Simultaneous Detection of Adenosine-to-Inosine Editing and N6-Methyladenosine at Identical RNA Sites through Deamination-Assisted Reverse Transcription Stalling.

Adenosine-to-inosine (A-to-I) editing and N6-methyladenosine (m6A) modifications are pivotal RNA modifications with widespread functional significance in physiological and pathological processes. Although significant effort has been dedicated to developing methodologies for identifying and quantifying these modifications, traditional approaches have often focused on each modification independently, neglecting the potential co-occurrence of A-to-I editing and m6A modifications at the same adenosine residues. This limitation has constrained our understanding of the intricate regulatory mechanisms governing RNA function and the interplay between different types of RNA modifications. To address this gap, we introduced an innovative technique called deamination-assisted reverse transcription stalling (DARTS), specifically designed for the simultaneous quantification of A-to-I editing and m6A at the same RNA sites. DARTS leverages the selective deamination activity of the engineered TadA-TadA8e protein, which converts adenosine residues to inosine, in combination with the unique property of Bst 2.0 DNA polymerase, which stalls when encountering inosine during reverse transcription. This approach enables the accurate quantification of A-to-I editing, m6A, and unmodified adenosine at identical RNA sites. The DARTS method is remarkable for its ability to directly quantify two distinct types of RNA modifications simultaneously, a capability that has remained largely unexplored in the field of RNA biology. By facilitating a comprehensive analysis of the co-occurrence and interaction between A-to-I editing and m6A modifications, DARTS opens new avenues for exploring the complex regulatory networks modulated by different RNA modifications.

Single-Base Resolution Detection of N6-Methyladenosine in RNA by Adenosine Deamination Sequencing.

N6-Methyladenosine (m6A) is one of the most abundant and prevalent natural modifications occurring in diverse RNA species. m6A plays a wide range of roles in physiological and pathological processes. Revealing the functions of m6A relies on the faithful detection of individual m6A sites in RNA. However, developing a simple method for the single-base resolution detection of m6A is still a challenging task. Herein, we report an adenosine deamination sequencing (AD-seq) technique for the facile detection of m6A in RNA at single-base resolution. The AD-seq approach capitalizes on the selective deamination of adenosine, but not m6A, by the evolved tRNA adenosine deaminase (TadA) variant of TadA8e or the dimer protein of TadA-TadA8e. In AD-seq, adenosine is deaminated by TadA8e or TadA-TadA8e to form inosine, which pairs with cytidine and is read as guanosine in sequencing. m6A resists deamination due to the interference of the methyl group at the N6 position of adenosine. Thus, the m6A base pairs with thymine and is still read as adenosine in sequencing. The differential readouts from A and m6A in sequencing can achieve the single-base resolution detection of m6A in RNA. Application of the proposed AD-seq successfully identified individual m6A sites in Escherichia coli 23S rRNA. Taken together, the proposed AD-seq allows simple and cost-effective detection of m6A at single-base resolution in RNA, which provides a valuable tool to decipher the functions of m6A in RNA.

Burden and Risk of Polychlorinated Naphthalenes in Chinese Human Milk and a Global Comparison of Human Exposure.

Polychlorinated naphthalenes (PCNs) are carcinogenic contaminants. Residues from historical production and ongoing unintentional releases from industrial thermal sources have led to the ubiquitous presence of PCNs in the environment. Our previous study has revealed that unintentional releases may be the main sources of PCNs in human milk from China. However, an assessment of PCN burden in human milk and exposure differences between historical residues and unintentional release exposure has not been conducted. In this study, we performed the first comparison of human exposure to PCNs and evaluated the differences between the estimated health risks from historical residues and unintentional releases. Three characteristic PCN congener patterns found in Chinese human milk specimens collected from 100 cities/counties can be considered characteristic of PCN exposures in regions with unintentional industrial releases as the main PCN sources. The health risk assessment suggested potential noncarcinogenic health effects in infants aged 0-6 months. The hazard index calculated for infants in Sweden indicates a strong impact of historical residues that nonetheless decreases over time, and a comparison of the hazard indices calculated for China and Ireland suggests that ongoing unintentional formation and release of PCNs from industrial processes should be a matter of public health concern.

Single-Nucleotide Resolution Mapping of N6-Methyladenine in Genomic DNA.

N6-Methyladenine (6mA) is a naturally occurring DNA modification in both prokaryotes and eukaryotes. Herein, we developed a deaminase-mediated sequencing (DM-seq) method for genome-wide mapping of 6mA at single-nucleotide resolution. The method capitalizes on the selective deamination of adenine, but not 6mA, in DNA mediated by an evolved adenine deaminase, ABE8e. By employing this method, we achieved genome-wide mapping of 6mA in Escherichia coli and in mammalian mitochondrial DNA (mtDNA) at single-nucleotide resolution. We found that the 6mA sites are mainly located in the GATC motif in the E. coli genome. We also identified 17 6mA sites in mtDNA of HepG2 cells, where all of the 6mA sites are distributed in the heavy strand of mtDNA. We envision that DM-seq will be a valuable tool for uncovering new functions of 6mA in DNA and for exploring its potential roles in mitochondria-related human diseases.

Occurrence and migration of phthalates in adhesive materials to fruits and vegetables.

Labels or tapes are widely used on fresh fruits and vegetables, which may contain phthalates (PAEs). There are few studies on the contamination pathway of PAEs from labels or tapes to food stuff. In this study, the concentrations of eleven PAEs in adhesive labels, tapes, labeled fruits and vegetables on the market were investigated. The eleven PAEs were detected with the total concentration of 7.44-30.51 mg/m2 in labels and tapes. Diethyl phthalate (DEP), di-n-butyl phthalate (DIBP), dimethyl phthalate (DMP), Bis (2-ethylhexyl) phthalate (DEHP) and di-n-butyl phthalate (DBP) had the highest detection frequency in adhesive material samples. The concentrations of PAEs in labeled fruits were higher than that in unlabeled fruits, especially in the peel, indicating PAEs could transfer from labels or tapes to fruits and vegetables. Furthermore, the migration behaviors of PAEs from labels or tapes to apples, avocados and celery were investigated. It was found that the PAEs could penetrate to apple and avocado pulp through the peel, resulting in the residue of the PAEs in the whole fruit. Unlike apple peel, the thick avocado peel was more difficultly penetrated by the PAEs. Due to the high lipid content, the PAEs distributed more evenly in avocado pulp than in apple pulp. The migrations up to a maximum of 4.16 mg/kg were found for butyl benzyl phthalate (BBP) in avocado peel and up to a maximum of 0.188 mg/kg in avocado pulp. The average migration of the PAEs in celery ranged from 0.3 to 26.1 µg/kg in three days and the low migration might result from the rough surface and less contacting area. These findings suggest that the use of labels or tapes in direct contact may increase the risk of PAEs exposure to humans through fruits and vegetables.

Polychlorinated naphthalenes in human milk: Health risk assessment to nursing infants and source analysis.

Polychlorinated naphthalenes are teratogenic environmental contaminants. Mother milk is the most important food for nursing infants. The World Health Organization actively promotes breastfeeding for its immunological, psychological, and economic advantages. We firstly measured concentrations of polychlorinated naphthalenes in human milk from 19 provinces in China and estimated their potential health risks to nursing infants and their possible sources. Concentrations ranged from 211.07 to 2497.43 pg/g lipid. The high prevalence of highly toxic hexachlorinated naphthalenes (Hexa-CN66/67) in human milk samples indicated a higher health risk in the sampling areas. Cancer risk posed to nursing infants was not significant, but potential non-carcinogenic adverse health effects were suggested and should be emphasized in some sampling areas. Unintentional emission of polychlorinated naphthalenes from industries that employ thermal processes appears to be the main source for PCNs in human milk in most sampling areas. Correlation analysis also suggested PCNs as impurities in polychlorinated biphenyl mixtures as a previously unrecognized source of polychlorinated naphthalenes in human milk.