Effects of Strain Differences, Humidity Changes, and Saliva Contamination on the Inactivation of SARS-CoV-2 by Ion Irradiation.

One of the methods to inactivate viruses is to denature viral proteins using released ions. However, there have been no reports detailing the effects of changes in humidity or contamination with body fluids on the inactivation of viruses. This study investigated the effects of humidity changes and saliva contamination on the efficacy of SARS-CoV-2 inactivation with ions using multiple viral strains. Virus solutions with different infectious titers were dropped onto a circular nitrocellulose membrane and irradiated with ions from 10 cm above the membrane. After the irradiation of ions for 60, 90, and 120 min, changes in viral infectious titers were measured. The effect of ions on virus inactivation under different humidity conditions was also examined using virus solutions containing 90% mixtures of saliva collected from 10 people. A decrease in viral infectivity was observed over time for all strains, but ion irradiation further accelerated the decrease in viral infectivity. Ion irradiation can inactivate all viral strains, but at 80% humidity, the effect did not appear until 90 min after irradiation. The presence of saliva protected the virus from drying and maintained infectiousness for a longer period compared with no saliva. In particular, the Omicron strain retained its infectivity titer longer than the other strains. Ion irradiation demonstrated a consistent reduction in the number of infectious viruses when compared to the control across varying levels of humidity and irradiation periods. This underscores the notable effectiveness of irradiation, even when the reduction effect is as modest as 50%, thereby emphasizing its crucial role in mitigating the rapid dissemination of SARS-CoV-2.

ICLAMP: a novel technique to explore adenosine deamination via inosine chemical labeling and affinity molecular purification.

Recent developments in sequencing and bioinformatics have advanced our understanding of adenosine-to-inosine (A-to-I) RNA editing. Surprisingly, recent analyses have revealed the capability of adenosine deaminase acting on RNA (ADAR) to edit DNA:RNA hybrid strands. However, edited inosines in DNA remain largely unexplored. A precise biochemical method could help uncover these potentially rare DNA editing sites. We explore maleimide as a scaffold for inosine labeling. With fluorophore-conjugated maleimide, we were able to label inosine in RNA or DNA. Moreover, with biotin-conjugated maleimide, we purified RNA and DNA containing inosine. Our novel technique of inosine chemical labeling and affinity molecular purification offers substantial advantages and provides a versatile platform for further discovery of A-to-I editing sites in RNA and DNA.

Development of Epstein-Barr virus-associated gastric cancer: Infection, inflammation, and oncogenesis.

Epstein-Barr virus (EBV)-associated gastric cancer (EBVaGC) cells originate from a single-cell clone infected with EBV. However, more than 95% of patients with gastric cancer have a history of Helicobacter pylori (H. pylori) infection, and H. pylori is a major causative agent of gastric cancer. Therefore, it has long been argued that H. pylori infection may affect the development of EBVaGC, a subtype of gastric cancer. Atrophic gastrointestinal inflammation, a symptom of H. pylori infection, is observed in the gastric mucosa of EBVaGC. Therefore, it remains unclear whether H. pylori infection is a cofactor for gastric carcinogenesis caused by EBV infection or whether H. pylori and EBV infections act independently on gastric cancer formation. It has been reported that EBV infection assists in the onco-genesis of gastric cancer caused by H. pylori infection. In contrast, several studies have reported that H. pylori infection accelerates tumorigenesis initiated by EBV infection. By reviewing both clinical epidemiological and experimental data, we reorganized the role of H. pylori and EBV infections in gastric cancer formation.

Regulation of A-to-I RNA editing and stop codon recoding to control selenoprotein expression during skeletal myogenesis.

Selenoprotein N (SELENON), a selenocysteine (Sec)-containing protein with high reductive activity, maintains redox homeostasis, thereby contributing to skeletal muscle differentiation and function. Loss-of-function mutations in SELENON cause severe neuromuscular disorders. In the early-to-middle stage of myoblast differentiation, SELENON maintains redox homeostasis and modulates endoplasmic reticulum (ER) Ca2+ concentration, resulting in a gradual reduction from the middle-to-late stages due to unknown mechanisms. The present study describes post-transcriptional mechanisms that regulate SELENON expression during myoblast differentiation. Part of an Alu element in the second intron of SELENON pre-mRNA is frequently exonized during splicing, resulting in an aberrant mRNA that is degraded by nonsense-mediated mRNA decay (NMD). In the middle stage of myoblast differentiation, ADAR1-mediated A-to-I RNA editing occurs in the U1 snRNA binding site at 5' splice site, preventing Alu exonization and producing mature mRNA. In the middle-to-late stage of myoblast differentiation, the level of Sec-charged tRNASec decreases due to downregulation of essential recoding factors for Sec insertion, thereby generating a premature termination codon in SELENON mRNA, which is targeted by NMD.

Adenosine-to-inosine RNA editing in neurological development and disease.

Adenosine-to-inosine (A-to-I) editing is one of the most prevalent post-transcriptional RNA modifications in metazoan. This reaction is catalysed by enzymes called adenosine deaminases acting on RNA (ADARs). RNA editing is involved in the regulation of protein function and gene expression. The numerous A-to-I editing sites have been identified in both coding and non-coding RNA transcripts. These editing sites are also found in various genes expressed in the central nervous system (CNS) and play an important role in neurological development and brain function. Aberrant regulation of RNA editing has been associated with the pathogenesis of neurological and psychiatric disorders, suggesting the physiological significance of RNA editing in the CNS. In this review, we discuss the current knowledge of editing on neurological disease and development.

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.

Complete chemical structures of human mitochondrial tRNAs.

Mitochondria generate most cellular energy via oxidative phosphorylation. Twenty-two species of mitochondrial (mt-)tRNAs encoded in mtDNA translate essential subunits of the respiratory chain complexes. mt-tRNAs contain post-transcriptional modifications introduced by nuclear-encoded tRNA-modifying enzymes. They are required for deciphering genetic code accurately, as well as stabilizing tRNA. Loss of tRNA modifications frequently results in severe pathological consequences. Here, we perform a comprehensive analysis of post-transcriptional modifications of all human mt-tRNAs, including 14 previously-uncharacterized species. In total, we find 18 kinds of RNA modifications at 137 positions (8.7% in 1575 nucleobases) in 22 species of human mt-tRNAs. An up-to-date list of 34 genes responsible for mt-tRNA modifications are provided. We identify two genes required for queuosine (Q) formation in mt-tRNAs. Our results provide insight into the molecular mechanisms underlying the decoding system and could help to elucidate the molecular pathogenesis of human mitochondrial diseases caused by aberrant tRNA modifications.

The RNA-binding protein QKI-7 recruits the poly(A) polymerase GLD-2 for 3' adenylation and selective stabilization of microRNA-122.

MicroRNA-122 (miR-122) is highly expressed in hepatocytes, where it plays an important role in regulating cholesterol and fatty acid metabolism, and it is also a host factor required for hepatitis C virus replication. miR-122 is selectively stabilized by 3' adenylation mediated by the cytoplasmic poly(A) polymerase GLD-2 (also known as PAPD4 or TENT2). However, it is unclear how GLD-2 specifically stabilizes miR-122. Here, we show that QKI7 KH domain-containing RNA binding (QKI-7), one of three isoforms of the QKI proteins, which are members of the signal transduction and activation of RNA (STAR) family of RNA-binding proteins, is involved in miR-122 stabilization. QKI down-regulation specifically decreased the steady-state level of mature miR-122, but did not affect the pre-miR-122 level. We also found that QKI-7 uses its C-terminal region to interact with GLD-2 and its QUA2 domain to associate with the RNA-induced silencing complex protein Argonaute 2 (Ago2), indicating that the GLD-2-QKI-7 interaction recruits GLD-2 to Ago2. QKI-7 exhibited specific affinity to miR-122 and significantly promoted GLD-2-mediated 3' adenylation of miR-122 in vitro Taken together, our findings indicate that miR-122 binds Ago2-interacting QKI-7, which recruits GLD-2 for 3' adenylation and stabilization of miR-122.

Transcriptome-wide identification of A-to-I RNA editing sites using ICE-seq.

In A-to-I RNA editing, adenosine is converted to inosine in double-stranded regions of RNAs. Inosine, an abundant epitranscriptomic mark, contributes to a wide range of biological processes by regulating gene expression post-transcriptionally. To understand the effect of A-to-I RNA editing on regulation of the epitranscriptome, accurate mapping of inosines is necessary. To this end, we established a biochemical method called inosine chemical erasing sequencing (ICE-seq) that enables unbiased and reliable identification of A-to-I RNA editing sites throughout the transcriptome. Here, we describe our updated protocol for ICE-seq in the human transcriptome.

Isolation, Structural Elucidation, and Liquid Chromatography-Mass Spectrometry Analysis of Steroidal Glycosides from Polygonatum odoratum.

The rhizomes of Polygonatum odoratum represent a traditional Chinese medicine and functional food. A phytochemical investigation resulted in the isolation of eight steroidal glycosides (1-8), including two new compounds, polygonatumosides F (1) and G (2). The structures were elucidated by spectroscopic data and chemical reactions. Compound 7 showed antiproliferation activity against human hepatocellular carcinoma cell line HepG2 (IC50 of 3.2 µM). The chemical profile and contents of steroidal glycosides of P. odoratum rhizomes collected at different dates and geographical locations were also investigated, indicating that the rational harvest of P. odoratum in spring and autumn is preferable to obtain higher levels of steroidal glycosides. Compounds 1 and 7 showed the highest contents in all P. odoratum samples and have potential to serve as chemotaxonomic and chemical markers for quality control of this important plant material. 14-Hydroxylation may be a key step for the biosynthesis of compounds 1-7.

RNA editing enzyme ADAR2 is a mediator of neuropathic pain after peripheral nerve injury.

Transcriptional and post-translational regulations are important in peripheral nerve injury-induced neuropathic pain, but little is known about the role of post-transcriptional modification. Our objective was to determine the possible effect of adenosine deaminase acting on RNA (ADAR) enzymes, which catalyze post-transcriptional RNA editing, in tactile allodynia, a hallmark of neuropathic pain. Seven days after L5 spinal nerve transection (SNT) in adult mice, we found an increase in ADAR2 expression and a decrease in ADAR3 expression in the injured, but not in the uninjured, dorsal root ganglions (DRGs). These changes were accompanied by elevated levels of editing at the D site of the serotonin (5-hydroxytryptamine) 2C receptor (5-HT2CR), at the I/V site of coatomer protein complex subunit α (COPA), and at the R/G site of AMPA receptor subunit GluA2 in the injured DRG. Compared to Adar2+/+/Gria2R/R littermate controls, Adar2-/-/Gria2R/R mice completely lacked the increased editing of 5-HT2CR, COPA, and GluA2 transcripts in the injured DRG and showed attenuated tactile allodynia after SNT. Furthermore, the antidepressant fluoxetine inhibited neuropathic allodynia after injury and reduced the COPA I/V site editing in the injured DRG. These findings suggest that ADAR2 is a mediator of injury-induced tactile allodynia and thus a potential therapeutic target for the treatment of neuropathic pain.-Uchida, H., Matsumura, S., Okada, S., Suzuki, T., Minami, T., Ito, S. RNA editing enzyme ADAR2 is a mediator of neuropathic pain after peripheral nerve injury.

Regulation of gene expression via retrotransposon insertions and the noncoding RNA 4.5S RNAH.

Short interspersed elements (SINEs) comprise a significant portion of mammalian genomes and regulate gene expression through a variety of mechanisms. Here, we show that Myodonta clade-specific 4.5S RNAH (4.5SH), an abundant nuclear noncoding RNA that is highly homologous to the retrotransposon SINE B1, controls the expression of reporter gene that contains the antisense insertion of SINE B1 via nuclear retention. The depletion of endogenous 4.5SH with antisense oligonucleotides neutralizes the nuclear retention and changes the subcellular distribution of the reporter transcripts containing the antisense SINE B1 insertion. Importantly, endogenous transcripts with antisense SINE B1 were increased in the cytoplasm after knockdown of 4.5SH, leading to a decrease in cellular growth. We propose a tentative hypothesis that the amplification of the 4.5SH cluster in specific rodent species might delineate their evolutionary direction via the regulation of genes containing the antisense insertion of SINE B1.

Biochemical and Transcriptome-Wide Identification of A-to-I RNA Editing Sites by ICE-Seq.

Inosine (I) is a modified adenosine (A) in RNA. In Metazoa, I is generated by hydrolytic deamination of A, catalyzed by adenosine deaminase acting RNA (ADAR) in a process called A-to-I RNA editing. A-to-I RNA editing affects various biological processes by modulating gene expression. In addition, dysregulation of A-to-I RNA editing results in pathological consequences. I on RNA strands is converted to guanosine (G) during cDNA synthesis by reverse transcription. Thus, the conventional method used to identify A-to-I RNA editing sites compares cDNA sequences with their corresponding genomic sequences. Combined with deep sequencing, this method has been applied to transcriptome-wide screening of A-to-I RNA editing sites. This approach, however, produces a large number of false positives mainly owing to mapping errors. To address this issue, we developed a biochemical method called inosine chemical erasing (ICE) to reliably identify genuine A-to-I RNA editing sites. In addition, we applied the ICE method combined with RNA-seq, referred to as ICE-seq, to identify transcriptome-wide A-to-I RNA editing sites. In this chapter, we describe the detailed protocol for ICE-seq, which can be applied to various sources and taxa.

Transcriptome-wide identification of adenosine-to-inosine editing using the ICE-seq method.

Inosine (I), a modified base found in the double-stranded regions of RNA in metazoans, has various roles in biological processes by modulating gene expression. Inosine is generated from adenosine (A) catalyzed by ADAR (adenosine deaminase acting on RNA) enzymes in a process called A-to-I RNA editing. As inosine is converted to guanosine (G) by reverse transcription, the editing sites can be identified by simply comparing cDNA sequences with the corresponding genomic sequence. One approach to screening I sites is by deep sequencing based on A-to-G conversion from genomic sequence to cDNA; however, this approach produces a high rate of false positives because it cannot efficiently eliminate G signals arising from inevitable mapping errors. To address this issue, we developed a biochemical method to identify inosines called inosine chemical erasing (ICE), which is based on cyanoethylation combined with reverse transcription. ICE was subsequently combined with deep sequencing (ICE-seq) for the reliable identification of transcriptome-wide A-to-I editing sites. Here we describe a protocol for the practical application of ICE-seq, which can be completed within 22 d, and which allows the accurate identification of transcriptome-wide A-to-I RNA editing sites.

A biochemical landscape of A-to-I RNA editing in the human brain transcriptome.

Inosine is an abundant RNA modification in the human transcriptome and is essential for many biological processes in modulating gene expression at the post-transcriptional level. Adenosine deaminases acting on RNA (ADARs) catalyze the hydrolytic deamination of adenosines to inosines (A-to-I editing) in double-stranded regions. We previously established a biochemical method called "inosine chemical erasing" (ICE) to directly identify inosines on RNA strands with high reliability. Here, we have applied the ICE method combined with deep sequencing (ICE-seq) to conduct an unbiased genome-wide screening of A-to-I editing sites in the transcriptome of human adult brain. Taken together with the sites identified by the conventional ICE method, we mapped 19,791 novel sites and newly found 1258 edited mRNAs, including 66 novel sites in coding regions, 41 of which cause altered amino acid assignment. ICE-seq detected novel editing sites in various repeat elements as well as in short hairpins. Gene ontology analysis revealed that these edited mRNAs are associated with transcription, energy metabolism, and neurological disorders, providing new insights into various aspects of human brain functions.