Identification of exceptionally potent adenosine deaminases RNA editors from high body temperature organisms.

The most abundant form of RNA editing in metazoa is the deamination of adenosines into inosines (A-to-I), catalyzed by ADAR enzymes. Inosines are read as guanosines by the translation machinery, and thus A-to-I may lead to protein recoding. The ability of ADARs to recode at the mRNA level makes them attractive therapeutic tools. Several approaches for Site-Directed RNA Editing (SDRE) are currently under development. A major challenge in this field is achieving high on-target editing efficiency, and thus it is of much interest to identify highly potent ADARs. To address this, we used the baker yeast Saccharomyces cerevisiae as an editing-naïve system. We exogenously expressed a range of heterologous ADARs and identified the hummingbird and primarily mallard-duck ADARs, which evolved at 40-42°C, as two exceptionally potent editors. ADARs bind to double-stranded RNA structures (dsRNAs), which in turn are temperature sensitive. Our results indicate that species evolved to live with higher core body temperatures have developed ADAR enzymes that target weaker dsRNA structures and would therefore be more effective than other ADARs. Further studies may use this approach to isolate additional ADARs with an editing profile of choice to meet specific requirements, thus broadening the applicability of SDRE.

The Profiles and Functions of RNA Editing Sites Associated with High-Altitude Adaptation in Goats.

High-altitude environments dramatically influenced the genetic evolution of vertebrates. However, little is known about the role of RNA editing on high-altitude adaptation in non-model species. Here, we profiled the RNA editing sites (RESs) of heart, lung, kidney, and longissimus dorsi muscle from Tibetan cashmere goats (TBG, 4500 m) and Inner Mongolia cashmere goats (IMG, 1200 m) to reveal RNA editing-related functions of high-altitude adaptation in goats. We identified 84,132 high-quality RESs that were unevenly distributed across the autosomes in TBG and IMG, and more than half of the 10,842 non-redundant editing sites were clustered. The majority (62.61%) were adenosine-to-inosine (A-to-I) sites, followed by cytidine-to-uridine (C-to-U) sites (19.26%), and 32.5% of them had a significant correlation with the expression of catalytic genes. Moreover, A-to-I and C-to-U RNA editing sites had different flanking sequences, amino acid mutations, and alternative splicing activity. TBG had higher editing levels of A-to-I and C-to-U than IMG in the kidney, whereas a lower level was found in the longissimus dorsi muscle. Furthermore, we identified 29 IMG and 41 TBG population-specific editing sites (pSESs) and 53 population-differential editing sites (pDESs) that were functionally involved in altering RNA splicing or recoding protein products. It is worth noting that 73.3% population-differential, 73.2% TBG-specific, and 80% IMG-specific A-to-I sites were nonsynonymous sites. Moreover, the pSESs and pDESs editing-related genes play critical functions in energy metabolisms such as ATP binding molecular function, translation, and adaptive immune response, which may be linked to goat high-altitude adaptation. Our results provide valuable information for understanding the adaptive evolution of goats and studying plateau-related diseases.

Profiling RNA Editing in Single Cells.

RNA editing is a widespread molecular phenomenon occurring in a variety of organisms. In humans, it mainly involves the deamination of adenosine to inosine (A-to-I) in double-stranded RNAs by ADAR enzymes. A-to-I RNA editing has been investigated in different tissues as well as in diverse experimental and pathological conditions. By contrast, its biological role in single cells has not been explored in depth. Recent methodologies for cell sorting in combination with deep sequencing technologies have enabled the study of eukaryotic transcriptomes at single cell resolution, paving the way to the profiling of their epitranscriptomic dynamics.Here we describe a step-by-step protocol to detect and characterize A-to-I events occurring in publicly available single-cell RNAseq experiments from human alpha and beta pancreatic cells.

Effects of different levels of inosine-5'-monophosphate (5'-IMP) supplementation on the growth performance and meat quality of finishing pigs (75 to 100 kg).

This study aimed to assess the effects of dietary supplementation of inosine-5'-monophosphate (5'-IMP) on energy efficiency, growth performance, carcass characteristics, meat quality, oxidative status, and biochemical profile of blood plasma in finishing pigs. Fifty-four crossbred castrated male pigs were distributed in a randomized block design consisting of nine blocks, with six treatments per block and one animal per treatment per block. Experimental diets were as follows: positive control diet (PC, 3300 kcal ME/kg), negative control diet (NC, 3200 kcal ME/kg), and four diets prepared by supplementing the NC diet with 0.050%, 0.100%, 0.150%, or 0.200% 5'-IMP. Based on regression analysis, supplementation with 0.129% 5'-IMP increased average daily weight gain (1.30 kg). Backfat thickness, pH45minutes and redness of m. Longissimus Lumborum (LL) increased linearly with 5'-IMP supplementation level. Drip loss and pH at 24 h post-slaughter had a quadratic response to 5'-IMP supplementation. It is concluded that 5'-IMP supplementation positively influenced growth performance, carcass characteristics and LL meat quality in finishing barrows.

Diagnostic kit based on halloysite nanoclay-ionic liquid nanocomposite modified electrode for electrochemical determination of cancer biomarker.

Nucleic acid hybridization is occurred between the selective single-stranded nucleic acid sequence and its target sequence, which is one of the essential procedure for electrochemical detection of nucleic acid. microRNA-21 (miRNA-21) is known as a biomarker in various cancers. The determination of miRNA-21 was attained through by hybridization of inosine substituted miRNA-21 specific DNA probe (Pinosine) with its target miRNA-21. In this study, the surface of pencil graphite electrode (PGE) was firstly modified with halloysite nanoclay-ionic liquid (HNT/IL) nanocomposite. The characterization of surface was performed by Scanning Electron Microscope (SEM) images and Energy Dispersive X-Ray Analysis (EDX) analysis, and the differences at surface modifications were also shown by electrochemical methods with electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). For sensitive and selective determination of miRNA-21, Pinosine and target miRNA concentration, immobilization and hybridization time were optimized by using HNT/IL modified PGE in combination with differential pulse voltammetry (DPV). The detection limit was achieved as 0.17 µg/mL (equals to 23.69 nM) in the linear range of 0.25-2 µg/mL miRNA-21. The selectivity of voltammetric method based on HNT/IL-PGE developed for miRNA-21 was examined in the presence of mismatch (MM) and non-complementary (NC) sequences. Because miRNA-21 is over-expressed in cancer cells, it has been tested in total RNA samples isolated from cancer cell line (breast cancer cell line, MCF-7). In the total RNA samples obtained from MCF-7, the detection limit was calculated as 0.28 µg/mL in the linear range of 1-4 µg/mL. Besides, the healthy cell line (human embryonic kidney cell line, HEK-293) was used as a control group and the results obtained by MCF-7 total RNA samples were compared to the results using HEK-293 total RNA samples in terms of miRNA-21 level.

RNA editing of microtubule-associated protein tau circular RNAs promotes their translation and tau tangle formation.

Aggregation of the microtubule-associated protein tau characterizes tauopathies, including Alzheimer's disease and frontotemporal lobar degeneration (FTLD-Tau). Gene expression regulation of tau is complex and incompletely understood. Here we report that the human tau gene (MAPT) generates two circular RNAs (circRNAs) through backsplicing of exon 12 to either exon 7 (12→7 circRNA) or exon 10 (12→10 circRNA). Both circRNAs lack stop codons. The 12→7 circRNA contains one start codon and is translated in a rolling circle, generating a protein consisting of multimers of the microtubule-binding repeats R1-R4. For the 12→10 circRNA, a start codon can be introduced by two FTLD-Tau mutations, generating a protein consisting of multimers of the microtubule-binding repeats R2-R4, suggesting that mutations causing FTLD may act in part through tau circRNAs. Adenosine to inosine RNA editing dramatically increases translation of circRNAs and, in the 12→10 circRNA, RNA editing generates a translational start codon by changing AUA to AUI. Circular tau proteins self-aggregate and promote aggregation of linear tau proteins. Our data indicate that adenosine to inosine RNA editing initiates translation of human circular tau RNAs, which may contribute to tauopathies.

Spatiotemporal and genetic regulation of A-to-I editing throughout human brain development.

Posttranscriptional RNA modifications by adenosine-to-inosine (A-to-I) editing are abundant in the brain, yet elucidating functional sites remains challenging. To bridge this gap, we investigate spatiotemporal and genetically regulated A-to-I editing sites across prenatal and postnatal stages of human brain development. More than 10,000 spatiotemporally regulated A-to-I sites were identified that occur predominately in 3' UTRs and introns, as well as 37 sites that recode amino acids in protein coding regions with precise changes in editing levels across development. Hyper-edited transcripts are also enriched in the aging brain and stabilize RNA secondary structures. These features are conserved in murine and non-human primate models of neurodevelopment. Finally, thousands of cis-editing quantitative trait loci (edQTLs) were identified with unique regulatory effects during prenatal and postnatal development. Collectively, this work offers a resolved atlas linking spatiotemporal variation in editing levels to genetic regulatory effects throughout distinct stages of brain maturation.

Structural basis for sequence-independent substrate selection by eukaryotic wobble base tRNA deaminase ADAT2/3.

The essential deamination of adenosine A34 to inosine at the wobble base is the individual tRNA modification with the greatest effects on mRNA decoding, empowering a single tRNA to translate three different codons. To date, many aspects of how eukaryotic deaminases specifically select their multiple substrates remain unclear. Here, using cryo-EM, we present the structure of a eukaryotic ADAT2/3 deaminase bound to a full-length tRNA, revealing that the enzyme distorts the anticodon loop, but in contrast to the bacterial enzymes, selects its substrate via sequence-independent contacts of eukaryote-acquired flexible or intrinsically unfolded motifs distal from the conserved catalytic core. A gating mechanism for substrate entry to the active site is identified. Our multi-step tRNA recognition model yields insights into how RNA editing by A34 deamination evolved, shaped the genetic code, and directly impacts the eukaryotic proteome.

Pathogenic variant c.1052T>A (p.Leu351Gln) in adenosine deaminase 2 impairs secretion and elevates type I IFN responsive gene expression.

Background: Adenosine deaminase 2 (ADA2) is a homodimeric, extracellular enzyme and putative growth factor that is produced by cells of the myeloid lineage and, catalytically, deaminates extracellular adenosine to inosine. Loss-of-(catalytic)-function variants in the ADA2 gene are associated with Deficiency of ADA2 (DADA2), an autosomal recessive disease associated with an unusually broad range of inflammatory manifestations including vasculitis, hematological defects and cytopenia. Previous work by our group led to the identification of ADA2 variants of novel association with DADA2, among which was a unique c.1052T>A (p.Leu351Gln; herein referred to as L351Q) variant located in the catalytic domain of the protein. Methods: Mammalian (Flp-IN CHO) cells were engineered to stably express wild-type ADA2 and ADA2 protein variants, including the pathogenic L351Q variant identified in DADA2 patients. An enzyme assay and immunoblotting were used to assess ADA2 catalytic activity and secretion, respectively, and the outcome of experimentally induced inhibition of protein processing (Golgi transport and N-linked glycosylation) was assessed. Reverse transcription quantitative real-time PCR (RT-qPCR) was applied to determine the relative expression of Type I Interferon stimulated genes (ISGs), IFIT3 and IRF7. Results: In addition to abrogating catalytic activity, the L351Q variant impaired secretion of L351Q ADA2 resulting in an intracellular accumulation of L351Q ADA2 protein that was not observed in cells expressing wild-type ADA2 or other ADA2 protein variants. Retention of L351Q ADA2 was not attributable to impaired glycosylation on neighboring asparagine residues and did not impact cell growth or integrity. Constitutive expression of Type I ISGs IFIT3 and IRF7 was observed in cells expressing L351Q ADA2. Conclusions: The impaired secretion of L351Q ADA2 may be an important factor leading to the severe phenotype observed in patients with this variant further emphasizing the importance of assessing impacts beyond catalytic activity when evaluating genotype-phenotype relationships in DADA2.

Unveiling the functional and evolutionary landscape of RNA editing in chicken using genomics and transcriptomics.

The evolutionary and functional features of RNA editing are well studied in mammals, cephalopods, and insects, but not in birds. Here, we integrated transcriptomic and whole-genomic analyses to exhaustively characterize the expansive repertoire of adenosine-to-inosine (A-to-I) RNA editing sites (RESs) in the chicken. In addition, we investigated the evolutionary status of the chicken editome as a potential mechanism of domestication. We detected the lowest editing level in the liver of chickens, compared to muscles in humans, and found higher editing activity and specificity in the brain than in non-neural tissues, consistent with the brain's functional complexity. To a certain extent, specific editing activity may account for the specific functions of tissues. Our results also revealed that sequences critical to RES secondary structures remained conserved within avian evolution. Furthermore, the RNA editome was shaped by purifying selection during chicken domestication and most RESs may have served as a selection pool for a few functional RESs involved in chicken domestication, including evolution of nervous and immune systems. Regulation of RNA editing in chickens by adenosine deaminase acting on RNA (ADAR) enzymes may be affected by non-ADAR factors whose expression levels changed widely after ADAR knockdown. Collectively, we provide comprehensive lists of candidate RESs and non-ADAR-editing regulators in the chicken, thus contributing to our current understanding of the functions and evolution of RNA editing in animals.

Immunosuppressive lncRNA LINC00624 promotes tumor progression and therapy resistance through ADAR1 stabilization.

BACKGROUND: Despite the success of HER2-targeted therapy in achieving prolonged survival in approximately 50% of treated individuals, treatment resistance is still an important challenge for HER2+ breast cancer (BC) patients. The influence of both adaptive and innate immune responses on the therapeutic outcomes of HER2+BC patients has been extensively demonstrated. METHODS: Long non-coding RNAs expressed in non-pathological complete response (pCR) HER2 positive BC were screened and validated by RNA-seq. Survival analysis were made by Kaplan-Meier method. Cell death assay and proliferation assay were performed to confirm the phenotype of LINC00624. RT-qPCR and western blot were used to assay the IFN response. Xenograft mouse model were used for in vivo confirmation of anti-neu treatment resistance. RNA pull-down and immunoblot were used to confirm the interaction of ADAR1 and LINC00624. ADAR1 recombinant protein were purified from baculovirus expression system. B16-OVA cells were used to study antigen presentation both in vitro and in vivo. Flow cytometry was used to determine the tumor infiltrated immune cells of xenograft model. Antisense oligonucleotides (ASOs) were used for in vivo treatment. RESULTS: In this study, we found that LINC00624 blocked the antitumor effect of HER2- targeted therapy both in vitro and in vivo by inhibiting type I interferon (IFN) pathway activation. The double-stranded RNA-like structure of LINC00624 can bind and be edited by the adenosine (A) to inosine (I) RNA-editing enzyme adenosine deaminase RNA specific 1 (ADAR1), and this editing has been shown to release the growth inhibition and attenuate the innate immune response caused by the IFN response. Notably, LINC00624 promoted the stabilization of ADAR1 by inhibiting its ubiquitination-induced degradation triggered by ß-TrCP. In contrast, LINC00624 inhibited major histocompatibility complex (MHC) class I antigen presentation and limited CD8+T cell infiltration in the cancer microenvironment, resulting in immune checkpoint blockade inhibition and anti-HER2 treatment resistance mediated through ADAR1. CONCLUSIONS: In summary, these results suggest that LINC00624 is a cancer immunosuppressive lncRNA and targeting LINC00624 through ASOs in tumors expressing high levels of LINC00624 has great therapeutic potential in future clinical applications.

Suppressed renoprotective purines in COVID-19 patients with acute kidney injury.

Acute kidney injury (AKI) is common in patients hospitalized for COVID-19, complicating their clinical course and contributing to worse outcomes. Animal studies show that adenosine, inosine and guanosine protect the kidney against some types of AKI. However, until now there was no evidence in patients supporting the possibility that abnormally low kidney levels of adenosine, inosine and guanosine contribute to AKI. Here, we addressed the question as to whether these renoprotective purines are altered in the urine of COVID-19 patients with AKI. Purines were measured by employing ultra-high-performance liquid chromatography-tandem mass spectrometry with stable-isotope-labeled internal standards for each purine of interest. Compared with COVID-19 patients without AKI (n = 23), COVID-19 patients with AKI (n = 20) had significantly lower urine levels of adenosine (P < 0.0001), inosine (P = 0.0008), and guanosine (P = 0.0008) (medians reduced by 85%, 48% and 61%, respectively) and lower levels (P = 0.0003; median reduced by 67%) of the 2nd messenger for A2A and A2B adenosine receptors, i.e., 3',5'-cAMP. Moreover, in COVID-19 patients with AKI, urine levels of 8-aminoguanine (endogenous inhibitor of inosine and guanosine metabolism) were nearly abolished (P < 0.0001). In contrast, the "upstream" precursors of renoprotective purines, namely 5'-AMP and 5'-GMP, were not significantly altered in COVID-19 patients with AKI, suggesting defective conversion of these precursors by CD73 (converts 5'-AMP to adenosine and 5'-GMP to guanosine). These findings imply that an imbalance in renoprotective purines may contribute to AKI in COVID-19 patients and that pharmacotherapy targeted to restore levels of renoprotective purines may attenuate the risk of AKI in susceptible patients with COVID-19.

Response of circulating metabolites to an oral glucose challenge and risk of cardiovascular disease and mortality in the community.

BACKGROUND: New biomarkers to identify cardiovascular disease (CVD) risk earlier in its course are needed to enable targeted approaches for primordial prevention. We evaluated whether intraindividual changes in blood metabolites in response to an oral glucose tolerance test (OGTT) may provide incremental information regarding the risk of future CVD and mortality in the community. METHODS: An OGTT (75 g glucose) was administered to a subsample of Framingham Heart Study participants free from diabetes (n = 361). Profiling of 211 plasma metabolites was performed from blood samples drawn before and 2 h after OGTT. The log2(post/pre) metabolite levels (Δmetabolites) were related to incident CVD and mortality in Cox regression models adjusted for age, sex, baseline metabolite level, systolic blood pressure, hypertension treatment, body mass index, smoking, and total/high-density lipoprotein cholesterol. Select metabolites were related to subclinical cardiometabolic phenotypes using Spearman correlations adjusted for age, sex, and fasting metabolite level. RESULTS: Our sample included 42% women, with a mean age of 56 ± 9 years and a body mass index of 30.2 ± 5.3 kg/m2. The pre- to post-OGTT changes (Δmetabolite) were non-zero for 168 metabolites (at FDR ≤ 5%). A total of 132 CVD events and 144 deaths occurred during median follow-up of 24.9 years. In Cox models adjusted for clinical risk factors, four Δmetabolites were associated with incident CVD (higher glutamate and deoxycholate, lower inosine and lysophosphatidylcholine 18:2) and six Δmetabolites (higher hydroxyphenylacetate, triacylglycerol 56:5, alpha-ketogluturate, and lower phosphatidylcholine 32:0, glucuronate, N-monomethyl-arginine) were associated with death (P < 0.05). Notably, baseline metabolite levels were not associated with either outcome in models excluding Δmetabolites. The Δmetabolites exhibited varying cross-sectional correlation with subclinical risk factors such as visceral adiposity, insulin resistance, and vascular stiffness, but overall relations were modest. Significant Δmetabolites included those with established roles in cardiometabolic disease (e.g., glutamate, alpha-ketoglutarate) and metabolites with less defined roles (e.g., glucuronate, lipid species). CONCLUSIONS: Dynamic changes in metabolite levels with an OGTT are associated with incident CVD and mortality and have potential relevance for identifying CVD risk earlier in its development and for discovering new potential therapeutic targets.

Effects of Donepezil Treatment on Brain Metabolites, Gut Microbiota, and Gut Metabolites in an Amyloid Beta-Induced Cognitive Impairment Mouse Pilot Model.

Accumulated clinical and biomedical evidence indicates that the gut microbiota and their metabolites affect brain function and behavior in various central nervous system disorders. This study was performed to investigate the changes in brain metabolites and composition of the fecal microbial community following injection of amyloid ß (Aß) and donepezil treatment of Aß-injected mice using metataxonomics and metabolomics. Aß treatment caused cognitive dysfunction, while donepezil resulted in the successful recovery of memory impairment. The Aß + donepezil group showed a significantly higher relative abundance of Verrucomicrobia than the Aß group. The relative abundance of 12 taxa, including Blautia and Akkermansia, differed significantly between the groups. The Aß + donepezil group had higher levels of oxalate, glycerol, xylose, and palmitoleate in feces and oxalate, pyroglutamic acid, hypoxanthine, and inosine in brain tissues than the Aß group. The levels of pyroglutamic acid, glutamic acid, and phenylalanine showed similar changes in vivo and in vitro using HT-22 cells. The major metabolic pathways in the brain tissues and gut microbiota affected by Aß or donepezil treatment of Aß-injected mice were related to amino acid pathways and sugar metabolism, respectively. These findings suggest that alterations in the gut microbiota might influence the induction and amelioration of Aß-induced cognitive dysfunction via the gut-brain axis. This study could provide basic data on the effects of Aß and donepezil on gut microbiota and metabolites in an Aß-induced cognitive impairment mouse model.

ADAR regulates APOL1 via A-to-I RNA editing by inhibition of MDA5 activation in a paradoxical biological circuit.

APOL1 risk variants are associated with increased risk of kidney disease in patients of African ancestry, but not all individuals with the APOL1 high-risk genotype develop kidney disease. As APOL1 gene expression correlates closely with the degree of kidney cell injury in both cell and animal models, the mechanisms regulating APOL1 expression may be critical determinants of risk allele penetrance. The APOL1 messenger RNA includes Alu elements at the 3' untranslated region that can form a double-stranded RNA structure (Alu-dsRNA) susceptible to posttranscriptional adenosine deaminase acting on RNA (ADAR)-mediated adenosine-to-inosine (A-to-I) editing, potentially impacting gene expression. We studied the effects of ADAR expression and A-to-I editing on APOL1 levels in podocytes, human kidney tissue, and a transgenic APOL1 mouse model. In interferon-γ (IFN-γ)-stimulated human podocytes, ADAR down-regulates APOL1 by preventing melanoma differentiation-associated protein 5 (MDA5) recognition of dsRNA and the subsequent type I interferon (IFN-I) response. Knockdown experiments showed that recognition of APOL1 messenger RNA itself is an important contributor to the MDA5-driven IFN-I response. Mathematical modeling suggests that the IFN-ADAR-APOL1 network functions as an incoherent feed-forward loop, a biological circuit capable of generating fast, transient responses to stimuli. Glomeruli from human kidney biopsies exhibited widespread editing of APOL1 Alu-dsRNA, while the transgenic mouse model closely replicated the edited sites in humans. APOL1 expression in mice was inversely correlated with Adar1 expression under IFN-γ stimuli, supporting the idea that ADAR regulates APOL1 levels in vivo. ADAR-mediated A-to-I editing is an important regulator of APOL1 expression that could impact both penetrance and severity of APOL1-associated kidney disease.

Chemical Probe-Based Nanopore Sequencing to Selectively Assess the RNA Modifications.

Nanopore direct RNA sequencing (dRNA-Seq) reads reveal RNA modifications through consistent error profiles specific to a modified nucleobase. However, a null data set is required to identify actual RNA modification-associated errors for distinguishing it from confounding highly intrinsic sequencing errors. Here, we reveal that inosine creates a signature mismatch error in dRNA-Seq reads and obviates the need for a null data set by harnessing the selective reactivity of acrylonitrile for validating the presence of actual inosine modifications. Selective reactivity of acrylonitrile toward inosine altered multiple dRNA-Seq parameters like signal intensity and trace value. We also deduced the stoichiometry of inosine modification through deviation in signal intensity and trace value using this chemical biology approach. Furthermore, we devised Nano ICE-Seq, a protocol to overcome the low coverage issue associated with direct RNA sequencing. Taken together, our chemical probe-based approach may facilitate the knockout-free detection of disease-associated RNA modifications in clinical scenarios.

Novel amides of mycophenolic acid and some heterocyclic derivatives as immunosuppressive agents.

The group of 18 new amide derivatives of mycophenolic acid (MPA) and selected heterocyclic amines was synthesised as potential immunosuppressive agents functioning as inosine-5'-monophosphate dehydrogenase (IMPDH) uncompetitive inhibitors. The synthesis of 14 of them employed uronium-type activating system (TBTU/HOBt/DIPEA) while 4 of them concerned phosphonic acid anhydride method (T3P/Py) facilitating amides to be obtained in moderate to excellent yields without the need of phenolic group protection. Most of optimised protocols did not require complicated reaction work-ups, including chromatographic, solvent-consuming methods. The biological activity assay was performed on the T-Jurkat cell line and peripheral mononuclear blood cells (PBMCs) which are both dedicated for antiproliferative activity determination. Each of designed derivatives was characterised by reduced cytotoxicity and benzoxazole analogue (A2) revealed the most promising activity. Subsequently, an observed structure-activity relationship was discussed.

Inhibition of UBA6 by inosine augments tumour immunogenicity and responses.

Anti-cancer immunity and response to immune therapy is influenced by the metabolic states of the tumours. Immune checkpoint blockade therapy (ICB) is known to involve metabolic adaptation, however, the mechanism is not fully known. Here we show, by metabolic profiling of plasma samples from melanoma-bearing mice undergoing anti-PD1 and anti-CTLA4 combination therapy, that higher levels of purine metabolites, including inosine, mark ICB sensitivity. Metabolic profiles of ICB-treated human cancers confirm the association between inosine levels and ICB sensitivity. In mouse models, inosine supplementation sensitizes tumours to ICB, even if they are intrinsically ICB resistant, by enhancing T cell-mediated cytotoxicity and hence generating an immunologically hotter microenvironment. We find that inosine directly inhibits UBA6 in tumour cells, and lower level of UBA6 makes the tumour more immunogenic and this is reflected in favourable outcome following ICB therapy in human melanomas. Transplanted mouse melanoma and breast cancer cells with genetic ablation of Uba6 show higher sensitivity to ICB than wild type tumours. Thus, we provide evidence of an inosine-regulated UBA6-dependent pathway governing tumour-intrinsic immunogenicity and hence sensitivity to immune checkpoint inhibition, which might provide targets to overcome ICB resistance.

The Combined Escherichia coli Nissle 1917 and Tryptophan Treatment Modulates Immune and Metabolome Responses to Human Rotavirus Infection in a Human Infant Fecal Microbiota-Transplanted Malnourished Gnotobiotic Pig Model.

Human rotavirus (HRV) is a major cause of childhood diarrhea in developing countries where widespread malnutrition contributes to the decreased oral vaccine efficacy and increased prevalence of other enteric infections, which are major concerns for global health. Neonatal gnotobiotic (Gn) piglets closely resemble human infants in their anatomy, physiology, and outbred status, providing a unique model to investigate malnutrition, supplementations, and HRV infection. To understand the molecular signatures associated with immune enhancement and reduced diarrheal severity by Escherichia coli Nissle 1917 (EcN) and tryptophan (TRP), immunological responses and global nontargeted metabolomics and lipidomics approaches were investigated on the plasma and fecal contents of malnourished pigs transplanted with human infant fecal microbiota and infected with virulent (Vir) HRV. Overall, EcN + TRP combined (rather than individual supplement action) promoted greater and balanced immunoregulatory/immunostimulatory responses associated with greater protection against HRV infection and disease in malnourished humanized piglets. Moreover, EcN + TRP treatment upregulated the production of several metabolites with immunoregulatory/immunostimulatory properties: amino acids (N-acetylserotonin, methylacetoacetyl-CoA), lipids (gamma-butyrobetaine, eicosanoids, cholesterol-sulfate, sphinganine/phytosphingosine, leukotriene), organic compound (biliverdin), benzenoids (gentisic acid, aminobenzoic acid), and nucleotides (hypoxathine/inosine/xanthine, cytidine-5'-monophosphate). Additionally, the levels of several proinflammatory metabolites of organic compounds (adenosylhomocysteine, phenylacetylglycine, urobilinogen/coproporphyrinogen) and amino acid (phenylalanine) were reduced following EcN + TRP treatment. These results suggest that the EcN + TRP effects on reducing HRV diarrhea in neonatal Gn pigs were at least in part due to altered metabolites, those involved in lipid, amino acid, benzenoids, organic compounds, and nucleotide metabolism. Identification of these important mechanisms of EcN/TRP prevention of HRV diarrhea provides novel targets for therapeutics development. IMPORTANCE Human rotavirus (HRV) is the most common cause of viral gastroenteritis in children, especially in developing countries, where the efficacy of oral HRV vaccines is reduced. Escherichia coli Nissle 1917 (EcN) is used to treat enteric infections and ulcerative colitis while tryptophan (TRP) is a biomarker of malnutrition, and its supplementation can alleviate intestinal inflammation and normalize intestinal microbiota in malnourished hosts. Supplementation of EcN + TRP to malnourished humanized gnotobiotic piglets enhanced immune responses and resulted in greater protection against HRV infection and diarrhea. Moreover, EcN + TRP supplementation increased the levels of immunoregulatory/immunostimulatory metabolites while decreasing the production of proinflammatory metabolites in plasma and fecal samples. Profiling of immunoregulatory and proinflammatory biomarkers associated with HRV perturbations will aid in the identification of treatments against HRV and other enteric diseases in malnourished children.