Metabolomic Analysis Reveals Association between Decreased Ovarian Reserve and In Vitro Fertilization Outcomes.

In vitro fertilization (IVF) is a highly effective treatment for infertility; however, it poses challenges for women with decreased ovarian reserve (DOR). Despite the importance of understanding the impact of DOR on IVF outcomes, limited research has explored this relationship, particularly using omics approaches. Hence, we conducted a study to investigate the association between DOR and IVF outcomes, employing a metabolomic approach. We analyzed serum samples from 207 women undergoing IVF treatment, including 89 with DOR and 118 with normal ovarian reserve (NOR). Our findings revealed that DOR was significantly associated with unfavorable IVF outcomes, characterized by a reduced oocyte count, lower embryo quality, and decreased rates of pregnancy and live births. Furthermore, we identified 82 metabolites that displayed significant alterations in DOR patients, impacting diverse metabolic pathways. Notably, a distinct panel of metabolites, including palmitic acid, stearic acid, LysoPC(9:0(CHO)/0:0), PC(18:0/9:0(CHO)), and PC(16:0/9:0(CHO)), exhibited discriminatory power between the DOR and NOR groups, showcasing a strong correlation with IVF outcomes. These findings emphasize the crucial role of metabolomic disruptions in influencing IVF outcomes among women with DOR.

Whole-Genome Mapping of Epigenetic Modification of 5-Formylcytosine at Single-Base Resolution by Chemical Labeling Enrichment and Deamination Sequencing.

DNA cytosine methylation (5-methylcytosine, 5mC) is a predominant epigenetic modification that plays a critical role in a variety of biological and pathological processes in mammals. In active DNA demethylation, the 10-11 translocation (TET) dioxygenases can sequentially oxidize 5mC to generate three modified forms of cytosine, 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). Beyond being a demethylation intermediate, recent studies have shown that 5fC has regulatory functions in gene expression and chromatin organization. While some methods have been developed to detect 5fC, genome-wide mapping of 5fC at base resolution is still highly desirable. Herein, we propose a chemical labeling enrichment and deamination sequencing (CLED-seq) method for detecting 5fC in genomic DNA at single-base resolution. The CLED-seq method utilizes selective labeling and enrichment of 5fC-containing DNA fragments, followed by deamination mediated by apolipoprotein B mRNA-editing catalytic polypeptide-like 3A (APOBEC3A or A3A) and sequencing. In the CLED-seq process, while all C, 5mC, and 5hmC are interpreted as T during sequencing, 5fC is still read as C, enabling the precise detection of 5fC in DNA. Using the proposed CLED-seq method, we accomplished genome-wide mapping of 5fC in mouse embryonic stem cells. The mapping study revealed that promoter regions enriched with 5fC overlapped with H3K4me1, H3K4me3, and H3K27ac marks. These findings suggest a correlation between 5fC marks and active gene expression in mESCs. In conclusion, CLED-seq is a straightforward, bisulfite-free method that offers a valuable tool for detecting 5fC in genomes at a single-base resolution.

Advancing Glucose Conjugated Gibberellins Discovery: A Structure-Oriented Screening and Identification Method for Unraveling Gibberellin Metabolites in Plants.

Gibberellins (GAs) play a pivotal role in modulating plant growth and development. Glucose-conjugated gibberellins (Glc-GAs), a prevalent conjugated form of GAs, regulate intracellular GA levels by the coupling and decoupling of glucose groups. However, the diversity of Glc-GAs identified within individual species remains limited, hinting at a multitude of yet undiscovered gibberellin metabolites. This lacuna poses considerable impediments to research efforts dedicated to comprehensively delineating the GA metabolic pathway. In this study, we developed a structure-oriented screening and identification method for Glc-GAs in plant species by employing LC-MS/MS coupled with chemical derivatization. Through the application of chemical derivatization technique, carboxyl groups on Glc-GAs were labeled which effectively enhanced the sensitivity and selectivity of mass spectrometry detection for these compounds. Concurrently, the integration of mass spectrometry fragmentation and chromatographic retention behavior facilitated the efficient screening and identification of potential Glc-GAs. With this strategy, we screened and identified 12 potential Glc-GAs from six plant species. These findings expand the Glc-GA diversity in plants and contribute to understanding GA metabolic pathways.

[Alignment method for metabolite chromatographic peaks using an N-acyl glycine retention index system].

Peak alignment is a crucial data-processing step in untargeted metabolomics analysis that aims to integrate metabolite data from multiple liquid chromatography-mass spectrometry (LC-MS) batches for enhanced comparability and reliability. However, slight variations in the chromatographic separation conditions can result in retention time (RT) shifts between consecutive analyses, adversely affecting peak alignment accuracy. In this study, we present a retention index (RI)-based chromatographic peak-shift correction (CPSC) strategy to address RT shifts and align chromatographic peaks for metabolomics studies. A series of N-acyl glycine homologues (C2-C23) was synthesized as calibrants, and an LC RI system was established. This system effectively corrected RT shifts arising from variations in flow rate, gradient elution, instrument systems, and chromatographic columns. Leveraging the RI system, we successfully adjusted the RT of raw data to mitigate RT shifts and then implemented the Joint Aligner algorithm for peak alignment. We assessed the accuracy of the RI-based CPSC strategy using pooled human fecal samples as a test model. Notably, the application of the RI-based CPSC strategy to a long-term dataset spanning 157 d as an illustration revealed a significant enhancement in peak alignment accuracy from 15.5% to 80.9%, indicating its ability to substantially improve peak-alignment precision in multibatch LC-MS analyses.

AlkB-Facilitated Demethylation Enables Quantitative and Site-Specific Detection of Dual Methylation of Adenosine in RNA.

RNA molecules undergo various chemical modifications that play critical roles in a wide range of biological processes. N6,N6-Dimethyladenosine (m6,6A) is a conserved RNA modification and is essential for the processing of rRNA. To gain a deeper understanding of the functions of m6,6A, site-specific and accurate quantification of this modification in RNA is indispensable. In this study, we developed an AlkB-facilitated demethylation (AD-m6,6A) method for the site-specific detection and quantification of m6,6A in RNA. The N6,N6-dimethyl groups in m6,6A can cause reverse transcription to stall at the m6,6A site, resulting in truncated cDNA. However, we found that Escherichia coli AlkB demethylase can effectively demethylate m6,6A in RNA, generating full-length cDNA from AlkB-treated RNA. By quantifying the amount of full-length cDNA produced using quantitative real-time PCR, we were able to achieve site-specific detection and quantification of m6,6A in RNA. Using the AD-m6,6A method, we successfully detected and quantified m6,6A at position 1851 of 18S rRNA and position 937 of mitochondrial 12S rRNA in human cells. Additionally, we found that the level of m6,6A at position 1007 of mitochondrial 12S rRNA was significantly reduced in lung tissues from sleep-deprived mice compared with control mice. Overall, the AD-m6,6A method provides a valuable tool for easy, accurate, quantitative, and site-specific detection of m6,6A in RNA, which can aid in uncovering the functions of m6,6A in human diseases.

Rapid LC-MS/MS detection of 25-hydroxyvitamin D in dried blood spots.

BACKGROUND: The detection of 25-hydroxyvitamin D (25OHD) from dried blood spots (DBS) has been widely studied. However, the existing pretreatment methods suffer from limitations in terms of throughput (usually exceeding 2 h), complexity (involving liquid-liquid extraction or solid-phase extraction), and contamination (including multiple steps of organic solvent evaporation). RESULTS: We first released 25OHD from DBS samples by 50% acetonitrile solution through ultrasonication. Subsequently, the cold-induced phase separation technique was introduced for in-situ concentration and purification. Afterward, the PTAD derivatization of 25OHD was performed directly, profiting from the high acetonitrile content in the concentrated solution. In the end, the resulting solution was submitted to LC-MS/MS for quantification. The established LC-MS/MS methodology possessed favorable analytical performance, possessing lower limit of quantification of 1 ng/mL pointing to plasma, accuracy of 86.8-110.1% and imprecision of 5.4-16.8%. Method comparison with plasma samples demonstrated that over 93% of the detections met the acceptance limit for cross-validation of ±20%. SIGNIFICANCE AND NOVELTY: The novel sample preparation can be finished within 15 min and eliminated the traditional steps of extraction and organic solvent evaporation. Based on this high-throughput, reliable and applicable LC-MS/MS method, the detection of 25OHD in DBS samples can be better achieved for clinical patients and researchers with relevant demands.

A dataset of branched fatty acid esters of hydroxy fatty acids diversity in foods.

Branched fatty acid esters of hydroxy fatty acids (FAHFAs) are a class of bioactive lipids that show therapeutic potential for diabetes, anti-cancer and inflammation. These FAHFAs can be obtained through dietary intake, potentially improving human health. However, there is currently inadequate knowledge regarding the presence and variety of FAHFAs in different foods. Herein, we profile FAHFAs from 12 typical food samples and 4 medicinal food samples with the aid of our previous established chemical isotope labeling-assisted liquid chromatography-mass spectrometry method and build a comprehensive dataset of FAHFA diversity. The dataset comprised a total of 1207 regioisomers belonging to 298 different families, with over 100 families being newly discovered for the first time. Therefore, our findings contribute valuable insights into the molecular diversity and presence of FAHFA in a range of foods. This dataset serves as a foundation for further exploration of the nutritional and medicinal functions of FAHFAs.

Determination of vitamin D metabolites in various biological samples through an improved chemical derivatization assisted liquid chromatography-tandem mass spectrometry approach.

Vitamin D (VD) metabolites are involved in a variety of important metabolic processes and physiological effects in organisms. Profiling of VD metabolites favors a deep understanding of the physiological role of VD. However, VD metabolites are difficult to detect due to their high chemical structural rigidity, structural similarity, and low sensitivities under liquid chromatography-tandem mass spectrometry (LC-MS). Herein, we present a chemical derivatization assisted LC-MS/MS strategy for the detection of VDs, in which 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) is employed to derivatize the conjugated diene of VD metabolites and provides sensitizing reporters for MS detection. After PTAD derivatization, the sensitivities of seven VD metabolites increased by 24-276 folds, with the limits of detection ranging from 3 to 20 pg mL-1. Using this method, we achieved a sensitive and accurate quantification of 7 VD metabolites (vitamin D2, vitamin D3, 25-hydroxyvitamin D2, 25-hydroxyvitamin D3, 1,25-dihydroxyvitamin D2, 1,25-dihydroxyvitamin D3, and 1,24,25-trihydroxyvitamin D3) of the VD metabolic pathway in different trace biological samples, including human serum, mouse tissues (namely liver, kidney, lung, and spleen), and cells. We believe that the present method can provide a promising tool for an in-depth analysis of VD metabolism.

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.

Novel Peak Shift Correction Method Based on the Retention Index for Peak Alignment in Untargeted Metabolomics.

Peak alignment is a crucial step in liquid chromatography-mass spectrometry (LC-MS)-based large-scale untargeted metabolomics workflows, as it enables the integration of metabolite peaks across multiple samples, which is essential for accurate data interpretation. Slight differences or fluctuations in chromatographic separation conditions, however, can cause the chromatographic retention time (RT) shift between consecutive analyses, ultimately affecting the accuracy of peak alignment between samples. Here, we introduce a novel RT shift correction method based on the retention index (RI) and apply it to peak alignment. We synthesized a series of N-acyl glycine (C2-C23) homologues via the amidation reaction between glycine with normal saturated fatty acids (C2-C23) as calibrants able to respond proficiently in both mass spectrometric positive- and negative-ion modes. Using these calibrants, we established an N-acyl glycine RI system. This RI system is capable of covering a broad chromatographic space and addressing chromatographic RT shift caused by variations in flow rate, gradient elution, instrument systems, and LC separation columns. Moreover, based on the RI system, we developed a peak shift correction model to enhance peak alignment accuracy. Applying the model resulted in a significant improvement in the accuracy of peak alignment from 15.5 to 80.9% across long-term data spanning a period of 157 days. To facilitate practical application, we developed a Python-based program, which is freely available at https://github.com/WHU-Fenglab/RI-based-CPSC.

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.

Uncovering the Carboxylated Metabolome in Gut Microbiota-Host Co-metabolism: A Chemical Derivatization-Molecular Networking Approach.

Gut microbiota-host co-metabolites serve as essential mediators of communication between the host and gut microbiota. They provide nutrient sources for host cells and regulate gut microenvironment, which are associated with a variety of diseases. Analysis of gut microbiota-host co-metabolites is of great significance to explore the host-gut microbiota interaction. In this study, we integrated chemical derivatization, liquid chromatography-mass spectrometry, and molecular networking (MN) to establish a novel CD-MN strategy for the analysis of carboxylated metabolites in gut microbial-host co-metabolism. Using this strategy, 261 carboxylated metabolites from mouse feces were detected, which grouped to various classes including fatty acids, bile acids, N-acyl amino acids, benzoheterocyclic acids, aromatic acids, and other unknown small-scale molecular clusters in MN. Based on the interpretation of the bile acid cluster, a novel type of phenylacetylated conjugates of host bile acids was identified, which were mediated by gut microbiota and exhibited a strong binding ability to Farnesoid X receptor and Takeda G protein-coupled receptor 5. Our proposed strategy offers a promising platform for uncovering carboxylated metabolites in gut microbial-host co-metabolism.

Chemical isotope labeling assisted liquid chromatography-mass spectrometry method for simultaneous analysis of central carbon metabolism intermediates.

Central carbon metabolism pathway (CCM) is one of the most important metabolic pathways in all living organisms and play crucial function in aspect of organism life. However, the simultaneous detection of CCM intermediates remains challenging. Here, we developed a chemical isotope labeling combined with LC-MS method for simultaneous determination of CCM intermediates with high coverage and accuracy. By chemical derivatization with 2-(diazo-methyl)-N-methyl-N-phenyl-benzamide (2-DMBA) and d5-2-DMBA, all CCM intermediates obtain better separation and accurate quantification at a single LC-MS run. The obtained limits of detection of CCM intermediates ranged from 5 to 36 pg/mL. Using this method, we achieved simultaneous and accurate quantification of 22 CCM intermediates in different biological samples. Take account of the high detection sensitivity of the developed method, this method was further applied to the quantification of CCM intermediates at single-cell level. Finally, 21 CCM intermediates were detected in 1000 HEK-293T cells and 9 CCM intermediates were detected in mouse kidney glomeruli optical slice samples (10∼100 cells).

Enzymatic Cleavage-Mediated Extension Stalling Enables Accurate Recognition and Quantification of Locus-Specific Uracil Modification in DNA.

Chemical modifications in DNA have profound influences on the structures and functions of DNA. Uracil, a naturally occurring DNA modification, can originate from the deamination of cytosine or arise from misincorporation of dUTP into DNA during DNA replication. Uracil in DNA will imperil genomic stability due to their potential in producing detrimental mutations. An in-depth understanding of the functions of uracil modification requires the accurate determination of its site as well as content in genomes. Herein, we characterized that a new member of the uracil-DNA glycosylase (UDG) family enzyme (UdgX-H109S) could selectively cleave both uracil-containing single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA). Based on this unique property of UdgX-H109S, we developed an enzymatic cleavage-mediated extension stalling (ECES) method for the locus-specific detection and quantification of uracil in genomic DNA. In the ECES method, UdgX-H109S specifically recognizes and cleaves the N-glycosidic bond of uracil from dsDNA and generates an apurinic/apyrimidinic (AP) site, which could be broken by APE1 to form a one-nucleotide gap. The specific cleavage by UdgX-H109S is then evaluated and quantified by qPCR. With the developed ECES approach, we demonstrated that the level of uracil at position Chr4:50566961 in genomic DNA of breast cancer tissues was significantly decreased. Collectively, the ECES method has been proved to be accurate and reproducible in the locus-specific quantification of uracil in genomic DNA from biological and clinical samples.

Phthalylglycyl Chloride as a Derivatization Agent for UHPLC-MS/MS Determination of Adrenaline, Dopamine and Octopamine in Urine.

Dopamine, adrenaline and octopamine are small polar molecules that play a vital role in regulatory systems. In this paper, phthalylglycyl chloride was proposed as a derivatization agent for octopamine, adrenaline and dopamine determination in urine for the first time. The derivatization procedure facilitated the use of reversed-phase liquid chromatography with positive electrospray ionization-high-resolution mass spectrometry. An LC-HRMS method was developed that provided quantification limits of 5 ng/mL and detection limits of 1.5 ng/mL for all analytes. The 95-97% yield of derivates was observed after a 10 min derivatization with phthalylglycyl chloride at pH 6.5 and 30 °C. The proposed method was successfully applied to the analysis of human urine samples. The obtained results were compared with those of conventional derivatization procedures with 9-fluorenyl-methoxycarbonyl chloride and dansyl chloride.

In-depth profiling of di(2-ethylhexyl) phthalate metabolic footprints in rats using click chemistry-mass spectrometry probes.

Di(2-ethylhexyl) phthalate (DEHP), the most widely used plasticizers in the world, has been regarded as an endocrine disrupting chemical with serious adverse health outcomes. Accumulating evidence strongly suggests that the undesirable biological effects of DEHP are meditated by its metabolites rather than itself. However, the metabolic footprints of DEHP in vivo are still unclear. Here we developed a click chemistry-assisted mass spectrometry (CC-MS) strategy for in-depth profiling DEHP metabolites in rats. An alkyne-modified DEHP analogue (alkyne-DEHP) was synthesized as a tracer for in vivo tracing, and a pair of MS probes (4-azido-nphenylbenzamide, 4-ANPA, and its deuterated reagent d5-4-ANPA) were prepared to specifically label the alkyne-DEHP metabolites, and prominently improve their detection sensitivity and selectivity. Using the CC-MS strategy, we successfully screened 247 alkyne-DEHP metabolites from rat urine, feces, and serum, including many unrevealed metabolites, such as oxidized phthalate diester metabolites and glucuronides of phthalate monoester metabolites. The discovery of new DEHP metabolites provides additional insights for understanding the metabolism of DEHP, which may be beneficial in exploring the mechanism underlying DEHP induced-toxicity in the future.

Luteolin Attenuates Hypertension via Inhibiting NF-κB-Mediated Inflammation and PI3K/Akt Signaling Pathway in the Hypothalamic Paraventricular Nucleus.

BACKGROUND: Luteolin is widely distributed among a number of vegetal species worldwide. The pharmacological effects of luteolin are diverse and amongst antioxidant, free radical scavenging, and anti-inflammatory activities. Preliminary study showed that luteolin can ameliorate hypertension. However, the precise mechanism needs further investigation. There is no evidence that luteolin affects the paraventricular nucleus of the hypothalamus (PVN), a brain nucleus associated with a critical neural regulator of blood pressure. Our main aim was to explore the effect of luteolin on the PI3K/Akt/NF-κB signaling pathway within the PVN of hypertensive rats. METHODS: spontaneously hypertensive rats (SHRs) and corresponding normotensive control rats, the Wistar Kyoto (WKY) rats were divided into four groups and subsequently treated for 4 weeks with bilateral PVN injections of either luteolin (20 µg/0.11 µL, volume: 0.11 µL/h) or vehicle (artificial cerebrospinal fluid). RESULTS: luteolin infusion to the PVN significantly decreased some hemodynamic parameters including the mean arterial pressure (MAP), heart rate (HR), circulating plasma norepinephrine (NE) and epinephrine (EPI). Additionally, there was a decrease in the expressions of the phosphatidylinositol 3-kinase (p-PI3K) and phosphorylated protein kinase-B (p-AKT), levels of reactive oxygen species (ROS), NAD(P)H oxidase subunit (NOX2, NOX4) in the PVN of SHRs. Meanwhile, the expression of inflammatory cytokines and the activity of nuclear factor κB (NF-κB) p65 in the PVN of SHRs were lowered. Furthermore, immunofluorescence results showed that injection of luteolin in the PVN reduced the expression of tyrosine hydroxylase (TH), and increased that of superoxide dismutase (SOD1) and the 67-kDa isoform of glutamate decarboxylase (GAD67) in the PVN of SHRs. CONCLUSION: Our novel findings revealed that luteolin lowered hypertension via inhibiting NF-κB-mediated inflammation and PI3K/Akt signaling pathway in the PVN.

A strategy for screening and identification of new amino acid-conjugated bile acids with high coverage by liquid chromatography-mass spectrometry.

Bile acids (BAs) are a class of vital gut microbiota-host cometabolites, and they play an important role in maintaining gut microbiota-host metabolic homeostasis. Very recently, a new mechanism of BA anabolic metabolism mediated by gut microbiota (BA-amino acid conjugation) has been revealed, which provides a perspective for the research on BA metabolism and gut metabolome. In this study, we established a polarity-switching multiple reaction monitoring mass spectrometry-based screening method to mine amino acid-conjugated bile acids (AA-BAs) derived from host-gut microbiota co-metabolism. In addition, a retention time-based annotation strategy was further proposed to identify the AA-BA isomers and epimers. Using the developed methods, we successfully screened 118 AA-BA conjugates from mouse and human feces, 28 of them were confirmed by standards, and 62 putatively identified based on their predicted retention times. Moreover, we observed that the levels of most AA-BAs were significantly downregulated in the feces of chronic sleep deprivation mice, suggesting that the AA-BA metabolism was closely related to the physiological state of the host.

Quantitative and site-specific detection of inosine modification in RNA by acrylonitrile labeling-mediated elongation stalling.

RNA molecules contain diverse modifications that play crucial roles in a wide variety of biological processes. Inosine is one of the most prevalent modifications in RNA and dysregulation of inosine is correlated with many human diseases. Herein, we established an acrylonitrile labeling-mediated elongation stalling (ALES) method for quantitative and site-specific detection of inosine in RNA from biological samples. In ALES method, inosine is selectively cyanoethylated with acrylonitrile to form N1-cyanoethylinosine (ce1I) through a Michael addition reaction. The N1-cyanoethyl group of ce1I compromises the hydrogen bond between ce1I and other nucleobases, leading to the stalling of reverse transcription at original inosine site. This specific property of stalling at inosine site could be evaluated by subsequent real-time quantitative PCR (qPCR). With the proposed ALES method, we found the significantly increased level of inosine at position Chr1:63117284 of Ino80dos RNA of multiple tissues from sleep-deprived mice compared to the control mice. This is the first report on the investigation of inosine modification in sleep-deprived mice, which may open up new direction for deciphering insomnia from RNA modifications. In addition, we found the decreased level of inosine at GluA2 Q/R site (Chr4:157336723) in glioma tissues, indicating the decreased level of inosine at GluA2 Q/R site may serve as potential indicator for the diagnosis of glioma. Taken together, the proposed ALES method is capable of quantitative and site-specific detection of inosine in RNA, which provides a valuable tool to uncover the functions of inosine in human diseases.

Engineered APOBEC3C Sequencing Enables Bisulfite-Free and Direct Detection of DNA Methylation at a Single-Base Resolution.

DNA methylation (5-methylcytosine, 5mC) is the most important epigenetic modification in mammals. Deciphering the roles of 5mC relies on the quantitative detection of 5mC at the single-base resolution. Bisulfite sequencing (BS-seq) is the most often employed technique for mapping 5mC in DNA. However, bisulfite treatment may cause serious degradation of input DNA due to the harsh reaction conditions. Here, we engineered the human apolipoprotein B mRNA-editing catalytic polypeptide-like 3C (A3C) protein to endow the engineered A3C (eA3C) protein with differential deamination activity toward cytosine and 5mC. By the virtue of the unique property of eA3C, we proposed an engineered A3C sequencing (EAC-seq) method for the bisulfite-free and quantitative mapping of 5mC in DNA at the single-base resolution. In EAC-seq, the eA3C protein can deaminate C but not 5mC, which is employed to differentiate C and 5mC in sequencing. Using the EAC-seq method, we quantitatively detected 5mC in genomic DNA of lung cancer tissue. In contrast to the harsh reaction conditions of BS-seq, which could lead to significant degradation of DNA, the whole procedure of EAC-seq is carried out under mild conditions, thereby preventing DNA damage. Taken together, the EAC-seq approach is bisulfite-free and straightforward, making it an invaluable tool for the quantitative detection of 5mC in limited DNA at the single-base resolution.