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Hematopoietic system aging is characterized by both hematopoietic stem cell (HSC) and niche degeneration resulting in myeloid lineage-biased differentiation, reduced B cell and T cell lymphopoiesis, increased HSC mobilization, and fat deposition in the bone marrow. Both alterations in RNA splicing and editing during HSC aging contribute to increased myeloid lineage skewing and inflammation-responsive transcription factors, underscoring the importance of epitranscriptomic mechanisms in the acquisition of an age-related phenotype.

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The role of adenosine deaminase acting on RNA1 (ADAR1) in colorectal cancer (CRC) is poorly understood. This study investigated the roles and underlying molecular mechanisms of ADAR1 and its isoforms, explored the correlations between ADAR1 expression and the immune microenvironment and anticancer drug sensitivity, and examined the potential synergy of using ADAR1 expression and clinical parameters to determine the prognosis of CRC patients. CRC samples showed significant upregulation of ADAR1, and high ADAR1 expression was correlated with poor prognosis. Silencing ADAR1 inhibited the proliferation, invasion, and migration of CRC cells and induced ferroptosis by suppressing FAK/AKT activation, and the results of rescue assays were consistent with these mechanisms. Both ADAR1-p110 and ADAR1-p150 were demonstrated to regulate the FAK/AKT pathway, with ADAR1-p110 playing a particularly substantial role. In evaluating the prognosis of CRC patients, combining ADAR1 expression with clinical parameters produced a substantial synergistic effect. The in vivo tumorigenesis of CRC was significantly inhibited by silencing ADAR1. Furthermore, ADAR1 expression was positively correlated with tumor mutational burden (TMB) and microsatellite status (p < 0.05), indicating that ADAR1 plays a complex role in CRC immunotherapy. In conclusion, ADAR1 plays oncogenic roles in CRC both in vitro and in vivo, potentially by inhibiting ferroptosis via downregulation of the FAK/AKT pathway. Thus, ADAR1 serves as a potential prognostic biomarker and a promising target for CRC therapy.

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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.

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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.

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Transposable elements (TEs) are of interest as immunomodulators for cancer therapies. TEs can fold into dsRNAs that trigger the interferon response. Here, we investigated the effect of different HDAC inhibitors (HDACIs) on the expression of TEs in leiomyosarcoma cells. Our data show that endogenous retroviruses (ERVs), especially ERV1 elements, are upregulated after treatment with HDAC1/2/3-specific inhibitors. Surprisingly, the interferon response was not activated. We observed an increase in A-to-I editing of upregulated ERV1. This could have an impact on the stability of dsRNAs and the activation of the interferon response. We also found that H3K27ac levels are increased in the LTR12 subfamilies, which could be regulatory elements controlling the expression of proapoptotic genes such as TNFRSF10B. In summary, we provide a detailed characterization of TEs modulation in response to HDACIs and suggest the use of HDACIs in combination with ADAR inhibitors to induce cell death and support immunotherapy in cancer.

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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.

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The National Institute of Health R25 Research Education Program was evaluated in the second year of implementation. Twelve mentors and 20 underrepresented minority students (URMs) scholars from partnerships and collaborations among five colleges and universities were added to the program to provide a more diverse research experience. Findings reveal that 100% of research mentors agree that the approachableness and accessibility of the program coordinator were beneficial in achieving mentorship goals and objectives. In addition, 85% of the students strongly agreed that the presentation of their research findings and the weekly reflection on goals, identification of accomplishments, and obstacles through the individual development plan were very effective. Of the 23 successfully tracked students for 2 years, six URMs (26.09%) obtained a bachelor's degree and were admitted into a graduate program; two were directly admitted to a PhD program in biomedical sciences.

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Adenosine deaminases acting on RNA (ADARs) are enzymes that catalyze the hydrolytic deamination of adenosine to inosine. The editing feature of ADARs has garnered much attention as a therapeutic tool to repurpose ADARs to correct disease-causing mutations at the mRNA level in a technique called site-directed RNA editing (SDRE). Administering a short guide RNA oligonucleotide that hybridizes to a mutant sequence forms the requisite dsRNA substrate, directing ADARs to edit the desired adenosine. However, much is still unknown about ADARs' selectivity and sequence-specific effects on editing. Atomic-resolution structures can help provide additional insight to ADARs' selectivity and lead to novel guide RNA designs. Indeed, recent structures of ADAR domains have expanded our understanding on RNA binding and the base-flipping catalytic mechanism. These efforts have enabled the rational design of improved ADAR guide strands and advanced the therapeutic potential of the SDRE approach. While no full-length structure of any ADAR is known, this review presents an exposition of the structural basis for function of the different ADAR domains, focusing on human ADAR2. Key insights are extrapolated to human ADAR1, which is of substantial interest because of its widespread expression in most human tissues.

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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.

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It is well established that sevoflurane exposure leads to widespread neuronal cell death in the developing brain. Adenosine deaminase acting on RNA-1 (ADAR1) dependent adenosine-to-inosine (A-to-I) RNA editing is dynamically regulated throughout brain development. The current investigation is designed to interrogate the contributed role of ADAR1 in developmental sevoflurane neurotoxicity. Herein, we provide evidence to show that developmental sevoflurane priming triggers neuronal pyroptosis, apoptosis and necroptosis (PANoptosis), and elicits the release of inflammatory factors including IL-1ß, IL-18, TNF-α and IFN-γ. Additionally, ADAR1-P150, but not ADAR1-P110, depresses cellular PANoptosis and inflammatory response by competing with Z-DNA/RNA binding protein 1 (ZBP1) for binding to Z-RNA in the presence of sevoflurane. Further investigation demonstrates that ADAR1-dependent A-to-I RNA editing mitigates developmental sevoflurane-induced neuronal PANoptosis. To restore RNA editing, we utilize adeno-associated virus (AAV) to deliver engineered circular ADAR-recruiting guide RNAs (cadRNAs) into cells, which is capable of recruiting endogenous adenosine deaminases to promote cellular A-to-I RNA editing. As anticipated, AAV-cadRNAs diminishes sevoflurane-induced cellular Z-RNA production and PANoptosis, which could be abolished by ADAR1-P150 shRNA transfection. Moreover, AAV-cadRNAs delivery ameliorates developmental sevoflurane-induced spatial and emotional cognitive deficits without influence on locomotor activity. Taken together, these results illustrate that ADAR1-P150 exhibits a prominent role in preventing ZBP1-dependent PANoptosis through A-to-I RNA editing in developmental sevoflurane neurotoxicity. Application of engineered cadRNAs to rectify the compromised ADAR1-dependent A-to-I RNA editing provides an inspiring direction for possible clinical preventions and therapeutics.

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RNA binding proteins play essential roles in coordinating germline gene expression and development in all organisms. Here, we report that loss of ADR-2, a member of the Adenosine DeAminase acting on RNA (ADAR) family of RNA binding proteins and the sole adenosine-to-inosine RNA editing enzyme in C. elegans, can improve fertility in multiple genetic backgrounds. First, we show that loss of RNA editing by ADR-2 restores normal embryo production to subfertile animals that transgenically express a vitellogenin (yolk protein) fusion to green fluorescent protein. Using this phenotype, a high-throughput screen was designed to identify RNA binding proteins that when depleted yield synthetic phenotypes with loss of adr-2. The screen uncovered a genetic interaction between ADR-2 and SQD-1, a member of the heterogenous nuclear ribonucleoprotein (hnRNP) family of RNA binding proteins. Microscopy, reproductive assays, and high-throughput sequencing reveal that sqd-1 is essential for the onset of oogenesis and oogenic gene expression in young adult animals, and that loss of adr-2 can counteract the effects of loss of sqd-1 on gene expression and rescue the switch from spermatogenesis to oogenesis. Together, these data demonstrate that ADR-2 can contribute to the suppression of fertility and suggest novel roles for both RNA editing-dependent and independent mechanisms in regulating embryogenesis.

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Adenosine-to-inosine (A-to-I) RNA editing is an important post-transcriptional modification mediated by the adenosine deaminases acting on RNA (ADAR) family of enzymes, expanding the transcriptome by altering selected nucleotides A to I in RNA molecules. Recently, A-to-I editing has been explored for correcting disease-causing mutations in RNA using therapeutic guide oligonucleotides to direct ADAR editing at specific sites. Humans have two active ADARs whose preferences and specificities are not well understood. To investigate their substrate specificity, we introduced hADAR1 and hADAR2, respectively, into Schizosaccharomyces pombe (S. pombe), which lacks endogenous ADARs, and evaluated their editing activities in vivo. Using transcriptome sequencing of S. pombe cultured at optimal growth temperature (30 °C), we identified 483 A-to-I high-confident editing sites for hADAR1 and 404 for hADAR2, compared with the non-editing wild-type control strain. However, these sites were mostly divergent between hADAR1 and hADAR2-expressing strains, sharing 33 common sites that are less than 9% for each strain. Their differential specificity for substrates was attributed to their differential preference for neighboring sequences of editing sites. We found that at the -3-position relative to the editing site, hADAR1 exhibits a tendency toward T, whereas hADAR2 leans toward A. Additionally, when varying the growth temperature for hADAR1- and hADAR2-expressing strains, we observed increased editing sites for them at both 20 and 35 °C, compared with them growing at 30 °C. However, we did not observe a significant shift in hADAR1 and hADAR2's preference for neighboring sequences across three temperatures. The vast changes in RNA editing sites at lower and higher temperatures were also observed for hADAR2 previously in budding yeast, which was likely due to the influence of RNA folding at these different temperatures, among many other factors. We noticed examples of longer lengths of dsRNA around the editing sites that induced editing at 20 or 35 °C but were absent at the other two temperature conditions. We found genes' functions can be greatly affected by editing of their transcripts, for which over 50% of RNA editing sites for both hADAR1 and hADAR2 in S. pombe were in coding sequences (CDS), with more than 60% of them resulting in amino acid changes in protein products. This study revealed the extensive differences in substrate selectivity between the two active human ADARS, i.e., ADAR1 and ADAR2, and provided novel insight when utilizing the two different enzymes for in vivo treatment of human genetic diseases using the RNA editing approach.

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The APOBEC/AID family is known for its mutator activity, and recent evidence also supports the potential impact of ADARs. Furthermore, the mutator impacts of APOBEC/ADAR mutations have not yet been investigated. Assessment of pancancer TCGA exomes identified enriched somatic variants among exomes with nonsynonymous APOBEC1, APOBEC3B, APOBEC3C, ADAR, and ADARB1 mutations, compared to exomes with synonymous ones. Principal component (PC) analysis reduced the number of potential players to eight in cancer exomes/genomes, and to five in cancer types. Multivariate regression analysis was used to assess the impact of the PCs on each COSMIC mutational signature among pancancer exomes/genomes and particular cancers, identifying several novel links, including SBS17b, SBS18, and ID7 mainly determined by APOBEC1 mRNA levels; SBS40, ID1, and ID2 by age; SBS3 and SBS16 by APOBEC3A/APOBEC3B mRNA levels; ID5 and DBS9 by DNA repair/replication (DRR) defects; and SBS7a-d, SBS38, ID4, ID8, ID13, and DBS1 by ultraviolet (UV) radiation/ADARB1 mRNA levels. APOBEC/ADAR mutations appeared to potentiate the impact of DRR defects on several mutational signatures, and some factors seemed to inversely affect certain signatures. These findings potentially implicate certain APOBEC/ADAR mutations/mRNA levels in distinct mutational signatures, particularly APOBEC1 mRNA levels in aging-related signatures and ADARB1 mRNA levels in UV radiation-related signatures.

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Patients with amyotrophic lateral sclerosis (ALS) do not develop oculomotor disturbances and vesicorectal dysfunction until end-stage disease owing to the survival of certain motor neurons (MNs), including oculomotor neurons and MNs within Onuf's nucleus. In sporadic ALS, adenosine deaminase acting on RNA 2 (ADAR2)-mediated editing of GluA2 mRNA at the Q/R site is compromised in lower MNs. We previously developed genetically modified mice with a conditional knockout of ADAR2 in cholinergic neurons (ADAR2flox/flox/VAChT-Cre, Fast; AR2). These mice displayed slow and progressive lower motor neuron death with TAR DNA-binding protein 43 (TDP-43) pathology, attributable to insufficient editing at the GluA2 Q/R site due to ADAR2 deficiency. MN death was more common in fast-fatigable MNs owing to differential vulnerability under conditions of ADAR2 deficiency. Although facial and hypoglossal nerves were impaired in AR2 mice, cell death did not occur within the oculomotor nerve nucleus, as observed in patients with sporadic ALS. Since the basis for avoiding cystorectal damage in ALS is unknown, we compared the features of Onuf's nucleus MNs in 12-month-old AR2 mice with those in age-matched wild-type mice. Although the number of MNs was not significantly lower in AR2 mice, the neurons exhibited a shrunken morphology and TDP-43 pathology. Onuf's nucleus MNs could survive in an ADAR2-deficient state and mainly included fast fatigue-resistant (FR) and slow (S) MNs. In summary, FR and S MNs show increased resilience to ADAR2 deficiency, potentially participating in an important neuronal death avoidance mechanism in ALS.

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Adenosine Deaminases Acting on RNA (ADARs) are evolutionarily conserved enzymes known to convert adenosine to inosine in double-stranded RNAs and participate in host-virus interactions. Conducting a meta-analysis of available transcriptome data, we identified and characterised eight ADAR transcripts in Chlamys farreri, a farmed marine scallop susceptible to Acute viral necrosis virus (AVNV) infections and mortality outbreaks. Accordingly, we identified six ADAR genes in the Zhikong scallop genome, revised previous gene annotations, and traced alternative splicing variants. In detail, each ADAR gene encodes a unique combination of functional domains, always including the Adenosine deaminase domain, RNA binding domains and, in one case, two copies of a Z-DNA binding domain. After phylogenetic analysis, five C. farreri ADARs clustered in the ADAR1 clade along with sequences from diverse animal phyla. Gene expression analysis indicated CF051320 as the most expressed ADAR, especially in the eye and male gonad. The other four ADAR1 genes and one ADAR2 gene exhibited variable expression levels, with CF105370 and CF051320 significantly increasing during early scallop development. ADAR-mediated single-base editing, evaluated across adult C. farreri tissues and developmental stages, was mainly detectable in intergenic regions (83 % and 85 %, respectively). Overall, the expression patterns of the six ADAR genes together with the editing and hyper-editing values computed on scallops RNA-seq samples support the adaptive value of ADAR1-mediated editing, particularly in the pre-settling larval stages.

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BACKGROUND: RNA-seq has brought forth significant discoveries regarding aberrations in RNA processing, implicating these RNA variants in a variety of diseases. Aberrant splicing and single nucleotide variants (SNVs) in RNA have been demonstrated to alter transcript stability, localization, and function. In particular, the upregulation of ADAR, an enzyme that mediates adenosine-to-inosine editing, has been previously linked to an increase in the invasiveness of lung adenocarcinoma cells and associated with splicing regulation. Despite the functional importance of studying splicing and SNVs, the use of short-read RNA-seq has limited the community's ability to interrogate both forms of RNA variation simultaneously. RESULTS: We employ long-read sequencing technology to obtain full-length transcript sequences, elucidating cis-effects of variants on splicing changes at a single molecule level. We develop a computational workflow that augments FLAIR, a tool that calls isoform models expressed in long-read data, to integrate RNA variant calls with the associated isoforms that bear them. We generate nanopore data with high sequence accuracy from H1975 lung adenocarcinoma cells with and without knockdown of ADAR. We apply our workflow to identify key inosine isoform associations to help clarify the prominence of ADAR in tumorigenesis. CONCLUSIONS: Ultimately, we find that a long-read approach provides valuable insight toward characterizing the relationship between RNA variants and splicing patterns.

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Epitranscriptomics is a field that delves into post-transcriptional changes. Among these modifications, the conversion of adenosine to inosine, traduced as guanosine (A>I(G)), is one of the known RNA-editing mechanisms, catalyzed by ADARs. This type of RNA editing is the most common type of editing in mammals and contributes to biological diversity. Disruption in the A>I(G) RNA-editing balance has been linked to diseases, including several types of cancer. Drug resistance in patients with cancer represents a significant public health concern, contributing to increased mortality rates resulting from therapy non-responsiveness and disease progression, representing the greatest challenge for researchers in this field. The A>I(G) RNA editing is involved in several mechanisms over the immunotherapy and genotoxic drug response and drug resistance. This review investigates the relationship between ADAR1 and specific A>I(G) RNA-edited sites, focusing particularly on breast cancer, and the impact of these sites on DNA damage repair and the immune response over anti-cancer therapy. We address the underlying mechanisms, bioinformatics, and in vitro strategies for the identification and validation of A>I(G) RNA-edited sites. We gathered databases related to A>I(G) RNA editing and cancer and discussed the potential clinical and research implications of understanding A>I(G) RNA-editing patterns. Understanding the intricate role of ADAR1-mediated A>I(G) RNA editing in breast cancer holds significant promise for the development of personalized treatment approaches tailored to individual patients' A>I(G) RNA-editing profiles.

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Measures of life outlook in older adults have been investigated in connection to pain, as both pain management and outlook are important factors of successful aging. We hypothesized that higher pain is associated with lower optimism among community-dwelling older adults. We utilized data from the UC San Diego Successful Aging Evaluation (SAGE), a prospective longitudinal cohort study initiated in 2010, to evaluate the relationship between pain and optimism in 378 community-dwelling adults aged ≥50 years. We used the revised Life Orientation Test (LOT-R) to measure optimism and three pain subscales-PROMIS Pain Interference, PROMIS Pain Intensity, and MOS 36-Item Short-Form Health Survey (SF-36)-as pain measures. Regression analyses reveal negative relationships between pain and optimism for all three pain scales, with regression coefficients of -0.277 (p < .0001), -0.246 (p < .0001), and 0.269 (p < .0001) respectively. This indicates value in considering physical and psychological elements in future intervention research to promote healthy aging.

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We examined the relationship between subjective and objective sleep outcomes and loneliness in older women at risk for Alzheimer's disease (AD). Our sample consisted of 39 participants (aged 65+) with mild cognitive deficits who completed the UCLA Loneliness Scale, the Pittsburgh Sleep Quality Index (PSQI), and an at home sleep test, to determine presence of obstructive sleep apnea. Based on sleep quality scores, individuals categorized as "poor sleepers" had significantly higher loneliness scores than "good sleepers." However, total loneliness scores did not significantly differ between groups with or without sleep apnea. We found that higher loneliness was significantly associated to lower habitual sleep efficiency and sleep duration and was also influenced by use of sleep medication. Our findings suggest that increased loneliness relates to worse subjective sleep quality, but not to sleep apnea. These findings suggest that combined interventions targeting loneliness and sleep quality may be important for older women.

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Adenosine deaminase acting on RNA 1 (ADAR1) converts adenosine to inosine in double-stranded RNA (dsRNA) molecules, a process known as A-to-I editing. ADAR1 deficiency in humans and mice results in profound inflammatory diseases characterised by the spontaneous induction of innate immunity. In cells lacking ADAR1, unedited RNAs activate RNA sensors. These include melanoma differentiation-associated gene 5 (MDA5) that induces the expression of cytokines, particularly type I interferons (IFNs), protein kinase R (PKR), oligoadenylate synthase (OAS), and Z-DNA/RNA binding protein 1 (ZBP1). Immunogenic RNAs 'defused' by ADAR1 may include transcripts from repetitive elements and other long duplex RNAs. Here, we review these recent fundamental discoveries and discuss implications for human diseases. Some tumours depend on ADAR1 to escape immune surveillance, opening the possibility of unleashing anticancer therapies with ADAR1 inhibitors.