LC-MS-based metabolomics reveals metabolite dynamic changes of beef after superchilling early post-mortem.

To explore the underlying mechanisms by which superchilling (SC, -3 °C within 5 h of slaughter) improves beef tenderness, an untargeted metabolomics strategy was employed. M. Longissimus lumborum (LL) muscles from twelve beef carcasses were assigned to either SC or very fast chilling (VFC, 0 °C within 5 h of slaughter) treatments, with conventional chilling (CC, 0 âˆ¼ 4 °C until 24 h post-mortem) serving as the control (6 per group). Biochemical properties and metabolites were investigated during the early post-mortem period. The results showed that the degradation of µ-calpain and caspase 3 occurred earlier in SC treated sample, which might be attributed to the accelerated accumulation of free Ca2+. The metabolomic profiles of samples from the SC and CC treatments were clearly distinguished based on partial least squares-discriminant analysis (PLS-DA) at each time point. It is noteworthy that more IMP and 4-hydroxyproline were found in the comparison between SC and CC treatments. According to the results of metabolic pathways analysis and the correlation analysis between traits related to tenderness and metabolites with significant differences (SC vs. CC), it can be suggested that the tenderization effect of the SC treatment may be related to the alteration of arginine and proline metabolism, and purine metabolism in the early post-mortem phase.

Inosine-Induced Base Pairing Diversity during Reverse Transcription.

A-to-I editing catalyzed by adenosine deaminase acting on RNAs impacts numerous physiological and biochemical processes that are essential for cellular functions and is a big contributor to the infectivity of certain RNA viruses. The outcome of this deamination leads to changes in the eukaryotic transcriptome functionally resembling A-G transitions since inosine preferentially pairs with cytosine. Moreover, hyper-editing or multiple A to G transitions in clusters were detected in measles virus. Inosine modifications either directly on viral RNA or on cellular RNA can have antiviral or pro-viral repercussions. While many of the significant roles of inosine in cellular RNAs are well understood, the effects of hyper-editing of A to I on viral polymerase activity during RNA replication remain elusive. Moreover, biological strategies such as molecular cloning and RNA-seq for transcriptomic interrogation rely on RT-polymerase chain reaction with little to no emphasis placed on the first step, reverse transcription, which may reshape the sequencing results when hypermodification is present. In this study, we systematically explore the influence of inosine modification, varying the number and position of inosines, on decoding outcomes using three different reverse transcriptases (RTs) followed by standard Sanger sequencing. We find that inosine alone or in clusters can differentially affect the RT activity. To gain structural insights into the accommodation of inosine in the polymerase site of HIV-1 reverse transcriptase (HIV-1-RT) and how this structural context affects the base pairing rules for inosine, we performed molecular dynamics simulations of the HIV-1-RT. The simulations highlight the importance of the protein-nucleotide interaction as a critical factor in deciphering the base pairing behavior of inosine clusters. This effort sets the groundwork for decrypting the physiological significance of inosine and linking the fidelity of reverse transcriptase and the possible diverse transcription outcomes of cellular RNAs and/or viral RNAs where hyper-edited inosines are present in the transcripts.

Discovery of a Novel Inhibitor of Human Purine Nucleoside Phosphorylase by a Simple Hydrophilic Interaction Liquid Chromatography Enzymatic Assay.

Human purine nucleoside phosphorylase (HsPNP) belongs to the purine salvage pathway of nucleic acids. Genetic deficiency of this enzyme triggers apoptosis of activated T-cells due to the accumulation of deoxyguanosine triphosphate (dGTP). Therefore, potential chemotherapeutic applications of human PNP inhibitors include the treatment of T-cell leukemia, autoimmune diseases and transplant tissue rejection. In this report, we present the discovery of novel HsPNP inhibitors by coupling experimental and computational tools. A simple, inexpensive, direct and non-radioactive enzymatic assay coupled to hydrophilic interaction liquid chromatography and UV detection (LC-UV using HILIC as elution mode) was developed for screening HsPNP inhibitors. Enzymatic activity was assessed by monitoring the phosphorolysis of inosine (Ino) to hypoxanthine (Hpx) by LC-UV. A small library of 6- and 8-substituted nucleosides was synthesized and screened. The inhibition potency of the most promising compound, 8-aminoinosine (4), was quantified through Ki and IC50 determinations. The effect of HsPNP inhibition was also evaluated in vitro through the study of cytotoxicity on human T-cell leukemia cells (CCRF-CEM). Docking studies were also carried out for the most potent compound, allowing further insights into the inhibitor interaction at the HsPNP active site. This study provides both new tools and a new lead for developing novel HsPNP inhibitors.

Quantitative Analysis of Adenosine-to-Inosine RNA Editing.

The conversion of adenosine to inosine (A to I) by RNA editing represents a common posttranscriptional mechanism for diversification of both the transcriptome and proteome, and is a part of the cellular response for innate immune tolerance. Due to its preferential base-pairing with cytosine (C), inosine (I) is recognized as guanosine (G) by reverse transcriptase, as well as the cellular splicing and translation machinery. A-to-I editing events appear as A-G discrepancies between genomic DNA and cDNA sequences. Molecular analyses of RNA editing have leveraged these nucleoside differences to quantify RNA editing in ensemble populations of RNA transcripts and within individual cDNAs using high-throughput sequencing or Sanger sequencing-derived analysis of electropherogram peak heights. Here, we briefly review and compare these methods of RNA editing quantification, as well as provide experimental protocols by which such analyses may be achieved.

Discovering A-to-I RNA Editing Through Chemical Methodology "ICE-seq".

RNA editing of adenosines to inosines contributes to a wide range of biological processes by regulating gene expression post-transcriptionally. To understand the effect, accurate mapping of inosines is necessary. The most conventional method to identify an editing site is to compare the cDNA sequence with its corresponding genomic sequence. However, this method has a high false discovery rate because guanosine signals, due to experimental errors or noise in the obtained sequences, contaminate genuine inosine signals detected as guanosine. To ensure high accuracy, we developed the Inosine Chemical Erasing (ICE) method to accurately and biochemically identify inosines in RNA strands utilizing inosine cyanoethylation and reverse transcription-PCR. Furthermore, we applied this technique to next-generation sequencing technology, called ICE-seq, to conduct an unbiased genome-wide screening of A-to-I editing sites in the transcriptome.

ALU A-to-I RNA Editing: Millions of Sites and Many Open Questions.

Alu elements are repetitive short interspersed elements prevalent in the primate genome. These repeats account for over 10% of the genome with more than a million highly similar copies. A direct outcome of this is an enrichment in long structures of stable dsRNA, which are the target of adenosine deaminases acting on RNAs (ADARs), the enzymes catalyzing A-to-I RNA editing. Indeed, A-to-I editing by ADARs is extremely abundant in primates: over a hundred million editing sites exist in their genomes. However, despite the radical increase in ADAR targets brought on by the introduction of Alu elements, the few evolutionary conserved editing sites manage to retain their editing levels. Here, we review and discuss the cost of having an unusual amount of dsRNA and editing in the transcriptome, as well as the opportunities it presents, which possibly contributed to accelerating primate evolution.

RNA Editing in Human and Mouse Tissues.

Adenosine-to-inosine (A-to-I) RNA editing is a fundamental posttranscriptional mechanism that greatly diversifies the transcriptome in many living organisms, including mammals. Multiple studies have demonstrated the importance of this process not just in normal development and physiology but also in various human diseases. Importantly, the precise editing level of a site may have downstream consequences on cellular behavior. Hence, the editing levels should be quantified as accurately as possible. In this chapter, we describe how to examine RNA editing in human and mouse tissues. The rapid development of next-generation sequencing technologies is affording us an unprecedented ability to accurately measure the editing levels of numerous sites simultaneously. Our experimental workflow includes the harvesting of high-quality RNA samples and the construction of different high-throughput sequencing libraries. We also delineate the computational steps needed to analyze the sequencing data from an Illumina platform.

Detection of A-to-I Hyper-edited RNA Sequences.

Following A-to-I editing of double-stranded RNA (dsRNA) molecules, sequencing reactions interpret the edited inosine (I) as guanosine (G). For this reason, current methods to detect A-to-I editing sites work to align RNA sequences to their reference DNA sequence in order to reveal A-to-G mismatches. However, areas with heavily edited reads produce dense clusters of A-to-G mismatches that hinder alignment, and complicate correct identification of the sites. The presented approach employs prudent alignment and examination of excessive mismatch events, enabling high-accuracy detection of hyper-edited reads and sites.

Proteome Diversification by RNA Editing.

RNA editing is an RNA modification that alters the RNA sequence relative to its genomic blueprint. The most common type of RNA editing is A-to-I editing by double-stranded RNA-specific adenosine deaminase (ADAR) enzymes. Editing of a protein-coding region within the RNA molecule may result in non-synonymous substitutions, leading to a modified protein product. These editing sites, also known as "recoding" sites, contribute to the complexity and diversification of the proteome. Recent computational transcriptomic studies have identified thousands of recoding sites in multiple species, many of which are conserved within (but not usually across) lineages and have functional and evolutionary importance. In this chapter we describe the recoding phenomenon across species, consider its potential utility for diversity and adaptation, and discuss its evolution.

MicroRNA Editing Detection and Function: A Combined In Silico and Experimental Approach for the Identification and Validation of Putative Oncogenic Targets.

MicroRNAs (miRNAs) are a class of ~22 nt noncoding RNAs playing essential roles in the post-transcriptional regulation of gene expression, cell proliferation, and cell differentiation and are often found deregulated in several diseases including cancer.The A-to-I RNA editing, mediated by ADAR enzymes, is a diffuse post-transcriptional mechanism that converts the genetically coded adenosine (A) into inosine (I) at the RNA level. Among different RNA targets, the ADAR enzymes can also edit miRNA precursors. Specifically, a single nucleotide change (A/I) lying within the mature miRNA can alter the miRNA binding specificity and redirect the edited miRNA to a different mRNA target. In several cancer types a consistent deregulation of A-to-I RNA editing machinery also involves important miRNAs (either oncomiRs or tumor-suppressor miRNAs). Herein we describe a combined in silico and experimental approach for the detection of edited miRNAs and the identification and validation of their target genes potentially involved in cancer progression or invasion.

Circadian movement behaviours and metabolism differences of the Pacific abalone Haliotis discus hannai.

Circadian rhythm is the most important and universal biological rhythm in marine organisms. In this research, the movement behaviour of abalone (Haliotis discus hannai) was continuously monitored under a light cycle of 12 L:12D. It was found that the cumulative movement distance and cumulative movement time of abalone reached was highest from 00:00-03:00 h. The minimum values of maximum movement velocity occurred between 21:00-00:00 h, and a significant circadian cosine rhythm was exhibited during these periods (P < 0.05). Metabolomic analysis of cerebral ganglions of abalone was conducted at 06:00 h (6 M), 14:00 h (14 M), and 22:00 h (22 M) and 380, 385, and 315 metabolites with significant differences were identified in 6 M vs 14 M, 14 M vs 22 M, and 6 M vs 22 M, respectively (P < 0.05). With the alternation of day and night, the expression levels of phosphatidylcholine, 5-HT, N-acetyl-5-hydroxytryptamine, indole-3-acetaldehyde, hypoxanthine, and deoxyinosine declined significantly, while those of Lysophosphatidylcholines (lysoPC) (20: 5 (5Z, 8Z, 11Z, 14Z, 17Z)), lysoPC (22: 4 (7Z, 10Z, 13Z, 16Z)), lysoPC (16: 1 (9Z) / 0: 0), phosphatidylethanolamine (PE) (18: 1 (11Z) 22: 2 (13Z, 16Z)), and guanosine 5'-phosphate rose significantly. These 11 metabolites can be used as differential metabolic markers. These findings not only quantitatively describe the circadian movement behaviours of abalone, but also provide an initial analysis of the circadian mechanism of the physiological metabolic conversion of abalone, which in turn provides guidelines for light control and feeding strategy for use in aquaculture production.

Chemical Profiling of A-to-I RNA Editing Using a Click-Compatible Phenylacrylamide.

Straightforward methods for detecting adenosine-to-inosine (A-to-I) RNA editing are key to a better understanding of its regulation, function, and connection with disease. We address this need by developing a novel reagent, N-(4-ethynylphenyl)acrylamide (EPhAA), and illustrating its ability to selectively label inosine in RNA. EPhAA is synthesized in a single step, reacts rapidly with inosine, and is "click"-compatible, enabling flexible attachment of fluorescent probes at editing sites. We first validate EPhAA reactivity and selectivity for inosine in both ribonucleosides and RNA substrates, and then apply our approach to directly monitor in vitro A-to-I RNA editing activity using recombinant ADAR enzymes. This method improves upon existing inosine chemical-labeling techniques and provides a cost-effective, rapid, and non-radioactive approach for detecting inosine formation in RNA. We envision this method will improve the study of A-to-I editing and enable better characterization of RNA modification patterns in different settings.

Metabolomic analysis of serum and myocardium in compensated heart failure after myocardial infarction.

AIMS: To determine the metabolic adaptations to compensated heart failure using a reproducible model of myocardial infarction and an unbiased metabolic screen. To address the limitations in sample availability and model variability observed in preclinical and clinical metabolic investigations of heart failure. MAIN METHODS: Metabolomic analysis was performed on serum and myocardial tissue from rabbits after myocardial infarction (MI) was induced by cryo-injury of the left ventricular free wall. Rabbits followed for 12 weeks after MI exhibited left ventricular dilation and depressed systolic function as determined by echocardiography. Serum and tissue from the viable left ventricular free wall, interventricular septum and right ventricle were analyzed using a gas chromatography time of flight mass spectrometry-based untargeted metabolomics assay for primary metabolites. KEY FINDINGS: Unique results included: a two- three-fold increase in taurine levels in all three ventricular regions of MI rabbits and similarly, the three regions had increased inosine levels compared to sham controls. Reduced myocardial levels of myo-inositol in the myocardium of MI animals point to altered phospholipid metabolism and membrane receptor function in heart failure. Metabolite profiles also provide evidence for responses to oxidative stress and an impairment in TCA cycle energy production in the failing heart. SIGNIFICANCE: Our results revealed metabolic changes during compensated cardiac dysfunction and suggest potential targets for altering the progression of heart failure.

Detection of Inosine on Transfer RNAs without a Reverse Transcription Reaction.

Inosine at the "wobble" position (I34) is one of the few essential posttranscriptional modifications in tRNAs (tRNAs). It results from the deamination of adenosine and occurs in bacteria on tRNAArgACG and in eukarya on six or seven additional tRNA substrates. Because inosine is structurally a guanosine analogue, reverse transcriptases recognize it as a guanosine. Most methods used to examine the presence of inosine rely on this phenomenon and detect the modified base as a change in the DNA sequence that results from the reverse transcription reaction. These methods, however, cannot always be applied to tRNAs because reverse transcription can be compromised by the presence of other posttranscriptional modifications. Here we present SL-ID (splinted ligation-based inosine detection), a reverse transcription-free method for detecting inosine based on an I34-dependent specific cleavage of tRNAs by endonuclease V, followed by a splinted ligation and polyacrylamide gel electrophoresis analysis. We show that the method can detect I34 on different tRNA substrates and can be applied to total RNA derived from different species, cell types, and tissues. Here we apply the method to solve previous controversies regarding the modification status of mammalian tRNAArgACG.

The observed variation in the purine composition of food after soaking in sake lees.

In this study, we investigated the alterations in the purine composition of swordfish prepared using a traditional Japanese processing method of soaking in sake lees. These alterations are the byproducts of the yeast fermentation of rice-koji and are renowned for enhancing the umami nature of food. Using a conventional assay method for hydrolyzing all of the purines into four bases and our developed method for simultaneously analyzing purines, we observed the alterations in four purine bases in the soaked sake lees and swordfish. The findings showed that the total purine content, and hypoxanthine-related and guanine-related purines in swordfish decreased after soaking in sake lees. We also analyzed the free purine composition and showed that the ratio of IMP in swordfish was decreased by soaking, while that of inosine in sake lees was increased by soaking swordfish in it.

[Study on quality control of Holotrichia diomphalia larvae by TLC and HPLC].

The TLC method was established for identification of Holotricha diomphalia larvae and the HPLC method was used to determine the content of inosine and guanosine in H. diomphalia larvae. The HPLC analysis was performed on a Waters HSS T3（4.6 mm×250 mm, 5 µm） column of with mobile phase consisting of acetonitrile (A) and 0.08% trifluoroacetic acid (B) in gradient elution. The detection wavelength was 260 nm. The flow rate was 1.0 mL·min⁻¹. The column temperature was 30 °C. As a result, TLC identification method had a good reproducibility and highly specificity. The linear equations of inosine and guanosine were in good linear range (r>0.999 8). The average recovery of inosine and guanosine was 96.53% (RSD=1.6%), 99.71% (RSD=2.7%). The method is simple, accurate and reproducible, which can provide a basis for quality standard improvement H. diomphalia larvae.

Tudor staphylococcal nuclease is a structure-specific ribonuclease that degrades RNA at unstructured regions during microRNA decay.

Tudor staphylococcal nuclease (TSN) is an evolutionarily conserved ribonuclease in eukaryotes that is composed of five staphylococcal nuclease-like domains (SN1-SN5) and a Tudor domain. TSN degrades hyper-edited double-stranded RNA, including primary miRNA precursors containing multiple I•U and U•I pairs, and mature miRNA during miRNA decay. However, how TSN binds and degrades its RNA substrates remains unclear. Here, we show that the C. elegans TSN (cTSN) is a monomeric Ca2+-dependent ribonuclease, cleaving RNA chains at the 5'-side of the phosphodiester linkage to produce degraded fragments with 5'-hydroxyl and 3'-phosphate ends. cTSN degrades single-stranded RNA and double-stranded RNA containing mismatched base pairs, but is not restricted to those containing multiple I•U and U•I pairs. cTSN has at least two catalytic active sites located in the SN1 and SN3 domains, since mutations of the putative Ca2+-binding residues in these two domains strongly impaired its ribonuclease activity. We further show by small-angle X-ray scattering that rice osTSN has a flexible two-lobed structure with open to closed conformations, indicating that TSN may change its conformation upon RNA binding. We conclude that TSN is a structure-specific ribonuclease targeting not only single-stranded RNA, but also unstructured regions of double-stranded RNA. This study provides the molecular basis for how TSN cooperates with RNA editing to eliminate duplex RNA in cell defense, and how TSN selects and degrades RNA during microRNA decay.

Deoxyinosine triphosphate induces MLH1/PMS2- and p53-dependent cell growth arrest and DNA instability in mammalian cells.

Deoxyinosine (dI) occurs in DNA either by oxidative deamination of a previously incorporated deoxyadenosine residue or by misincorporation of deoxyinosine triphosphate (dITP) from the nucleotide pool during replication. To exclude dITP from the pool, mammals possess specific hydrolysing enzymes, such as inosine triphosphatase (ITPA). Previous studies have shown that deficiency in ITPA results in cell growth suppression and DNA instability. To explore the mechanisms of these phenotypes, we analysed ITPA-deficient human and mouse cells. We found that both growth suppression and accumulation of single-strand breaks in nuclear DNA of ITPA-deficient cells depended on MLH1/PMS2. The cell growth suppression of ITPA-deficient cells also depended on p53, but not on MPG, ENDOV or MSH2. ITPA deficiency significantly increased the levels of p53 protein and p21 mRNA/protein, a well-known target of p53, in an MLH1-dependent manner. Furthermore, MLH1 may also contribute to cell growth arrest by increasing the basal level of p53 activity.

Analysis of Trinitrophenylated Adenosine and Inosine by Capillary Electrophoresis and γ-Cyclodextrin-Enhanced Fluorescence Detection.

Monitoring molecules such as adenosine (Ado) and inosine (Ino) in the central nervous system has enabled the field of neuroscience to correlate molecular concentrations dynamics to neurological function, behavior, and disease. In vivo sampling techniques are commonly used to monitor these dynamics; however, many techniques are limited by the sensitivity and sample volume requirements of currently available detection methods. Here, we present a novel capillary electrophoresis-laser-induced fluorescence detection (CE-LIF) method that analyzes Ado and Ino by derivatization with 2,4,6-trinitrobenzenesulfonic acid to form fluorescent trinitrophenylated complexes of Ado (TNP-Ado) and Ino (TNP-Ino). These complexes exhibit ∼25-fold fluorescence enhancement upon the formation of inclusion complexes with γ-cyclodextrin (γ-CD). Association constants were determined as 4600 M(-1) for Ado and 1000 M(-1) for Ino by CE-LIF. The structure of the TNP-Ado:γ-CD complex was determined by 2D nuclear magnetic resonance (NMR) spectroscopy. Optimal trinitrophenylation reaction conditions and CE-LIF parameters were determined and resulted in the limit of detection of 1.6 µM for Ado and 4 µM for Ino. Ado and Ino were simultaneously quantified in homogenized rat forebrain samples to illustrate application of the technique. Simulated biological samples, desalted by ultrafiltration in the presence γ-CD, were concentrated on-capillary by large-volume sample stacking (LVSS) to achieve detection limits of 32 and 38 nM for TNP-Ado and TNP-Ino, respectively.

Triplex-forming peptide nucleic acid modified with 2-aminopyridine as a new tool for detection of A-to-I editing.

RNA editing from adenosine to inosine (A-to-I editing) is one of the mechanisms that regulate and diversify the transcriptome. Here, a triplex-forming peptide nucleic acid (PNA) modified with a 2-aminopyridine nucleobase was applied for the recognition of the A-to-I editing event in double-stranded RNAs. The triplex-forming PNA enabled sequence-specific detection of single nucleobase editing at sub-nanomolar concentration.