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1.
Sci Rep ; 12(1): 12887, 2022 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-35902652

RESUMO

In observational studies, serum urate positively associates with cardiometabolic and kidney diseases. We analyzed data from a randomised placebo-controlled trial to determine whether moderate hyperuricemia induced by inosine affects cardiometabolic and kidney function markers. One hundred and twenty post-menopausal women were recruited into a 6-month randomised, double-blind, placebo-controlled trial of inosine for bone health. Change from baseline in the following pre-specified endpoints was analyzed: body mass index; blood pressure; lipid profile; C-reactive protein; fasting glucose; insulin; HbA1c; serum creatinine; and estimated glomerular filtration rate (eGFR). Despite increases in serum urate levels (+ 0.17 mmol/L at week 6, P < 0.0001), no significant between-group differences were observed in cardiometabolic markers, with the exception of lower fasting glucose concentrations with inosine at week 19. In the inosine group, change in serum urate correlated with change in serum creatinine (r = 0.41, P = 0.0012). However, there was no between-group difference in serum creatinine values. Over the entire study period, there was no significant difference in eGFR (ANCOVA P = 0.13). Reduction in eGFR was greater in the inosine group at Week 13 (mean difference - 4.6 mL/min/1.73 m2, false detection rate P = 0.025), with no between-group difference in eGFR at other time points. These data indicate that increased serum urate does not negatively influence body mass index, blood pressure, lipid profile, or glycaemic control. Serum urate changes associated with inosine intake correlate with changes in serum creatinine, but this does not lead to clinically important reduction in kidney function over 6 months.Clinical trial registration number: Australia and New Zealand Clinical Trials Registry (ACTRN12617000940370), registered 30/06/2017.


Assuntos
Doenças Cardiovasculares , Ácido Úrico , Biomarcadores , Doenças Cardiovasculares/tratamento farmacológico , Creatinina , Suplementos Nutricionais , Método Duplo-Cego , Feminino , Glucose , Humanos , Inosina , Rim , Lipídeos
2.
Int J Mol Sci ; 23(14)2022 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-35887219

RESUMO

Acute kidney injury (AKI) is a common complication of severe human diseases, resulting in increased morbidity and mortality as well as unfavorable long-term outcomes. Although the mammalian kidney is endowed with an amazing capacity to recover from AKI, little progress has been made in recent decades to facilitate recovery from AKI. To elucidate the early repair mechanisms after AKI, we employed the zebrafish pronephros injury model. Since damaged cells release large amounts of ATP and ATP-degradation products to signal apoptosis or necrosis to neighboring cells, we examined how depletion of purinergic and adenosine receptors impacts the directed cell migration that ensues immediately after a laser-induced tubular injury. We found that depletion of the zebrafish adenosine receptors adora1a, adora1b, adora2aa, and adora2ab significantly affected the repair process. Similar results were obtained after depletion of the purinergic p2ry2 receptor, which is highly expressed during zebrafish pronephros development. Released ATP is finally metabolized to inosine by adenosine deaminase. Depletion of zebrafish adenosine deaminases ada and ada2b interfered with the repair process; furthermore, combinations of ada and ada2b, or ada2a and ada2b displayed synergistic effects at low concentrations, supporting the involvement of inosine signaling in the repair process after a tubular injury. Our findings suggest that nucleotide-dependent signaling controls immediate migratory responses after tubular injury.


Assuntos
Injúria Renal Aguda , Peixe-Zebra , Injúria Renal Aguda/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Movimento Celular , Humanos , Inosina , Mamíferos/metabolismo , Nucleotídeos , Receptores Purinérgicos P1/metabolismo , Receptores Purinérgicos P2Y2 , Peixe-Zebra/metabolismo
3.
Molecules ; 27(14)2022 Jul 21.
Artigo em Inglês | MEDLINE | ID: mdl-35889517

RESUMO

Neurodegenerative diseases, such as Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), and multiple sclerosis (MS), currently represent major unmet medical needs. Therefore, novel therapeutic strategies are needed in order to improve patients' quality of life and prognosis. Since oxidative stress can be strongly involved in neurodegenerative diseases, the potential use of inosine, known for its antioxidant properties, in this context deserves particular attention. The protective action of inosine treatment could be mediated by its metabolite urate. Here, we review the current preclinical and clinical studies investigating the use of inosine in AD, PD, ALS, and MS. The most important properties of inosine seem to be its antioxidant action and its ability to raise urate levels and to increase energetic resources by improving ATP availability. Inosine appears to be generally safe and well tolerated; however, the possible formation of kidney stones should be monitored, and data on its effectiveness should be further explored since, so far, they have been controversial. Overall, inosine could be a promising potential strategy in the management of neurodegenerative diseases, and additional studies are needed in order to further investigate its safety and efficacy and its use as a complementary therapy along with other approved drugs.


Assuntos
Doença de Alzheimer , Esclerose Amiotrófica Lateral , Esclerose Múltipla , Doenças Neurodegenerativas , Doença de Parkinson , Doença de Alzheimer/tratamento farmacológico , Esclerose Amiotrófica Lateral/tratamento farmacológico , Antioxidantes/metabolismo , Antioxidantes/uso terapêutico , Humanos , Inosina/uso terapêutico , Esclerose Múltipla/tratamento farmacológico , Doenças Neurodegenerativas/metabolismo , Doença de Parkinson/tratamento farmacológico , Qualidade de Vida , Ácido Úrico/metabolismo
4.
Nutrients ; 14(14)2022 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-35889786

RESUMO

Inosine is a type of purine nucleoside, which is considered to a physiological energy source, and exerts a widely range of anti-inflammatory efficacy. The TLR4/MyD88/NF-κB signaling pathway is essential for preventing host oxidative stresses and inflammation, and represents a promising target for host-directed strategies to improve some forms of disease-related inflammation. In the present study, the results showed that inosine pre-intervention significantly suppressed the pulmonary elevation of pro-inflammatory cytokines (including tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß)), malondialdehyde (MDA), nitric oxide (NO), and reactive oxygen species (ROS) levels, and restored the pulmonary catalase (CAT), glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), and myeloperoxidase (MPO) activities (p < 0.05) in lipopolysaccharide (LPS)-treated mice. Simultaneously, inosine pre-intervention shifted the composition of the intestinal microbiota by decreasing the ratio of Firmicutes/Bacteroidetes, elevating the relative abundance of Tenericutes and Deferribacteres. Moreover, inosine pretreatment affected the TLR4/MyD88/NF-κB signaling pathway in the pulmonary inflammatory response, and then regulated the expression of pulmonary iNOS, COX2, Nrf2, HO-1, TNF-α, IL-1ß, and IL-6 levels. These findings suggest that oral administration of inosine pretreatment attenuates LPS-induced pulmonary inflammatory response by regulating the TLR4/MyD88/NF-κB signaling pathway, and ameliorates intestinal microbiota disorder.


Assuntos
Lesão Pulmonar Aguda , Inosina , NF-kappa B , Lesão Pulmonar Aguda/induzido quimicamente , Lesão Pulmonar Aguda/tratamento farmacológico , Animais , Inflamação , Inosina/uso terapêutico , Lipopolissacarídeos/efeitos adversos , Camundongos , Fator 88 de Diferenciação Mieloide/metabolismo , NF-kappa B/metabolismo , Transdução de Sinais , Receptor 4 Toll-Like/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
5.
Nature ; 607(7920): 784-789, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35859175

RESUMO

The RNA-editing enzyme adenosine deaminase acting on RNA 1 (ADAR1) limits the accumulation of endogenous immunostimulatory double-stranded RNA (dsRNA)1. In humans, reduced ADAR1 activity causes the severe inflammatory disease Aicardi-Goutières syndrome (AGS)2. In mice, complete loss of ADAR1 activity is embryonically lethal3-6, and mutations similar to those found in patients with AGS cause autoinflammation7-12. Mechanistically, adenosine-to-inosine (A-to-I) base modification of endogenous dsRNA by ADAR1 prevents chronic overactivation of the dsRNA sensors MDA5 and PKR3,7-10,13,14. Here we show that ADAR1 also inhibits the spontaneous activation of the left-handed Z-nucleic acid sensor ZBP1. Activation of ZBP1 elicits caspase-8-dependent apoptosis and MLKL-mediated necroptosis of ADAR1-deficient cells. ZBP1 contributes to the embryonic lethality of Adar-knockout mice, and it drives early mortality and intestinal cell death in mice deficient in the expression of both ADAR and MAVS. The Z-nucleic-acid-binding Zα domain of ADAR1 is necessary to prevent ZBP1-mediated intestinal cell death and skin inflammation. The Zα domain of ADAR1 promotes A-to-I editing of endogenous Alu elements to prevent dsRNA formation through the pairing of inverted Alu repeats, which can otherwise induce ZBP1 activation. This shows that recognition of Alu duplex RNA by ZBP1 may contribute to the pathological features of AGS that result from the loss of ADAR1 function.


Assuntos
Adenosina Desaminase , Inflamação , Proteínas de Ligação a RNA , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Adenosina/metabolismo , Adenosina Desaminase/química , Adenosina Desaminase/deficiência , Adenosina Desaminase/metabolismo , Animais , Apoptose , Doenças Autoimunes do Sistema Nervoso , Caspase 8/metabolismo , Humanos , Inflamação/metabolismo , Inflamação/prevenção & controle , Inosina/metabolismo , Intestinos/patologia , Camundongos , Necroptose , Malformações do Sistema Nervoso , Edição de RNA , RNA de Cadeia Dupla , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Pele/patologia
6.
J Pharm Biomed Anal ; 219: 114886, 2022 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-35715372

RESUMO

Purine and pyrimidine metabolism are vital metabolic pathways in the development, proliferation or repairment of cells or tissues associated with various diseases. Here, a simple, all-in-one injection hydrophilic interaction liquid chromatography-tandem mass spectrometry method was developed for simultaneous determination of 20 metabolites: adenine, adenosine, deoxyadenosine, adenosine 5'-monophosphate, cyclic adenosine monophosphate, hypoxanthine, xanthine, inosine, deoxyinosine, xanthosine, xanthosine 5'-monophosphate and uric acid, which are products of purine metabolism; uridine, deoxyuridine, uridine 5'-monophosphate and uracil, are products of pyrimidine metabolism; and corticosterone, methionine, acetylcholine and serotonin. To minimize interference of endogenous molecules in sample matrixes, a combination of activated carbon adsorption and a serum substitute matrix (5% bovine serum albumin in phosphate buffered saline) was utilized and jointly applied. The sensitivity, linearity, stability, precision, accuracy and extraction recovery were evaluated, and the method was demonstrated to be accurate, sensitive and reliable. An analytical strategy was successfully applied to quantitatively determine 20 metabolite levels in the serum and hippocampus of mice with chronic social defeat stress-induced depression. The results showed greatly perturbed purine metabolism in the depressed mice, which was primarily characterized by dramatic increases in hypoxanthine, xanthine and inosine in serum and reduced levels of adenine, adenosine and adenosine 5'-monophosphate in the hippocampus. These findings suggest that this novel strategy can facilitate the quantitative analysis of adenine and other purine and pyrimidine metabolites in tissue and serum and exhibits great potential in the exploration of metabolism-related mechanisms of relevant diseases.


Assuntos
Purinas , Espectrometria de Massas em Tandem , Adenina/metabolismo , Adenosina , Monofosfato de Adenosina , Animais , Cromatografia Líquida de Alta Pressão/métodos , Hipocampo/metabolismo , Hipoxantinas , Inosina , Camundongos , Purinas/metabolismo , Pirimidinas
7.
Nat Methods ; 19(7): 833-844, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35697834

RESUMO

Inosine is a prevalent RNA modification in animals and is formed when an adenosine is deaminated by the ADAR family of enzymes. Traditionally, inosines are identified indirectly as variants from Illumina RNA-sequencing data because they are interpreted as guanosines by cellular machineries. However, this indirect method performs poorly in protein-coding regions where exons are typically short, in non-model organisms with sparsely annotated single-nucleotide polymorphisms, or in disease contexts where unknown DNA mutations are pervasive. Here, we show that Oxford Nanopore direct RNA sequencing can be used to identify inosine-containing sites in native transcriptomes with high accuracy. We trained convolutional neural network models to distinguish inosine from adenosine and guanosine, and to estimate the modification rate at each editing site. Furthermore, we demonstrated their utility on the transcriptomes of human, mouse and Xenopus. Our approach expands the toolkit for studying adenosine-to-inosine editing and can be further extended to investigate other RNA modifications.


Assuntos
Nanoporos , RNA , Adenosina/genética , Animais , Inosina/genética , Camundongos , RNA/genética , RNA/metabolismo , Edição de RNA , Análise de Sequência de RNA
8.
Anal Chem ; 94(24): 8740-8747, 2022 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-35678728

RESUMO

RNA molecules contain diverse modifications that play crucial roles in a wide variety of biological processes. Adenosine-to-inosine (A-to-Ino) RNA editing is one of the most prevalent modifications among all types of RNA. Abnormal A-to-InoRNA editing has been demonstrated to be associated with many human diseases. Identification of A-to-Ino editing sites is indispensable to deciphering their biological roles. Herein, by employing the unique property of human endonuclease V (hEndoV), we proposed a hEndoV-mediated sequencing (hEndoV-seq) method for the single-base resolution detection of A-to-InoRNA editing sites. In this approach, the terminal 3'OH of RNA is first blocked by 3'-deoxyadenosine (3'-deoxy-A). Specific cleavage of Ino sites by hEndoV protein produces new terminal 3'OH, which can be identified by sequencing analysis, and therefore offers the site-specific detection of Ino in RNA. The principle of hEndoV-seq is straightforward and the analytical procedure is simple. No chemical reaction is involved in the sequencing library preparation. The whole procedure in hEndoV-seq is carried out under mild conditions and RNA is not prone to degradation. Taken together, the proposed hEndoV-seq method is capable of site-specific identification of A-to-Ino editing in RNA, which provides a valuable tool for elucidating the functions of A-to-Ino editing in RNA.


Assuntos
Edição de RNA , RNA , Adenosina/metabolismo , Endonucleases/metabolismo , Humanos , Inosina , RNA/metabolismo
9.
Artigo em Inglês | MEDLINE | ID: mdl-35680173

RESUMO

INTRODUCTION: Antioxidants may have positive impact on diabetic polyneuropathy (DPN), presumably due to alleviation of oxidative stress. We aimed to evaluate the efficacy and safety of combination of antioxidants: succinic acid, inosine, nicotinamide, and riboflavin (SINR) in the treatment of DPN. RESEARCH DESIGN AND METHODS: In a double-blind, placebo-controlled clinical trial, men and women aged 45-74 years with type 2 diabetes and symptomatic DPN, with initial Total Symptom Score (TSS) ˃5, were randomized into experimental (n=109) or placebo (n=107) group. Patients received study medication/placebo intravenously for 10 days, followed by oral administration for 75 days. Statistical significance was defined as a two-tailed p<0.05. RESULTS: In SINR group, mean TSS change after 12 weeks was -2.65 (±1.46) vs -1.73 (±1.51) in the placebo group (p<0.0001; t-test). Reduction of symptoms in the SINR group was achieved regardless of hemoglobin A1c levels, but better results were observed in patients with initial TSS <7.5. The analysis of TSS subscores revealed statistically significant between-group differences by dynamics of the intensity of paresthesia and of numbness starting from day 11 (p=0.035 and p=0.001, respectively; mixed model); by day 57, statistically significant between-group differences were detected also by dynamics of burning intensity (p=0.005; mixed model). Study limitations are small effect size, moderate proportion of patients with severe DPN symptoms, subjective assessment of outcomes, exclusion of participants who received injectable glucose-lowering medications other than insulins, and patients with uncontrolled and type 1 diabetes. CONCLUSIONS: The combination of SINR effectively alleviates DPN symptoms in patients with type 2 diabetes. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov Registry (NCT04649203; Unique Protocol ID: CTF-III-DM-2019).


Assuntos
Diabetes Mellitus Tipo 2 , Neuropatias Diabéticas , Antioxidantes/uso terapêutico , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/tratamento farmacológico , Neuropatias Diabéticas/tratamento farmacológico , Feminino , Humanos , Inosina/uso terapêutico , Masculino , NAD/uso terapêutico , Niacinamida/efeitos adversos , Riboflavina/efeitos adversos , Ácido Succínico/uso terapêutico
10.
Protein J ; 41(3): 381-393, 2022 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-35674860

RESUMO

A class of plant defense and storage proteins, including Putranjiva roxburghii PNP protein (PRpnp), belongs to PNP-UDP family. The PRpnp and related plant proteins contain a disrupted PNP-UDP domain as revealed in previous studies. In PRpnp, the insert disrupting the domain contains the trypsin inhibitory site. In the present work, we analyzed native PRpnp (nPRpnp) complex formation with trypsin and inosine using SAXS experiments and established its dual functionality. Results indicated a relatively compact nPRpnp:Inosine structure, whereas trypsin complex showed conformational changes/flexibility. nPRpnp also exhibited a strong anti-cancer activity toward breast cancer (MCF-7), prostate cancer (DU-145) and hepatocellular carcinoma (HepG2) cell lines. MCF-7 and DU-145 were more sensitive to nPRpnp treatment as compared to HepG2. However, nPRpnp treatment showed no effect on the viability of HEK293 cells indicating that nPRpnp is specific for targeting the viability of only cancer cells. Further, acridine orange, DAPI and DNA fragmentation studies showed that cytotoxic effect of nPRpnp is mediated through induction of apoptosis as evident from the apoptosis-associated morphological changes and nuclear fragmentation observed after PRpnp treatment of cancer cells. These results suggest that PRpnp has the potential to be used as an anticancer agent. This is first report of anticancer activity as well as SAXS-based analysis for a PNP enzyme with trypsin inhibitory activity.


Assuntos
Antineoplásicos , Magnoliopsida , Neoplasias , Antineoplásicos/farmacologia , Apoptose , Células HEK293 , Células Hep G2 , Humanos , Inosina/farmacologia , Células MCF-7 , Magnoliopsida/química , Masculino , Neoplasias/tratamento farmacológico , Proteínas de Plantas/farmacologia , Espalhamento a Baixo Ângulo , Tripsina/metabolismo , Difosfato de Uridina/farmacologia , Difração de Raios X
11.
Nat Commun ; 13(1): 2540, 2022 05 09.
Artigo em Inglês | MEDLINE | ID: mdl-35534484

RESUMO

Epitranscriptomic features, such as single-base RNA editing, are sources of transcript diversity in cancer, but little is understood in terms of their spatial context in the tumour microenvironment. Here, we introduce spatial-histopathological examination-linked epitranscriptomics converged to transcriptomics with sequencing (Select-seq), which isolates regions of interest from immunofluorescence-stained tissue and obtains transcriptomic and epitranscriptomic data. With Select-seq, we analyse the cancer stem cell-like microniches in relation to the tumour microenvironment of triple-negative breast cancer patients. We identify alternative splice variants, perform complementarity-determining region analysis of infiltrating T cells and B cells, and assess adenosine-to-inosine base editing in tumour tissue sections. Especially, in triple-negative breast cancer microniches, adenosine-to-inosine editome specific to different microniche groups is identified.


Assuntos
Adenosina Desaminase , Neoplasias de Mama Triplo Negativas , Adenosina/genética , Adenosina Desaminase/genética , Humanos , Inosina/genética , Células-Tronco Neoplásicas , Microambiente Tumoral/genética
12.
Molecules ; 27(9)2022 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-35566345

RESUMO

The antibiotic resistance rates of Klebsiella pneumoniae have been steadily increasing in recent years. Nevertheless, the metabolic features of the drug-resistant Klebsiella pneumoniae and its associated benefits for bacterial pathogenicity are far from expounded. This study aims to unravel the unique physiological and metabolic properties specific to drug-resistant K. pneumoniae. Using scanning electron microscopy (SEM), we observed a thicker extracellular mucus layer around a drug-resistant K. pneumonia strain (Kp-R) than a drug-sensitive K. pneumonia strain (Kp-S). Kp-R also produced more capsular polysaccharide (CPS) and biofilm, and appeared to have a significant competitive advantage when co-cultured with Kp-S. Moreover, Kp-R was easier to adhere to and invade A549 epithelial cells than Kp-S but caused less cell-viability damage according to cell counting kit-8 (CCK-8) tests. Immunofluorescence revealed that both Kp-R and Kp-S infection destroyed the tight junctions and F-actin of epithelial cells, while the damage caused by Kp-S was more severe than Kp-R. We detected the extracellular metabolites secreted by the two strains with UHPLC-Q-TOF MS to explore the critical secretion products. We identified 16 predominant compounds that were differentially expressed. Among them, inosine increased the viability of epithelial cells in a dose-dependent manner, and an A2AR antagonist can abolish such enhancement. D-mannose, which was secreted less in Kp-R, inhibited the viability of A549 cells in the range of low doses. These findings provide potential targets and research strategies for preventing and treating drug-resistant K. pneumoniae infections.


Assuntos
Infecções por Klebsiella , Klebsiella pneumoniae , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Células Epiteliais , Humanos , Inosina , Infecções por Klebsiella/tratamento farmacológico , Infecções por Klebsiella/microbiologia , Pulmão , Manose/farmacologia , Testes de Sensibilidade Microbiana
13.
Int J Mol Sci ; 23(9)2022 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-35563631

RESUMO

Adenosine-to-inosine RNA editing is a system of post-transcriptional modification widely distributed in metazoans which is catalyzed by ADAR enzymes and occurs mostly in double-stranded RNA (dsRNA) before splicing. This type of RNA editing changes the genetic code, as inosine generally pairs with cytosine in contrast to adenosine, and this expectably modulates RNA splicing. We review the interconnections between RNA editing and splicing in the context of human cancer. The editing of transcripts may have various effects on splicing, and resultant alternatively spliced isoforms may be either tumor-suppressive or oncogenic. Dysregulated RNA splicing in cancer often causes the release of excess amounts of dsRNA into cytosol, where specific dsRNA sensors provoke antiviral-like responses, including type I interferon signaling. These responses may arrest cell division, causing apoptosis and, externally, stimulate antitumor immunity. Thus, small-molecule spliceosome inhibitors have been shown to facilitate the antiviral-like signaling and are considered to be potential cancer therapies. In turn, a cytoplasmic isoform of ADAR can deaminate dsRNA in cytosol, thereby decreasing its levels and diminishing antitumor innate immunity. We propose that complete or partial inhibition of ADAR may enhance the proapoptotic and cytotoxic effects of splicing inhibitors and that it may be considered a promising addition to cancer therapies targeting RNA splicing.


Assuntos
Adenosina Desaminase , Neoplasias , Adenosina/metabolismo , Adenosina Desaminase/genética , Adenosina Desaminase/metabolismo , Antivirais , Humanos , Inosina/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/genética , Splicing de RNA , RNA de Cadeia Dupla/genética , Proteínas de Ligação a RNA/metabolismo
14.
Nat Commun ; 13(1): 2997, 2022 05 30.
Artigo em Inglês | MEDLINE | ID: mdl-35637184

RESUMO

Posttranscriptional adenosine-to-inosine modifications amplify the functionality of RNA molecules in the brain, yet the cellular and genetic regulation of RNA editing is poorly described. We quantify base-specific RNA editing across three major cell populations from the human prefrontal cortex: glutamatergic neurons, medial ganglionic eminence-derived GABAergic neurons, and oligodendrocytes. We identify more selective editing and hyper-editing in neurons relative to oligodendrocytes. RNA editing patterns are highly cell type-specific, with 189,229 cell type-associated sites. The cellular specificity for thousands of sites is confirmed by single nucleus RNA-sequencing. Importantly, cell type-associated sites are enriched in GTEx RNA-sequencing data, edited ~twentyfold higher than all other sites, and variation in RNA editing is largely explained by neuronal proportions in bulk brain tissue. Finally, we uncover 661,791 cis-editing quantitative trait loci across thirteen brain regions, including hundreds with cell type-associated features. These data reveal an expansive repertoire of highly regulated RNA editing sites across human brain cell types and provide a resolved atlas linking cell types to editing variation and genetic regulatory effects.


Assuntos
Inosina , Edição de RNA , Encéfalo/metabolismo , Humanos , Inosina/genética , Inosina/metabolismo , Locos de Características Quantitativas/genética , RNA/metabolismo , Edição de RNA/genética
15.
Am J Physiol Cell Physiol ; 322(6): C1110-C1116, 2022 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-35508185

RESUMO

Fibroblasts play an important role in the pathogenic mechanisms of several socially significant diseases, including pulmonary and cardiovascular fibrosis, liver cirrhosis, systemic sclerosis, progressive kidney disease. The alterations of the epitranscriptome, including more than 170 distinct posttranscriptional RNA modifications or editing events, justified their investigation as an important modulator of fibrosis. Recent development of high-throughput methods allows the identification of RNA modification sites and their mechanistic aspect in the fibrosis development. The most common RNA modification is methylation of N6-adenosine deposited by the m6A methyltransferase complex (METTL3/14/16, WTAP, KIAA1429, and RBM15/15B), erased by demethylases (FTO and ALKBH5), and recognized by binding proteins (e.g., YTHDF1/2/3, YTHDC1/2, IGF2BP1/2/3, etc.). Adenosine to inosine (A-to-I) RNA editing is another abundant editing event converting adenosine to inosine in double-stranded RNA regions through the action of the adenosine deaminase (ADAR) proteins. Last but not least, 5-methylcytosine (m5C) regulates the stability and translation of mRNAs. All those RNA modifications have been observed in mRNA as well as the noncoding regions of pre-mRNA and noncoding RNAs (ncRNAs) and demonstrated to be involved in fibrosis in different cellular and animal models. This Mini-Review focuses on the latest research on epitranscriptomic marks related to fibroblast biology and fibrosis as well as elucidates the future research directions in this context.


Assuntos
Adenosina , RNA , Adenosina/genética , Adenosina/metabolismo , Animais , Fibroblastos/metabolismo , Fibrose , Inosina/genética , RNA/genética , RNA/metabolismo , RNA Mensageiro/metabolismo
16.
Sci Rep ; 12(1): 6408, 2022 04 18.
Artigo em Inglês | MEDLINE | ID: mdl-35436992

RESUMO

Inosine triphosphate pyrophosphatases (ITPases) are ubiquitous house-cleaning enzymes that specifically recognize deaminated purine nucleotides and catalyze their hydrolytic cleavage. In this work, we have characterized the Trypanosoma brucei ITPase ortholog (TbITPA). Recombinant TbITPA efficiently hydrolyzes (deoxy)ITP and XTP nucleotides into their respective monophosphate form. Immunolocalization analysis performed in bloodstream forms suggests that the primary role of TbITPA is the exclusion of deaminated purines from the cytosolic nucleoside triphosphate pools. Even though ITPA-knockout bloodstream parasites are viable, they are more sensitive to inhibition of IMP dehydrogenase with mycophenolic acid, likely due to an expansion of IMP, the ITP precursor. On the other hand, TbITPA can also hydrolyze the activated form of the antiviral ribavirin although in this case, the absence of ITPase activity in the cell confers protection against this nucleoside analog. This unexpected phenotype is dependant on purine availability and can be explained by the fact that ribavirin monophosphate, the reaction product generated by TbITPA, is a potent inhibitor of trypanosomal IMP dehydrogenase and GMP reductase. In summary, the present study constitutes the first report on a protozoan inosine triphosphate pyrophosphatase involved in the removal of harmful deaminated nucleotides from the cytosolic pool.


Assuntos
Nucleotídeos , Trypanosoma brucei brucei , IMP Desidrogenase , Inosina , Inosina Trifosfato , Pirofosfatases/genética , Ribavirina/farmacologia
17.
Poult Sci ; 101(6): 101829, 2022 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-35385823

RESUMO

Chicken breed is one of the key factors that influence meat quality. The quality attributes of breast meat from commercial broiler (CB), Thai native chicken (NC, Leung Hang Khao), and the crossbred Korat chicken (KC) were investigated via synchrotron radiation-based Fourier transform infrared (SR-FTIR) microspectroscopy, Fourier transform Raman (FT-Raman) spectroscopy, and physicochemical analysis. The protein and carbonyl contents of KC and NC meats were higher than that of CB meat, but the lipid content was lower (P < 0.05). CB meat was characterized by high moisture, lightness (L*), and presence of taste-active nucleotides, namely, inosine 5'-monophosphate (IMP) and guanosine 5'-monophosphate (GMP). Moreover, NC meat had the highest insoluble collagen and inosine contents (P < 0.05). The predominant protein secondary structures of KC and NC meats were ß-turns and random coils, whereas α-helices were mainly found in CB meat. Based on principal component analysis, the meat quality and spectra were clearly separated by breeds. The high moisture and lipid content of meat corresponded to O-H stretching (3,203 cm-1) and C-H stretching (2,854 cm-1) in the FT-Raman spectra, whereas PO2- stretching (1,240 cm-1), measured via SR-FTIR, was well correlated with the IMP content. In addition, the FT-Raman wavenumber of 934 cm-1, indicating C-C stretching, was correlated with high water-holding capacity (WHC) in KC meat. The quality of meat from slow- and fast-growing chickens significantly varies. Vibrational spectroscopy is a powerful technique that provides insightful molecular information correlated with various meat attributes.


Assuntos
Galinhas , Inosina Monofosfato , Animais , Inosina , Inosina Monofosfato/análise , Lipídeos/química , Carne/análise , Análise Espectral/veterinária
18.
Int J Biol Sci ; 18(6): 2472-2483, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35414795

RESUMO

Epitranscriptomic changes caused by adenosine-to-inosine (A-to-I) RNA editing contribute to the pathogenesis of human cancers; however, only a small fraction of the millions editing sites detected so far has clear functionality. To facilitate more in-depth studies on the editing, this paper offers REIA (http://bioinfo-sysu.com/reia), an interactive web server that analyses and visualizes the association between human cancers and A-to-I RNA editing sites (RESs). As a comprehensive database, REIA curates not only 8,447,588 RESs from 9,895 patients across 34 cancers, where 33 are from TCGA and 1 from GEO, but also 13 different types of multi-omic data for the cancers. As an interactive server, REIA provides various options for the user to specify the interested sites, to browse their annotation/editing level/profile in cancer, and to compare the difference in multi-omic features between editing and non-editing groups. From the editing profiles, REIA further detects 658 peptides that are supported by mass spectrum data but not yet covered in any prior works.


Assuntos
Neoplasias , Edição de RNA , Adenosina/genética , Adenosina/metabolismo , Humanos , Inosina/genética , Inosina/metabolismo , Neoplasias/genética , RNA , Edição de RNA/genética
19.
Life Sci ; 300: 120569, 2022 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-35472453

RESUMO

Huntington's disease (HD) is an autosomal dominant inherited neurodegenerative disease characterized by involuntary bizarre movements, psychiatric symptoms, dementia, and early death. Several studies suggested neuroprotective activities of inosine; however its role in HD is yet to be elucidated. The current study aimed to demonstrate the neuroprotective effect of inosine in 3-nitropropionic acid (3-NP)-induced neurotoxicity in rats while investigating possible underlying mechanisms. Rats were randomly divided into five groups; group 1 received i.p. injections of 1% DMSO, whereas groups 2, 3, 4, and 5 received 3-NP (10 mg/kg, i.p.) for 14 days, concomitantly with inosine (200 mg/kg., i.p.) in groups 3, 4, and 5, SCH58261, a selective adenosine 2A receptor (A2AR) antagonist, (0.05 mg/kg, i.p.) in group 4, and PD98059, an extracellular signal-regulated kinase (ERK) inhibitor, (0.3 mg/kg, i.p.) in group 5. Treatment with inosine mitigated 3-NP-induced motor abnormalities and body weight loss. Moreover, inosine boosted the striatal brain-derived neurotrophic factor (BDNF) level, p-tropomyosin receptor kinase B (TrKB), p-ERK, and p-cAMP response element-binding protein (CREB) expression, which subsequently suppressed oxidative stress biomarkers (malondialdehyde and nitric oxide) and pro-inflammatory cytokines (tumor necrosis factor alpha and interleukin-1ß) and replenished the glutathione content. Similarly, histopathological analyses revealed decreased striatal injury score, the expression of the glial fibrillary acidic protein, and neuronal loss after inosine treatment. These effects were attenuated by the pre-administration of SCH58261 or PD98059. In conclusion, inosine attenuated 3-NP-induced HD-like symptoms in rats, at least in part, via the activation of the A2AR/BDNF/TrKB/ERK/CREB signaling pathway.


Assuntos
Doença de Huntington , Doenças Neurodegenerativas , Fármacos Neuroprotetores , Animais , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Fator B do Complemento/metabolismo , Fator B do Complemento/farmacologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Doença de Huntington/induzido quimicamente , Doença de Huntington/tratamento farmacológico , Doença de Huntington/metabolismo , Inosina/farmacologia , Fármacos Neuroprotetores/uso terapêutico , Nitrocompostos , Propionatos/farmacologia , Ratos , Transdução de Sinais
20.
Anal Chem ; 94(17): 6436-6440, 2022 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-35435665

RESUMO

Aptamers are widely used in small molecule detection applications due to their specificity, stability, and cost effectiveness. One key challenge in utilizing aptamers in sensors is matching the binding affinity of the aptamer to the desired concentration range for analyte detection. The most common methods for modulating affinity have inherent limitations, such as the likelihood of drastic changes in aptamer folding. Here, we propose that substituting guanosine for inosine at specific locations in the aptamer sequence provides a less perturbative approach to modulating affinity. Inosine is a naturally occurring nucleotide that results from hydrolytic deamination of adenosine, and like guanine, it base pairs with cytosine. Using the well-studied cocaine binding aptamer, we systematically replaced guanosine with inosine and were able to generate sequences having a range of binding affinities from 230 nM to 80 µM. Interestingly, we found that these substitutions could also modulate the specificity of the aptamers, leading to a range of binding affinities for structurally related analytes. Analysis of folding stability via melting temperature shows that, as expected, aptamer structure is impacted by guanosine-to-inosine substitutions. The ability to tune binding affinity and specificity through guanosine-to-inosine substitution provides a convenient and reliable approach for rapidly generating aptamers for diverse biosensing applications.


Assuntos
Aptâmeros de Nucleotídeos , Adenosina , Aptâmeros de Nucleotídeos/química , Guanosina , Inosina
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