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PubMed1994

Mechanisms and origins of RNA editing.

RNA editing is an essential post-transcriptional process that has been identified in an increasing number of eukaryotic organisms. In the past year, progress has been made in the development of in vitro systems to study the mechanism of RNA editing. Analysis of nucleotide insertion/deletion editing in trypanosome mitochondria has revealed the existence of putative editing intermediates in vivo and in vitro. The development of an in vitro editing system for mammalian apolipoprotein B mRNA has allowed the elucidation of both the sequence requirements and the biochemical mechanism of this form of RNA editing. In addition, recent work has underscored the diversity of RNAs whose structure and function are altered by post-translational editing reactions.

Animals↗
PubMed2001

Molecular mechanisms of apolipoprotein B mRNA editing.

A site-specific post-transcriptional cytidine to uridine deamination reaction is responsible for the production of apolipoprotein B48 in the mammalian small intestine. The molecular machinery responsible for apolipoprotein B RNA editing consists of apobec-1, an RNA-specific cytidine deaminase that functions in conjunction with a recently identified protein referred to as ACF/ASP. These proteins together represent the minimal editing enzyme, although other proteins may associate with the enzyme complex. Apobec-1 is a member of a supergene family of cytidine deaminases, with several homologs recently identified in the human genome. ACF/ASP is novel, and emerging information reveals interesting clues to its role in the apolipoprotein B RNA editing enzyme complex.

Apolipoproteins B↗
PubMed2024

Hepatitis B Virus-KMT2B Integration Drives Hepatic Oncogenic Processes in a Human Gene-edited Induced Pluripotent Stem Cells-derived Model.

BACKGROUND & AIMS: Hepatitis B virus (HBV)-DNA integration into the host genome contributes to hepatocellular carcinoma (HCC) development. KMT2B is the second most frequent locus of HBV-DNA integration in HCC; however, its role and function remain unclear. We aimed to clarify the impact of HBV-KMT2B integration in HCC development using a human genome-edited induced pluripotent stem cell (iPSCs) model. METHODS: Based on the genetic information on HBV-KMT2B integration in HCC, we determined its complete DNA sequence and transcript variants. To exclude the effect of other oncogenic mutations, we reproduced HBV integration in healthy donor iPSCs with an intact genome and analyzed its effects using iPSC-derived hepatic progenitor cells (HPCs) and hepatocytes (iPS-Heps). RESULTS: The reproduced HBV-KMT2B integration significantly upregulated the proliferation of hepatic cells. Comprehensive transcriptional and epigenetic analyses revealed enhanced expression of cell cycle-related genes in hepatic cells with HBV-KMT2B integration based on perturbation of histone 3 lysine 4 tri-methylation (H3K4me3), mimicking that in the original HCC sample. Long-read RNA-sequence detected the common KMT2B transcript variants in the HCC sample and HPCs. Overexpression of the truncated variant significantly enhanced proliferation of hepatic cells, whereas HBV-KMT2B fusion transcripts did not enhance proliferation. HBV-KMT2B-integrated HPCs exhibited replication stress and DNA damage, indicating that our model initiated the process of hepatocarcinogenesis due to abnormally promoted KMT2B function. CONCLUSIONS: Our disease model using genetically engineered iPSCs provides the first insight into both the KMT2B function in HCC development and the oncogenic processes by HBV-KMT2B integration. We clarified the novel oncogenic mechanism in HBV-related HCC due to aberrant KMT2B function.

Humans↗
PubMed2003

A site-specific factor interacts directly with its cognate RNA editing site in chloroplast transcripts.

RNA editing involves a variety of genetic systems and occurs by different mechanisms. In higher plant chloroplasts, specific sites of some transcripts are subject to C-to-U conversion. We have previously shown that site-specific trans-acting factors for psbE and petB mRNA editing bind corresponding cis-elements, which are located 5 nucleotides upstream from the editing site. Here we report that, by using mRNAs labeled either at the center of the upstream cis-element or at the editing site, the site-specific factors can be cross-linked with nucleotides at both positions. Mutations of nucleotides in the proximal region of the editing site revealed a correlation between editing activity and cross-linking efficiency of factors with the editing site, even though cross-linking with the upstream cis-element was unaffected. These observations suggest that the site-specific factor binds stably to the upstream ciselement, whereas it interacts weakly with the editing site. This finding raises the intriguing possibility that the site-specific factor is involved in both site-determination and C-to-U conversion in chloroplast RNA editing.

Base Sequence↗
PubMed1996

Occurrence of silent RNA editing in chloroplasts: its species specificity and the influence of environmental and developmental conditions.

We have identified three new C-to-U RNA editing sites, one in atpF and two in atpA transcripts from tobacco chloroplasts. Two of them lead to amino acid substitutions to restore the conserved amino acid found in the corresponding genes of other plants. However, one editing site in the atpA transcript was found to take place partially at the third base of a serine codon (CUC_ to CUU_), thus not leading to an amino acid substitution. This is the first report of silent editing in chloroplasts. The extent of silent editing depends on plastid stage and light conditions, while editing as another site (found 4 nt upstream from the silent editing site) takes place constitutively even in non-photosynthetic cultured cells and bleached white seedlings grown in the presence of spectinomycin and streptomycin. In pea and spinach, despite a conservation in sequence, no editing at the site corresponding to the silent site in tobacco was found. This observation suggests that the silent editing detected in this study is species-specific.

Amino Acid Sequence↗
PubMed2006

Spectral editing in solid-state MAS NMR of quadrupolar nuclei using selective satellite inversion.

A sensitivity enhancement method based on selective adiabatic inversion of a satellite transition has been employed in a (pi/2)CT-(pi)ST1-(pi/2)CT spectral editing sequence to both enhance and resolve multisite NMR spectra of quadrupolar nuclei. In addition to a total enhancement of 2.5 times for spin 3/2 nuclei, enhancements up to 2.0 times is reported for the edited sites in a mixture of rubidium salts.

Algorithms↗
PubMed2026

CasY7: An optimized Cas12i system for enhanced genome editing in monocot crops.

The CRISPR-Cas12 family nucleases, particularly the Cas12i subtypes, are considered promising alternatives to Cas9 for genome editing in plants. We previously developed a new Cas12i variant, CasY7, which has been successfully applied in clinical trials; its performance in plants remains to be investigated. Initial testing in stable transgenic maize and rice showed that the codon-optimized CasY7 (pCasY7e1) achieved average editing efficiencies of 58.7% and 62.3% across five target sites, respectively, outperforming the typical Cpf1 (pCpf1) control that targets the same sites. To further enhance activity, we fused T5 exonuclease to CasY7 (pCasY7e2), which shifted mutation profiles toward larger deletions, and subsequently integrated an MS2 aptamer into the crRNA scaffold (pCasY7e3). The optimized pCasY7e3 system increased editing efficiencies to 87.7% in maize and 82.9% in rice-approximately 2.7-fold higher than pCpf1. We further demonstrated multiplexed editing in maize, generating biallelic dwarf mutants, and validated functionality in hexaploid wheat with editing efficiencies up to 58.8%. Overall, our comprehensive validation across 942 transgenic plants confirmed robust editing in maize, rice, and wheat, establishing CasY7 as a high-efficiency addition to the CRISPR toolkit.

Zea mays↗
PubMed2025

Gut microbiota: a hidden player in polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is an endocrine disorder that affects reproductive-aged women worldwide, causing hormonal imbalances and ovarian dysfunction. PCOS affects metabolic health and increases the risk of obesity, insulin resistance, and cardiovascular disease, in addition to infertility. This review delves deeper into the connections of gut microbiota with PCOS pathophysiology, particularly into its impact on hormone metabolism, obesity, inflammation, and insulin resistance by way of short-chain fatty acids, lipopolysaccharides, and gut-brain axis. Studies also show that changes in the metabolic processes and immune responses are seen in changes in the gut microbiota in PCOS subjects, such as changes in the Bacteroidetes and Firmicutes groups. Some bacteria, like Escherichia and Shigella, have been associated with dysbiosis in patients with PCOS, leading to systemic inflammation and changed hormone levels, which further worsen the clinical symptoms. Therapeutic interventions targeting the gut microbiota comprise probiotics, prebiotics, and fecal microbiota transplantation; these have potential to alleviate the symptoms of PCOS. Other precision microbiome-based therapies include postbiotics, and CRISPR-Cas9 genome editing, which are relatively new avenues toward precision treatment. This complex interlink of gut microbiota and PCOS pathophysiology will open the avenues for possible treatments for hormonal imbalances and metabolic problems that characterize these complex disorders. The review here focuses on the requirement of further studies to be able to elucidate the specific pathways relating gut microbiota dysregulation to PCOS and, thus, improve microbiome-based therapies for better clinical outcomes in affected individuals.

Humans↗
PubMed1997

PolyPhred: automating the detection and genotyping of single nucleotide substitutions using fluorescence-based resequencing.

Fluorescence-based sequencing is playing an increasingly important role in efforts to identify DNA polymorphisms and mutations of biological and medical interest. The application of this technology in generating the reference sequence of simple and complex genomes is also driving the development of new computer programs to automate base calling (Phred), sequence assembly (Phrap) and sequence assembly editing (Consed) in high throughput settings. In this report we describe a new computer program known as PolyPhred that automatically detects the presence of heterozygous single nucleotide substitutions by fluorescencebased sequencing of PCR products. Its operations are integrated with the use of the Phred, Phrap and Consed programs and together these tools generate a high throughput system for detecting DNA polymorphisms and mutations by large scale fluorescence-based resequencing. Analysis of sequences containing known DNA variants demonstrates that the accuracy of PolyPhred with single pass data is >99% when the sequences are generated with fluorescent dye-labeled primers and approximately 90% for those prepared with dye-labeled terminators.

Animals↗
PubMed1994

Dimeric structure of a human apolipoprotein B mRNA editing protein and cloning and chromosomal localization of its gene.

Apolipoprotein B (apoB) mRNA editing consists of a posttranscriptional C-->U conversion involving the first base of the codon CAA encoding glutamine-2153 to UAA, a stop codon, in apoB mRNA. Using a cloned rat cDNA as a probe, we cloned the cDNA and genomic sequences of the gene for a human apoB mRNA editing protein. Expression of the cDNA in HepG2 cells results in editing of the intracellular apoB mRNA. By fluorescence in situ hybridization, we localized the gene for the editing protein to chromosome band 12p13.1-p13.2. By Northern blot analysis, it was shown that the human editing protein mRNA is expressed exclusively in the small intestine. The cDNA sequence predicts a translation product of 236-aa residues. By attaching an epitope tag sequence to the C terminus of the editing protein, we examined the polymerization state of the editing protein synthesized in vitro. We found that the editing protein undergoes spontaneous polymerization. The migration of the human apoB mRNA editing protein on an HPLC column and the stoichiometry of polymeric epitope-tagged to untagged protein indicate that the protein exists as a dimer. Dimerization does not require glycosylation of a consensus N-linked glycosylation sequence present in the protein and is not mediated by disulfide bridge formation. The human apoB mRNA editing protein is a cytidine deaminase showing structural homology to some known mammalian and bacteriophage deoxycytidylate deaminases. The latter enzymes exist as homopolymers. The fact that the apoB mRNA editing protein also exists as a homodimer has important implications for the mechanism of apoB mRNA editing in humans.

APOBEC-1 Deaminase↗
PubMed1995

Editing efficiency of hepatitis delta virus RNA is related to the course of infection in woodchucks.

Based on evidence in vitro which shows that the small form of hepatitis delta virus (HDV) antigen (S-HDAg) initiates virus replication, whereas the long form (L-HDAg) encoded by the edited L-genome inhibits replication, we first put forward the hypothesis that HDV RNA editing efficiency, i.e. the intracellular L/S-genome ratio, could be a determining factor on the course of the infection. In order to analyse the precise sequence of events after infection, woodchuck carriers of woodchuck hepatitis virus (WHV) were superinfected with HDV and sequential changes in HDV RNA editing efficiency were analysed in relation to the duration of viraemia. Our findings show that: (1) in both transiently and persistently viraemic woodchucks, the percentage of L-genome is higher at the early stage of onset of the disease than at the late stage; (2) at the early stage of onset the percentage of L-genome is higher in cases with transient viraemia than in those with persistent viraemia; (3) a relatively greater decrease in L-genome is seen later in transiently viraemic animals than in those that remain persistently viraemic. In view of the above findings in vivo and other supporting evidence in vitro, we propose a hypothesis for the pathogenesis of HDV. This hypothesis predicts the outcome of acute infection and we suggest a gene therapeutic approach for this disease based on the intracellular accumulation (or increase) of the L-genome.

Animals↗
PubMed2001

A comparison of published head and neck stage groupings in carcinomas of the tonsillar region.

BACKGROUND: The combination of T, N, and M classifications into stage groupings was designed to facilitate a number of activities including: the estimation of prognosis and the comparison of therapeutic interventions among similar groups of cases. The authors tested the UICC/AJCC 5th edition stage grouping and seven other TNM-based groupings proposed for head and neck cancer to determine their ability to meet these expectations in a specific site: carcinoma of the tonsillar region. METHODS: The authors defined four criteria to assess each stage grouping scheme: 1) The subgroups defined by T and N comprising a given group within a grouping scheme have similar survival rates (hazard consistency); 2) The survival rates differ across the groups (hazard discrimination); 3) The prediction of cure is high (outcome prediction); and 4) The distribution of patients among the groups is balanced. The authors identified or derived a measure for each criterion and the findings were summarized using a scoring system. The range of scores was from 0 (best) to 7 (worst). Data were from a retrospective chart review on 642 cases of carcinoma of the tonsillar region treated with radiotherapy for cure at the Princess Margaret Hospital from 1970-1991. None of the patients had distant metastases. RESULTS: The scheme proposed by Synderman and Wagner, which was published in Otolaryngology Head and Neck Surgery in 1995 (vol.112, pages 691-4), scored best at 1.2. The UICC/AJCC scheme scored worst at 6.1. The hazard consistency ranged from a 3.1% average survival difference to 6.7% across the 8 schemes. The hazard discrimination measure varied by 28% from the best to worst scheme. Prediction varied by up to almost twofold across the schemes assessed. The distribution of patients varied from expected by between 0.13% and 0.57%. CONCLUSION: UICC/AJCC stage groupings were defined without empirical investigation. When tested, this scheme did not perform as well as any of seven empirically-derived schemes the authors evaluated. The results of the current study suggest that the usefulness of the TNM system can be enhanced by optimizing the design of stage groupings through empirical investigation.

Carcinoma, Squamous Cell↗
PubMed2002

Prognostic importance of tumor size for localized conventional (clear cell) renal cell carcinoma: assessment of TNM T1 and T2 tumor categories and comparison with other prognostic parameters.

BACKGROUND: The T1 and T2 classifications of the International Union Against Cancer TNM classification system for renal cell carcinoma are based on primary tumor size, and in various editions of the classification, the cut points between T1 and T2 have been amended to provide clinical utility. In the current edition, the T1/T2 cut point is less than or equal to and greater than 7 cm. and more recently a subdivision of the T1 classification (less than or equal to and < 4 cm) has been proposed to identify patients suitable for partial nephrectomy. This study investigates the prognostic significance of tumor size in a series of organ-confined clear cell renal cell carcinomas. METHODS: One hundred thirty cases of organ-confined clear cell renal cell carcinomas, with a minimum of 5 years' follow-up, were identified from the New South Wales Cancer Registry. Tumor size was compared with survival using the method of Kaplan and Meier for TNM size categories, and proportional hazards regression was used for assessing size as a continuous variable. Proportional hazards regression also was used for multivariable comparisons of size and other prognostic parameters (Fuhrman grade, AgNOR score, and Ki-67 index) against survival. RESULTS: Of 116 cases for which tumor dimension was recorded, 25 patients had died of cancer-related causes. Primary tumor size ranged from 12 to 140 mm (mean, 57.3 mm). The association between survival and size was significant irrespective of the TNM classification and was also significant when size was modeled continuously (P = 0.000125, hazard of death increased by 3.51 times for each doubling of tumor size). On univariate analysis, Fuhrman grade (P = 0.04) and AgNOR score (P = 0.015) were associated with survival; however, on multivariate analysis only tumor size retained significance. CONCLUSIONS: Although the cut point of T1 and T2 TNM categories and the proposed T1 subdivision cut point correlate with survival, our finding that size is a continuous variable indicates that as a prognostic parameter for clear cell renal cell carcinoma, primary tumor size is relative rather than indicative.

Adult↗
PubMed2001

A comparison of published head and neck stage groupings in carcinomas of the oral cavity.

BACKGROUND: The combination of T, N, and M classifications into stage groupings is meant to facilitate a number of activities, including the estimation of prognosis and the comparison of therapeutic interventions among similar groups of cases. We tested the UICC/AJCC 5th edition stage grouping and seven other TNM-based groupings proposed for head and neck cancer for their ability to meet these expectations in a specific site: carcinomas of the oral cavity. METHODS: We defined four criteria to assess each grouping scheme: (1) the subgroups defined by T, N, and M that make up a given group within a grouping scheme have similar survival rates (hazard consistency); (2) the survival rates differ among the groups (hazard discrimination); (3) the prediction of cure is high (outcome prediction); and (4) the distribution of patients among the groups is balanced. We identified or derived a measure for each criterion, and the findings were summarized by use of a scoring system. The range of scores was from 0 (best) to 7 (worst). The data are population based from a prospectively gathered series in Southern Norway, with 556 patients diagnosed from 1983 through 1995. Clinical stage assignment was used, and the outcome of interest was cause-specific survival. RESULTS: Summary scores across the eight schemes ranged from 1.66 for TANIS-3 to 6.50 for UICC/AJCC-5. The TANIS-7 staging scheme performed best on the hazard consistency criterion. The Kiricuta scheme performed best on the hazard discrimination criterion. Synderman predicted outcome best overall and Berg produced the most balanced distribution of cases among its groups. CONCLUSIONS: UICC/AJCC stage groupings were defined without empirical investigation. When tested, this scheme did not perform as well as any of seven empirically derived schemes we evaluated. Our results suggest that the usefulness of the TNM system could be enhanced by optimizing the design of stage groupings through empirical investigation.

Carcinoma, Squamous Cell↗
PubMed1990

Cancer incidence correlations: genital, urinary and some tobacco-related cancers.

Bivariate correlations between sex-specific age-adjusted incidence rates of 10 genital and urinary tract cancer sites and lung cancer were calculated. The data base used was that published in the 5th edition of the series Cancer incidence in five continents. The purpose of the analyses was to test the consistency of the data with the hypothesis of a common risk factor for genital cancers and other cancers of the urinary tract and for lung and genital cancers. The results indicate that the incidence of cervical cancer is strongly correlated with that of cancer of the penis, of other cancers of the female genital tract and of chorioepithelioma. In countries at low/intermediate risk for cervical cancer (mostly developed Western countries), the incidence of cervical cancer is also correlated with that of lung cancer in males. The incidence of male lung cancer is, moreover, strongly correlated with cancers of the prostate, penis, testis, bladder and other urinary tract cancers. These analyses support the views that: urogenital cancers in both sexes might share (a) common risk factor(s), which may be certain types of human papilloma-virus; and that cigarette smoking is likely to be a risk factor for most of these cancers in countries where the use of tobacco has been widespread for a long time. Other suggestive etiological conclusions about less well-studied cancer sites are also presented.

Age Factors↗
PubMed2000

Identification and phylogenetic comparison of Salem virus, a novel paramyxovirus of horses.

A virus that could not be identified as a previously known equine virus was isolated from the mononuclear cells of a horse. Electron microscopy revealed enveloped virions with nucleocapsid structures characteristic of viruses in the Paramyxoviridae family. The virus failed to hemabsorb chicken or guinea pig red blood cells and lacked neuraminidase activity. Two viral genes were isolated from a cDNA expression library. Multiple sequence alignments of one gene indicated an average identity of 45% as compared to Morbillivirus N protein sequences. A weaker relationship was found with Tupaia paramyxovirus (TPMV) and Hendra virus (HeV) N proteins. In the second gene, multiple open reading frames (ORFs) were identified, corresponding to the arrangement of the P, V, and C ORFs in the Morbillivirus and Respirovirus viruses. Short stretches in the C-terminal regions of the P and C proteins showed limited homologies to viruses in the Morbillivirus genus but no obvious relationship to viruses in other genera. The V ORF translation product contained a highly conserved, cysteine-rich domain that is common to most viruses in the Paramyxovirinae subfamily. Sequencing of P gene cDNA clones confirmed the use of a cotranscriptional editing mechanism for the regulation of P/V expression. Based on the location of its origin it has been named Salem virus (SalV).

Amino Acid Sequence↗