabilitation unit in Brisbane, Australia, called a 'community care unit' (CCU). A pragmatic approach to grounded theory was taken in the analysis of the transcripts of semistructured interviews with eight staff. Convenience sampling was used, and there was representation of junior and senior staff across nursing, allied health, and non-clinical support roles. Four key themes emerged from the analysis: (i) rehabilitation is different to treatment; (ii) the CCU is a positive transitional space; (iii) they (consumers) have to be ready to engage; and (iv) recovery is central to rehabilitation practice. Staff understandings of recovery in rehabilitation work were complex and included consideration of both personal and clinical recovery concepts. Rehabilitation readiness was considered important to the ability to deliver recovery-oriented care; however, the shared role of staff in maintaining engagement was acknowledged. Threats to recovery-oriented rehabilitation practice included staff burnout and external pressure to accept consumers who are not ready. The reality of working at a community-based recovery-oriented rehabilitation unit is complex. Active vigilance is needed to maintain a focus on recovery and rehabilitation. Leadership needs to focus on reducing burnout and in adapting these services to emergent needs. © 2016 Australian College of Mental Health Nurses Inc.

Detection of Protein Interactions in T3S Systems Using Yeast Two-Hybrid Analysis.

PubMed

Nilles, Matthew L

2017-01-01

Two-hybrid systems, sometimes termed interaction traps, are genetic systems designed to find and analyze interactions between proteins. The most common systems are yeast based (commonly Saccharomyces cerevisae) and rely on the functional reconstitution of the GAL4 transcriptional activator. Reporter genes, such as the lacZ gene of Escherichia coli (encodes β-galactosidase), are placed under GAL4-dependent transcriptional control to provide quick and reliable detection of protein interactions. In this method the use of a yeast-based two-hybrid system is described to study protein interactions between components of type III secretion systems.

The establishment of polarized membrane traffic in Xenopus laevis embryos.

PubMed

Roberts, S J; Leaf, D S; Moore, H P; Gerhart, J C

1992-09-01

Delineation of apical and basolateral membrane domains is a critical step in the epithelialization of the outer layer of cells in the embryo. We have examined the initiation of polarized membrane traffic in Xenopus and show that membrane traffic is not polarized in oocytes but polarized membrane domains appear at first cleavage. The following proteins encoded by injected RNA transcripts were used as markers to monitor membrane traffic: (a) VSV G, a transmembrane glycoprotein preferentially inserted into the basolateral surface of polarized epithelial cells; (b) GThy-1, a fusion protein of VSV G and Thy-1 that is localized to the apical domains of polarized epithelial cells; and (c) prolactin, a peptide hormone that is not polarly secreted. In immature oocytes, there is no polarity in the expression of VSV G or GThy-1, as shown by the constitutive expression of both proteins at the surface in the animal and vegetal hemispheres. At meiotic maturation, membrane traffic to the surface is blocked; the plasma membrane no longer accepts the vesicles synthesized by the oocyte (Leaf, D. L., S. J. Roberts, J. C. Gerhart, and H.-P. Moore. 1990. Dev. Biol. 141:1-12). When RNA transcripts are injected after fertilization, VSV G is expressed only in the internal cleavage membranes (basolateral orientation) and is excluded from the outer surface (apical orientation, original oocyte membrane). In contrast, GThy-1 and prolactin, when expressed in embryos, are inserted or released at both the outer membrane derived from the oocyte and the inner cleavage membranes. Furthermore, not all of the cleavage membrane comes from an embryonic pool of vesicles--some of the cleavage membrane comes from vesicles synthesized during oogenesis. Using prolactin as a marker, we found that a subset of vesicles synthesized during oogenesis was only released after fertilization. However, while embryonic prolactin was secreted from both apical and basolateral surfaces, the secretion of oogenic prolactin was polarized. Oogenic prolactin was secreted only into the blastocoel (from the cleavage membrane), none could be detected in the external medium (from the original oocyte membrane). These results provide the first direct evidence that the oocyte synthesizes a cache of vesicles for specific recruitment to the embryonic cleavage membranes which are polarized beginning with the first cleavage division.

Flexible DNA binding of the BTB/POZ-domain protein FBI-1.

PubMed

Pessler, Frank; Hernandez, Nouria

2003-08-01

POZ-domain transcription factors are characterized by the presence of a protein-protein interaction domain called the POZ or BTB domain at their N terminus and zinc fingers at their C terminus. Despite the large number of POZ-domain transcription factors that have been identified to date and the significant insights that have been gained into their cellular functions, relatively little is known about their DNA binding properties. FBI-1 is a BTB/POZ-domain protein that has been shown to modulate HIV-1 Tat trans-activation and to repress transcription of some cellular genes. We have used various viral and cellular FBI-1 binding sites to characterize the interaction of a POZ-domain protein with DNA in detail. We find that FBI-1 binds to inverted sequence repeats downstream of the HIV-1 transcription start site. Remarkably, it binds efficiently to probes carrying these repeats in various orientations and spacings with no particular rotational alignment, indicating that its interaction with DNA is highly flexible. Indeed, FBI-1 binding sites in the adenovirus 2 major late promoter, the c-fos gene, and the c-myc P1 and P2 promoters reveal variously spaced direct, inverted, and everted sequence repeats with the consensus sequence G(A/G)GGG(T/C)(C/T)(T/C)(C/T) for each repeat.

A long antisense RNA in plant chloroplasts.

PubMed

Georg, J; Honsel, A; Voss, B; Rennenberg, H; Hess, W R

2010-05-01

Based on computational prediction of RNA secondary structures, a long antisense RNA (asRNA) was found in chloroplasts of Arabidopsis, Nicotiana tabacum and poplar, which occurs in two to three major transcripts. Mapping of primary 5' ends, northern hybridizations and quantitative real-time reverse transcription polymerase chain reaction (qPCR) experiments demonstrated that these transcripts originate from a promoter that is typical for the plastid-encoded RNA polymerase and are over their full length in antisense orientation to the gene ndhB and therefore were designated asRNA_ndhB. The asRNA_ndhB transcripts predominantly accumulate in young leaves and at physiological growth temperatures. Two nucleotide positions in the mRNA that are subject to C-to-U RNA editing and which were previously found to be sensitive to elevated temperatures are covered by asRNA_ndhB. Nevertheless, the correlation between the accumulation of asRNA_ndhB and RNA editing appeared weak in a temperature shift experiment. With asRNA_ndhB, we describe the first asRNA of plant chloroplasts that covers RNA editing sites, as well as a group II intron splice acceptor site, and that is under developmental control, raising the possibility that long asRNAs could be involved in RNA maturation or the control of RNA stability.

Measurement of the orientation of buffer-gas-cooled, electrostatically-guided ammonia molecules

NASA Astrophysics Data System (ADS)

Steer, Edward W.; Petralia, Lorenzo S.; Western, Colin M.; Heazlewood, Brianna R.; Softley, Timothy P.

2017-02-01

The extent to which the spatial orientation of internally and translationally cold ammonia molecules can be controlled as molecules pass out of a quadrupole guide and through different electric field regions is examined. Ammonia molecules are collisionally cooled in a buffer gas cell, and are subsequently guided by a three-bend electrostatic quadrupole into a detection chamber. The orientation of ammonia molecules is probed using (2 + 1) resonance-enhanced multiphoton ionisation (REMPI), with the laser polarisation axis aligned both parallel and perpendicular to the time-of-flight axis. Even with the presence of a near-zero field region, the ammonia REMPI spectra indicate some retention of orientation. Monte Carlo simulations propagating the time-dependent Schrödinger equation in a full basis set including the hyperfine interaction enable the orientation of ammonia molecules to be calculated - with respect to both the local field direction and a space-fixed axis - as the molecules pass through different electric field regions. The simulations indicate that the orientation of ∼95% of ammonia molecules in JK =11 could be achieved with the application of a small bias voltage (17 V) to the mesh separating the quadrupole and detection regions. Following the recent combination of the buffer gas cell and quadrupole guide apparatus with a linear Paul ion trap, this result could enable one to examine the influence of molecular orientation on ion-molecule reaction dynamics and kinetics.

Development of detection and recognition of orientation of geometric and real figures.

PubMed

Stein, N L; Mandler, J M

1975-06-01

Black and white kindergarten and second-grade children were tested for accuracy of detection and recognition of orientation and location changes in pictures of real-world and geometric figures. No differences were found in accuracy of recognition between the 2 kinds of pictures, but patterns of verbalization differed on specific transformations. Although differences in accuracy were found between kindergarten and second grade on an initial recognition task, practice on a matching-to-sample task eliminated differences on a second recognition task. Few ethnic differences were found on accuracy of recognition, but significant differences were found in amount of verbal output on specific transformations. For both groups, mention of orientation changes was markedly reduced when location changes were present.

Specific inhibition of aphthovirus infection by RNAs transcribed from both the 5' and the 3' noncoding regions.

PubMed Central

Gutiérrez, A; Martínez-Salas, E; Pintado, B; Sobrino, F

1994-01-01

RNA molecules containing the 3' terminal region of foot-and-mouth disease virus (FMDV) RNA in both antisense and sense orientations were able to inhibit viral FMDV translation and infective particle formation in BHK-21 cells following comicroinjection or cotransfection with infectious viral RNA. Antisense, but not sense, transcripts from the 5' noncoding region including the proximal element of the internal ribosome entry site and the two functional initiation AUGs were also inhibitory, both in in vitro translation and in vivo in comicroinjected or cotransfected BHK-21 cells. This effect was not observed with nonrelated RNA transcripts from lambda phage. The inhibitions found were permanent, sequence specific, and dose dependent; an inverse correlation between the length of the transcript and the extent of the antiviral effect was seen. In all cases, the extent of inhibition increased when viral RNAs and transcripts were allowed to reanneal before transfection, concomitant with a decrease in the doses required. The antiviral effect was specific for FMDV, since transcripts failed to inhibit infective particle formation by other picornavirus, such as encephalomyocarditis virus. These results indicate that the ability of RNA transcripts to inhibit viral multiplication depends on their efficient hybridization with target regions on the viral genome. Furthermore, cells transfected with the 5'1as transcript, which is complementary to the 5' noncoding region, showed a significant reduction of plaque-forming ability during the course of a natural infection. RNA 5'1as was able to inhibit FMDV RNA translation in vitro, suggesting that the inhibitions observed are mediated by a blockage of the viral translation initiation. Conversely, hybridization of short sequences of both sense and antisense transcripts from the 3' end induces distortion of predicted highly ordered structural motifs, which could be required for the synthesis of negative-stranded viral RNA, and correlates with inhibition of viral propagation. Images PMID:7933126

Specific inhibition of aphthovirus infection by RNAs transcribed from both the 5' and the 3' noncoding regions.

PubMed

Gutiérrez, A; Martínez-Salas, E; Pintado, B; Sobrino, F

1994-11-01

RNA molecules containing the 3' terminal region of foot-and-mouth disease virus (FMDV) RNA in both antisense and sense orientations were able to inhibit viral FMDV translation and infective particle formation in BHK-21 cells following comicroinjection or cotransfection with infectious viral RNA. Antisense, but not sense, transcripts from the 5' noncoding region including the proximal element of the internal ribosome entry site and the two functional initiation AUGs were also inhibitory, both in in vitro translation and in vivo in comicroinjected or cotransfected BHK-21 cells. This effect was not observed with nonrelated RNA transcripts from lambda phage. The inhibitions found were permanent, sequence specific, and dose dependent; an inverse correlation between the length of the transcript and the extent of the antiviral effect was seen. In all cases, the extent of inhibition increased when viral RNAs and transcripts were allowed to reanneal before transfection, concomitant with a decrease in the doses required. The antiviral effect was specific for FMDV, since transcripts failed to inhibit infective particle formation by other picornavirus, such as encephalomyocarditis virus. These results indicate that the ability of RNA transcripts to inhibit viral multiplication depends on their efficient hybridization with target regions on the viral genome. Furthermore, cells transfected with the 5'1as transcript, which is complementary to the 5' noncoding region, showed a significant reduction of plaque-forming ability during the course of a natural infection. RNA 5'1as was able to inhibit FMDV RNA translation in vitro, suggesting that the inhibitions observed are mediated by a blockage of the viral translation initiation. Conversely, hybridization of short sequences of both sense and antisense transcripts from the 3' end induces distortion of predicted highly ordered structural motifs, which could be required for the synthesis of negative-stranded viral RNA, and correlates with inhibition of viral propagation.

Archaeal amoA and ureC genes and their transcriptional activity in the Arctic Ocean

NASA Astrophysics Data System (ADS)

Pedneault, Estelle; Galand, Pierre E.; Potvin, Marianne; Tremblay, Jean-Éric; Lovejoy, Connie

2014-04-01

Thaumarchaeota and the gene encoding for a subunit of ammonia monooxygenase (amoA) are ubiquitous in Polar Seas, and some Thaumarchaeota also have a gene coding for ureC, diagnostic for urease. Using quantitative PCR we investigated the occurrence of genes and transcripts of ureC and amoA in Arctic samples from winter, spring and summer. AmoA genes, ureC genes and amoA transcripts were always present, but ureC transcripts were rarely detected. Over a 48 h light manipulation experiment amoA transcripts persisted under light and dark conditions, but not ureC transcripts. In addition, maxima for amoA transcript were nearer the surface compared to amoA genes. Clone libraries using DNA template recovered shallow and deep amoA clades but only the shallow clade was recovered from cDNA (from RNA). These results imply environmental control of amoA expression with direct or indirect light effects, and rare ureC expression despite its widespread occurrence in the Arctic Ocean.

Evidence that Mediator is essential for Pol II transcription, but is not a required component of the preinitiation complex in vivo.

PubMed

Petrenko, Natalia; Jin, Yi; Wong, Koon Ho; Struhl, Kevin

2017-07-12

The Mediator complex has been described as a general transcription factor, but it is unclear if it is essential for Pol II transcription and/or is a required component of the preinitiation complex (PIC) in vivo. Here, we show that depletion of individual subunits, even those essential for cell growth, causes a general but only modest decrease in transcription. In contrast, simultaneous depletion of all Mediator modules causes a drastic decrease in transcription. Depletion of head or middle subunits, but not tail subunits, causes a downstream shift in the Pol II occupancy profile, suggesting that Mediator at the core promoter inhibits promoter escape. Interestingly, a functional PIC and Pol II transcription can occur when Mediator is not detected at core promoters. These results provide strong evidence that Mediator is essential for Pol II transcription and stimulates PIC formation, but it is not a required component of the PIC in vivo.

Mechanisms of two-color laser-induced field-free molecular orientation.

PubMed

Spanner, Michael; Patchkovskii, Serguei; Frumker, Eugene; Corkum, Paul

2012-09-14

Two mechanisms of two-color (ω+2ω) laser-induced field-free molecular orientation, based on the hyperpolarizability and ionization depletion, are explored and compared. The CO molecule is used as a computational example. While the hyperpolarizability mechanism generates small amounts of orientation at intensities below the ionization threshold, ionization depletion quickly becomes the dominant mechanism as soon as ionizing intensities are reached. Only the ionization mechanism leads to substantial orientation (e.g., on the order of ≳0.1). For intensities typical of laser-induced molecular alignment and orientation experiments, the two mechanisms lead to robust, characteristic timings of the field-free orientation wave-packet revivals relative to the alignment revivals and the revival time. The revival timings can be used to detect the active orientation mechanism experimentally.

[The establishment and application of internal quality control system for real-time quantitative PCR detection of BCR-ABL (P210) transcript levels].

PubMed

Zhong, C Q; He, N; Hua, M Q; Wei, X D; Ma, D X; Ji, C Y

2016-09-14

Objective: To set internal quality control system of BCR-ABL (P210) transcript levels for real-time quantitative PCR (RQ-PCR). Methods: Using K562 cells and HL-60 cells, we prepared high- and low-level BCR-ABL internal quality control substance. The BCR-ABL (P210) transcript levels of internal quality control substance have been determined for 184 times together with clinical samples from August 2013 to October 2015. The slope rate, intercept and correlation coefficient of standard curve were calculated according to different reagent lots (lots number 20130303, 20131212, 20140411 and 20150327 are called R1、R2、R3 and R4 for short respectively), and the detection results of quality control substance were calculated according to different reagent lots and quality control substance lots (lots number 20130725, 20140611 are called Q1、Q2 for short respectively). Then the results were analyzed by Levey-Jennings quality control chart combined with Westgard multi-rules theory. Results: ①We analyzed the slope rate and intercept of standard curve. Fifty-three times of the R1 reagent detection, 80 times of the R3 reagent detection and 14 times of the R4 reagent detection were all under control. For 37 times detection of R2 reagent, the slope rate was out of control for 6 times. It was lower than x - s for the 2-8 tests and upper the average for the 12-37 tests. The intercept was out of control for 9 times, upper the x + s for the 1-8 tests and lower the average for the 12-37 tests. ② According to the detection results of quality control substance, for Q1 quality control substance, 49 tests by R1 reagent were under control, and 1 out of 23 tests by R2 reagent was out of control. For Q2 quality control substance, 14 tests by R2 reagent detection, 72 tests by R3 reagent detection and 14 tests by R4 reagent were all under control. Conclusion: The preparation of high- and low-level quality control substance using K562 and HL-60 cells was convenient and the detection results were reliable and stable. The application of quality control substance combined with slope rate and intercept in the internal quality control may contribute to quality assurance for quantitative detection of BCR-ABL (P210) transcript levels.

E6-associated transcription patterns in human papilloma virus 16-positive cervical tissues.

PubMed

Lin, Kezhi; Lu, Xulian; Chen, Jun; Zou, Ruanmin; Zhang, Lifang; Xue, Xiangyang

2015-01-01

The change in transcription pattern induced by post-transcriptional RNA splicing is an important mechanism in the regulation of the early gene expression of human papilloma virus (HPV). The present study was conducted to establish a method to specifically amplify HPV-16 E6-associated transcripts. The E6-related transcripts from 63 HPV-16-positive cervical tumor tissue samples were amplified, consisting of eight cases of low-risk intraepithelial lesions, 38 cases of high-risk intraepithelial lesions and 17 cases of cervical cancer (CxCa). The appropriate amplified segments were recovered following agarose gel electrophoresis, and subjected to further sequencing and sequence alignment analysis. Six groups of E6 transcription patterns were identified from HPV-16-positive cervical tumor tissue, including five newly-discovered transcripts. Different HPV-16 E6-associated transcription patterns were detected during the development of CxCa. Over the course of the progression of the low-grade squamous intraepithelial lesions to CxCa, the specific HPV-16 E6-associated transcription patterns and the dominant transcripts were all different. As indicated by this study, the transcription pattern of the E6 early gene of HPV-16 was closely associated with the stages of cervical carcinogenesis, and may also be involved in the development of CxCa.

Divergent transcription is associated with promoters of transcriptional regulators

PubMed Central

2013-01-01

Background Divergent transcription is a wide-spread phenomenon in mammals. For instance, short bidirectional transcripts are a hallmark of active promoters, while longer transcripts can be detected antisense from active genes in conditions where the RNA degradation machinery is inhibited. Moreover, many described long non-coding RNAs (lncRNAs) are transcribed antisense from coding gene promoters. However, the general significance of divergent lncRNA/mRNA gene pair transcription is still poorly understood. Here, we used strand-specific RNA-seq with high sequencing depth to thoroughly identify antisense transcripts from coding gene promoters in primary mouse tissues. Results We found that a substantial fraction of coding-gene promoters sustain divergent transcription of long non-coding RNA (lncRNA)/mRNA gene pairs. Strikingly, upstream antisense transcription is significantly associated with genes related to transcriptional regulation and development. Their promoters share several characteristics with those of transcriptional developmental genes, including very large CpG islands, high degree of conservation and epigenetic regulation in ES cells. In-depth analysis revealed a unique GC skew profile at these promoter regions, while the associated coding genes were found to have large first exons, two genomic features that might enforce bidirectional transcription. Finally, genes associated with antisense transcription harbor specific H3K79me2 epigenetic marking and RNA polymerase II enrichment profiles linked to an intensified rate of early transcriptional elongation. Conclusions We concluded that promoters of a class of transcription regulators are characterized by a specialized transcriptional control mechanism, which is directly coupled to relaxed bidirectional transcription. PMID:24365181

The salt-responsive transcriptome of chickpea roots and nodules via deepSuperSAGE

PubMed Central

2011-01-01

Background The combination of high-throughput transcript profiling and next-generation sequencing technologies is a prerequisite for genome-wide comprehensive transcriptome analysis. Our recent innovation of deepSuperSAGE is based on an advanced SuperSAGE protocol and its combination with massively parallel pyrosequencing on Roche's 454 sequencing platform. As a demonstration of the power of this combination, we have chosen the salt stress transcriptomes of roots and nodules of the third most important legume crop chickpea (Cicer arietinum L.). While our report is more technology-oriented, it nevertheless addresses a major world-wide problem for crops generally: high salinity. Together with low temperatures and water stress, high salinity is responsible for crop losses of millions of tons of various legume (and other) crops. Continuously deteriorating environmental conditions will combine with salinity stress to further compromise crop yields. As a good example for such stress-exposed crop plants, we started to characterize salt stress responses of chickpeas on the transcriptome level. Results We used deepSuperSAGE to detect early global transcriptome changes in salt-stressed chickpea. The salt stress responses of 86,919 transcripts representing 17,918 unique 26 bp deepSuperSAGE tags (UniTags) from roots of the salt-tolerant variety INRAT-93 two hours after treatment with 25 mM NaCl were characterized. Additionally, the expression of 57,281 transcripts representing 13,115 UniTags was monitored in nodules of the same plants. From a total of 144,200 analyzed 26 bp tags in roots and nodules together, 21,401 unique transcripts were identified. Of these, only 363 and 106 specific transcripts, respectively, were commonly up- or down-regulated (>3.0-fold) under salt stress in both organs, witnessing a differential organ-specific response to stress. Profiting from recent pioneer works on massive cDNA sequencing in chickpea, more than 9,400 UniTags were able to be linked to UniProt entries. Additionally, gene ontology (GO) categories over-representation analysis enabled to filter out enriched biological processes among the differentially expressed UniTags. Subsequently, the gathered information was further cross-checked with stress-related pathways. From several filtered pathways, here we focus exemplarily on transcripts associated with the generation and scavenging of reactive oxygen species (ROS), as well as on transcripts involved in Na+ homeostasis. Although both processes are already very well characterized in other plants, the information generated in the present work is of high value. Information on expression profiles and sequence similarity for several hundreds of transcripts of potential interest is now available. Conclusions This report demonstrates, that the combination of the high-throughput transcriptome profiling technology SuperSAGE with one of the next-generation sequencing platforms allows deep insights into the first molecular reactions of a plant exposed to salinity. Cross validation with recent reports enriched the information about the salt stress dynamics of more than 9,000 chickpea ESTs, and enlarged their pool of alternative transcripts isoforms. As an example for the high resolution of the employed technology that we coin deepSuperSAGE, we demonstrate that ROS-scavenging and -generating pathways undergo strong global transcriptome changes in chickpea roots and nodules already 2 hours after onset of moderate salt stress (25 mM NaCl). Additionally, a set of more than 15 candidate transcripts are proposed to be potential components of the salt overly sensitive (SOS) pathway in chickpea. Newly identified transcript isoforms are potential targets for breeding novel cultivars with high salinity tolerance. We demonstrate that these targets can be integrated into breeding schemes by micro-arrays and RT-PCR assays downstream of the generation of 26 bp tags by SuperSAGE. PMID:21320317

The salt-responsive transcriptome of chickpea roots and nodules via deepSuperSAGE.

PubMed

Molina, Carlos; Zaman-Allah, Mainassara; Khan, Faheema; Fatnassi, Nadia; Horres, Ralf; Rotter, Björn; Steinhauer, Diana; Amenc, Laurie; Drevon, Jean-Jacques; Winter, Peter; Kahl, Günter

2011-02-14

The combination of high-throughput transcript profiling and next-generation sequencing technologies is a prerequisite for genome-wide comprehensive transcriptome analysis. Our recent innovation of deepSuperSAGE is based on an advanced SuperSAGE protocol and its combination with massively parallel pyrosequencing on Roche's 454 sequencing platform. As a demonstration of the power of this combination, we have chosen the salt stress transcriptomes of roots and nodules of the third most important legume crop chickpea (Cicer arietinum L.). While our report is more technology-oriented, it nevertheless addresses a major world-wide problem for crops generally: high salinity. Together with low temperatures and water stress, high salinity is responsible for crop losses of millions of tons of various legume (and other) crops. Continuously deteriorating environmental conditions will combine with salinity stress to further compromise crop yields. As a good example for such stress-exposed crop plants, we started to characterize salt stress responses of chickpeas on the transcriptome level. We used deepSuperSAGE to detect early global transcriptome changes in salt-stressed chickpea. The salt stress responses of 86,919 transcripts representing 17,918 unique 26 bp deepSuperSAGE tags (UniTags) from roots of the salt-tolerant variety INRAT-93 two hours after treatment with 25 mM NaCl were characterized. Additionally, the expression of 57,281 transcripts representing 13,115 UniTags was monitored in nodules of the same plants. From a total of 144,200 analyzed 26 bp tags in roots and nodules together, 21,401 unique transcripts were identified. Of these, only 363 and 106 specific transcripts, respectively, were commonly up- or down-regulated (>3.0-fold) under salt stress in both organs, witnessing a differential organ-specific response to stress.Profiting from recent pioneer works on massive cDNA sequencing in chickpea, more than 9,400 UniTags were able to be linked to UniProt entries. Additionally, gene ontology (GO) categories over-representation analysis enabled to filter out enriched biological processes among the differentially expressed UniTags. Subsequently, the gathered information was further cross-checked with stress-related pathways. From several filtered pathways, here we focus exemplarily on transcripts associated with the generation and scavenging of reactive oxygen species (ROS), as well as on transcripts involved in Na+ homeostasis. Although both processes are already very well characterized in other plants, the information generated in the present work is of high value. Information on expression profiles and sequence similarity for several hundreds of transcripts of potential interest is now available. This report demonstrates, that the combination of the high-throughput transcriptome profiling technology SuperSAGE with one of the next-generation sequencing platforms allows deep insights into the first molecular reactions of a plant exposed to salinity. Cross validation with recent reports enriched the information about the salt stress dynamics of more than 9,000 chickpea ESTs, and enlarged their pool of alternative transcripts isoforms. As an example for the high resolution of the employed technology that we coin deepSuperSAGE, we demonstrate that ROS-scavenging and -generating pathways undergo strong global transcriptome changes in chickpea roots and nodules already 2 hours after onset of moderate salt stress (25 mM NaCl). Additionally, a set of more than 15 candidate transcripts are proposed to be potential components of the salt overly sensitive (SOS) pathway in chickpea. Newly identified transcript isoforms are potential targets for breeding novel cultivars with high salinity tolerance. We demonstrate that these targets can be integrated into breeding schemes by micro-arrays and RT-PCR assays downstream of the generation of 26 bp tags by SuperSAGE.

Developmentally regulated expression of APG-1, a member of heat shock protein 110 family in murine male germ cells.

PubMed

Kaneko, Y; Kimura, T; Nishiyama, H; Noda, Y; Fujita, J

1997-04-07

Apg-1 encodes a heat shock protein belonging to the heat shock protein 110 family, and is inducible by a 32 degrees C to 39 degrees C heat shock. Northern blot analysis of the testis from immature and adult mice, and of the purified germ cells revealed the quantitative change of the apg-1 transcripts during germ cell development. By in situ hybridization histochemistry the expressions of the apg-1 transcripts were detected in germ cells at specific stages of development including spermatocytes and spermatids. Although heat-induction of the apg-1 transcripts was observed in W/Wv mutant testis lacking germ cells, it was not detected in wild-type testis nor in the purified germ cells. Thus, the apg-1 expression is not heat-regulated but developmentally regulated in germ cells, suggesting that APG-1 plays a role in normal development of germ cells.

Pigment Production Analysis in Human Melanoma Cells.

PubMed

Hopkin, Amelia Soto; Paterson, Elyse K; Ruiz, Rolando; Ganesan, Anand K

2016-05-25

The human epidermal melanocyte is a highly specialized pigmented cell that serves to protect the epidermis from ultraviolet (UV) damage through the production of melanin, or melanogenesis. Misregulation in melanogenesis leading to either hyper- or hypo-pigmentation is found in human diseases such as malasma and vitiligo. Current therapies for these diseases are largely unsuccessful and the need for new therapies is necessary. In order to identify genes and or compounds that can alter melanogenesis, methods are required that can detect changes in pigment production as well as expression of key melanogenesis transcription factors and enzymes. Here we describe methods to detect changes in melanogenesis in a human melanoma cell line, MNT-1, by (1) analyzing pigment production by measuring the absorbance of melanin present by spectrophotometry, (2) analyzing transcript expression of potent regulators of melanogenesis by qunatitative reverse-transcription (RT)PCR and (3) analyzing protein expression of potent regulators of melanogenesis by Western blot (WB).

Rapid Detection of Prunus Necrotic Ringspot Virus by Reverse Transcription-cross-priming Amplification Coupled with Nucleic Acid Test Strip Cassette.

PubMed

Huo, Ya-Yun; Li, Gui-Fen; Qiu, Yan-Hong; Li, Wei-Min; Zhang, Yong-Jiang

2017-11-23

Prunus necrotic ringspot virus (PNRSV) is one of the most devastating viruses to Prunus spp. In this study, we developed a diagnostic system RT-CPA-NATSC, wherein reverse transcription-cross-priming amplification (RT-CPA) is coupled with nucleic acid test strip cassette (NATSC), a vertical flow (VF) visualization, for PNRSV detection. The RT-CPA-NATSC assay targets the encoding gene of the PNRSV coat protein with a limit of detection of 72 copies per reaction and no cross-reaction with the known Prunus pathogenic viruses and viroids, demonstrating high sensitivity and specificity. The reaction is performed on 60 °C and can be completed less than 90 min with the prepared template RNA. Field sample test confirmed the reliability of RT-CPA-NATSC, indicating the potential application of this simple and rapid detection method in routine test of PNRSV.

Diterpenes biochemical profile and transcriptional analysis of cytochrome P450s genes in leaves, roots, flowers, and during Coffea arabica L. fruit development.

PubMed

Ivamoto, Suzana T; Sakuray, Leonardo M; Ferreira, Lucia P; Kitzberger, Cíntia S G; Scholz, Maria B S; Pot, David; Leroy, Thierry; Vieira, Luiz G E; Domingues, Douglas S; Pereira, Luiz F P

2017-02-01

Lipids are among the major chemical compounds present in coffee beans, and they affect the flavor and aroma of the coffee beverage. Coffee oil is rich in kaurene diterpene compounds, mainly cafestol (CAF) and kahweol (KAH), which are related to plant defense mechanisms and to nutraceutical and sensorial beverage characteristics. Despite their importance, the final steps of coffee diterpenes biosynthesis remain unknown. To understand the molecular basis of coffee diterpenes biosynthesis, we report the content dynamics of CAF and KAH in several Coffea arabica tissues and the transcriptional analysis of cytochrome P450 genes (P450). We measured CAF and KAH concentrations in leaves, roots, flower buds, flowers and fruit tissues at seven developmental stages (30-240 days after flowering - DAF) using HPLC. Higher CAF levels were detected in flower buds and flowers when compared to fruits. In contrast, KAH concentration increased along fruit development, peaking at 120 DAF. We did not detect CAF or KAH in leaves, and higher amounts of KAH than CAF were detected in roots. Using P450 candidate genes from a coffee EST database, we performed RT-qPCR transcriptional analysis of leaves, flowers and fruits at three developmental stages (90, 120 and 150 DAF). Three P450 genes (CaCYP76C4, CaCYP82C2 and CaCYP74A1) had transcriptional patterns similar to CAF concentration and two P450 genes (CaCYP71A25 and CaCYP701A3) have transcript accumulation similar to KAH concentration. These data warrant further investigation of these P450s as potential candidate genes involved in the final stages of the CAF and KAH biosynthetic pathways. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Recent Advancements in DNA Damage-Transcription Crosstalk and High-Resolution Mapping of DNA Breaks.

PubMed

Vitelli, Valerio; Galbiati, Alessandro; Iannelli, Fabio; Pessina, Fabio; Sharma, Sheetal; d'Adda di Fagagna, Fabrizio

2017-08-31

Until recently, DNA damage arising from physiological DNA metabolism was considered a detrimental by-product for cells. However, an increasing amount of evidence has shown that DNA damage could have a positive role in transcription activation. In particular, DNA damage has been detected in transcriptional elements following different stimuli. These physiological DNA breaks are thought to be instrumental for the correct expression of genomic loci through different mechanisms. In this regard, although a plethora of methods are available to precisely map transcribed regions and transcription start sites, commonly used techniques for mapping DNA breaks lack sufficient resolution and sensitivity to draw a robust correlation between DNA damage generation and transcription. Recently, however, several methods have been developed to map DNA damage at single-nucleotide resolution, thus providing a new set of tools to correlate DNA damage and transcription. Here, we review how DNA damage can positively regulate transcription initiation, the current techniques for mapping DNA breaks at high resolution, and how these techniques can benefit future studies of DNA damage and transcription.

CTCF-Mediated Human 3D Genome Architecture Reveals Chromatin Topology for Transcription.

PubMed

Tang, Zhonghui; Luo, Oscar Junhong; Li, Xingwang; Zheng, Meizhen; Zhu, Jacqueline Jufen; Szalaj, Przemyslaw; Trzaskoma, Pawel; Magalska, Adriana; Wlodarczyk, Jakub; Ruszczycki, Blazej; Michalski, Paul; Piecuch, Emaly; Wang, Ping; Wang, Danjuan; Tian, Simon Zhongyuan; Penrad-Mobayed, May; Sachs, Laurent M; Ruan, Xiaoan; Wei, Chia-Lin; Liu, Edison T; Wilczynski, Grzegorz M; Plewczynski, Dariusz; Li, Guoliang; Ruan, Yijun

2015-12-17

Spatial genome organization and its effect on transcription remains a fundamental question. We applied an advanced chromatin interaction analysis by paired-end tag sequencing (ChIA-PET) strategy to comprehensively map higher-order chromosome folding and specific chromatin interactions mediated by CCCTC-binding factor (CTCF) and RNA polymerase II (RNAPII) with haplotype specificity and nucleotide resolution in different human cell lineages. We find that CTCF/cohesin-mediated interaction anchors serve as structural foci for spatial organization of constitutive genes concordant with CTCF-motif orientation, whereas RNAPII interacts within these structures by selectively drawing cell-type-specific genes toward CTCF foci for coordinated transcription. Furthermore, we show that haplotype variants and allelic interactions have differential effects on chromosome configuration, influencing gene expression, and may provide mechanistic insights into functions associated with disease susceptibility. 3D genome simulation suggests a model of chromatin folding around chromosomal axes, where CTCF is involved in defining the interface between condensed and open compartments for structural regulation. Our 3D genome strategy thus provides unique insights in the topological mechanism of human variations and diseases. Copyright © 2015 Elsevier Inc. All rights reserved.

DNA and RNA editing of retrotransposons accelerate mammalian genome evolution.

PubMed

Knisbacher, Binyamin A; Levanon, Erez Y

2015-04-01

Genome evolution is commonly viewed as a gradual process that is driven by random mutations that accumulate over time. However, DNA- and RNA-editing enzymes have been identified that can accelerate evolution by actively modifying the genomically encoded information. The apolipoprotein B mRNA editing enzymes, catalytic polypeptide-like (APOBECs) are potent restriction factors that can inhibit retroelements by cytosine-to-uridine editing of retroelement DNA after reverse transcription. In some cases, a retroelement may successfully integrate into the genome despite being hypermutated. Such events introduce unique sequences into the genome and are thus a source of genomic innovation. adenosine deaminases that act on RNA (ADARs) catalyze adenosine-to-inosine editing in double-stranded RNA, commonly formed by oppositely oriented retroelements. The RNA editing confers plasticity to the transcriptome by generating many transcript variants from a single genomic locus. If the editing produces a beneficial variant, the genome may maintain the locus that produces the RNA-edited transcript for its novel function. Here, we discuss how these two powerful editing mechanisms, which both target inserted retroelements, facilitate expedited genome evolution. © 2015 New York Academy of Sciences.

Genome-wide Determinants of Proviral Targeting, Clonal Abundance and Expression in Natural HTLV-1 Infection

PubMed Central

Melamed, Anat; Laydon, Daniel J.; Gillet, Nicolas A.; Tanaka, Yuetsu; Taylor, Graham P.; Bangham, Charles R. M.

2013-01-01

The regulation of proviral latency is a central problem in retrovirology. We postulate that the genomic integration site of human T lymphotropic virus type 1 (HTLV-1) determines the pattern of expression of the provirus, which in turn determines the abundance and pathogenic potential of infected T cell clones in vivo. We recently developed a high-throughput method for the genome-wide amplification, identification and quantification of proviral integration sites. Here, we used this protocol to test two hypotheses. First, that binding sites for transcription factors and chromatin remodelling factors in the genome flanking the proviral integration site of HTLV-1 are associated with integration targeting, spontaneous proviral expression, and in vivo clonal abundance. Second, that the transcriptional orientation of the HTLV-1 provirus relative to that of the nearest host gene determines spontaneous proviral expression and in vivo clonal abundance. Integration targeting was strongly associated with the presence of a binding site for specific host transcription factors, especially STAT1 and p53. The presence of the chromatin remodelling factors BRG1 and INI1 and certain host transcription factors either upstream or downstream of the provirus was associated respectively with silencing or spontaneous expression of the provirus. Cells expressing HTLV-1 Tax protein were significantly more frequent in clones of low abundance in vivo. We conclude that transcriptional interference and chromatin remodelling are critical determinants of proviral latency in natural HTLV-1 infection. PMID:23555266

Variations in the non-coding transcriptome as a driver of inter-strain divergence and physiological adaptation in bacteria

PubMed Central

Kopf, Matthias; Klähn, Stephan; Scholz, Ingeborg; Hess, Wolfgang R.; Voß, Björn

2015-01-01

In all studied organisms, a substantial portion of the transcriptome consists of non-coding RNAs that frequently execute regulatory functions. Here, we have compared the primary transcriptomes of the cyanobacteria Synechocystis sp. PCC 6714 and PCC 6803 under 10 different conditions. These strains share 2854 protein-coding genes and a 16S rRNA identity of 99.4%, indicating their close relatedness. Conserved major transcriptional start sites (TSSs) give rise to non-coding transcripts within the sigB gene, from the 5′UTRs of cmpA and isiA, and 168 loci in antisense orientation. Distinct differences include single nucleotide polymorphisms rendering promoters inactive in one of the strains, e.g., for cmpR and for the asRNA PsbA2R. Based on the genome-wide mapped location, regulation and classification of TSSs, non-coding transcripts were identified as the most dynamic component of the transcriptome. We identified a class of mRNAs that originate by read-through from an sRNA that accumulates as a discrete and abundant transcript while also serving as the 5′UTR. Such an sRNA/mRNA structure, which we name ‘actuaton’, represents another way for bacteria to remodel their transcriptional network. Our findings support the hypothesis that variations in the non-coding transcriptome constitute a major evolutionary element of inter-strain divergence and capability for physiological adaptation. PMID:25902393

Rapid and Sensitive Salmonella Typhi Detection in Blood and Fecal Samples Using Reverse Transcription Loop-Mediated Isothermal Amplification.

PubMed

Fan, Fenxia; Yan, Meiying; Du, Pengcheng; Chen, Chen; Kan, Biao

2015-09-01

Typhoid fever caused by Salmonella enterica serovar Typhi remains a significant public health problem in developing countries. Although the main method for diagnosing typhoid fever is blood culture, the test is time consuming and not always able to detect infections. Thus, it is very difficult to distinguish typhoid from other infections in patients with nonspecific symptoms. A simple and sensitive laboratory detection method remains necessary. The purpose of this study is to establish and evaluate a rapid and sensitive reverse transcription-based loop-mediated isothermal amplification (RT-LAMP) method to detect Salmonella Typhi infection. In this study, a new specific gene marker, STY1607, was selected to develop a STY1607-RT-LAMP assay; this is the first report of specific RT-LAMP detection assay for typhoid. Human-simulated and clinical blood/stool samples were used to evaluate the performance of STY1607-RT-LAMP for RNA detection; this method was compared with STY1607-LAMP, reverse transcription real-time polymerase chain reaction (rRT-PCR), and bacterial culture methods for Salmonella Typhi detection. Using mRNA as the template, STY1607-RT-LAMP exhibited 50-fold greater sensitivity than STY1607-LAMP for DNA detection. The STY1607-RT-LAMP detection limit is 3 colony-forming units (CFU)/mL for both the pure Salmonella Typhi samples and Salmonella Typhi-simulated blood samples and was 30 CFU/g for the simulated stool samples, all of which were 10-fold more sensitive than the rRT-PCR method. RT-LAMP exhibited improved Salmonella Typhi detection sensitivity compared to culture methods and to rRT-PCR of clinical blood and stool specimens from suspected typhoid fever patients. Because it can be performed without sophisticated equipment or skilled personnel, RT-LAMP is a valuable tool for clinical laboratories in developing countries. This method can be applied in the clinical diagnosis and care of typhoid fever patients as well as for a quick public health response.

Automatically Detecting Likely Edits in Clinical Notes Created Using Automatic Speech Recognition

PubMed Central

Lybarger, Kevin; Ostendorf, Mari; Yetisgen, Meliha

2017-01-01

The use of automatic speech recognition (ASR) to create clinical notes has the potential to reduce costs associated with note creation for electronic medical records, but at current system accuracy levels, post-editing by practitioners is needed to ensure note quality. Aiming to reduce the time required to edit ASR transcripts, this paper investigates novel methods for automatic detection of edit regions within the transcripts, including both putative ASR errors but also regions that are targets for cleanup or rephrasing. We create detection models using logistic regression and conditional random field models, exploring a variety of text-based features that consider the structure of clinical notes and exploit the medical context. Different medical text resources are used to improve feature extraction. Experimental results on a large corpus of practitioner-edited clinical notes show that 67% of sentence-level edits and 45% of word-level edits can be detected with a false detection rate of 15%. PMID:29854187

Detection and quantification of Epstein-Barr virus EBER1 in EBV-infected cells by fluorescent in situ hybridization and flow cytometry

NASA Technical Reports Server (NTRS)

Stowe, R. P.; Cubbage, M. L.; Sams, C. F.; Pierson, D. L.; Barrett, A. D.

1998-01-01

A rapid and highly sensitive fluorescent in situ hybridization (FISH) assay was developed to detect Epstein Barr virus (EBV)-infected cells in peripheral blood. Multiple fluorescein-labeled antisense oligonucleotide probes were designed to hybridize to the EBER1 transcript, which is highly expressed in latently infected cells. After a rapid (30 min) hybridization, the cells were analyzed by flow cytometry. EBER1 was detected in several positive control cell lines that have variable numbers of EBV genome copies. No EBER1 was detected in two known EBV-negative cell lines. Northern blot analyses confirmed the presence and quantity of EBER1 transcripts in each cell line. This method was used to quantify the number of EBV-infected cells in peripheral blood from a patient with chronic mononucleosis. These results indicate that EBV-infected cells can be detected at the single cell level, and that this assay can be used to quantify the number of EBV-infected cells in clinical samples.

Simultaneous Detection of Four Foodborne Viruses in Food Samples Using a One-Step Multiplex Reverse Transcription PCR.

PubMed

Lee, Shin-Young; Kim, Mi-Ju; Kim, Hyun-Joong; Jeong, KwangCheol Casey; Kim, Hae-Yeong

2018-02-28

A one-step multiplex reverse transcription PCR (RT-PCR) method comprising six primer sets (for the detection of norovirus GI and GII, hepatitis A virus, rotavirus, and astrovirus) was developed to simultaneously detect four kinds of pathogenic viruses. The size of the PCR products for norovirus GI and GII, hepatitis A virus (VP3/VP1 and P2A regions), rotavirus, and astrovirus were 330, 164, 244, 198, 629, and 449 bp, respectively. The RT-PCR with the six primer sets showed specificity for the pathogenic viruses. The detection limit of the developed multiplex RT-PCR, as evaluated using serially diluted viral RNAs, was comparable to that of one-step single RT-PCR. Moreover, this multiplex RT-PCR was evaluated using food samples such as water, oysters, lettuce, and vegetable product. These food samples were artificially spiked with the four kinds of viruses in diverse combinations, and the spiked viruses in all food samples were detected successfully.

Evidence for instantaneous e-vector detection in the honeybee using an associative learning paradigm

PubMed Central

Sakura, Midori; Okada, Ryuichi; Aonuma, Hitoshi

2012-01-01

Many insects use the polarization pattern of the sky for obtaining compass information during orientation or navigation. E-vector information is collected by a specialized area in the dorsal-most part of the compound eye, the dorsal rim area (DRA). We tested honeybees' capability of learning certain e-vector orientations by using a classical conditioning paradigm with the proboscis extension reflex. When one e-vector orientation (CS+) was associated with sugar water, while another orientation (CS−) was not rewarded, the honeybees could discriminate CS+ from CS−. Bees whose DRA was inactivated by painting did not learn CS+. When ultraviolet (UV) polarized light (350 nm) was used for CS, the bees discriminated CS+ from CS−, but no discrimination was observed in blue (442 nm) or green light (546 nm). Our data indicate that honeybees can learn and discriminate between different e-vector orientations, sensed by the UV receptors of the DRA, suggesting that bees can determine their flight direction from polarized UV skylight during foraging. Fixing the bees' heads during the experiments did not prevent learning, indicating that they use an ‘instantaneous’ algorithm of e-vector detection; that is, the bees do not need to actively scan the sky with their DRAs (‘sequential’ method) to determine e-vector orientation. PMID:21733901

Mental-orientation: A new approach to assessing patients across the Alzheimer's disease spectrum.

PubMed

Peters-Founshtein, Gregory; Peer, Michael; Rein, Yanai; Kahana Merhavi, Shlomzion; Meiner, Zeev; Arzy, Shahar

2018-05-21

This study aims to assess the role of mental-orientation in the diagnosis of mild cognitive impairment and Alzheimer's disease using a novel task. A behavioral study (Experiment 1) compared the mental-orientation task to standard neuropsychological tests in patients across the Alzheimer's disease spectrum. A functional MRI study (Experiment 2) in young adults compared activations evoked by the mental-orientation and standard-orientation tasks as well as their overlap with brain regions susceptible to Alzheimer's disease pathology. The mental-orientation task differentiated mild cognitively impaired and healthy controls at 95% accuracy, while the Addenbrooke's Cognitive Examination, Mini-Mental State Examination and standard-orientation achieved 74%, 70% and 50% accuracy, respectively. Functional MRI revealed the mental-orientation task to preferentially recruit brain regions exhibiting early Alzheimer's-related atrophy, unlike the standard-orientation test. Mental-orientation is suggested to play a key role in Alzheimer's disease, and consequently in early detection and follow-up of patients along the Alzheimer's disease spectrum. (PsycINFO Database Record (c) 2018 APA, all rights reserved).

Nondestructive testing based on scanning-from-heating approach: application to nonthrough defect detection and fiber orientation assessment

NASA Astrophysics Data System (ADS)

Belkacemi, Mohamed; Stolz, Christophe; Mathieu, Alexandre; Lemaitre, Guillaume; Massich, Joan; Aubreton, Olivier

2015-11-01

Today, industries ensure the quality of their manufactured products through computer vision techniques and nonconventional imaging. Three-dimensional (3-D) scanners and nondestructive testing (NDT) systems are commonly used independently for such applications. Furthermore, these approaches combined constitute hybrid systems, providing a 3-D reconstruction and NDT analysis. These systems, however, suffer from drawbacks such as errors during the data fusion and higher cost for manufacturers. In an attempt to solve these problems, a single active thermography system based on scanning-from-heating is proposed in this paper. In addition to 3-D digitization of the object, our contributions are twofold: (1) the nonthrough defect detection for a homogeneous metallic object and (2) fiber orientation assessment for a long fiber composite material. The experiments on steel and aluminum plates show that our method achieves the detection of nonthrough defects. Additionally, the estimation of the fiber orientation is evaluated on carbon-fiber composite material.

A group filter algorithm for sea mine detection

NASA Astrophysics Data System (ADS)

Cobb, J. Tory; An, Myoung; Tolimieri, Richard

2005-06-01

Automatic detection of sea mines in coastal regions is a difficult task due to the highly variable sea bottom conditions present in the underwater environment. Detection systems must be able to discriminate objects which vary in size, shape, and orientation from naturally occurring and man-made clutter. Additionally, these automated systems must be computationally efficient to be incorporated into unmanned underwater vehicle (UUV) sensor systems characterized by high sensor data rates and limited processing abilities. Using noncommutative group harmonic analysis, a fast, robust sea mine detection system is created. A family of unitary image transforms associated to noncommutative groups is generated and applied to side scan sonar image files supplied by Naval Surface Warfare Center Panama City (NSWC PC). These transforms project key image features, geometrically defined structures with orientations, and localized spectral information into distinct orthogonal components or feature subspaces of the image. The performance of the detection system is compared against the performance of an independent detection system in terms of probability of detection (Pd) and probability of false alarm (Pfa).

Automatic detection of kidney in 3D pediatric ultrasound images using deep neural networks

NASA Astrophysics Data System (ADS)

Tabrizi, Pooneh R.; Mansoor, Awais; Biggs, Elijah; Jago, James; Linguraru, Marius George

2018-02-01

Ultrasound (US) imaging is the routine and safe diagnostic modality for detecting pediatric urology problems, such as hydronephrosis in the kidney. Hydronephrosis is the swelling of one or both kidneys because of the build-up of urine. Early detection of hydronephrosis can lead to a substantial improvement in kidney health outcomes. Generally, US imaging is a challenging modality for the evaluation of pediatric kidneys with different shape, size, and texture characteristics. The aim of this study is to present an automatic detection method to help kidney analysis in pediatric 3DUS images. The method localizes the kidney based on its minimum volume oriented bounding box) using deep neural networks. Separate deep neural networks are trained to estimate the kidney position, orientation, and scale, making the method computationally efficient by avoiding full parameter training. The performance of the method was evaluated using a dataset of 45 kidneys (18 normal and 27 diseased kidneys diagnosed with hydronephrosis) through the leave-one-out cross validation method. Quantitative results show the proposed detection method could extract the kidney position, orientation, and scale ratio with root mean square values of 1.3 +/- 0.9 mm, 6.34 +/- 4.32 degrees, and 1.73 +/- 0.04, respectively. This method could be helpful in automating kidney segmentation for routine clinical evaluation.

Lateral interactions and speed of information processing in highly functioning multiple sclerosis patients.

PubMed

Nagy, Helga; Bencsik, Krisztina; Rajda, Cecília; Benedek, Krisztina; Janáky, Márta; Beniczky, Sándor; Kéri, Szabolcs; Vécsei, László

2007-06-01

Visual impairment is a common feature of multiple sclerosis. The aim of this study was to investigate lateral interactions in the visual cortex of highly functioning patients with multiple sclerosis and to compare that with basic visual and neuropsychologic functions. Twenty-two young, visually unimpaired multiple sclerosis patients with minimal symptoms (Expanded Disability Status Scale <2) and 30 healthy controls subjects participated in the study. Lateral interactions were investigated with the flanker task, during which participants were asked to detect the orientation of a low-contrast Gabor patch (vertical or horizontal), flanked with 2 collinear or orthogonal Gabor patches. Stimulus exposure time was 40, 60, 80, and 100 ms. Digit span forward/backward, digit symbol, verbal fluency, and California Verbal Learning Test procedures were used for background neuropsychologic assessment. Results revealed that patients with multiple sclerosis showed intact visual contrast sensitivity and neuropsychologic functions, whereas orientation detection in the orthogonal condition was significantly impaired. At 40-ms exposure time, collinear flankers facilitated the orientation detection performance of the patients resulting in normal performance. In conclusion, the detection of briefly presented, low-contrast visual stimuli was selectively impaired in multiple sclerosis. Lateral interactions between target and flankers robustly facilitated target detection in the patient group.

A nuclear gene for the iron-sulfur subunit of mitochondrial complex II is specifically expressed during Arabidopsis seed development and germination.

PubMed

Elorza, Alvaro; Roschzttardtz, Hannetz; Gómez, Isabel; Mouras, Armand; Holuigue, Loreto; Araya, Alejandro; Jordana, Xavier

2006-01-01

Three nuclear genes, SDH2-1, SDH2-2 and SDH2-3, encode the essential iron-sulfur subunit of mitochondrial complex II in Arabidopsis thaliana. SDH2-1 and SDH2-2 probably arose via a recent duplication event and we reported that both are expressed in all organs from adult plants. In contrast, transcripts from SDH2-3 were not detected. Here we present data demonstrating that SDH2-3 is specifically expressed during seed development. SDH2-3 transcripts appear during seed maturation, persist through desiccation, are abundant in dry seeds and markedly decline during germination. Analysis of transgenic Arabidopsis plants carrying the SDH2-3 promoter fused to the beta-glucuronidase reporter gene shows that the SDH2-3 promoter is activated in the embryo during maturation, from the bent-cotyledon stage. beta-Glucuronidase expression correlates with the appearance of endogenous SDH2-3 transcripts, suggesting that control of this nuclear gene is achieved through transcriptional regulation. Furthermore, progressive deletions of this promoter identified a 159 bp region (-223 to -65) important for SDH2-3 transcriptional activation in seeds. Interestingly, the SDH2-3 promoter remains active in embryonic tissues during germination and post-germinative growth, and is turned off in vegetative tissues (true leaves). In contrast to SDH2-3 transcripts, SDH2-1 and SDH2-2 transcripts are barely detected in dry seeds and increase during germination and post-germinative growth. The opposite expression patterns of SDH2 nuclear genes strongly suggest that during germination the embryo-specific SDH2-3 is replaced by SDH2-1 or SDH2-2 in mitochondrial complex II.

Cloning, tissue distribution and effects of fasting on pituitary adenylate cyclase-activating polypeptide in largemouth bass

NASA Astrophysics Data System (ADS)

Li, Shengjie; Han, Linqiang; Bai, Junjie; Ma, Dongmei; Quan, Yingchun; Fan, Jiajia; Jiang, Peng; Yu, Lingyun

2015-03-01

Pituitary adenylate cyclase activating polypeptide (PACAP) has a wide range of biological functions. We cloned the full-length cDNAs encoding PACAP and PACAP-related peptide (PRP) from the brain of largemouth bass ( Micropterus salmoides) and used real-time quantitative PCR to detect PRP-PACAP mRNA expression. The PRP-PACAP cDNA has two variants expressed via alternative splicing: a long form, which encodes both PRP and PACAP, and a short form, which encodes only PACAP. Sequence analysis results are consistent with a higher conservation of PACAP than PRP peptide sequences. The expression of PACAP-long and PACAP-short transcripts was highest in the forebrain, followed by the medulla, midbrain, pituitary, stomach, cerebellum, intestine, and kidney; however, these transcripts were either absent or were weakly expressed in the muscle, spleen, gill, heart, fatty tissue, and liver. The level of PACAP-short transcript expression was significantly higher than expression of the long transcript in the forebrain, cerebella, pituitary and intestine, but lower than that of the long transcript in the stomach. PACAP-long and PACAP-short transcripts were first detected at the blastula stage of embryogenesis, and the level of expression increased markedly between the muscular contraction stage and 3 d post hatch (dph). The expression of PACAP-long and PACAP-short transcripts decreased significantly in the brain following 4 d fasting compared with the control diet group. The down-regulation effect was enhanced as fasting continued. Conversely, expression levels increased significantly after 3 d of re-feeding. Our results suggest that PRP-PACAP acts as an important factor in appetite regulation in largemouth bass.

Estimations of BCR-ABL/ABL transcripts by quantitative PCR in chronic myeloid leukaemia after allogeneic bone marrow transplantation and donor lymphocyte infusion.

PubMed

Otazú, Ivone B; Tavares, Rita de Cassia B; Hassan, Rocío; Zalcberg, Ilana; Tabak, Daniel G; Seuánez, Héctor N

2002-02-01

Serial assays of qualitative (multiplex and nested) and quantitative PCR were carried out for detecting and estimating the level of BCR-ABL transcripts in 39 CML patients following bone marrow transplantation. Seven of these patients, who received donor lymphocyte infusions (DLIs) following to relapse, were also monitored. Quantitative estimates of BCR-ABL transcripts were obtained by co-amplification with a competitor sequence. Estimates of ABL transcripts were used, an internal control and the ratio BCR-ABL/ABL was thus estimated for evaluating the kinetics of residual clones. Twenty four patients were followed shortly after BMT; two of these patients were in cytogenetic relapse coexisting with very high BCR-ABL levels while other 22 were in clinical, haematologic and cytogenetic remission 2-42 months after BMT. In this latter group, seven patients showed a favourable clinical-haematological progression in association with molecular remission while in 14 patients quantitative PCR assays indicated molecular relapse that was not associated with an early cytogenetic-haematologic relapse. BCR-ABL/ABL levels could not be correlated with presence of GVHD in 24 patients after BMT. In all seven patients treated with DLI, high levels of transcripts were detected at least 4 months before the appearance of clinical haematological relapse. Following DLI, five of these patients showed decreasing transcript levels from 2 to 5 logs between 4 and 12 months. In eight other patients studied long after BMT, five showed molecular relapse up to 117 months post-BMT and only one showed cytogenetic relapse. Our findings indicated that quantitative estimates of BCR-ABL transcripts were valuable for monitoring minimal residual disease in each patient.

In planta assays involving epigenetically silenced genes reveal inhibition of cytosine methylation by genistein

PubMed Central

2012-01-01

Background Cytosine methylation is involved in epigenetic control of gene expression in a wide range of organisms. An increasing number of examples indicate that changing the frequency of cytosine methylation in the genome is a feasible tool to engineer novel traits in plants. Although demethylating effects of compounds have been analyzed in human cultured cells in terms of suppressing cancer, their effect in plant cells has not been analyzed extensively. Here, we developed in planta assay systems to detect inhibition of cytosine methylation using plants that contain a transgene transcriptionally silenced by an epigenetic mechanism. Results Seeds of two transgenic plants were used: a petunia line that has been identified as a revertant of the co-suppression of the chalcone synthase-A (CHS-A) gene and contains CHS-A transgenes whose transcription is repressed; Nicotiana benthamiana plants that contain the green fluorescent protein (GFP) reporter gene whose transcription is repressed through virus-induced transcriptional gene silencing. Seeds of these plants were sown on a medium that contained a demethylating agent, either 5-azacytidine or trichostatin A, and the restoration of the transcriptionally active state of the transgene was detected in seedlings. Using these systems, we found that genistein, a major isoflavonoid compound, inhibits cytosine methylation, thus restoring transgene transcription. Genistein also restored the transcription of an epigenetically silenced endogenous gene in Arabidopsis plants. Conclusions Our assay systems allowed us to assess the inhibition of cytosine methylation, in particular of maintenance of methylation, by compounds in plant cells. These results suggest a novel role of flavonoids in plant cells and that genistein is useful for modifying the epigenetic state of plant genomes. PMID:22424588

Global Identification and Characterization of Transcriptionally Active Regions in the Rice Genome

PubMed Central

Stolc, Viktor; Deng, Wei; He, Hang; Korbel, Jan; Chen, Xuewei; Tongprasit, Waraporn; Ronald, Pamela; Chen, Runsheng; Gerstein, Mark; Wang Deng, Xing

2007-01-01

Genome tiling microarray studies have consistently documented rich transcriptional activity beyond the annotated genes. However, systematic characterization and transcriptional profiling of the putative novel transcripts on the genome scale are still lacking. We report here the identification of 25,352 and 27,744 transcriptionally active regions (TARs) not encoded by annotated exons in the rice (Oryza. sativa) subspecies japonica and indica, respectively. The non-exonic TARs account for approximately two thirds of the total TARs detected by tiling arrays and represent transcripts likely conserved between japonica and indica. Transcription of 21,018 (83%) japonica non-exonic TARs was verified through expression profiling in 10 tissue types using a re-array in which annotated genes and TARs were each represented by five independent probes. Subsequent analyses indicate that about 80% of the japonica TARs that were not assigned to annotated exons can be assigned to various putatively functional or structural elements of the rice genome, including splice variants, uncharacterized portions of incompletely annotated genes, antisense transcripts, duplicated gene fragments, and potential non-coding RNAs. These results provide a systematic characterization of non-exonic transcripts in rice and thus expand the current view of the complexity and dynamics of the rice transcriptome. PMID:17372628

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wu, Wei; Shih, Wei-Heng; Shih, Wan Y., E-mail: shihwy@drexel.edu

We have examined the mechanism of the detection resonance frequency shift, Δf/f, of a 1370 μm long and 537 μm wide [Pb(Mg{sub 1/3}Nb{sub 2/3})O{sub 3}]{sub 0.65}[PbTiO{sub 3}]{sub 0.35} (PMN-PT) piezoelectric plate sensor (PEPS) made of a 8-μm thick PMN-PT freestanding film. The Δf/f of the PEPS was monitored in a three-step binding model detections of (1) binding of maleimide-activated biotin to the sulfhydryl on the PEPS surface followed by (2) binding of streptavidin to the bound biotin and (3) subsequent binding of biotinylated probe deoxyribonucleic acid to the bound streptavidin. We used a PMN-PT surrogate made of the same 8-μm thick PMN-PTmore » freestanding film that the PEPS was made of but was about 1 cm in length and width to carry out crystalline orientation study using X-ray diffraction (XRD) scan around the (002)/(200) peaks after each of the binding steps. The result of the XRD studies indicated that each binding step caused the crystalline orientation of the PMN-PT thin layer to switch from the vertical (002) orientation to the horizontal (200) orientation, and most of the PEPS detection Δf/f was due to the change in the lateral Young's modulus of the PMN-PT thin layer as a result of the crystalline orientation change.« less

Mitosis-associated repression in development.

PubMed

Esposito, Emilia; Lim, Bomyi; Guessous, Ghita; Falahati, Hanieh; Levine, Michael

2016-07-01

Transcriptional repression is a pervasive feature of animal development. Here, we employ live-imaging methods to visualize the Snail repressor, which establishes the boundary between the presumptive mesoderm and neurogenic ectoderm of early Drosophila embryos. Snail target enhancers were attached to an MS2 reporter gene, permitting detection of nascent transcripts in living embryos. The transgenes exhibit initially broad patterns of transcription but are refined by repression in the mesoderm following mitosis. These observations reveal a correlation between mitotic silencing and Snail repression. We propose that mitosis and other inherent discontinuities in transcription boost the activities of sequence-specific repressors, such as Snail. © 2016 Esposito et al.; Published by Cold Spring Harbor Laboratory Press.

COPS: Detecting Co-Occurrence and Spatial Arrangement of Transcription Factor Binding Motifs in Genome-Wide Datasets

PubMed Central

Lohmann, Ingrid

2012-01-01

In multi-cellular organisms, spatiotemporal activity of cis-regulatory DNA elements depends on their occupancy by different transcription factors (TFs). In recent years, genome-wide ChIP-on-Chip, ChIP-Seq and DamID assays have been extensively used to unravel the combinatorial interaction of TFs with cis-regulatory modules (CRMs) in the genome. Even though genome-wide binding profiles are increasingly becoming available for different TFs, single TF binding profiles are in most cases not sufficient for dissecting complex regulatory networks. Thus, potent computational tools detecting statistically significant and biologically relevant TF-motif co-occurrences in genome-wide datasets are essential for analyzing context-dependent transcriptional regulation. We have developed COPS (Co-Occurrence Pattern Search), a new bioinformatics tool based on a combination of association rules and Markov chain models, which detects co-occurring TF binding sites (BSs) on genomic regions of interest. COPS scans DNA sequences for frequent motif patterns using a Frequent-Pattern tree based data mining approach, which allows efficient performance of the software with respect to both data structure and implementation speed, in particular when mining large datasets. Since transcriptional gene regulation very often relies on the formation of regulatory protein complexes mediated by closely adjoining TF binding sites on CRMs, COPS additionally detects preferred short distance between co-occurring TF motifs. The performance of our software with respect to biological significance was evaluated using three published datasets containing genomic regions that are independently bound by several TFs involved in a defined biological process. In sum, COPS is a fast, efficient and user-friendly tool mining statistically and biologically significant TFBS co-occurrences and therefore allows the identification of TFs that combinatorially regulate gene expression. PMID:23272209

Analysis of the grape MYB R2R3 subfamily reveals expanded wine quality-related clades and conserved gene structure organization across Vitis and Arabidopsis genomes

PubMed Central

Matus, José Tomás; Aquea, Felipe; Arce-Johnson, Patricio

2008-01-01

Background The MYB superfamily constitutes the most abundant group of transcription factors described in plants. Members control processes such as epidermal cell differentiation, stomatal aperture, flavonoid synthesis, cold and drought tolerance and pathogen resistance. No genome-wide characterization of this family has been conducted in a woody species such as grapevine. In addition, previous analysis of the recently released grape genome sequence suggested expansion events of several gene families involved in wine quality. Results We describe and classify 108 members of the grape R2R3 MYB gene subfamily in terms of their genomic gene structures and similarity to their putative Arabidopsis thaliana orthologues. Seven gene models were derived and analyzed in terms of gene expression and their DNA binding domain structures. Despite low overall sequence homology in the C-terminus of all proteins, even in those with similar functions across Arabidopsis and Vitis, highly conserved motif sequences and exon lengths were found. The grape epidermal cell fate clade is expanded when compared with the Arabidopsis and rice MYB subfamilies. Two anthocyanin MYBA related clusters were identified in chromosomes 2 and 14, one of which includes the previously described grape colour locus. Tannin related loci were also detected with eight candidate homologues in chromosomes 4, 9 and 11. Conclusion This genome wide transcription factor analysis in Vitis suggests that clade-specific grape R2R3 MYB genes are expanded while other MYB genes could be well conserved compared to Arabidopsis. MYB gene abundance, homology and orientation within particular loci also suggests that expanded MYB clades conferring quality attributes of grapes and wines, such as colour and astringency, could possess redundant, overlapping and cooperative functions. PMID:18647406

Detection of Coconut cadang-cadang viroid (CCCVd) in oil palm by reverse transcription loop-mediated isothermal amplification (RT-LAMP).

PubMed

Thanarajoo, Sathis Sri; Kong, Lih Ling; Kadir, Jugah; Lau, Wei Hongi; Vadamalai, Ganesan

2014-06-01

A reverse transcription loop-mediated isothermal amplification (RT-LAMP) detected Coconut cadang-cadang viroid (CCCVd) within 60 min at 60 °C in total nucleic acid extracted from oil palm leaves infected with CCCVd. Positive reactions showed colour change from orange to green in the reaction mix after the addition of fluorescent reagent, and a laddering pattern band on 2% agarose gel electrophoresis. Conventional RT-PCR with LAMP primers produced amplicons with a sequence identical to the 297-nt CCCVd oil palm variant with the primers being specific for CCCVd and not for other viroids such as PSTVd and CEVd. RT-LAMP was found to be rapid and specific for detecting oil palm CCCVd. Copyright © 2014 Elsevier B.V. All rights reserved.

Propidium monoazide reverse transcription PCR and RT-qPCR for detecting infectious enterovirus and norovirus

EPA Science Inventory

Presently there is no established cell line or small animal model that allows for the detection of infectious human norovirus. Current methods based on RT-PCR and RT-qPCR detect both infectious and non-infectious virus and thus the conclusions that may be drawn regarding the publ...

Evaluation of a sensitive reverse transcription PCR-enzymelinked immunosorbent assay for detection of hepatitis A virus in oysters (Saccostrea glomerata) on the east coast of the Gulf of Thailand.

PubMed

Intamaso, Uraiwan; Ketkhunthod, Sitthisak

2014-05-01

Hepatitis A virus (HAV) contamination in food can lead to major health problems. We developed a combination reverse transcription (RT) PCR method plus enzyme-linked immunosorbent assay (ELISA) to detect HAV in fresh oysters harvested along the east coast of the Gulf of Thailand. Viral nucleic acid was extracted via the glycine-arginine-polyethylene glycol method followed by RT-PCR amplification with specifically designed primers against HAV and an ELISA to detect the digoxigenin-labeled RT-PCR products. The ELISA in concert with the RT-PCR protocol further increased the detection sensitivity by 100-fold for the HAV genome and 10-fold in artificially contaminated oysters. The overall sensitivity of the RT-PCR in combination with the ELISA was 31.88 pg and 16 PFU/g, respectively. The ELISA increases the specificity of the RT-PCR assay for detecting naturally occurring HAV in oysters. This combined RT-PCR-ELISA approach is a practical and sensitive method for HAV detection and can be utilized in routine screening for HAV in shellfish.

Rapid and sensitive detection of human astrovirus in water samples by loop-mediated isothermal amplification with hydroxynaphthol blue dye

PubMed Central

2014-01-01

Background The aim of this paper was to develop a reverse transcription loop-mediated isothermal amplification (RT-LAMP) method for rapid, sensitive and inexpensive detection of astrovirus. Results The detection limit of LAMP using in vitro RNA transcripts was 3.6×10 copies·μL-1, which is as sensitive as the presently used PCR assays. However, the LAMP products could be identified as different colors with the naked eye following staining with hydroxynaphthol blue dye (HNB). No cross-reactivity with other gastroenteric viruses (rotavirus and norovirus) was observed, indicating the relatively high specificity of LAMP. The RT-LAMP method with HNB was used to effectively detect astrovirus in reclaimed water samples. Conclusions The LAMP technique described in this study is a cheap, sensitive, specific and rapid method for the detection of astrovirus. The RT-LAMP method can be simply applied for the specific detection of astrovirus and has the potential to be utilized in the field as a screening test. PMID:24524254

Cross-subtype Detection of HIV-1 Using Reverse Transcription and Recombinase Polymerase Amplification

PubMed Central

Lillis, Lorraine; Lehman, Dara A.; Siverson, Joshua B.; Weis, Julie; Cantera, Jason; Parker, Mathew; Piepenburg, Olaf; Overbaugh, Julie; Boyle, David S.

2016-01-01

A low complexity diagnostic test that rapidly and reliably detects HIV infection in infants at the point of care could facilitate early treatment, improving outcomes. However, many infant HIV diagnostics can only be performed in laboratory settings. Recombinase polymerase amplification (RPA) is an isothermal amplification technology that can rapidly amplify proviral DNA from multiple subtypes of HIV-1 in under twenty minutes without complex equipment. In this study we added reverse transcription (RT) to RPA to allow detection of both HIV-1 RNA and DNA. We show that this RT-RPA HIV-1 assay has a limit of detection of 10 to 30 copies of an exact sequence matched DNA or RNA, respectively. In addition, at 100 copies of RNA or DNA, the assay detected 171 of 175 (97.7 %) sequence variants that represent all the major subtypes and recombinant forms of HIV-1 Groups M and O. This data suggests that the application of RT-RPA for the combined detection of HIV-1 viral RNA and proviral DNA may prove a highly sensitive tool for rapid and accurate diagnosis of infant HIV. PMID:26821087

A homogeneous, Anti-dsDNA antibody-based assay for multicolor detection of cancer stem cell transcription factors.

PubMed

Ma, Jiehua; Shi, Hai; Zhang, Meiling; Li, Chao; Xiang, Yang; Liu, Ping

2018-10-31

Cancer stem cells (CSCs) are responsible for maintaining tumor growth, metastasis and recurrence. The high expression of cancer stem cell transcription factors (Oct4, Sox2 and Nanog) is a valuable prognostic factor, suggesting a higher risk of tumor recurrence and metastasis. So, the development of a convenient and cost-effective method for multiplex assay of these transcription factors (TFs) is highly required. In this work, we have proposed a universal homogeneous assay for multicolor detection of these TFs based on anti-dsDNA antibody-decorated Fe 3 O 4 magnetite nanoparticles (aadMNPs). In the presence of analytes, the dye-labeled dsDNAs are bound by specific TFs, which will inhibit the interactions between the dsDNAs and aadMNPs, generating higher fluorescence that may provide signal readout for the immunosensing process. By using the proposed method, Oct4 can be determined in a linear range from 3 to 1200 ng/mL with a detection limit of 0.035 ng/mL. Furthermore, we have presented assays for the sensitive, selective and rapid detection of Oct4, Sox2 and Nanog in cell extract, as well as the analysis of binding affinity of the mutated binding sequences. This work may provide potential applications in clinical CSCs detections, and may open new opportunity for the study of nucleotide polymorphisms in TF binding sites. Copyright © 2018 Elsevier B.V. All rights reserved.

Spatio-temporal pattern of sylvatic rabies in the Sultanate of Oman, 2006-2010.

PubMed

Hussain, Muhammad Hammad; Ward, Michael P; Body, Mohammed; Al-Rawahi, Abdulmajeed; Wadir, Ali Awlad; Al-Habsi, Saif; Saqib, Muhammad; Ahmed, Mohammed Sayed; Almaawali, Mahir Gharib

2013-07-01

Rabies was first reported in the Sultanate of Oman is 1990. We analysed passive surveillance data (444 samples) collected and reported between 2006 and 2010. During this period, between 45 and 75% of samples submitted from suspect animals were subsequently confirmed (fluorescent antibody test, histopathology and reverse transcription PCR) as rabies cases. Overall, 63% of submitted samples were confirmed as rabies cases. The spatial distribution of species-specific cases were similar (centred in north-central Oman with a northeast-southwest distribution), although fox cases had a wider distribution and an east-west orientation. Clustering of cases was detected using interpolation, local spatial autocorrelation and scan statistical analysis. Several local government areas (wilayats) in north-central Oman were identified where higher than expected numbers of laboratory-confirmed rabies cases were reported. For fox rabies, more clusters (local spatial autocorrelation analysis) and a larger clustered area (scan statistical analysis) were detected. In Oman, monthly reports of fox rabies cases were highly correlated (rSP>0.5) with reports of camel, cattle, sheep and goat rabies. The best-fitting ARIMA model included a seasonality component. Fox rabies cases reported 6 months previously best explained rabies reported cases in other animal species. Despite likely reporting bias, results suggest that rabies exists as a sylvatic cycle of transmission in Oman and an opportunity still exists to prevent establishment of dog-mediated rabies. Copyright © 2013 Elsevier B.V. All rights reserved.

Uniformity under in vitro conditions: Changes in the phenotype of cancer cell lines derived from different medulloblastoma subgroups.

PubMed

Chlapek, Petr; Zitterbart, Karel; Kren, Leos; Filipova, Lenka; Sterba, Jaroslav; Veselska, Renata

2017-01-01

Medulloblastoma comprises four main subgroups (WNT, SHH, Group 3 and Group 4) originally defined by transcriptional profiling. In primary medulloblastoma tissues, these groups are thought to be distinguishable using the immunohistochemical detection of β-catenin, filamin A, GAB1 and YAP1 protein markers. To investigate the utility of these markers for in vitro studies using medulloblastoma cell lines, immunoblotting and indirect immunofluorescence were employed for the detection of β-catenin, filamin A, GAB1 and YAP1 in both DAOY and D283 Med reference cell lines and the panel of six medulloblastoma cell lines derived in our laboratory from the primary tumor tissues of known molecular subgroups. Immunohistochemical detection of these markers was performed on formalin-fixed paraffin-embedded tissue of the matching primary tumors. The results revealed substantial divergences between the primary tumor tissues and matching cell lines in the immunoreactivity pattern of medulloblastoma-subgroup-specific protein markers. Regardless of the molecular subgroup of the primary tumor, all six patient-derived medulloblastoma cell lines exhibited a uniform phenotype: immunofluorescence showed the nuclear localization of YAP1, accompanied by strong cytoplasmic positivity for β-catenin and filamin A, as well as weak positivity for GAB1. The same immunoreactivity pattern was also found in both DAOY and D283 Med reference medulloblastoma cell lines. Therefore, we can conclude that various medulloblastoma cell lines tend to exhibit the same characteristics of protein marker expression under standard in vitro conditions. Such a finding emphasizes the importance of the analyses of primary tumors in clinically oriented medulloblastoma research and the urgent need to develop in vitro models of improved clinical relevance, such as 3D cultures and organotypic slice cultures.

Antiandrogens act as selective androgen receptor modulators at the proteome level in prostate cancer cells.

PubMed

Brooke, Greg N; Gamble, Simon C; Hough, Michael A; Begum, Shajna; Dart, D Alwyn; Odontiadis, Michael; Powell, Sue M; Fioretti, Flavia M; Bryan, Rosie A; Waxman, Jonathan; Wait, Robin; Bevan, Charlotte L

2015-05-01

Current therapies for prostate cancer include antiandrogens, inhibitory ligands of the androgen receptor, which repress androgen-stimulated growth. These include the selective androgen receptor modulators cyproterone acetate and hydroxyflutamide and the complete antagonist bicalutamide. Their activity is partly dictated by the presence of androgen receptor mutations, which are commonly detected in patients who relapse while receiving antiandrogens, i.e. in castrate-resistant prostate cancer. To characterize the early proteomic response to these antiandrogens we used the LNCaP prostate cancer cell line, which harbors the androgen receptor mutation most commonly detected in castrate-resistant tumors (T877A), analyzing alterations in the proteome, and comparing these to the effect of these therapeutics upon androgen receptor activity and cell proliferation. The majority are regulated post-transcriptionally, possibly via nongenomic androgen receptor signaling. Differences detected between the exposure groups demonstrate subtle changes in the biological response to each specific ligand, suggesting a spectrum of agonistic and antagonistic effects dependent on the ligand used. Analysis of the crystal structures of the AR in the presence of cyproterone acetate, hydroxyflutamide, and DHT identified important differences in the orientation of key residues located in the AF-2 and BF-3 protein interaction surfaces. This further implies that although there is commonality in the growth responses between androgens and those antiandrogens that stimulate growth in the presence of a mutation, there may also be influential differences in the growth pathways stimulated by the different ligands. This therefore has implications for prostate cancer treatment because tumors may respond differently dependent upon which mutation is present and which ligand is activating growth, also for the design of selective androgen receptor modulators, which aim to elicit differential proteomic responses dependent upon cellular context. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Antiandrogens Act as Selective Androgen Receptor Modulators at the Proteome Level in Prostate Cancer Cells*

PubMed Central

Brooke, Greg N.; Gamble, Simon C.; Hough, Michael A.; Begum, Shajna; Dart, D. Alwyn; Odontiadis, Michael; Powell, Sue M.; Fioretti, Flavia M.; Bryan, Rosie A.; Waxman, Jonathan; Wait, Robin; Bevan, Charlotte L.

2015-01-01

Current therapies for prostate cancer include antiandrogens, inhibitory ligands of the androgen receptor, which repress androgen-stimulated growth. These include the selective androgen receptor modulators cyproterone acetate and hydroxyflutamide and the complete antagonist bicalutamide. Their activity is partly dictated by the presence of androgen receptor mutations, which are commonly detected in patients who relapse while receiving antiandrogens, i.e. in castrate-resistant prostate cancer. To characterize the early proteomic response to these antiandrogens we used the LNCaP prostate cancer cell line, which harbors the androgen receptor mutation most commonly detected in castrate-resistant tumors (T877A), analyzing alterations in the proteome, and comparing these to the effect of these therapeutics upon androgen receptor activity and cell proliferation. The majority are regulated post-transcriptionally, possibly via nongenomic androgen receptor signaling. Differences detected between the exposure groups demonstrate subtle changes in the biological response to each specific ligand, suggesting a spectrum of agonistic and antagonistic effects dependent on the ligand used. Analysis of the crystal structures of the AR in the presence of cyproterone acetate, hydroxyflutamide, and DHT identified important differences in the orientation of key residues located in the AF-2 and BF-3 protein interaction surfaces. This further implies that although there is commonality in the growth responses between androgens and those antiandrogens that stimulate growth in the presence of a mutation, there may also be influential differences in the growth pathways stimulated by the different ligands. This therefore has implications for prostate cancer treatment because tumors may respond differently dependent upon which mutation is present and which ligand is activating growth, also for the design of selective androgen receptor modulators, which aim to elicit differential proteomic responses dependent upon cellular context. PMID:25693800

The mitochondrial genome of Hydra oligactis (Cnidaria, Hydrozoa) sheds new light on animal mtDNA evolution and cnidarian phylogeny.

PubMed

Kayal, Ehsan; Lavrov, Dennis V

2008-02-29

The 16,314-nuceotide sequence of the linear mitochondrial DNA (mtDNA) molecule of Hydra oligactis (Cnidaria, Hydrozoa)--the first from the class Hydrozoa--has been determined. This sequence contains genes for 13 energy pathway proteins, small and large subunit rRNAs, and methionine and tryptophan tRNAs, as is typical for cnidarians. All genes have the same transcriptional orientation and their arrangement in the genome is similar to that of the jellyfish Aurelia aurita. In addition, a partial copy of cox1 is present at one end of the molecule in a transcriptional orientation opposite to the rest of the genes, forming a part of inverted terminal repeat characteristic of linear mtDNA and linear mitochondrial plasmids. The sequence close to at least one end of the molecule contains several homonucleotide runs as well as small inverted repeats that are able to form strong secondary structures and may be involved in mtDNA maintenance and expression. Phylogenetic analysis of mitochondrial genes of H. oligactis and other cnidarians supports the Medusozoa hypothesis but also suggests that Anthozoa may be paraphyletic, with octocorallians more closely related to the Medusozoa than to the Hexacorallia. The latter inference implies that Anthozoa is paraphyletic and that the polyp (rather than a medusa) is the ancestral body type in Cnidaria.

Automated correction of improperly rotated diffusion gradient orientations in diffusion weighted MRI.

PubMed

Jeurissen, Ben; Leemans, Alexander; Sijbers, Jan

2014-10-01

Ensuring one is using the correct gradient orientations in a diffusion MRI study can be a challenging task. As different scanners, file formats and processing tools use different coordinate frame conventions, in practice, users can end up with improperly oriented gradient orientations. Using such wrongly oriented gradient orientations for subsequent diffusion parameter estimation will invalidate all rotationally variant parameters and fiber tractography results. While large misalignments can be detected by visual inspection, small rotations of the gradient table (e.g. due to angulation of the acquisition plane), are much more difficult to detect. In this work, we propose an automated method to align the coordinate frame of the gradient orientations with that of the corresponding diffusion weighted images, using a metric based on whole brain fiber tractography. By transforming the gradient table and measuring the average fiber trajectory length, we search for the transformation that results in the best global 'connectivity'. To ensure a fast calculation of the metric we included a range of algorithmic optimizations in our tractography routine. To make the optimization routine robust to spurious local maxima, we use a stochastic optimization routine that selects a random set of seed points on each evaluation. Using simulations, we show that our method can recover the correct gradient orientations with high accuracy and precision. In addition, we demonstrate that our technique can successfully recover rotated gradient tables on a wide range of clinically realistic data sets. As such, our method provides a practical and robust solution to an often overlooked pitfall in the processing of diffusion MRI. Copyright © 2014 Elsevier B.V. All rights reserved.

Cluster of Genes That Encode Positive and Negative Elements Influencing Filament Length in a Heterocyst-Forming Cyanobacterium

PubMed Central

Merino-Puerto, Victoria; Herrero, Antonia

2013-01-01

The filamentous, heterocyst-forming cyanobacteria perform oxygenic photosynthesis in vegetative cells and nitrogen fixation in heterocysts, and their filaments can be hundreds of cells long. In the model heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120, the genes in the fraC-fraD-fraE operon are required for filament integrity mainly under conditions of nitrogen deprivation. The fraC operon transcript partially overlaps gene all2395, which lies in the opposite DNA strand and ends 1 bp beyond fraE. Gene all2395 produces transcripts of 1.35 kb (major transcript) and 2.2 kb (minor transcript) that overlap fraE and whose expression is dependent on the N-control transcription factor NtcA. Insertion of a gene cassette containing transcriptional terminators between fraE and all2395 prevented production of the antisense RNAs and resulted in an increased length of the cyanobacterial filaments. Deletion of all2395 resulted in a larger increase of filament length and in impaired growth, mainly under N2-fixing conditions and specifically on solid medium. We denote all2395 the fraF gene, which encodes a protein restricting filament length. A FraF-green fluorescent protein (GFP) fusion protein accumulated significantly in heterocysts. Similar to some heterocyst differentiation-related proteins such as HglK, HetL, and PatL, FraF is a pentapeptide repeat protein. We conclude that the fraC-fraD-fraE←fraF gene cluster (where the arrow indicates a change in orientation), in which cis antisense RNAs are produced, regulates morphology by encoding proteins that influence positively (FraC, FraD, FraE) or negatively (FraF) the length of the filament mainly under conditions of nitrogen deprivation. This gene cluster is often conserved in heterocyst-forming cyanobacteria. PMID:23813733

Requirement of Multiple cis-Acting Elements in the Human Cytomegalovirus Major Immediate-Early Distal Enhancer for Viral Gene Expression and Replication

PubMed Central

Meier, Jeffery L.; Keller, Michael J.; McCoy, James J.

2002-01-01

We have shown previously that the human cytomegalovirus (HCMV) major immediate-early (MIE) distal enhancer is needed for MIE promoter-dependent transcription and viral replication at low multiplicities of infection (MOI). To understand how this region works, we constructed and analyzed a series of HCMVs with various distal enhancer mutations. We show that the distal enhancer is composed of at least two parts that function independently to coordinately activate MIE promoter-dependent transcription and viral replication. One such part is contained in a 47-bp segment that has consensus binding sites for CREB/ATF, SP1, and YY1. At low MOI, these working parts likely function in cis to directly activate MIE gene expression, thus allowing viral replication to ensue. Three findings support the view that these working parts are likely cis-acting elements. (i) Deletion of either part of a bisegmented distal enhancer only slightly alters MIE gene transcription and viral replication. (ii) Reversing the distal enhancer’s orientation largely preserves MIE gene transcription and viral replication. (iii) Placement of stop codons at −300 or −345 in all reading frames does not impair MIE gene transcription and viral replication. Lastly, we show that these working parts are dispensable at high MOI, partly because of compensatory stimulation of MIE promoter activity and viral replication that is induced by a virion-associated component(s) present at a high viral particle/cell ratio. We conclude that the distal enhancer is a complex multicomponent cis-acting region that is required to augment both MIE promoter-dependent transcription and HCMV replication. PMID:11739696

Nucleus detection using gradient orientation information and linear least squares regression

NASA Astrophysics Data System (ADS)

Kwak, Jin Tae; Hewitt, Stephen M.; Xu, Sheng; Pinto, Peter A.; Wood, Bradford J.

2015-03-01

Computerized histopathology image analysis enables an objective, efficient, and quantitative assessment of digitized histopathology images. Such analysis often requires an accurate and efficient detection and segmentation of histological structures such as glands, cells and nuclei. The segmentation is used to characterize tissue specimens and to determine the disease status or outcomes. The segmentation of nuclei, in particular, is challenging due to the overlapping or clumped nuclei. Here, we propose a nuclei seed detection method for the individual and overlapping nuclei that utilizes the gradient orientation or direction information. The initial nuclei segmentation is provided by a multiview boosting approach. The angle of the gradient orientation is computed and traced for the nuclear boundaries. Taking the first derivative of the angle of the gradient orientation, high concavity points (junctions) are discovered. False junctions are found and removed by adopting a greedy search scheme with the goodness-of-fit statistic in a linear least squares sense. Then, the junctions determine boundary segments. Partial boundary segments belonging to the same nucleus are identified and combined by examining the overlapping area between them. Using the final set of the boundary segments, we generate the list of seeds in tissue images. The method achieved an overall precision of 0.89 and a recall of 0.88 in comparison to the manual segmentation.

Oriented xenon hydride molecules in the gas phase

NASA Astrophysics Data System (ADS)

Buck, Udo; Fárník, Michal

The production of the xenon hydride molecules HXeX with X = I and Cl in the gas phase is reviewed. These molecules are generated by the photolysis of the hydrogen halide HI and HCl molecules on the surface of large xenon Xen clusters. Molecular dynamics simulations show that the flexible H atoms react with the heavy XeX moiety and form the desired molecules with nearly no rotational motion. They are observed by photodissociation with subsequent detection of the kinetic energy of the H atom fragment. During the generating process, the cluster starts to evaporate and the hydride molecule is left essentially free. For further discrimination against the H atom fragments from HX, the HXeX molecules are oriented in a combined pulsed laser field and a weak electrostatic field. The three topics which represent the background of our experiments are briefly reviewed: the nature and generation of rare gas hydrides, the alignment and orientation of molecules in electric fields, and the photodissociation of selected molecules in rare gas clusters. The conditions for detecting them in the gas phase are discussed. This is the trade off between the stability, which requires high electron affinity, and the conditions for orientation, which necessitate large polarizability anisotropies and dipole moments. Finally the prospects of detecting other classes of molecules are discussed.

Seismotectonic, structural, volcanologic, and geomorphic study of New Zealand; indigenous forest assessment in New Zealand; mapping, land use, and environmental studies in New Zealand, volume 2

NASA Technical Reports Server (NTRS)

Probine, M. C.; Suggate, R. P.; Mcgreevy, M. G.; Stirling, I. F. (Principal Investigator)

1977-01-01

The author has identified the following significant results. Ship detection via LANDSAT MSS data was demonstrated. In addition, information on ship size, orientation, and movement was obtained. Band 7 was used for the initial detection followed by confirmation on other MSS bands. Under low turbidity, as experienced in open seas, the detection of ships 100 m long was verified and detection of ships down to 30 m length theorized. High turbidity and sea state inhibit ship detection by decreasing S/N ratios. The radiance effect from snow of local slope angles and orientation was also studied. Higher radiance values and even overloading in three bands were recorded for the sun-facing slope. Local hot spots from solar reflection appear at several locations along transect D-C in Six Mile Creek Basin during September 1976.

Violence detection based on histogram of optical flow orientation

NASA Astrophysics Data System (ADS)

Yang, Zhijie; Zhang, Tao; Yang, Jie; Wu, Qiang; Bai, Li; Yao, Lixiu

2013-12-01

In this paper, we propose a novel approach for violence detection and localization in a public scene. Currently, violence detection is considerably under-researched compared with the common action recognition. Although existing methods can detect the presence of violence in a video, they cannot precisely locate the regions in the scene where violence is happening. This paper will tackle the challenge and propose a novel method to locate the violence location in the scene, which is important for public surveillance. The Gaussian Mixed Model is extended into the optical flow domain in order to detect candidate violence regions. In each region, a new descriptor, Histogram of Optical Flow Orientation (HOFO), is proposed to measure the spatial-temporal features. A linear SVM is trained based on the descriptor. The performance of the method is demonstrated on the publicly available data sets, BEHAVE and CAVIAR.

Detection of EGFR Gene Mutation by Mutation-oriented LAMP Method.

PubMed

Matsumoto, Naoyuki; Kumasaka, Akira; Ando, Tomohiro; Komiyama, Kazuo

2018-04-01

Epidermal growth factor receptor (EGFR) is a target of molecular therapeutics for non-small cell lung cancer. EGFR gene mutations at codons 746-753 promote constitutive EGFR activation and result in worst prognosis. However, these mutations augment the therapeutic effect of EGFR-tyrosine kinase inhibitor. Therefore, the detection of EGFR gene mutations is important for determining treatment planning. The aim of the study was to establish a method to detect EGFR gene mutations at codons 746-753. EGFR gene mutation at codons 746-753 in six cancer cell lines were investigated. A loop-mediated isothermal amplification (LAMP)-based procedure was developed, that employed peptide nucleic acid to suppress amplification of the wild-type allele. This mutation-oriented LAMP can amplify the DNA fragment of the EGFR gene with codons 746-753 mutations within 30 min. Moreover, boiled cells can work as template resources. Mutation oriented-LAMP assay for EGFR gene mutation is sensitive on extracted DNA. This procedure would be capable of detecting EGFR gene mutation in sputum, pleural effusion, broncho-alveolar lavage fluid or trans-bronchial lung biopsy by chair side. Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Imaging Transcriptional Regulation of Eukaryotic mRNA Genes: Advances and Outlook.

PubMed

Yao, Jie

2017-01-06

Regulation of eukaryotic transcription in vivo occurs at distinct stages. Previous research has identified many active or repressive transcription factors (TFs) and core transcription components and studied their functions in vitro and in vivo. Nonetheless, how individual TFs act in concert to regulate mRNA gene expression in a single cell remains poorly understood. Direct observation of TF assembly and disassembly and various biochemical reactions during transcription of a single-copy gene in vivo is the ideal approach to study this problem. Research in this area requires developing novel techniques for single-cell transcription imaging and integrating imaging studies into understanding the molecular biology of transcription. In the past decade, advanced cell imaging has enabled unprecedented capabilities to visualize individual TF molecules, to track single transcription sites, and to detect individual mRNA in fixed and living cells. These studies have raised several novel insights on transcriptional regulation such as the "hit-and-run" model and transcription bursting that could not be obtained by in vitro biochemistry analysis. At this point, the key question is how to achieve deeper understandings or discover novel mechanisms of eukaryotic transcriptional regulation by imaging transcription in single cells. Meanwhile, further technical advancements are likely required for visualizing distinct kinetic steps of transcription on a single-copy gene in vivo. This review article summarizes recent progress in the field and describes the challenges and opportunities ahead. Copyright © 2016 Elsevier Ltd. All rights reserved.

Characterizing Feature Matching Performance Over Long Time Periods (Author’s Manuscript)

DTIC Science & Technology

2015-01-05

older imagery. These applications, including approaches to geo-location, geo- orientation [13], geo-tagging [16], landmark recognition [23], image... orientation between features is less than 10 degrees. We calculate the percent of features from the reference image that fit into each of these three...always because the key point detection algorithm did not find feature points at the same locations and orientation . 5. Conclusions In this paper, we offer

Solea senegalensis sperm cryopreservation: New insights on sperm quality

PubMed Central

Riesco, Marta F.; Oliveira, Catarina; Soares, Florbela; Gavaia, Paulo J.; Dinis, María T.

2017-01-01

Cryopreservation of Senegalese sole sperm can represent an alternative to overcome some reproductive problems of this species. However, it is important to guarantee the safe use of cryopreserved sperm by selecting an appropriate protocol according to a high demand quality need to be ensured. It has been demonstrated that traditional assays such as motility and viability do not provide enough information to identify specific damage caused by cryopreservation process (freezing and thawing). Specific tests, including lipid peroxidation and DNA damage, should be performed. In the present study, motility and lipid peroxidation were performed as specific tests allowing us to discard cryopreservation conditions such as methanol as internal cryoprotectant and bovine serum albumin as external cryoprotectant. In addition, a caspase 3/7 detection by flow cytometry was performed to analyze apoptosis activity in the best selected conditions. Moreover, new highly sensitive tests based on transcript number detection have recently been described in fish sperm cryopreservation. For this reason, a transcript level detection assay was performed on certain oxidative and chaperone genes related to fertilization ability and embryo development (hsp70, hsp90BB, hsp90AA, gpx) to select the best cryopreservation conditions. DMSO+ egg yolk proved to be the best cryoprotectant combination in terms of transcript level. This study describes an optimized cryopreservation protocol for Solea senegalensis sperm demonstrating for the first time that transcript degradation is the most sensitive predictor of cell status in this species after cryopreservation. PMID:29053706