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PubMed1997

A JAVA-based multimedia tool for clinical practice guidelines.

We have developed a specific language for the representation of Clinical Practice Guidelines (CPGs) and Windows C++ and platform independent JAVA applications for multimedia presentation and edition of electronically stored CPGs. This approach facilitates translation of guidelines and protocols from paper to computer-based flowchart representations. Users can navigate through the algorithm with a friendly user interface and access related multimedia information within the context of each clinical problem. CPGs can be stored in a computer server and distributed over the World Wide Web, facilitating dissemination, local adaptation, and use as a reference element in medical care. We have chosen the Agency for Health Care and Policy Research's heart failure guideline to demonstrate the capabilities of our tool.

Computer Communication Networks↗

PubMed2006

Reclassifying DIS-III-R alcohol use disorders to DSM-IV criteria in a sample of convicted impaired drivers.

OBJECTIVE: This study used data gathered from the Diagnostic Interview Schedule, Version Three, Revised (DIS-III-R), which calculated diagnoses based on the Diagnostic and Statistical Manual of Mental Disorders, Third Edition, Revised (DSM-III-R), criteria and rescored the data to be compatible with the criteria of the Fourth Edition of the DSM (DSM-IV) for lifetime alcohol abuse and dependence. METHOD: A psychologist reassigned questions from the DIS-III-R according to DSM-IV criteria. Another clinician evaluated the rescoring criteria and discrepancies were discussed and resolved. Using these criteria, SAS code was written to automate the rescoring of responses to DIS-III-R questions to DSM-IV diagnoses from a population of DWI offenders. RESULTS: There was a fair-to-good level of agreement between the DSM-III-R and rescored DSM-IV diagnoses (kappa = .65). Three hundred forty-eight subjects classified as alcohol dependent using DSMIII- R were reclassified as alcohol abuse in the DSM-IV rescore. Among subjects who were alcohol dependent based on DIS-III-R criteria, the distribution of DSM-IV diagnoses was similar across gender, age, and ethnic groups. There was no difference in agreement between DSMIII- R and the rescored DSM-IV diagnoses by age category. However, women and Hispanics had significantly higher weighted kappa statistics than men and non-Hispanic whites. CONCLUSIONS: Our rescoring results were consistent with earlier studies that compared DSM-III-R and DSM-IV diagnoses. Here, we offer an approach that may be useful to investigators who used the DIS-III-R in earlier studies. The DIS-III-R questions corresponding to DSM-IV criteria for alcohol abuse and alcohol dependence are on our Web site at www.bhrcs.org, along with the scoring algorithm.

Adult↗

PubMed2005

A single nucleotide polymorphism based approach for the identification and characterization of gene expression modulation using MassARRAY.

Single nucleotide polymorphisms (SNPs) are the most common form of genetic variation. Their abundance and the ease with which they can be assayed have lead to their use in applications beyond simple genotyping. One such application is the quantitative determination of transcript levels associated with distinct alleles or haplotypes found in promoters and coding regions of genes. These changes in expression due to allelic variation are often associated with additional genomic or transcript modifications such as DNA methylation or RNA editing. Here, we describe the use of an integrated genetic analysis platform, based on matrix-assisted laser desorption/ionisation-time-of-flight (MALDI-TOF) to first, discover coding SNPs (cSNPs); second, use these cSNPs to identify and analyze allele-specific expression; and third, from this knowledge to further analyze methylation patterns as a putative cause for the allele-specific expression. An established model involving allele-specific expression profiles of the human tumor protein 73 (TP73) gene is presented as an example to outline and validate data obtained from the MassARRAY platform. The availability of a single integrated platform to assay stable and dynamic variation at the genomic and transcript level greatly simplifies complex functional genomic studies.

5' Untranslated Regions↗

PubMed2004

Rapid evolution of RNA editing sites in a small non-essential plastid gene.

Chloroplast RNA editing proceeds by C-to-U transitions at highly specific sites. Here, we provide a phylogenetic analysis of RNA editing in a small plastid gene, petL, encoding subunit VI of the cytochrome b6f complex. Analyzing representatives from most major groups of seed plants, we find an unexpectedly high frequency and dynamics of RNA editing. High-frequency editing has previously been observed in plastid ndh genes, which are remarkable in that their mutational inactivation does not produce an obvious mutant phenotype. In order to test the idea that reduced functional constraints allow for more flexible evolution of RNA editing sites, we have created petL knockout plants by tobacco chloroplast transformation. We find that, in the higher plant tobacco, targeted inactivation of petL does not impair plant growth under a variety of conditions markedly contrasting the important role of petL in photosynthesis in the green alga Chlamydomonas reinhardtii. Together with a low number of editing sites in plastid genes that are essential to gene expression and photosynthetic activity, these data suggest that RNA editing sites may evolve more readily in those genes whose transitory loss of function can be tolerated. Accumulated evidence for this 'relative neutrality hypothesis for the evolution of plastid editing sites' is discussed.

Base Sequence↗

PubMed1994

A single homogeneous form of ATP6 protein accumulates in petunia mitochondria despite the presence of differentially edited atp6 transcripts.

Transcripts encoding ATP synthase subunit 6 (ATP6) in petunia mitochondria were shown to be edited at 15 sites, leading to 14 amino acid changes. Certain sites are partially edited, including a site that introduces a new translation termination codon that is 13 codons upstream of the genomically encoded stop codon. Transcripts lacking the new stop codon are present in an approximately 2.5:1 ratio to transcripts carrying the stop codon created by RNA editing. To investigate whether partially edited transcripts are represented as proteins, we generated an antibody against a 12-residue peptide that is specific for translation products of unedited transcripts. This antibody did not recognize any ATP6 protein in either total mitochondrial protein preparations or ATP6 samples purified by organic solvent extraction and reverse phase HPLC procedures. According to analysis by mass spectrometry, only one form of ATP6 protein accumulates in mitochondria despite the presence of abundant partially edited transcripts. Partially edited atp6 transcripts were associated with ribosomes, suggesting that a screening mechanism(s) acts cotranslationally or post-translationally to exclude the expression of incompletely edited transcripts.

Amino Acid Sequence↗

PubMed1987

Functional effects of cis-thymine glycol lesions on DNA synthesis in vitro.

Thymine glycol, a DNA lesion produced by ionizing radiation, has been introduced site specifically at high frequency into a synthetic oligonucleotide by chemical oxidation of the single thymine residue within the sequence. The lesion-containing template was then annealed to a complementary synthetic primer and used to study the effects of cis-thymine glycol lesions on DNA polymerase function in vitro. Synthesis by polymerase I (Klenow fragment), T4 DNA polymerase, and polymerase alpha 2 was arrested quantitatively at the site of the lesion. AMV reverse transcriptase was less inhibited and was able to synthesize past a significant fraction of the lesions. Changing the template base immediately 5' to thymine glycol from A to C did not significantly alter the pattern of synthesis arrest for any of the polymerases. The correct nucleotide, dAMP, was inserted opposite the lesion more than 90% of the time by all four polymerases, suggesting that thymine glycol forms a reasonably stable base pair with adenine. However, the 3'-5' exonuclease activity of polymerase I removed a 3'-terminal dAMP residue more rapidly from an A . thymine glycol base pair than from an A.T base pair. These results suggest that increased nucleotide turnover at the site of the lesion contributes to the inhibitory effects of thymine glycol lesions on DNA synthesis in vitro, at least for polymerases such as polymerase I that have intrinsic or associated editing exonuclease functions.

Base Sequence↗

PubMed2002

American Academy of Disability Evaluating Physicians (AADEP) position paper: complex regional pain syndrome I (RSD): impairment and disability issues.

OBJECTIVE: To provide an overview and methodology for the evaluation of impairment and functional residual capacity in complex regional pain syndrome I (CRPS I, RSD). This paper is intended to provide assistance to physicians asked to evaluate impairment- and disability-related issues and is not primarily geared to guide treatment of the CRPS I patient. METHOD: Conference and literature review by neurology, anesthesiology, pain medicine, physiatry, and disability evaluating physicians followed by description of issues, options, and recommendations based upon the committee's deliberations and the widely used AMA Guides to the Evaluation of Permanent Impairment, fourth and fifth editions. The authors present functional criteria for assessing the severity of CRPS. These criteria were developed by committee consensus opinion based on multidisciplinary clinical experience considering daily functional issues. They can be used in conjunction with CRPS-ADL Classes of Impairment in order to categorize an individual into a specific class for purposes of permanent impairment rating. Future reliability and validity studies of this rating scale are pending future use, acceptance and, hopefully, additional studies. RESULTS: Evaluators should perform a comprehensive assessment of patients with CRPS I to make an accurate diagnosis and exclude other conditions that could explain the symptoms and signs of the condition. While radiological, laboratory, and other diagnostic studies may be of assistance in making the diagnosis, in the final analysis, this is a clinical diagnosis. Impairment is based on objectively validated limitation in activities of daily living (ADL).

Journal Article↗

PubMed1993

Apolipoprotein B mRNA editing: the sequence to the event.

Editing of the mRNA coding for apolipoprotein B involves a cytidine to uridine transition at nucleotide 6666 and enables translation of two protein variants. The development of in vitro editing systems has led to the identification of three sequence requirements in this process. The mechanism for C-->U editing requires specific sequences for editing site recognition, positioning of the catalytic activity and enhancement of catalytic efficiency. The dependence of in vitro editing on extract proteins has focused future research in this field on the identification of factors involved in apoB mRNA editing and the role of these factors in the assembly of ribonucleoprotein editosomes.

Animals↗

PubMed1997

Kinetoplastid RNA editing: complexes and catalysts.

RNA editing in kinetoplastids involves post-transcriptional insertion and deletion of uridylates (Us) to produce mature mitochondrial mRNAs with sequences specified by trans acting small guide RNAs. In vitro studies indicate the reaction pathway involves endonucleolytic cleavage of the precursor mRNA at the editing site, uridylate addition or removal at the 3' end of the 5' cleavage product, followed by ligation to the 3' cleavage product. This editing is catalyzed by a macromolecular complex that is in the early stages of characterization. Recent studies have resolved the general mechanism of editing, and show that editing occurs in association with a macromolecular complex.

Animals↗

PubMed1994

Relationship between patient self-ratings and physician ratings of general health, depression, and anxiety.

OBJECTIVE: To examine the relationship between patient self-ratings and physician ratings of general health, depression, and anxiety and patient and physician ratings in comparison to Diagnostic Interview Schedule (DIS) diagnoses of depression and anxiety. DESIGN: Observational study. SETTING: A general medical outpatient clinic. PATIENTS: Clinic attendees. METHOD: Consecutive clinic attendees on randomly selected days completed a self-reported screening questionnaire for hypochondriasis, composed of the Whiteley Index and the Somatic Symptom Inventory. A random sample of patients (N = 79), 95% of whom had scores below the cutoff for hypochondriasis (n = 75) and 5% of whom had scores at or above the cutoff for hypochondriasis (N = 4), returned at a later date to complete a research battery consisting of self-reported questionnaires, structured and semistructured interviews, and a structured interview for diagnoses of anxiety and depression based on the DIS, which used criteria from the Diagnostic and Statistical Manual of Mental Disorders, Revised Third Edition. OUTCOME MEASURES: Responses to questionnaires and interviews. RESULTS: Agreement between patients and physicians was statistically significant concerning patients' physical health, depression, and anxiety. Based on one question, both physicians' and patients' ratings of depression and anxiety compared favorably with DIS diagnoses. By means of receiver operating characteristics analysis, values for the areas under the curve and their SEs were as follows: for depression, 0.789 +/- 0.075 for patient self-ratings and 0.825 +/- 0.054 for physician ratings; for anxiety, 0.734 +/- 0.058 for patient self-ratings and 0.667 +/- 0.065 for physician ratings. CONCLUSION: One simple question about a patient's status with respect to depression or anxiety is sufficient to detect these disorders with high sensitivity and specificity, yielding values comparable to those yielded by instruments consisting of many items. Asking patients to rate their own levels of depression and anxiety may constitute one portion of the family physician's diagnostic assessment for these conditions.

Anxiety Disorders↗

PubMed2026

Emerging trends in genome editing of wild animals.

Globally, nearly one million species are currently threatened with extinction, highlighting the need for more efficient solutions to biological conservation. Genome editing, which allows for faster and more precise changes in genomes, is a promising technique for boosting populations through facilitated adaptation, management of invasive or pathogenic populations, and potentially even facilitating the revival of extinct species. These approaches belong to a new field of research termed conservation biotechnology, which places a great responsibility on researchers and decision makers to ensure sustainability. In this paper, we have mapped the emerging trends in genome editing of wild animals. Current projects primarily focus on population control and de-extinction, with fewer initiatives aimed at preserving threatened species. We then explore four critical dimensions of conservation biotechnology: the technology itself, new perspectives on conservation practices, research organization, and governance and policy. Despite its potential, key questions remain-particularly whether genome editing can increase genetic diversity without causing unintended non-target impacts. Genome editing also provokes new perspectives on conservation practices where ecosystem-wide impact assessment, case-by-case evaluations, and post-release monitoring needs to be prioritized. Furthermore, conservation biotechnology is heavily funded through private funding showing varying stakeholder interest, which can lead to untraditional and less transparent research processes. Stakeholders, including local and indigenous people, are only to a certain degree involved, which may weaken inclusion of local knowledge and monitoring efforts. Finally, concerning governance and policy, there is an urgent need to develop more adequate regulation of conservation biotechnology, as environmental release of genome-edited animals challenges definitions and guidelines in current nature protection laws and GMO regulations. Based on our analysis, we outline key points for further investigation toward a more sustainable approach to conservation biotechnology.

Animals↗

PubMed1999

Editorial quality, diffusion and bibliometric indicators of the Revista Espaãnola de Enfermedades Digestivas.

AIM: To evaluate the editorial quality, diffusion, relevance of the scientific content, and the publication practices of the specialised journal Revista Española de Enfermedades Digestivas. MATERIALS AND METHODS: We checked 136 parameters based on ISO standards, the recommendations of scientific and editorial organisations, and studies of scientific editing and international publishing practices for biomedical journals. Diffusion was calculated using national and international databases, specialised libraries in Spain, and Internet sources. The analysis of the scientific content and publication practices was based on bibliometric indicators for the journal, authorship, and contributions. The sample for this study comprised six alternate issues of volume number 88 (1996), the last issue of this volume, and the first issue of volume 89 (1997). The samples used for the bibliometric analysis varied depending on the characteristics of specific indicators and the availability of information. RESULTS: The overall mean value for compliance with standards was 46.1%, while the real mean was calculated at 72.21%. The editorial procedures at the journal are similar to those of analogous international journals. The Revista Española de Enfermedades Digestivas is included in international databases of biomedical journals, and in the interdisciplinary international database SCI. It was found to be present in 70% of the medical libraries of Spanish universities, and in 73% of the hospital libraries studied. Bibliometric indicators showed co-authorship to be 5.5%; the origin of the authors grouped by province and by type of institutional affiliations showed 27.8% of all authors to be from Madrid, and that more were affiliated with general hospitals than with university hospitals. The mean delay between initial receipt of a manuscript and its publication was 300 days. Cocitation analysis gave the journal a central position amongst the 38 Spanish biomedical journals considered representative of the field. The journal's impact factor for 1996 was 0.260. CONCLUSIONS: The Revista Española de Enfermedades Digestivas is a high-quality vehicle of research results, and has acceptable internal editorial procedures. The journal is widely distributed, though its visibility on the Internet should be improved. Co-authorship is similar to that seen in other medical journals. Steps should be taken to make this journal better known within Spain, and to reduce the delay between the initial receipt and the final publication of manuscripts. Its impact factor is increasing steadily.

Authorship↗

PubMed1998

Demonstration of mRNA editing and localization of guide RNA genes in kinetoplast-mitochondria of the plant trypanosomatid Phytomonas serpens.

Maxicircle molecules of kDNA in several isolates of Phytomonas were detected by hybridization with the 12S rRNA gene probe from Leishmania tarentolae. The estimated size of maxicircles is isolate-specific and varies from 27 to 36 kb. Fully edited and polyadenylated mRNA for kinetoplast-encoded ribosomal protein S12 (RPS12) was found in the steady-state kinetoplast RNA isolated from Phytomonas serpens strain 1G. Two minicircles (1.45 kb) from this strain were also sequenced. Each minicircle contains two 120 bp conserved regions positioned 180 degrees apart, a region enriched with G and T bases and a variable region. One minicircle encodes a gRNA for the first block of editing of RPSl2 mRNA, and the other encodes a gRNA with unknown function. A gRNA gene for the second block of RPSl2 was found on a minicircle sequenced previously. On each minicircle, a gRNA gene is located in the variable region in a similar position and orientation with respect to the conserved regions.

Amino Acid Sequence↗

PubMed2026

A dual-dimensional CRISPR toolkit enables one-step high-efficiency multiplex genome editing in Komagataella phaffii.

Against the backdrop of green biomanufacturing, engineering methanol-utilizing Komagataella phaffii (K. phaffii) represents an effective strategy to expand the one carbon (C1) product profile and speed up the industrialization of C1-based bioeconomy. To address the technical challenges of low efficiency and cumbersome experimental procedures for multiplex gene editing and precise large-fragment integration during the reconstruction of complex metabolic pathways in K. phaffii, this study established a CRISPR toolkit - Efficient Multi-Gene Editing System 3.0 (EMGES 3.0) - which enabled one-step large-fragment integration coupled with multiplex gene knockout. EMGES 3.0 was constructed through the synergistic optimization of a repair-engineered chassis and an episomal CRISPR vector. For chassis engineering, five DNA repair modules: Δlig4 (DNA Ligase IV, non-homologous end joining end ligation), ppMRE11(The endogenous MRE11 gene from Pichia pastoris) overexpression (The Meiotic Recombination 11, DNA double-strand break end resection), Δrad9 (Radiation-Sensitive 9, DNA damage checkpoint regulation), Δmph1 (Mutator Phenotype Helicase 1, improvement of homologous recombinant strand extension), and PapRecT-PaSSB co-expression (stabilization of recombination intermediates) were integrated to generate the highly recombinogenic strain Y09. For vector engineering, cenARS was replaced by panARS and the endogenous promoter PGAP was employed to drive the double hammerhead ribozyme-single guide RNA-hepatitis delta virus ribozyme (double HH-sgRNA-HDV: dHgH)-mediated sgRNA expression, yielding the optimized vector Nov_pGAP_panARS_pLAT1_Cas9. These two features on K. phaffii together enhanced the EMGES 3.0 to a higher standard of transformation rate and editing efficiency. According to our results, EMGES 3.0 achieved dual-functional gene knockout efficiencies between 76.6% and 100%. For insertion of medium-long fragments (>4.5 kb), the efficiency achieved 93.3%. In addition, the one-step integration of ultra-long fragments (>16 kb) achieved 14.8%, which was reported for the first time. Furthermore, the efficiency of simultaneous long-fragment integration at three neutral loci reached 38.4% (>15 kb). We applied the system for one-step production of free fatty acids (FFAs, yield: 5.82 ∼ 7.30 mg/L/OD600) and resveratrol (yield: 1.14 ∼ 1.28 mg/L) using methanol as the sole carbon source. EMGES 3.0 provides a robust technical foundation for complex compounds biosynthesis and high-yield industrial strains, while also advancing K. phaffii as an industrial synthetic biology chassis for efficient C1 utilization.

CRISPR-Cas Systems↗

PubMed2001

Trypanosome RNA editing: simple guide RNA features enhance U deletion 100-fold.

Trypanosome RNA editing is a massive processing of mRNA by U deletion and U insertion, directed by trans-acting guide RNAs (gRNAs). A U deletion cycle and a U insertion cycle have been reproduced in vitro using synthetic ATPase (A6) pre-mRNA and gRNA. Here we examine which gRNA features are important for this U deletion. We find that, foremost, this editing depends critically on the single-stranded character of a few gRNA and a few mRNA residues abutting the anchor duplex, a feature not previously appreciated. That plus any base-pairing sequence to tether the upstream mRNA are all the gRNA needs to direct unexpectedly efficient in vitro U deletion, using either the purified editing complex or whole extract. In fact, our optimized gRNA constructs support faithful U deletion up to 100 times more efficiently than the natural gRNA, and they can edit the majority of mRNA molecules. This is a marked improvement of in vitro U deletion, in which previous artificial gRNAs were no more active than natural gRNA and the editing efficiencies were at most a few percent. Furthermore, this editing is not stimulated by most other previously noted gRNA features, including its potential ligation bridge, 3' OH moiety, any U residues in the tether, the conserved structure of the central region, or proteins that normally bind these regions. Our data also have implications about evolutionary forces active in RNA editing.

Animals↗

PubMed2005

2004 American College of Cardiology/American Heart Association guidelines for the management of patients with ST-elevation myocardial infarction: implications for emergency department practice.

The American College of Cardiology and the American Heart Association last published evidence-based guidelines for the management of ST-segment elevation myocardial infarction (STEMI) in 1999. In mid-2004, in recognition of the evolution and improvement of many of the most basic tenets of clinical management of STEMI since that time, an updated edition of the STEMI guidelines has been published. These guidelines offer many evidence-based recommendations that are pertinent to the out-of-hospital and emergency department care of STEMI patients, including initial evaluation, risk stratification, stabilizing management, and the choice between pharmacologic and mechanical revascularization. These are presented and discussed here.

Electrocardiography↗

PubMed2004

Proteolytic degradation of the nuclear isoform of uracil-DNA glycosylase occurs during the S phase of the cell cycle.

Uracil-DNA glycosylases are enzymes that remove uracil from DNA and initiate base-excision repair. These enzymes play a key role in maintaining genomic integrity by reducing the mutagenic events caused by G:C to A:T transition mutations. The recent finding that a family of RNA editing enzymes (APOBECs) can deaminate cytosine in DNA has raised the interest in these base-excision repair enzymes. This research focuses on the regulation of the nuclear isoform of uracil-DNA glycosylase, a 36000 Da protein that contains a unique 44 amino acid N-terminus. In synchronized HeLa cells, UDG1A protein levels decrease to barely detectable levels during the S phase of the cell cycle. Immunoblot analysis of immunoprecipitated or affinity-isolated UDG1A reveals ubiquitin-conjugated UDG1A when proteolysis is inhibited using N-acetyl-leu-leu-norleu-al or MG132, inhibitors of proteosomal dependent protein degradation. Transient transfection experiments, with histidine-tagged ubiquitin, were used to confirm that endogenous UDG1A is ubiquitinated in vivo. Addition of the nuclear export inhibitor, leptomycin B, prevents ubiquitination and degradation of UDG1A. This indicates that translocation from the nucleus may be a step in UDG1A turnover. Finally, UDG1A protein degradation is prevented when cells are incubated with the cyclin-dependent kinase inhibitor, roscovitine. These results suggest that protein phosphorylation and/or nuclear export participate in the post-translational regulation of UDG1A protein levels.

Cell Nucleus↗

PubMed2000

Functions and mechanisms of RNA editing.

RNA editing can be broadly defined as any site-specific alteration in an RNA sequence that could have been copied from the template, excluding changes due to processes such as RNA splicing and polyadenylation. Changes in gene expression attributed to editing have been described in organisms from unicellular protozoa to man, and can affect the mRNAs, tRNAs, and rRNAs present in all cellular compartments. These sequence revisions, which include both the insertion and deletion of nucleotides, and the conversion of one base to another, involve a wide range of largely unrelated mechanisms. Recent advances in the development of in vitro editing and transgenic systems for these varied modifications have provided a better understanding of similarities and differences between the biochemical strategies, regulatory sequences, and cellular factors responsible for such RNA processing events.

Amino Acid Sequence↗