Expansion of CAG repeats in certain genes is a known cause of several neurodegenerative diseases, but exact mechanism behind this is not yet fully understood. It is believed that the double-stranded RNA regions formed by CAG repeats could be harmful to the cell. This study aimed to test the hypothesis that these RNA regions might potentially interfere with ADAR RNA editing enzymes, leading to the reduced A-to-I editing of RNA and activation of the interferon response. We studied induced pluripotent stem cells (iPSCs) derived from the patients with Huntington\'s disease or ataxia type 17, as well as midbrain organoids developed from these cells. A targeted panel for next-generation sequencing was used to assess editing in the specific RNA regions. Differentiation of iPSCs into brain organoids led to increase in the ADAR2 gene expression and decrease in the expression of protein inhibitors of RNA editing. As a result, there was increase in the editing of specific ADAR2 substrates, which allowed identification of differential substrates of ADAR isoforms. However, comparison of the pathology and control groups did not show differences in the editing levels among the iPSCs. Additionally, brain organoids with 42-46 CAG repeats did not exhibit global changes. On the other hand, brain organoids with the highest number of CAG repeats in the huntingtin gene (76) showed significant decrease in the level of RNA editing of specific transcripts, potentially involving ADAR1. Notably, editing of the long non-coding RNA PWAR5 was nearly absent in this sample. It could be stated in conclusion that in most cultures with repeat expansion, the hypothesized effect on RNA editing was not confirmed.

Inosine is a nucleotide resulting from the deamination of adenosine in RNA. This chemical modification process, known as RNA editing, is typically mediated by a family of double-stranded RNA binding proteins named Adenosine Deaminase Acting on dsRNA (ADAR). While the presence of ADAR orthologs has been traced throughout the evolution of metazoans, the existence and extension of RNA editing have been characterized in a more limited number of animals so far. Undoubtedly, ADAR-mediated RNA editing plays a vital role in physiology, organismal development and disease, making the understanding of the evolutionary conservation of this phenomenon pivotal to a deep characterization of relevant biological processes. However, the lack of direct high-throughput methods to reveal RNA modifications at single nucleotide resolution limited an extended investigation of RNA editing. Nowadays, these methods have been developed, and appropriate bioinformatic pipelines are required to fully exploit this data, which can complement existing approaches to detect ADAR editing. Here, we review the current literature on the \"bioinformatics for inosine\" subject and we discuss future research avenues in the field.

BACKGROUND: Adenosine-to-inosine (A-to-I) RNA editing is a co-/post-transcriptional modification introducing A-to-G variations in RNAs. There is extensive discussion on whether the flexibility of RNA editing exerts a proteomic diversification role, or it just acts like hardwired mutations to correct the genomic allele. Eusocial insects evolved the ability to generate phenotypically differentiated individuals with the same genome, indicating the involvement of epigenetic/transcriptomic regulation.
METHODS: We obtained the genomes of 104 Hymenoptera insects and the transcriptomes of representative species. Comparative genomic analysis was performed to parse the evolutionary trajectory of a regulatory Ile > Met auto-recoding site in Adar gene.
RESULTS: At genome level, the pre-editing Ile codon is conserved across a node containing all eusocial hymenopterans. At RNA level, the editing events are confirmed in representative species and shows considerable condition-specificity. Compared to random expectation, the editable Ile codon avoids genomic substitutions to Met or to uneditable Ile codons, but does not avoid mutations to other unrelated amino acids.
CONCLUSIONS: The flexibility of Adar auto-recoding site in Hymenoptera is selectively maintained, supporting the flexible RNA editing hypothesis. We proposed a new angle to view the adaptation of RNA editing, providing another layer to explain the great phenotypical plasticity of eusocial insects.

BACKGROUND: Adenosine deaminase (ADA) in pleural fluid is a useful marker for diagnosing tuberculous pleurisy. However, recent studies have reported a lower specificity of pleural fluid ADA levels. We previously developed a diagnostic flowchart for patients with pleural fluid ADA ≥40 U/L, incorporating variables such as pleural fluid lactate dehydrogenase <825 U/L, predominant pleural fluid neutrophils or cell degeneration, and a pleural fluid ADA/total protein ratio <14. This flowchart was effective in distinguishing between tuberculous pleurisy and other diseases. Here, we conducted a validation analysis of this flowchart.
METHODS: We retrospectively collected data from 458 patients with pleural fluid ADA concentrations ≥40 U/L across eight institutions from January 2019 to December 2023. The diagnostic accuracy rate, sensitivity, and specificity of the diagnostic flowchart were analysed and compared to those in the original study.
RESULTS: Eighty-seven patients were diagnosed with tuberculous pleurisy, and 371 patients were diagnosed with other diseases. The diagnostic accuracy, sensitivity, and specificity for diagnosing tuberculous pleurisy were 77.7%, 86.2%, and 75.7%, respectively. Compared with that in the original study, the rate of tuberculous pleurisy was lower (19.0% vs. 44.5%, p < 0.001), but the diagnostic accuracy rates were not significantly different (p = 0.253). On the basis of the findings from this validation study, we have revised the flowchart to enhance its utility.
CONCLUSIONS: The diagnostic flowchart exhibited high diagnostic accuracy in this validation study, comparable to that in the original study. This validation confirms the effectiveness of the flowchart, even in settings with a low incidence of tuberculosis.

Adenosine to inosine (A-to-I) RNA editing by ADAR1 has been implicated in maintaining self-tolerance, preventing autoimmunity, and mediating antiviral immunity. Foreign viral double-stranded RNA triggers rapid interferon response and activates ADAR1 in the host immune system. Emerging data points to a role of ADAR1 A-to-I editing in the inflammatory response associated with severe COVID-19 disease. We identify A-to-I editing events within human whole transcriptome data from SARS-CoV-2 infected individuals, non-infected individuals, and individuals with other viral illnesses from nasopharyngeal swabs. High levels of RNA editing in host cells are associated with low SARS-CoV-2 viral load (p = 9.27 E-06), suggesting an inhibitory effect of ADAR1 on viral infection. Additionally, we find differentially expressed genes associated with RNA-modifications and interferon response. Single cell RNA-sequencing analysis of SARS-CoV-2 infected nasopharyngeal swabs reveals that cytotoxic CD8 T cells upregulate ADAR1 in COVID-19 positive samples (p = 0.0269). We further reveal ADAR1 expression increases with CD4 and CD8 T cell activation, and knockdown of ADAR1 leads to apoptosis and aberrant IL-2 secretion. Together, our data suggests A-to-I RNA editing is required to maintain healthy homeostasis of activated T cells to combat SARS-CoV-2 infection.

Adar null mutant mouse embryos die with aberrant double-stranded RNA (dsRNA)-driven interferon induction, and Adar Mavs double mutants, in which interferon induction is prevented, die soon after birth. Protein kinase R (Pkr) is aberrantly activated in Adar Mavs mouse pup intestines before death, intestinal crypt cells die, and intestinal villi are lost. Adar Mavs Eifak2 (Pkr) triple mutant mice rescue all defects and have long-term survival. Adenosine deaminase acting on RNA 1 (ADAR1) and PKR co-immunoprecipitate from cells, suggesting PKR inhibition by direct interaction. AlphaFold studies on an inhibitory PKR dsRNA binding domain (dsRBD)-kinase domain interaction before dsRNA binding and on an inhibitory ADAR1 dsRBD3-PKR kinase domain interaction on dsRNA provide a testable model of the inhibition. Wild-type or editing-inactive human ADAR1 expressed in A549 cells inhibits activation of endogenous PKR. ADAR1 dsRNA binding is required for, but is not sufficient for, PKR inhibition. Mutating the ADAR1 dsRBD3-PKR contact prevents co-immunoprecipitation, ADAR1 inhibition of PKR activity, and co-localization of ADAR1 and PKR in cells.

UNASSIGNED: To investigate the factors influencing accelerated aging in patients with type 2 diabetes mellitus (T2DM) and coronary heart disease (CHD).
UNASSIGNED: A total of 216 patients diagnosed with T2DM and CHD between August 2019 and August 2023 at Xuzhou Central Hospital were selected. Patients were divided into an aging group and a non-aging group, based on the positive or negative values of phenotypic age acceleration (PhenoAgeAccel). Logistic regression analysis was conducted. Variables that had a univariate analysis P< 0.05 were included in the multivariate analysis to identify factors influencing aging in patients with T2DM and CHD, and the area under the curve of the model was reported.
UNASSIGNED: This study included 216 patients, with 89 in the accelerated aging group, and 127 in the non-accelerated aging group. The average age of patients was 70.40 (95% CI: 69.10-71.69) years, with 137 males (63.4%). Compared with the non-accelerated aging group, patients in the accelerated aging group were older, with a higher proportion of males, and a higher prevalence of hypertension, stable angina pectoris, and unstable angina pectoris. Multivariate Logistic regression analysis indicated that the absolute value of neutrophils (NEUT#), urea (UREA), adenosine deaminase (ADA), and the triglyceride-glucose index (TyG) were risk factors for accelerated aging, while cholinesterase (CHE) was a protective factor. For each unit increase in NEUT#, UREA, ADA, and TyG, the risk of aging increased by 64%, 48%, 10%, and 789%, respectively. The overall area under the receiver operating characteristic (ROC) curve of the model in the training set was 0.894, with a 95% confidence interval (CI) of 0.851-0.938.
UNASSIGNED: NEUT#, CHE, UREA, ADA, and TyG are predictors of accelerated aging in patients with T2DM and CHD, with the model showing favorable overall predictive performance.

Vascular smooth muscle cells (VSMCs) play a critical role in maintaining vascular integrity. VSMC dysfunction leads to numerous vascular diseases. Adenosine deaminases acting on RNA 1 (ADAR1), an RNA editing enzyme, has shown both RNA editing and non-editing functions. Global deletion of ADAR1 causes embryonic lethality, but the phenotype of homozygous ADAR1 deletion specifically in SMCs (ADAR1sm-/-) remains to be determined. By crossing ADAR1fl/fl mice with Myh11-CreERT2 mice followed by Tamoxifen induction, we found that ADAR1sm-/- leads to lethality in adult mice 14 days after the induction. Gross examination revealed extensive hemorrhage and detrimental vascular damage in different organs. Histological analyses revealed destruction of artery structural integrity with detachment of elastin laminae from VSMCs in ADAR1sm-/- aortas. Furthermore, ADAR1sm-/- resulted in severe VSMC apoptosis and mitochondrial dysfunction. RNA sequencing analyses of ADAR1sm-/- aorta segments demonstrated profound transcriptional alteration of genes impacting vascular health including a decrease in fibrillin-1 expression. More importantly, ADAR1sm-/- disrupts the elastin and fibrillin-1 interaction, a molecular event essential for artery structure. Our results indicate that ADAR1 plays a critical role in maintaining SMC survival and vascular stability and resilience.

Adenosine deaminase 2 (ADA2) deficiency is an autosomal recessively inherited autoinflammatory disorder caused by loss-of-function mutations in the ADA2 gene. Although the pathogenesis involves the triggering of a proinflammatory cascade due to increased production of inflammatory cytokines such as tumor necrosis factor (TNF)-α and dysregulation of neutrophil extracellular trap formation resulting from an excess accumulation of extracellular adenosine, the pathogenetic mechanism still needs further clarification due to the broad clinical spectrum. In addition to the initially described vasculitis-related symptoms, hematological, immunological, and autoinflammatory symptoms are now well recognized. The diagnosis is made by demonstration of pathogenic variants of ADA2 with biallelic loss of function and identification of low plasma ADA2 catalytic activity. Currently, TNF-α inhibitors are the treatment of choice for controlling vasculitis manifestations and preventing strokes. However, in patients presenting with severe hematologic findings, TNF-α inhibitors are not the treatment of choice and hematopoietic stem cell transplantation has been shown to be successful in selected cases. Recombinant ADA2 protein and gene therapy are promising treatment modalities for the future. In conclusion, ADA2 deficiency has a broad phenotype and should be considered in the differential diagnosis of different clinical situations. In this review, we summarize the disease manifestations of ADA2 deficiency and available treatment options.
Adenozin deaminaz 2 (ADA2) eksikliği, ADA2 genindeki işlev kaybı mutasyonlarının neden olduğu otozomal resesif geçişli otoenflamatuvar bir hastalıktır. Patogenez, tümör nekroz faktörü (TNF)-alfa gibi enflamatuvar sitokinlerin üretiminin artması nedeniyle proinflamatuvar bir kaskadın tetiklenmesini ve ekstraselüler adenozinin aşırı birikiminden kaynaklanan nötrofil ekstraselüler tuzak oluşumu disregülasyon sürecini içermesine rağmen, geniş klinik spektrum nedeniyle patogenetik mekanizmanın hala daha fazla açıklığa kavuşturulması gerekmektedir. Başlangıçta tanımlanan vaskülitle ilişkili semptomlara ek olarak, hematolojik, immünolojik ve otoinflamatuar semptomlar da artık iyi tanınmaktadır. Tanı, ADA2’nin biallel fonksiyon kaybı ile patojenik varyantlarının gösterilmesi ve düşük plazma ADA2 katalitik aktivitesinin tanımlanması ile konur. Günümüzde TNF alfa inhibitörleri, vaskülit belirtilerini kontrol altına almak ve felçleri önlemek için tercih edilen tedavidir. Şiddetli hematolojik bulgularla başvuran hastalarda, TNF alfa inhibitörleri tercih edilen tedavi değildir ve hematopoetik kök hücre naklinin seçilmiş vakalarda başarılı olduğu gösterilmiştir. Rekombinant ADA2 proteini ve gen tedavisi gelecek için umut verici tedavi yöntemleridir. Sonuç olarak, ADA2 geniş bir fenotipe sahiptir ve farklı klinik durumlarda ayırıcı tanıda göz önünde bulundurulmalıdır. Bu derlemede,ADA2 eksikliğinin hastalık belirtilerini ve mevcut tedavi seçeneklerini özetlemeyi amaçladık.

RNA base editing relies on the introduction of adenosine-to-inosine changes into target RNAs in a highly programmable manner in order to repair disease-causing mutations. Here, we propose that RNA base editing could be broadly applied to perturb protein function by removal of regulatory phosphorylation and acetylation sites. We demonstrate the feasibility on more than 70 sites in various signaling proteins and identify key determinants for high editing efficiency and potent down-stream effects. For the JAK/STAT pathway, we demonstrate both, negative and positive regulation. To achieve high editing efficiency over a broad codon scope, we applied an improved version of the SNAP-ADAR tool. The transient nature of RNA base editing enables the comparably fast (hours to days), dose-dependent (thus partial) and reversible manipulation of regulatory sites, which is a key advantage over DNA (base) editing approaches. In summary, PTM interference might become a valuable field of application of RNA base editing.