ADAR-Mediated A>I(G) RNA Editing in the Genotoxic Drug Response of Breast Cancer.

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.

Development of a prognostic risk model of uveal melanoma based on N7-methylguanosine-related regulators.

BACKGROUND: Uveal melanoma (UVM) stands as the predominant type of primary intraocular malignancy among adults. The clinical significance of N7-methylguanosine (m7G), a prevalent RNA modifications, in UVM remains unclear. METHODS: Primary information from 80 UVM patients were analyzed as the training set, incorporating clinical information, mutation annotations and mRNA expression obtained from The Cancer Genome Atlas (TCGA) website. The validation set was carried out using Gene Expression Omnibus (GEO) database GSE22138 and GSE84976. Kaplan-Meier and Cox regression of univariate analyses were subjected to identify m7G-related regulators as prognostic genes. RESULT: A prognostic risk model comprising EIF4E2, NUDT16, SNUPN and WDR4 was established through Cox regression of LASSO. Evaluation of the model's predictability for UVM patients' prognosis by Receiver Operating Characteristic (ROC) curves in the training set, demonstrated excellent performance Area Under the Curve (AUC) > 0.75. The high-risk prognosis within the TCGA cohort exhibit a notable worse outcome. Additionally, an independent correlation between the risk score and overall survival (OS) among UVM patients were identified. External validation of this model was carried out using the validation sets (GSE22138 and GSE84976). Immune-related analysis revealed that patients with high score of m7G-related risk model exhibited elevated level of immune infiltration and immune checkpoint gene expression. CONCLUSION: We have developed a risk prediction model based on four m7G-related regulators, facilitating effective estimate UVM patients' survival by clinicians. Our findings shed novel light on essential role of m7G-related regulators in UVM and suggest potential novel targets for the diagnosis, prognosis and therapy of UVM.

Pan-Cancer Analysis Links Altered RNA m7G Methyltransferase Expression to Oncogenic Pathways, Immune Cell Infiltrations and Overall Survival.

BACKGROUND: N7-methylguanosine (m7G) modification is one of the most prevalent RNA modifications in humans. Dysregulated m7G modifications caused by aberrant expression of m7G writers contribute to cancer progression and result in worse patient survival in several human cancers. However, studies that systematically assess the frequency and clinical relevance of aberrant m7G writer expression in a pan-cancer cohort remain to be performed. AIMS: This study aims to systematically investigate the molecular alteration and clinical relevance of m7G methyltransferase in human cancers. METHODS: We analysed genome, transcriptome and clinical data from the Cancer Genome Atlas Research Network spanning 33 types of human cancers for aberrant changes in genes encoding m7G writers. RESULT: We demonstrate that m7G writers are dysregulated in human cancers and are associated predominantly with poorer survival. By dividing patients into those with high and low m7G scores, we show that a lower m7G score is generally associated with immune infiltration and better response to immunotherapy. CONCLUSION: Our analyses indicate the genetic alterations, expression patterns and clinical relevance of m7G writers across various cancers. This study provides insights into the potential utility of m7G writer expression as a cancer biomarker and proposes the possibility of targeting m7G writers for cancer therapy.

Exploring the role of m7G modification in Cancer: Mechanisms, regulatory proteins, and biomarker potential.

The dysregulation of N(7)-methylguanosine (m7G) modification is increasingly recognized as a key factor in the pathogenesis of cancers. Aberrant expression of these regulatory proteins in various cancers, including lung, liver, and bladder cancers, suggests a universal role in tumorigenesis. Studies have established a strong correlation between the expression levels of m7G regulatory proteins, such as Methyltransferase like 1 (METTL1) and WD repeat domain 4 (WDR4), and clinical parameters including tumor stage, grade, and patient prognosis. For example, in hepatocellular carcinoma, high METTL1 expression is associated with advanced tumor stage and poor prognosis. Similarly, WDR4 overexpression in colorectal cancer correlates with increased tumor invasiveness and reduced patient survival. This correlation underscores the potential of these proteins as valuable biomarkers for cancer diagnosis and prognosis. Additionally, m7G modification regulatory proteins influence cancer progression by modulating the expression of target genes involved in critical biological processes, including cell proliferation, apoptosis, migration, and invasion. Their ability to regulate these processes highlights their significance in the intricate network of molecular interactions driving tumor development and metastasis. Given their pivotal role in cancer biology, m7G modification regulatory proteins are emerging as promising therapeutic targets. Targeting these proteins could offer a novel approach to disrupt the malignant behavior of cancer cells and enhance treatment outcomes. Furthermore, their diagnostic and prognostic value could aid in the early detection of cancer and the selection of appropriate therapeutic strategies, ultimately enhancing patient management and survival rates. This review aims to explore the mechanisms of action of RNA m7G modification regulatory proteins in tumors and their potential applications in cancer progression and treatment. By delving into the roles of these regulatory proteins, we intend to provide a theoretical foundation for the development of novel cancer treatment strategies.

Moss-m7G: A Motif-Based Interpretable Deep Learning Method for RNA N7-Methlguanosine Site Prediction.

N-7methylguanosine (m7G) modification plays a crucial role in various biological processes and is closely associated with the development and progression of many cancers. Accurate identification of m7G modification sites is essential for understanding their regulatory mechanisms and advancing cancer therapy. Previous studies often suffered from insufficient research data, underutilization of motif information, and lack of interpretability. In this work, we designed a novel motif-based interpretable method for m7G modification site prediction, called Moss-m7G. This approach enables the analysis of RNA sequences from a motif-centric perspective. Our proposed word-detection module and motif-embedding module within Moss-m7G extract motif information from sequences, transforming the raw sequences from base-level into motif-level and generating embeddings for these motif sequences. Compared with base sequences, motif sequences contain richer contextual information, which is further analyzed and integrated through the Transformer model. We constructed a comprehensive m7G data set to implement the training and testing process to address the data insufficiency noted in prior research. Our experimental results affirm the effectiveness and superiority of Moss-m7G in predicting m7G modification sites. Moreover, the introduction of the word-detection module enhances the interpretability of the model, providing insights into the predictive mechanisms.

Abundance of Modifications in Mature miRNAs Revealed by LC-MS/MS Method Coupled with a Two-Step Hybridization Purification Strategy.

Accurate detection of endogenous miRNA modifications, such as N6-methyladenosine (m6A), 7-methylguanosine (m7G), and 5-methylcytidine (m5C), poses significant challenges, resulting in considerable uncertainty regarding their presence in mature miRNAs. In this study, we demonstrate for the first time that liquid chromatography coupled with a tandem mass spectrometry (LC-MS/MS) nucleoside analysis method is a practical tool for quantitatively analyzing human miRNA modifications. The newly designed liquid-solid two-step hybridization (LSTH) strategy enhances specificity for miRNA purification, while LC-MS/MS offers robust capability in recognizing modifications and sufficient sensitivity with detection limits ranging from attomoles to low femtomoles. Therefore, it provides a more reliable approach compared to existing techniques for revealing modifications in endogenous miRNAs. With this approach, we characterized m6A, m7G, and m5C modifications in miR-21-5p, Let-7a/e-5p, and miR-10a-5p isolated from cultured cells and observed unexpectedly low abundance (<1% at each site) of these modifications.

Global and single-nucleotide resolution detection of 7-methylguanosine in RNA.

RNA modifications, including N-7-methylguanosine (m7G), are pivotal in governing RNA stability and gene expression regulation. The accurate detection of internal m7G modifications is of paramount significance, given recent associations between altered m7G deposition and elevated expression of the methyltransferase METTL1 in various human cancers. The development of robust m7G detection techniques has posed a significant challenge in the field of epitranscriptomics. In this study, we introduce two methodologies for the global and accurate identification of m7G modifications in human RNA. We introduce borohydride reduction sequencing (Bo-Seq), which provides base resolution mapping of m7G modifications. Bo-Seq achieves exceptional performance through the optimization of RNA depurination and scission, involving the strategic use of high concentrations of NaBH4, neutral pH and the addition of 7-methylguanosine monophosphate (m7GMP) during the reducing reaction. Notably, compared to NaBH4-based methods, Bo-Seq enhances the m7G detection performance, and simplifies the detection process, eliminating the necessity for intricate chemical steps and reducing the protocol duration. In addition, we present an antibody-based approach, which enables the assessment of m7G relative levels across RNA molecules and biological samples, however it should be used with caution due to limitations associated with variations in antibody quality between batches. In summary, our novel approaches address the pressing need for reliable and accessible methods to detect RNA m7G methylation in human cells. These advancements hold the potential to catalyse future investigations in the critical field of epitranscriptomics, shedding light on the complex regulatory roles of m7G in gene expression and its implications in cancer biology.

The Effect of Fermentation on the Chemical Constituents of Gastrodia Tuber Hallimasch Powder (GTHP) Estimated by UHPLC-Q-Orbitrap HRMS and HPLC.

OBJECTIVE: To compare the effect of fermentation on the chemical constituents of Gastrodia Tuder Halimasch Powder (GTHP), to establish its fingerprinting and multicomponent content determination, and to provide a basis for the processing, handling, and clinical application of this herb. METHODS: Ultra-high-performance liquid chromatography-quadrupole-Orbitrap high-resolution mass spectrometry (UHPLC-Q-Orbitrap HRMS) was used to conduct a preliminary analysis of the chemical constituents in GTHP before and after fermentation. High-performance liquid chromatography (HPLC) was used to determine some major differential components of GTHP and establish fingerprints. Cluster analysis (CA), and principal component analysis (PCA) were employed for comprehensive evaluation. RESULTS: Seventy-nine compounds were identified, including flavonoids, organic acids, nucleosides, terpenoids, and others. The CA and PCA results showed that ten samples were divided into three groups. Through standard control and HPLC analysis, 10 compounds were identified from 22 peaks, namely uracil, guanosine, adenosine, 5-hydroxymethylfurfural (5-HMF), daidzin, genistin, glycitein, daidzein, genistein, and ergosterol. After fermentation, GTHP exhibited significantly higher contents of uracil, guanosine, adenosine, 5-hydroxymethylfurfural, and ergosterol and significantly lower genistein and daidzein contents. CONCLUSIONS: The UHPLC-Q-Orbitrap HRMS and HPLC methods can effectively identify a variety of chemical components before and after the fermentation of GTHP. This study provides a valuable reference for further research on the rational clinical application and quality control improvement of GTHP.

The causal relationship between genetically predicted blood metabolites and idiopathic pulmonary fibrosis: A bidirectional two-sample Mendelian randomization study.

BACKGROUND: Numerous metabolomic studies have confirmed the pivotal role of metabolic abnormalities in the development of idiopathic pulmonary fibrosis (IPF). Nevertheless, there is a lack of evidence on the causal relationship between circulating metabolites and the risk of IPF. METHODS: The potential causality between 486 blood metabolites and IPF was determined through a bidirectional two-sample Mendelian randomization (TSMR) analysis. A genome-wide association study (GWAS) involving 7,824 participants was performed to analyze metabolite data, and a GWAS meta-analysis involving 6,257 IPF cases and 947,616 control European subjects was conducted to analyze IPF data. The TSMR analysis was performed primarily with the inverse variance weighted model, supplemented by weighted mode, MR-Egger regression, and weighted median estimators. A battery of sensitivity analyses was performed, including horizontal pleiotropy assessment, heterogeneity test, Steiger test, and leave-one-out analysis. Furthermore, replication analysis and meta-analysis were conducted with another GWAS dataset of IPF containing 4,125 IPF cases and 20,464 control subjects. Mediation analyses were used to identify the mediating role of confounders in the effect of metabolites on IPF. RESULTS: There were four metabolites associated with the elevated risk of IPF, namely glucose (odds ratio [OR] = 2.49, 95% confidence interval [95%CI] = 1.13-5.49, P = 0.024), urea (OR = 6.24, 95% CI = 1.77-22.02, P = 0.004), guanosine (OR = 1.57, 95%CI = 1.07-2.30, P = 0.021), and ADpSGEGDFXAEGGGVR (OR = 1.70, 95%CI = 1.00-2.88, P = 0.0496). Of note, the effect of guanosine on IPF was found to be mediated by gastroesophageal reflux disease. Reverse Mendelian randomization analysis displayed that IPF might slightly elevate guanosine levels in the blood. CONCLUSION: Conclusively, hyperglycemia may confer a promoting effect on IPF, highlighting that attention should be paid to the relationship between diabetes and IPF, not solely to the diagnosis of diabetes. Additionally, urea, guanosine, and ADpSGEGDFXAEGGGVR also facilitate the development of IPF. This study may provide a reference for analyzing the potential mechanism of IPF and carry implications for the prevention and treatment of IPF.

Purine nucleosides replace cAMP in allosteric regulation of PKA in trypanosomatid pathogens.

Cyclic nucleotide binding domains (CNB) confer allosteric regulation by cAMP or cGMP to many signaling proteins, including PKA and PKG. PKA of phylogenetically distant Trypanosoma is the first exception as it is cyclic nucleotide-independent and responsive to nucleoside analogues (Bachmaier et al., 2019). Here, we show that natural nucleosides inosine, guanosine and adenosine are nanomolar affinity CNB ligands and activators of PKA orthologs of the important tropical pathogens Trypanosoma brucei, Trypanosoma cruzi, and Leishmania. The sequence and structural determinants of binding affinity, -specificity and kinase activation of PKAR were established by structure-activity relationship (SAR) analysis, co-crystal structures and mutagenesis. Substitution of two to three amino acids in the binding sites is sufficient for conversion of CNB domains from nucleoside to cyclic nucleotide specificity. In addition, a trypanosomatid-specific C-terminal helix (αD) is required for high affinity binding to CNB-B. The αD helix functions as a lid of the binding site that shields ligands from solvent. Selectivity of guanosine for CNB-B and of adenosine for CNB-A results in synergistic kinase activation at low nanomolar concentration. PKA pulldown from rapid lysis establishes guanosine as the predominant ligand in vivo in T. brucei bloodstream forms, whereas guanosine and adenosine seem to synergize in the procyclic developmental stage in the insect vector. We discuss the versatile use of CNB domains in evolution and recruitment of PKA for novel nucleoside-mediated signaling.

A multi-omics investigation of the lung injury induced by PM2.5 at environmental levels via the lung-gut axis.

Long-term exposure to fine particulate matter (PM2.5) posed injury for gastrointestinal and respiratory systems, ascribing with the lung-gut axis. However, the cross-talk mechanisms remain unclear. Here, we attempted to establish the response networks of lung-gut axis in mice exposed to PM2.5 at environmental levels. Male Balb/c mice were exposed to PM2.5 (dose of 0.1, 0.5, and 1.0 mg/kg) collected from Chengdu, China for 10 weeks, through intratracheally instillation, and examined the effect of PM2.5 on lung functions of mice. The changes of lung and gut microbiota and metabolic profiles of mice in different groups were determined. Furthermore, the results of multi-omics were conjointly analyzed to elucidate the primary microbes and the associated metabolites in lung and gut responsible for PM2.5 exposure. Accordingly, the cross-talk network and key pathways between lung-gut axis were established. The results indicated that exposed to PM2.5 0.1 mg/kg induced obvious inflammations in mice lung, while emphysema was observed at 1.0 mg/kg. The levels of metabolites guanosine, hypoxanthine, and hepoxilin B3 increased in the lung might contribute to lung inflammations in exposure groups. For microbiotas in lung, PM2.5 exposure significantly declined the proportions of Halomonas and Lactobacillus. Meanwhile, the metabolites in gut including L-tryptophan, serotonin, and spermidine were up-regulated in exposure groups, which were linked to the decreasing of Oscillospira and Helicobacter in gut. Via lung-gut axis, the activations of pathways including Tryptophan metabolism, ABC transporters, Serotonergic synapse, and Linoleic acid metabolism contributed to the cross-talk between lung and gut tissues of mice mediated by PM2.5. In summary, the microbes including Lactobacillus, Oscillospira, and Parabacteroides, and metabolites including hepoxilin B3, guanosine, hypoxanthine, L-tryptophan, and spermidine were the main drivers. In this lung-gut axis study, we elucidated some pro- and pre-biotics in lung and gut microenvironments contributed to the adverse effects on lung functions induced by PM2.5 exposure.

WDR4 promotes HCC pathogenesis through N7-methylguanosine by regulating and interacting with METTL1.

BACKGROUND: The N7-methylguanosine (m7G), a modification at defined internal positions within tRNAs and rRNAs, is correlated with tumor progression. Methyltransferase like 1 (METTL1)/ WD repeat domain 4 (WDR4) mediated tRNA m7G modification, which could alter many oncogenic mRNAs translation to promote progress of multiple cancer types. However, whether and how the internal mRNA m7G modification is involved in tumorigenesis remains unclear. METHODS: The immunohistochemistry assay was conducted to detect the expression of WDR4 and METTL1 in hepatocellular carcinoma (HCC) and the expression of both genes whether contributes to the prognosis of the survival rate of HCC patients. Then, CCK8, colony formation assays and tumor xenograft models were conducted to determine the effects of WDR4 on HCC cells in vitro and vivo. Besides, dot blot assay, m7G-MeRIP-seq and RNA-seq analysis were conducted to determine whether WDR4 contributes to m7G modification and underlying mechanism in HCC cells. Finally, rescue and CO-IP assay were conducted to explore whether WDR4 and METTL1 proteins form a complex in Huh7 cells. RESULTS: WDR4 modulates m7G modification at the internal sites of tumor-promoting mRNAs by forming the WDR4-METTL1 complex. WDR4 knockdown downregulated the expression of mRNA and protein levels of METTL1 gene and thus further modulate the formation of WDR4-METTL1 complex indirectly. METTL1 expression was markedly correlated with WDR4 expression in HCC tissues. HCC patients with high expression of both genes had a poor prognosis. CONCLUSIONS: WDR4 may contribute to HCC pathogenesis by interacting with and regulating the expression of METTL1 to synergistically modulate the m7G modification of target mRNAs in tumor cells.

Multi-omics reveals the testosterone promotion effect mechanism of Cordyceps Sobolifera on Leydig cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Cordyceps sobolifera (CS) has been traditionally utilized as an ethnic remedy for various health conditions, including chronic kidney diseases, anti-fatigue interventions, and management of chronic inflammation. Notably, CS is recognized for its substantial content of bioactive compounds, among which nucleosides prominently feature as constituents with diverse therapeutic advantages. AIM OF THE STUDY: This study aims to investigate the effects of CS on testosterone secretion in Leydig cells and explore the underlying mechanism. MATERIALS AND METHODS: Leydig cells were isolated from rat testes to establish a primary rat Leydig cells model. Cell proliferation and testosterone secretion were assessed via the methyl-piperidino-pyrazole (MTT) assay and enzyme-linked immunosorbent assay (ELISA), respectively. Samples earmarked for RNA sequencing (RNA-Seq) analysis facilitated the identification of significantly differentially expressed genes (DEGs), and we conducted Gene Ontology (GO)/Kyoto Encyclopedia of Genes and Genomes (KEGG) functional annotation and enrichment analyses. The veracity of our findings was validated through quantitative real time polymerase chain reaction (qRT-PCR) and western blotting. RESULTS: The results showed that CS and guanosine could promote Leydig cell proliferation and bolster testosterone secretion. Our integrative analysis of metabolomics and transcriptomics has unveiled the potential mechanisms governing testosterone synthesis. Specifically, metabolomics has illuminated striking correlations within cholesterol metabolism, and bile secretion. Concurrently, transcriptomics has underscored the pivotal roles played by the cyclic adenosine monophosphate (cAMP) signaling pathway and steroid hormone biosynthesis. Furthermore, our investigation has demonstrated CS's aptitude in elevating the expression of proteins and genes. Notably, our findings have elucidated that these effects can be mitigated by protein kinase A (PKA) and adenylate cyclase (AC) specific inhibitors. CONCLUSION: This study delineates the cAMP-PKA pathways as plausible mechanisms underpinning the testosterone-enhancing properties of CS, with guanosine emerging as a fundamental bioactive constituent.

HIV-1 RNAs whose transcription initiates from the third deoxyguanosine of GGG tract in the 5' long terminal repeat serve as a dominant genome for efficient provirus DNA formation.

Unspliced HIV-1 RNAs function as messenger RNAs for Gag or Gag-Pol polyproteins and progeny genomes packaged into virus particles. Recently, it has been reported that fate of the RNAs might be primarily determined, depending on transcriptional initiation sites among three consecutive deoxyguanosine residues (GGG tract) downstream of TATA-box in the 5' long terminal repeat (LTR). Although HIV-1 RNA transcription starts mostly from the first deoxyguanosine of the GGG tract and often from the second or third deoxyguanosine, RNAs beginning with one guanosine (G1-form RNAs), whose transcription initiates from the third deoxyguanosine, were predominant in HIV-1 particles. Despite selective packaging of G1-form RNAs into virus particles, its biological impact during viral replication remains to be determined. In this study, we revealed that G1-form RNAs are primarily selected as a template for provirus DNA rather than other RNAs. In competitions between HIV-1 and lentiviral vector transcripts in virus-producing cells, approximately 80% of infectious particles were found to generate provirus using HIV-1 transcripts, while lentiviral vector transcripts were conversely selected when we used HIV-1 mutants in which the third deoxyguanosine in the GGG tract was replaced with deoxythymidine or deoxycytidine (GGT or GGC mutants, respectively). In the other analyses of proviral sequences after infection with an HIV-1 mutant in which the GGG tract in 3' LTR was replaced with TTT, most proviral sequences of the GGG-tract region in 5' LTR were found to be TTG, which is reasonably generated using the G1-form transcripts. Our results indicate that the G1-form RNAs serve as a dominant genome to establish provirus DNA.IMPORTANCESince the promoter for transcribing HIV-1 RNA is unique, all viral elements including genomic RNA and viral proteins have to be generated by the unique transcripts through ingenious mechanisms including RNA splicing and frameshifting during protein translation. Previous studies suggested a new mechanism for diversification of HIV-1 RNA functions by heterogeneous transcriptional initiation site usage; HIV-1 RNAs whose transcription initiates from a certain nucleotide were predominant in virus particles. In this study, we established two methods to analyze heterogenous transcriptional initiation site usage by HIV-1 during viral infection and showed that RNAs beginning with one guanosine (G1-form RNAs), whose transcription initiates from the third deoxyguanosine of the GGG tract in 5' LTR, were primarily selected as viral genome in infectious particles and thus are used as a template to generate provirus for continuous replication. This study provides insights into the mechanism for diversification of unspliced RNA functions and requisites of lentivirus infectivity.

Forodesine Enhances Immune Responses through Guanosine-Mediated TLR7 Activation while Preventing Graft-versus-Host Disease.

Recent evidence indicates that specific types of nuclear acids, including guanosine and its derivatives, act as natural ligands for TLR7. This led us to hypothesize that purine nucleoside phosphorylase inhibitors not only can induce apoptosis of T cells but also can lead to TLR7 activation by accumulation of guanine nucleosides, in particular under systemic inflammation, where damaged tissues release a large amount of nucleotides. We demonstrate in the present study that a purine nucleoside phosphorylase inhibitor, forodesine, can reduce the disease severity and prolong the survival in a xenogeneic mouse model of graft-versus-host disease (GVHD). Guanine nucleosides were undetectable in mice during GVHD but increased significantly following forodesine treatment. Our in vitro experiments showed that forodesine enhanced guanosine-mediated cytokine production from APCs, including alveolar macrophages and plasmacytoid dendritic cells, through TLR7 signaling. Forodesine also enhanced Ag-presenting capacity, as demonstrated by increased CD8+ T cell proliferation and higher secretion of IFN-γ and IL-12p40 in an MLR with plasmacytoid dendritic cells. Furthermore, forodesine stimulated IFN-γ production from activated T cells in the presence of a low concentration of guanosine while inhibiting their proliferation and inducing apoptotic cell death. Although forodesine ameliorated GVHD severity, mice treated with forodesine showed significantly higher levels of multiple proinflammatory cytokines and chemokines in plasma, suggesting in vivo upregulation of TLR7 signaling. Our study suggests that forodesine may activate a wide range of immune cells, including T cells, through TLR7 stimulation while inhibiting GVHD by inducing apoptosis of T cells, after allogeneic hematopoietic stem cell transplant.

Extracellular Guanosine and Guanine Nucleotides Decrease Viability of Human Breast Cancer SKBR-3 Cells.

Though the physiological effects of adenosine and adenine nucleotides on purinergic receptors in cancer cells have been well studied, the influence of extracellular guanosine and guanine nucleotides on breast cancer cells remains unclear. Here, we show that extracellular guanosine and guanine nucleotides decrease the viability and proliferation of human breast cancer SKBR-3 cells. Treatment with guanosine or guanine nucleotides increased mitochondrial production of reactive oxygen species (ROS), and modified the cell cycle. Guanosine-induced cell death was suppressed by treatment with adenosine or the equilibrium nucleoside transporter (ENT) 1/2 inhibitor dipyridamole, but was not affected by adenosine receptor agonists or antagonists. These results suggest that guanosine inhibits adenosine uptake through ENT1/2, but does not antagonize adenosine receptors. In contrast, guanosine triphosphate (GTP)-induced cell death was suppressed not only by adenosine and dipyridamole, but also by the A1 receptor agonist 2-chloro-N6-cyclopentyladenosine (CCPA), suggesting that GTP-induced cell death is mediated in part by an antagonistic effect on adenosine A1 receptor. Thus, both guanosine and GTP induce apoptosis of breast cancer cells, but via at least partially different mechanisms.

Identification and validation of N7 -methylguanosine-associated gene NCBP1 as prognostic and immune-associated biomarkers in breast cancer patients.

We intend to evaluate the importance of N7 -methylguanosine (m7G) for the prognosis of breast cancer (BC). We gained 29 m7G-related genes from the published literature and among them, 16 m7G-related genes were found to have differential expression. Five differentially expressed genes (CYFIP1, EIF4E, EIF4E3, NCBP1 and WDR4) were linked to overall survival. This suggests that m7G-related genes might be prognostic or therapeutic targets for BC patients. We put the five genes to LASSO regression analysis to create a four-gene signature, including EIF4E, EIF4E3, WDR4 and NCBP1, that divides samples into two risky groups. Survival was drastically worsened in a high-risk group (p < 0.001). The signature's predictive capacity was demonstrated using ROC (10-year AUC 0.689; 10-year AUC 0.615; 3-year AUC 0.602). We found that immune status was significantly different between the two risk groups. In particular, NCBP1 also has a poor prognosis, with higher diagnostic value in ROC. NCBP1 also has different immune states according to its high or low expression. Meanwhile, knockdown of NCBP1 suppresses BC malignancy in vitro. Therefore, m7G RNA regulators are crucial participants in BC and four-gene mRNA levels are important predictors of prognosis. NCBP1 plays a critical target of m7G mechanism in BC.

Recent Advances in Adenosine-to-Inosine RNA Editing in Cancer.

RNA epigenetics, or epitranscriptome, is a growing group of RNA modifications historically classified into two categories: RNA editing and RNA modification. RNA editing is usually understood as post-transcriptional RNA processing (except capping, splicing and polyadenylation) that changes the RNA nucleotide sequence encoded by the genome. This processing can be achieved through the insertion or deletion of nucleotides or deamination of nucleobases, generating either standard nucleotides such as uridine (U) or the rare nucleotide inosine (I). Adenosine-to-inosine (A-to-I) RNA editing is the most prevalent type of RNA modification in mammals and is catalyzed by adenosine deaminase acting on the RNA (ADAR) family of enzymes that recognize double-stranded RNAs (dsRNAs). Inosine mimics guanosine (G) in base pairing with cytidine (C), thereby A-to-I RNA editing alters dsRNA secondary structure. Inosine is also recognized as guanosine by the splicing and translation machineries, resulting in mRNA alternative splicing and protein recoding. Therefore, A-to-I RNA editing is an important mechanism that causes and regulates "RNA mutations" in both normal physiology and diseases including cancer. In this chapter, we reviewed current paradigms and developments in the field of A-to-I RNA editing in the context of cancer.

Compatibility of Nucleobases Containing Pt(II) Complexes with Red Blood Cells for Possible Drug Delivery Applications.

The therapeutic advantages of some platinum complexes as major anticancer chemotherapeutic agents and of nucleoside analogue-based compounds as essential antiviral/antitumor drugs are widely recognized. Red blood cells (RBCs) offer a potential new strategy for the targeted release of therapeutic agents due to their biocompatibility, which can protect loaded drugs from inactivation in the blood, thus improving biodistribution. In this study, we evaluated the feasibility of loading model nucleobase-containing Pt(II) complexes into human RBCs that were highly stabilized by four N-donors and susceptible to further modification for possible antitumor/antiviral applications. Specifically, platinum-based nucleoside derivatives [PtII(dien)(N7-Guo)]2+, [PtII(dien)(N7-dGuo)]2+, and [PtII(dien)(N7-dGTP)] (dien = diethylenetriamine; Guo = guanosine; dGuo = 2'-deoxy-guanosine; dGTP = 5'-(2'-deoxy)-guanosine-triphosphate) were investigated. These Pt(II) complexes were demonstrated to be stable species suitable for incorporation into RBCs. This result opens avenues for the possible incorporation of other metalated nucleobases analogues, with potential antitumor and/or antiviral activity, into RBCs.

Identification of Bona Fide RNA Editing Sites: History, Challenges, and Opportunities.

Adenosine-to-inosine (A-to-I) RNA editing, catalyzed by the adenosine deaminase acting on the RNA (ADAR) family of enzymes of which there are three members (ADAR1, ADAR2, and ADAR3), is a major gene regulatory mechanism that diversifies the transcriptome. It is widespread in many metazoans, including humans. As inosine is interpreted by cellular machineries mainly as guanosine, A-to-I editing effectively gives A-to-G nucleotide changes. Depending on its location, an editing event can generate new protein isoforms or influence other RNA processing pathways. Researchers have found that ADAR-mediated editing performs diverse functions. For example, it enables living organisms such as cephalopods to adapt rapidly to fluctuating environmental conditions such as water temperature. In development, the loss of ADAR1 is embryonically lethal partly because endogenous double-stranded RNAs (dsRNAs) are no longer marked by inosines, which signal "self", and thus cause the melanoma differentiation-associated protein 5 (MDA5) sensor to trigger a deleterious interferon response. Hence, ADAR1 plays a key role in preventing aberrant activation of the innate immune system. Furthermore, ADAR enzymes have been implicated in myriad human diseases. Intriguingly, some cancer cells are known to exploit ADAR1 activity to dodge immune responses. However, the exact identities of immunogenic RNAs in different biological contexts have remained elusive. Consequently, there is tremendous interest in identifying inosine-containing RNAs in the cell.The identification of A-to-I RNA editing sites is dependent on the sequencing of nucleic acids. Technological and algorithmic advancements over the past decades have revolutionized the way editing events are detected. At the beginning, the discovery of editing sites relies on Sanger sequencing, a first-generation technology. Both RNA, which is reverse transcribed into complementary DNA (cDNA), and genomic DNA (gDNA) from the same source are analyzed. After sequence alignment, one would require an adenosine to be present in the genome but a guanosine to be detected in the RNA sample for a position to be declared as an editing site. However, an issue with Sanger sequencing is its low throughput. Subsequently, Illumina sequencing, a second-generation technology, was invented. By permitting the simultaneous interrogation of millions of molecules, it enables many editing sites to be identified rapidly. However, a key challenge is that the Illumina platform produces short sequencing reads that can be difficult to map accurately. To tackle the challenge, we and others developed computational workflows with a series of filters to discard sites that are likely to be false positives. When Illumina sequencing data sets are properly analyzed, A-to-G variants should emerge as the most dominant mismatch type. Moreover, the quantitative nature of the data allows us to build a comprehensive atlas of editing-level measurements across different biological contexts, providing deep insights into the spatiotemporal dynamics of RNA editing. However, difficulties remain in identifying true A-to-I editing sites in short protein-coding exons or in organisms and diseases where DNA mutations and genomic polymorphisms are prevalent and mostly unknown. Nanopore sequencing, a third-generation technology, promises to address the difficulties, as it allows native RNAs to be sequenced without conversion to cDNA, preserving base modifications that can be directly detected through machine learning. We recently demonstrated that nanopore sequencing could be used to identify A-to-I editing sites in native RNA directly. Although further work is needed to enhance the detection accuracy in single molecules from fewer cells, the nanopore technology holds the potential to revolutionize epitranscriptomic studies.