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PubMed1989

Heart rate variability in relation to prognosis after myocardial infarction: selection of optimal processing techniques.

Automatic analysis of heart rate variability from Holter recordings may be invalidated by beat recognition errors and recording artefact, necessitating filtering and editing of the computer-recognized RR interval sequence. Two new methods for heart rate variability analysis have been developed, based on an estimation of the width of the main peak of the frequency distribution curve of the computer-recognized normal-to-normal beat sequence. These methods are independent of a low level of recognition error and artefact, thus removing the need for operator-dependent, time-consuming editing. The value of the new methods (heart variability indices 1 and 2) in identifying patients with serious events (death and symptomatic, sustained documented ventricular tachycardia) during a 6-month follow-up after acute myocardial infarction was assessed in a case-control study comparing 20 patients who had experienced such events (Group I) with 20 patients who, following admission with acute myocardial infarction, had remained free of complications for greater than 6 months after discharge (Group II). Group II was selected to match Group I with regard to age, sex, infarct site, ejection fraction, and beta-blocker treatment. Analysis of the unfiltered computer-recognized normal-to-normal interval sequence showed that heart rate variability indices 1 and 2 were significantly lower (P less than 0.005, P less than 0.002) in those who had experienced events compared with those free from complications. Two other methods of expressing heart rate variability, including the standard deviation method, in combination with four different data-filtering techniques, gave less significant distinction between those with and without events during follow-up. It is concluded that using the methods described, reduced heart rate variability in patients at risk from death or sustained ventricular tachycardia after acute myocardial infarction can be detected automatically from unfiltered Holter tape recordings even in the presence of a low level of beat recognition error and recording artefact.

Aged↗
PubMed2004

Evidence-based strategies to promote physical activity among children, adolescents and young adults: review and update.

This paper updates evidence reviewed in the first edition of Getting Australia Active on effective physical activity (PA) intervention strategies among children, adolescents and young adults. Intervention studies published between 1999 and September 2003 were identified using electronic databases and hand searching. A total of 28 discrete studies were identified (31 papers). Six of nine studies reported significant effects on child or youth PA in school settings. Those that incorporated whole-of-school approaches including curriculum, policy and environmental strategies appeared to be more effective than those that incorporated curriculum-only approaches. Five of 10 studies with children and two of five studies with adolescents reported increased PA or decreased sedentary behaviour in other settings. Interventions that included contact with families generally appeared to be most effective. One study with adolescents provided some evidence of the potential effectiveness of interventions based in primary care. Two of four papers reported modest short-term results among young adults, including increased PA stage of change or a higher likelihood of being adequately physically active, but none showed any evidence of sustained increases in PA. There is an urgent need for additional studies examining interventions aimed at young adults. Across the three life stages, future studies should include objective PA measures, longer-term follow-up, larger sample sizes, a specific focus on PA (rather than weight) and culturally-specific strategies that build evidence in Australian populations. Future studies should target high risk groups and a broad range of settings and strategies focusing on reducing sedentary behaviours as well as increasing PA.

Adolescent↗
PubMed1997

Mitochondrial mRNA 3' cleavage/polyadenylation and RNA editing in Trypanosoma brucei are independent events.

The organization of the mitochondrial maxicircle genome of Trypanosoma brucei is unique in the close packing of the mRNA genes. For many of them, the 5' and 3' ends of adjacent transcripts overlap and formation of the proper 3' or 5' end can eliminate a portion of the coding sequence of the adjacent gene. Large, polycistronic transcripts have been detected. suggesting that mechanisms for precise cleavages at both 5' and 3' gene boundaries must exist. However, no common sequences near the ends of the mRNAs that could be candidates for control regions have been detected. In addition, nothing is known about how RNA editing interacts with and affects 5' and 3' processing and/or polyadenylation. Edited precursor transcripts have been detected, indicating that editing complexes can assemble prior to transcript cleavage. Because editing often initiates near the 3' end of the mRNA, the assembly of an editing complex in this region may influence the cleavage selection process. In order to determine the extent that RNA editing and 3' end-processing interact, RNAs were analyzed to determine the extent of editing in precursor RNAs and to determine if unedited transcripts can be cleaved and polyadenylated. Two overlapping RNA junctions were analyzed; the junction between NADH dehydrogenase (ND) subunit 7 and cytochrome oxidase (CO) subunit III, and the junction between CO subunit II and maxicircle unidentified reading frame (MURF) II. For both of these RNAs, editing affects restriction endonuclease recognition sequences, allowing us to analyze editing patterns by differential restriction digests. These analyses suggest that when the gRNA is supplied in trans, RNA editing and cleavage/polyadenylation are independent events and while they may influence one another, one event is not dependent on the other. Conversely, for the COII transcript, where the gRNA is located at the 3' end of the mRNA and appears to be supplied in cis, edited precursors were not detected. This suggests a requirement for a precise intramolecular interaction for COII editing that cannot form prior to 3' end-maturation.

Animals↗
PubMed1993

Trypanosoma brucei minicircles encode multiple guide RNAs which can direct editing of extensively overlapping sequences.

Small guide RNAs (gRNAs) may direct RNA editing in kinetoplastid mitochondria. We have characterized multiple gRNA genes from Trypanosoma brucei (EATRO 164), that can specify up to 30% of the editing of the COIII, ND7, ND8, and A6 mRNAs and we have also found that the non-translated region of edited COIII mRNA of strain (EATRO 164) differs from that of another strain. Several of the gRNAs specify overlapping regions of the same mRNA often specifying sequence beyond that required for an anchor duplex with the next gRNA. Some gRNAs have different sequence but specify identical editing of the same region of mRNA. These data indicate a complex gRNA population and consequent complex pattern of editing in T. brucei.

Animals↗
PubMed2004

A survey of RNA editing in human brain.

We have conducted a survey of RNA editing in human brain by comparing sequences of clones from a human brain cDNA library to the reference human genome sequence and to genomic DNA from the same individual. In the RNA sample from which the library was constructed, approximately 1:2000 nucleotides were edited out of >3 Mb surveyed. All edits were adenosine to inosine (A-->I) and were predominantly in intronic and in intergenic RNAs. No edits were found in translated exons and few in untranslated exons. Most edits were in high-copy-number repeats, usually Alus. Analysis of the genome in the vicinity of edited sequences strongly supports the idea that formation of intramolecular double-stranded RNA with an inverted copy underlies most A-->I editing. The likelihood of editing is increased by the presence of two inverted copies of a sequence within the same intron, proximity of the two sequences to each other (preferably within 2 kb), and by a high density of inverted copies in the vicinity. Editing exhibits sequence preferences and is less likely at an adenosine 3' to a guanosine and more likely at an adenosine 5' to a guanosine. Simulation by BLAST alignment of the double-stranded RNA molecules that underlie known edits indicates that there is a greater likelihood of A-->I editing at A:C mismatches than editing at other mismatches or at A:U matches. However, because A:U matches in double-stranded RNA are more common than all mismatches, overall the likely effect of editing is to increase the number of mismatches in double-stranded RNA.

Aged↗
PubMed2003

Identification of a mutation in editing of defective Newcastle disease virus recombinants that modulates P-gene mRNA editing and restores virus replication and pathogenicity in chicken embryos.

Editing of P-gene mRNA of Newcastle disease virus (NDV) enables the formation of two additional proteins (V and W) by inserting one or two nontemplated G residues at a conserved editing site (5'-AAAAAGGG). The V protein of NDV plays an important role in virus replication and is also a virulence factor presumably due to its ability to counteract the antiviral effects of interferon. A recombinant virus possessing a nucleotide substitution within the A-stretch (5'-AAgAAGGG) produced 20-fold-less V protein and, in consequence, was impaired in replication capacity and completely attenuated in pathogenicity for chicken embryos. However, in a total of seven serial passages, restoration of replication and pathogenic capacity in 9- to 11-day-old chicken embryos was noticed. Determining the sequence around the editing site of the virus at passage 7 revealed a C-to-U mutation at the second nucleotide immediately upstream of the 5'-A(5) stretch (5'-GuUAAgAAGGG). The V mRNA increased from an undetectable level at passage 5 to ca. 1 and 5% at passages 6 and 7, respectively. In addition, similar defects in another mutant possessing a different substitution mutation (5'-AAAcAGGG) were restored in an identical manner within a total of seven serial passages. Introduction of the above C-to-U mutation into the parent virus (5'-GuUAAAAAGGG) altered the frequency of P, V, and W mRNAs from 68, 28, and 4% to 15, 44, and 41%, respectively, demonstrating that the U at this position is a key determinant in modulating P-gene mRNA editing. The results indicate that this second-site mutation is required to compensate for the drop in edited mRNAs and consequently to restore the replication capacity, as well as the pathogenic potential, of editing-defective NDV recombinants.

Amino Acid Sequence↗
PubMed1999

Genetic analysis of cytochrome b5 from arachidonic acid-producing fungus, Mortierella alpina 1S-4: cloning, RNA editing and expression of the gene in Escherichia coli, and purification and characterization of the gene product.

Information on the amino acid sequences of the internal peptide fragments of cytochrome b5 from Mortierella hygrophila was used to prepare synthetic oligonucleotides as primers for the polymerase chain reaction. A 100-base DNA fragment was thus amplified, by using a genomic gene from Mortierella alpina 1S-4 as a template, which produced polyunsaturated fatty acids such as arachidonic acid. The amplified DNA fragment was used as the probe to clone both a 523-base cDNA fragment and a 2.1-kilobase SalI-NruI genomic fragment coding for the whole M. alpina 1S-4 cytochrome b5. On the basis of nucleotide sequences of both cytochrome b5 genomic gene and cDNA, the genomic cytochrome b5 gene was found to consist of four exons and three introns. A novel type of RNA editing, in which the cDNA included either guanine insertion or adenine-->guanine substitution at one base upstream of poly(A), was interestingly observed. The deduced amino acid sequence of M. alpina 1S-4 cytochrome b5 showed significant similarities with those of cytochrome b5s from other organisms such as rat, chicken, and yeast. The soluble form of the cytochrome b5 gene was expressed to 16% of the total soluble protein in Escherichia coli. The holo-cytochrome b5 accounted for 8% of the total cytochrome b5 in the transformants. The purified cytochrome b5 showed the oxidized and reduced absorbance spectra characteristic of fungal microsomal cytochrome b5.

Amino Acid Sequence↗
PubMed2001

Hepatitis delta virus minimal substrates competent for editing by ADAR1 and ADAR2.

A host-mediated RNA-editing event allows hepatitis delta virus (HDV) to express two essential proteins, the small delta antigen (HDAg-S) and the large delta antigen (HDAg-L), from a single open reading frame. One or several members of the ADAR (adenosine deaminases that act on RNA) family are thought to convert the adenosine to an inosine (I) within the HDAg-S amber codon in antigenomic RNA. As a consequence of replication, the UIG codon is converted to a UGG (tryptophan [W]) codon in the resulting HDAg-L message. Here, we used a novel reporter system to monitor the editing of the HDV amber/W site in the absence of replication. In cultured cells, we observed that both human ADAR1 (hADAR1) and hADAR2 were capable of editing the amber/W site with comparable efficiencies. We also defined the minimal HDV substrate required for hADAR1- and hADAR2-mediated editing. Only 24 nucleotides from the amber/W site were sufficient to enable efficient editing by hADAR1. Hence, the HDV amber/W site represents the smallest ADAR substrate yet identified. In contrast, the minimal substrate competent for hADAR2-mediated editing contained 66 nucleotides.

Adenosine Deaminase↗
PubMed2026

Expanding the scope of precision editing in seaweeds through the application of a novel CRISPR-associated nuclease 12a-aligned CRISPR system in Ulva prolifera.

Seaweeds, such as the fast-growing green alga Ulva prolifera, can be harnessed as valuable marine crops. The lack of scalable genome-editing tools hampers functional genomics to explore and elucidate algal molecular pathways with industrial importance. Here, we expanded precision genome modification in seaweeds by successfully demonstrating gene editing with a transgene-free AT-rich-targeting CRISPR-associated protein (Cas) system in U. prolifera. By evaluating various delivery buffers, comparing different Cas systems, and optimizing incubation temperatures, we determined suitable conditions for more widespread applicability of a novel Cas12a-aligned ST8 editor. We obtained >50 ST8-mediated knockout mutants of a toxin-based endogenous marker gene, UpAPT, at 28 °C post-delivery incubations. Our work diversified the applicable genome-editing tools in seaweeds, advancing algal functional genomics and providing more strategies to precisely target unexplored seaweed resources.

Ulva↗
PubMed2000

RNA editing of the human serotonin 5-HT(2C) receptor delays agonist-stimulated calcium release.

RNA encoding the human 5-HT(2C) receptor undergoes adenosine-to-inosine RNA editing events at five positions in the putative second intracellular loop, with a corresponding reduction in receptor/G-protein coupling. Agonist-stimulated calcium release was examined in NIH-3T3 fibroblasts stably expressing the nonedited human INI (hINI) or the edited hVSV or hVGV variants. We hypothesized that different receptor isoforms would show altered dynamics of agonist-induced calcium release. The three isoforms showed a rightward shift in agonist concentration-response curves for eliciting calcium release (EC(50) values: hINI, 2.2 nM; hVSV, 15 nM; hVGV, 49 nM). Additionally, the hVGV receptor showed a blunted and delayed [Ca(2+)](i) peak compared with the hINI or hVSV receptor isoforms. These distinctions in agonist-induced [Ca(2+)](i) release imply that edited 5-HT(2C) receptors may produce distinct physiological responses within the central nervous system.

3T3 Cells↗
PubMed2006

SNPeffect v2.0: a new step in investigating the molecular phenotypic effects of human non-synonymous SNPs.

UNLABELLED: Single nucleotide polymorphisms (SNPs) constitute the most fundamental type of genetic variation in human populations. About 75 000 of these reported variations cause an amino acid change in the translated protein. An important goal in genomic research is to understand how this variability affects protein function, and whether or not particular SNPs are associated to disease susceptibility. Accordingly, the SNPeffect database uses sequence- and structure-based bioinformatics tools to predict the effect of non-synonymous SNPs on the molecular phenotype of proteins. SNPeffect analyses the effect of SNPs on three categories of functional properties: (1) structural and thermodynamic properties affecting protein dynamics and stability (2) the integrity of functional and binding sites and (3) changes in posttranslational processing and cellular localization of proteins. The search interface of the database can be used to search specifically for polymorphisms that are predicted to cause a change in one of these properties. Now based on the Ensembl human databases, the SNPeffect database has been remodeled to better fit an automatically updatable structure. The current edition holds the molecular phenotype of 74 567 nsSNPs in 23 426 proteins. AVAILABILITY: SNPeffect can be accessed through http://snpeffect.vib.be.

Algorithms↗
PubMed1990

Pseudo-templated transcription by Escherichia coli RNA polymerase at a mutant promoter.

A G----T mutation at the start-point of transcription of the phage P22 sar promoter (sar + 1T) causes a novel defect in promoter clearance by Escherichia coli RNA polymerase (RNAP) in vitro. Under standard transcription conditions, in the presence of high concentrations of all four NTPs, the predominant products from this promoter are poly(U) chains of varying length. Because the mutation creates a run of four T: A base-pairs from - 1 to +3 (TGTT----TTTT), we propose that synthesis of poly(U) is pseudo-templated by the A4 stretch on the template strand. G----A and G----C mutations at position +1 do not cause pseudo-templated transcription. Several molecules of poly(U) are produced and released per sar+1T promoter-polymerase complex without dissociation of RNAP from the template DNA. The exponential relationship between yield and size of individual poly(U) species indicates that there is a constant probability that another U residue will be added to the nascent chain. Presumably, pseudo-templated transcription occurs by a slippage (stuttering) mechanism like that proposed to explain certain kinds of RNA editing in eukaryotic viral mRNAs.

Base Sequence↗
PubMed1996

Sequences directing C to U editing of the plastid psbL mRNA are located within a 22 nucleotide segment spanning the editing site.

In plastids, editing of an ACG codon to an AUG codon creates the translation initiation codon for the psbL and ndhD transcripts in tobacco. To identify the RNA segment required for psbL editing, chimeric kanamycin resistance genes were constructed containing psbL deletion derivatives, and tested in vivo for editing in transgenic plants. We report here that a 22 nucleotide segment is sufficient to direct efficient psbL editing, including 16 nucleotides upstream and five nucleotides downstream of the editing site. Mutation of the A nucleotide to a C upstream of the editing site completely abolished editing, while mutation of the downstream G to a C only reduced the editing efficiency. Out of the 22 nucleotide editing target sequence, the 16 upstream nucleotides were found to compete with the endogenous psbL transcript for a depletable trans-factor. To test whether editing of initiation codons involves a common trans-factor, a chimeric gene containing the ndhD editing site was expressed in tobacco plastids. As for psbL, editing of the ndhD site requires a depletable trans-factor. However, the ndhD trans-factor is distinct from that required for psbL editing. Distinct cissequences and trans-factor requirements for the psbL and ndhD editing sites indicate an individual recognition mechanism for the editing of plastid initiation codons.

Amino Acid Sequence↗
PubMed2003

Liver disintegration in the mouse embryo caused by deficiency in the RNA-editing enzyme ADAR1.

ADAR1 (adenosine deaminase acting on RNA-1) is widely expressed in mammals, but its biological role is unknown. We show here by gene targeting that ADAR1 selectively edits in vivo two of five closely spaced adenosines in the serotonin 5-hydroxytryptamine subtype 2C receptor pre-mRNA of nervous tissue; and hence, site-selective adenosine-to-inosine editing is indeed a function of ADAR1. Remarkably, homozygosity for two different null alleles of ADAR1 caused a consistent embryonic phenotype appearing early at embryonic day 11 and leading to death between embryonic days 11.5 and 12.5. This phenotype manifests a rapidly disintegrating liver structure, along with severe defects in definitive hematopoiesis, encompassing both erythroid and myeloid/granuloid progenitors as well as spleen colony-forming activity from the aorta-gonad-mesonephros region and fetal liver. Probably as a consequence of these developmental impairments, ADAR1-deficient embryonic stem cells failed to contribute to liver, bone marrow, spleen, thymus, and blood in adult chimeric mice. Thus, ADAR1 subserves critical steps in developing non-nervous tissue, which are likely to include transcript editing.

Adenosine Deaminase↗
PubMed2004

In vitro RNA editing systems from higher plant chloroplasts.

RNA editing in higher plant chloroplasts involves C-to-U conversion at ~30 specific sites. In vitro systems supporting accurate editing have been developed from tobacco and pea chloroplasts. mRNA substrates labeled with 32P at C residues to be edited provide sensitive detection of editing activity in vitro. The present systems allow the rapid identification of cis-elements using mutated mRNA substrates and trans-acting factors by ultraviolet crosslinking.

Base Sequence↗
PubMed1998

A comparison of staging systems for nasopharyngeal carcinoma.

BACKGROUND: The fifth edition of the American Joint Committee on Cancer staging manual defines new rules for classifying nasopharyngeal carcinoma. The authors tested the value of this new system by applying these rules retrospectively to their previously treated patients and comparing the results with those obtained using the fourth edition of the AJCC staging manual or the Ho staging system. METHODS: Information from 107 patients who had biopsy-proven squamous cell carcinoma of the nasopharynx that was treated in a constant fashion with definitive-intent radiation therapy alone at one institution provided the data base for this analysis. The extent of disease of each patient was staged according to the rules of 1) the fourth edition of the AJCC staging manual, 2) the Ho staging system, and 3) the fifth edition of the AJCC staging manual. RESULTS: The new system appears to be better than the two previous systems. It separated patients into cohorts of more equal size than did either of the other systems. It also correlated with outcome for the study population more appropriately than did the fourth edition of the AJCC staging manual or the Ho staging system. CONCLUSIONS: The fifth edition of the AJCC staging manual appears to be an improvement over the previous AJCC or Ho staging systems for the staging of nasopharyngeal carcinoma.

Carcinoma, Squamous Cell↗
PubMed1995

RNA-d2: a computer program for editing and display of RNA secondary structures.

RNA-d2 is a user-friendly program developed for interactively generating aesthetic and non-overlapping drawings of RNA secondary structures. It designed so that the drawings can be edited in a very natural and intuitive way, in order to emphasize structural homologies between several molecules, as well as the foldings themselves to update the base-pair sets according to new data. The program automatically produces a polygonal display in which the unpaired nucleotides are regularly positioned on circles and the stems harmoniously distributed on their periphery. RNA secondary structures can be encoded via the keyboard, but the program also automatically draws output files from thermodynamic prediction programs. The user interacts directly on different screen displays according to the editing functions. Rotation/translation of any subdomain and deletion of stems are performed on a coloured backbone view to make easier the identification of structural features, whereas addition of new base-pairings and numbering manipulation are realized on a complete polygonal view. Each modification is displayed in real time on the screen. When the display is obscured by numerous overlaps despite the colour code of the backbone view, an automatic function progressively straightens the subdomains which are highly compacted by very dissymmetric internal loops. RNA-d2 allows easy untangling and editing of RNA molecules > 1000 nucleotides long.

Algorithms↗