Dynamic changes in RNA m6A and 5 hmC influence gene expression programs during macrophage differentiation and polarisation.

RNA modifications are essential for the establishment of cellular identity. Although increasing evidence indicates that RNA modifications regulate the innate immune response, their role in monocyte-to-macrophage differentiation and polarisation is unclear. While m6A has been widely studied, other RNA modifications, including 5 hmC, remain poorly characterised. We profiled m6A and 5 hmC epitranscriptomes, transcriptomes, translatomes and proteomes of monocytes and macrophages at rest and pro- and anti-inflammatory states. Transcriptome-wide mapping of m6A and 5 hmC reveals enrichment of m6A and/or 5 hmC on specific categories of transcripts essential for macrophage differentiation. Our analyses indicate that m6A and 5 hmC modifications are present in transcripts with critical functions in pro- and anti-inflammatory macrophages. Notably, we also discover the co-occurrence of m6A and 5 hmC on alternatively-spliced isoforms and/or opposing ends of the untranslated regions (UTR) of mRNAs with key roles in macrophage biology. In specific examples, RNA 5 hmC controls the decay of transcripts independently of m6A. This study provides (i) a comprehensive dataset to interrogate the role of RNA modifications in a plastic system (ii) a resource for exploring different layers of gene expression regulation in the context of human monocyte-to-macrophage differentiation and polarisation, (iii) new insights into RNA modifications as central regulators of effector cells in innate immunity.

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.

Accelerated Aging of T-Cell Subsets Among ART-Naïve HIV-Infected Chinese Men Who have Sex with Men: A Case-Control Study.

BACKGROUND: Evidence of lymphopoiesis, exhaustion, and premature aging in Chinese patients with human immunodeficiency virus (HIV) is very limited. OBJECTIVE: To assess biological aging and immune senescence in Chinese healthy controls (HC) and ART-naïve HIV-infected men who have sex with men (MSM). METHODS: This case-control study was conducted in Beijing Ditan Hospital from March 2018 to June 2019. The percentages of naïve (TN), central memory (TCM), effector memory (TEM), and terminally differentiated memory (TemRA) subsets of CD4 and CD8 T cells were studied, along with markers of senescence (CD28-CD57+) and activation (HLA-DR+). Telomere length of naïve (CD45RA+) and memory (CD45RO+) CD8 T cells were quantified by real-time PCR. RESULTS: A total of 26 HIV-infected and 20 age-matched HC MSM were included. Compared to the HC group, the CD4/CD8 ratio of the HIV-infected group was significantly reduced (0.30 vs. 1.70, P<0.001); significant differences emerged among all CD8 but not CD4 T cell subsets (all P<0.05). In the HIV-infected group, the percentages of senescent cells (CD28-CD57+) in TN, TCM, TEM, and TemRA subsets of CD8 T cells were higher (all P<0.05); while a significant difference was only found in naïve CD4 T cells (P<0.05). HLA-DR expression was increased significantly in all CD4 and CD8 T cell subsets. Both naïve (CD45RA+) and memory (CD45RO+) CD8 T cells in this population had significantly shorter telomere lengths (P<0.01) compared to the HC group. CONCLUSION: HIV-infected MSM exhibit signs of accelerated immune senescence and biological aging, which particularly affects the CD8 T-cell subsets.

The characteristics of overseas imported COVID-19 cases and the effectiveness of screening strategies in Beijing, China.

BACKGROUND: In March 2020, the WHO declared the novel coronavirus outbreak a global pandemic. While great success in coronavirus disease 2019 (COVID-19) control has been achieved in China, imported cases have become a major challenge. This study aimed to describe the epidemiological and clinical characteristics of imported COVID-19 cases and to assess the effectiveness of screening strategies in Beijing, China. METHODS: This retrospective study included all imported cases transferred to Beijing Ditan Hospital from 29 February to 20 March 2020 who were screened by both chest computed tomography (CT) and reverse-transcriptase-polymerase chain reaction (RT-PCR) at the initial presentation. Demographic, clinical and laboratory data, in addition to chest CT imaging, were collected and analysed. RESULTS: In total, 2545 cases were included, among which 71 (2.8%) were finally diagnosed with laboratory-confirmed COVID-19. The majority 63 (88.7%) were from Europe. The most common initial symptoms were cough and fever, which accounted for 49.3% and 42.3%, respectively. Only four cases (5.6%) had lymphocytopenia, and thirteen cases (18.3%) demonstrated elevated levels of C-reactive protein (CRP). All cases had normal serum levels of procalcitonin (PCT). At initial presentation, among the 71 confirmed cases, 59 (83.1%) had a positive RT-PCR assay, and 35 (49.3%) had a positive chest CT. Twelve (16.9%) had a negative RT-PCR assay but a positive chest CT. CONCLUSIONS: A combination of RT-PCR and chest CT is an effective strategy for the screening of imported COVID-19 cases. Our findings provide important information and clinical evidence about the infection control of imported COVID-19 cases.

An enzyme-mediated bioorthogonal labeling method for genome-wide mapping of 5-hydroxymethyluracil.

DNA 5-hydroxymethyluracil (5hmU) is a thymine modification existing in the genomes of various organisms. The post-replicative formation of 5hmU occurs via hydroxylation of thymine by ten-eleven translocation (TET) dioxygenases in mammals and J-binding proteins (JBPs) in protozoans, respectively. In addition, 5hmU can also be generated through oxidation of thymine by reactive oxygen species or deamination of 5hmC by cytidine deaminase. While the biological roles of 5hmU have not yet been fully explored, determining its genomic location will highly assist in elucidating its functions. Herein, we report a novel enzyme-mediated bioorthogonal labeling method for selective enrichment of 5hmU in genomes. 5hmU DNA kinase (5hmUDK) was utilized to selectively install an azide (N3) group or alkynyl group into the hydroxyl moiety of 5hmU followed by incorporation of the biotin linker through click chemistry, which enabled the capture of 5hmU-containing DNA fragments via streptavidin pull-down. The enriched fragments were applied to deep sequencing to determine the genomic distribution of 5hmU. With this established enzyme-mediated bioorthogonal labeling strategy, we achieved the genome-wide mapping of 5hmU in Trypanosoma brucei. The method described here will allow for a better understanding of the functional roles and dynamics of 5hmU in genomes.

Impact of immune checkpoint molecules on FoxP3+ Treg cells and related cytokines in patients with acute and chronic brucellosis.

BACKGROUND: The immunoregulatory functions of regulatory T cells (Tregs) in the development and progression of some chronic infectious diseases are mediated by immune checkpoint molecules and immunosuppressive cytokines. However, little is known about the immunosuppressive functions of Tregs in human brucellosis, which is a major burden in low-income countries. In this study, expressions of immune checkpoint molecules and Treg-related cytokines in patients with acute and chronic Brucella infection were evaluated to explore their impact at different stages of infection. METHODS: Forty patients with acute brucellosis and 19 patients with chronic brucellosis admitted to the Third People's Hospital of Linfen in Shanxi Province between August 2016 and November 2017 were enrolled. Serum and peripheral blood mononuclear cells were isolated from patients before antibiotic treatment and from 30 healthy subjects. The frequency of Tregs (CD4+ CD25+ FoxP3+ T cells) and expression of CTLA-4, GITR, and PD-1 on Treg cells were detected by flow cytometry. Levels of Treg-related cytokines, including IL-35, TGF-ß1, and IL-10, were measured by customised multiplex cytokine assays using the Luminex platform. RESULTS: The frequency of Tregs was higher in chronic patients than in healthy controls (P = 0.026) and acute patients (P = 0.042); The frequency of CTLA-4+ Tregs in chronic patients was significantly higher than that in healthy controls (P = 0.011). The frequencies of GITR+ and PD-1+ Tregs were significantly higher in acute and chronic patients than in healthy controls (P < 0.05), with no significant difference between the acute and chronic groups (all P > 0.05). Serum TGF-ß1 levels were higher in chronic patients (P = 0.029) and serum IL-10 levels were higher in acute patients (P = 0.033) than in healthy controls. We detected weak correlations between serum TGF-ß1 levels and the frequencies of Tregs (R = 0.309, P = 0.031) and CTLA-4+ Tregs (R = 0.302, P = 0.035). CONCLUSIONS: Treg cell immunity is involved in the chronicity of Brucella infection and indicates the implication of Tregs in the prognosis of brucellosis. CTLA-4 and TGF-ß1 may contribute to Tregs-mediated immunosuppression in the chronic infection stage of a Brucella infection.

Soluble IL-2Rα correlates with imbalances of Th1/Th2 and Tc1/Tc2 cells in patients with acute brucellosis.

BACKGROUND: Previous studies showed that soluble IL-2Rα is an important marker of cellular immune activation and might be a marker of treatment efficacy for children with brucellosis. However, data regarding adult patients with brucellosis were unknown. The aim of study was to explore the potential role of serum sIL-2Rα evaluating treatment responses in adult patients with brucellosis, and T cell immune status was also examined. METHODS: During January 2016-April 2017, 30 patients with acute brucellosis from the Third People's Hospital of Linfen in Shanxi Province and Beijing Di Tan Hospital, and 28 healthy controls were included in this study. Peripheral blood samples were collected before and after six weeks of antibiotic treatment. Serum sIL-2Rα levels were measured by enzyme-linked immunosorbent assay, and the percentage of Th1, Th2, Tc1, Tc2, and Tregs was detected by flow cytometry after intracellular staining for cytokines (interferon-γ and interleukin-4) and Foxp3 in T lymphocytes from peripheral blood. The obtained data were analyzed with Wilcoxon ranked sum tests for paired values, Mann-Whitney U-tests for comparisons between patients and healthy controls, and Spearman rank tests for correlation analyses. RESULTS: Serum sIL-2Rα levels were significantly higher in patients than in controls (P = 0.001). A significant decline was observed in patients after the cessation of treatment (P < 0.001) and return to normal (P > 0.05). Th1, Tc1, Th2, and Tc2 cell frequencies were higher in patients than in healthy subjects (P < 0.05), while the Th1/Th2 and Tc1/Tc2 ratios were significantly lower (P = 0.0305 and 0.0005, respectively) and returned to normal levels after treatment. In patients with acute brucellosis, serum sIL-2Rα levels were negatively correlated with the Th1/Th2 ratio (r = - 0.478, P = 0.028), Tc1/Tc2 ratio (r = - 0.677, P = 0.001), and Tc1 percentage (r = - 0.516, P = 0.017). Serum sIL-2Rα and Tc2 percentages were positively correlated (r = 0.442, P = 0.045). CONCLUSIONS: Based on the correlations with Th1/Th2 and Tc1/Tc2 ratios, serum sIL-2Rα levels may reflect the immune response status. sIL-2Rα may be a marker for therapeutic efficacy in acute brucellosis.

Quantification and Single-Base Resolution Analysis of N1-Methyladenosine in mRNA by Ligation-Assisted Differentiation.

RNA modification, such as N1-methyladenosine (m1A), affects the secondary structure of RNA and its ability to recognize specific reader proteins. Methods for detecting site-specific m1A are in demand. We report here a ligation-assisted differentiation approach for quantitative detection of m1A in mRNA with single-base resolution. The methyl group in m1A disrupts the Watson-Crick base pairing with uridine, resulting in a lower ligation efficiency of certain ligases and lower amounts of ligation products. Detection of the ligation products using quantitative real-time PCR provided site-specific evaluation of m1A. We first screened appropriate ligase and found that T3 DNA ligase offered the best discrimination between m1A and adenosine. We successfully detected and quantified m1A at position 1674 of bromodomain containing 2 (BRD2) mRNA from HEK293T cells. In lung carcinoma tissues, the level of m1A at position 1674 of BRD2 mRNA was significantly decreased compared to the tumor-adjacent normal tissues, suggesting that site-specific m1A may be involved in carcinogenesis.

Chemical labeling - Assisted mass spectrometry analysis for sensitive detection of cytidine dual modifications in RNA of mammals.

RNA molecules carry diverse modifications that exert important influences in many cellular processes. In addition to the single modification occurring in either nucleobase or 2' hydroxyl of ribose in RNA, some dual modifications occur in both the nucleobase and 2' hydroxyl of ribose in RNA. 2'-O-methyl-5-methylcytidine (m5Cm), the dual modifications of cytidine, was first discovered from the tRNA of archaea. Recent studies identified that 2'-O-methyl-5-hydroxymethylcytidine (hm5Cm) and 2'-O-methyl-5-formylcytidine (f5Cm) were present in the anticodon of cytoplasmic tRNA of mammals. Similar to the series of single modification of cytidines of 5-methylcytosine (m5C), 5-hydroxymethylcytidine (hm5C), 5-formylcytidine (f5C), and 5-carboxylcytidine (ca5C) in nucleic acids, the dual modifications of m5Cm, hm5Cm, f5Cm and 2'-O-methyl-5-carboxylcytidine (ca5Cm) may also constitute the series of cytidine modifications in mammals. However, it is normally challenging to detect these modifications because of their low endogenous levels. Here, we established a method by chemical labeling-assisted liquid chromatography - electrospray ionization - tandem mass spectrometry (LC-ESI-MS/MS) analysis for the sensitive and simultaneous determination of all these four cytidine dual modifications, i.e., m5Cm, hm5Cm, f5Cm and ca5Cm. Three different labeling reagents (2-bromo-1-(3,4-dimeth oxyphenyl)-ethanone, BDMOPE; 2-bromo-1-(4-methoxyphenyl)-ethanone, BMOPE; 2-bromo-1-(4-diethylaminophenyl)-ethanone, BDEPE) were used for the chemical labeling. The results showed that the detection sensitivities of m5Cm, hm5Cm, f5Cm and ca5Cm increased up to 462 folds after chemical labeling. With the developed method, we achieved the simultaneous detection of m5Cm, hm5Cm and f5Cm in RNA of mammals. In addition, we found these cytidine dual modifications mainly exist in small RNA (<200 nt) and barely detected in other types of RNA. Moreover, we found that the levels of m5Cm in RNA of human lung carcinoma tissues significantly increased, while hm5Cm and f5Cm significantly decreased compared to tumor adjacent normal tissues. The significant changes of m5Cm, hm5Cm and f5Cm levels may serve as indicator for the detection and prognosis of lung cancer.

Chemical tagging for sensitive determination of uridine modifications in RNA.

The discovery of dynamic and reversible modifications in messenger RNA (mRNA) is opening new directions in RNA modification-mediated regulation of biological processes. Methylation is the most prevalent modification occurring in mRNA and the methyl group is mainly decorated in the adenine, cytosine, and guanine base or in the 2'-hydroxyl group of ribose. However, methylation of the uracil base (5-methyluridine, m5U) has not been discovered in mRNA of eukaryotes. In the current study, we established a method of N-cyclohexyl-N'-ß-(4-methylmorpholinium) ethylcarbodiimide p-toluenesulfonate (CMCT) labelling coupled with liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS/MS) analysis for the sensitive determination of uridine modifications in RNA. Our results demonstrated that the detection sensitivities of uridine modifications in RNA increased up to 1408 fold upon CMCT labelling. Using the developed method, we identified the distinct existence of m5U in mRNA of various mammalian cells and tissues. In addition, the stable isotope tracing monitored by mass spectrometry revealed that the methyl group of m5U originated from S-adenosyl-l-methionine (SAM). Our study expanded the list of modifications occurring in mRNA of mammals. Future work on transcriptome-wide mapping of m5U will further uncover the functional roles of m5U in mRNA of mammals.

Determination of RNA Hydroxylmethylation in Mammals by Mass Spectrometry Analysis.

RNA molecules harbor diverse chemical modifications that play important regulatory roles in a variety of biological processes. Up to date, more than 150 modifications have been identified in various RNA species. Most of these modifications occurring in nucleic acids are the methylation of nucleic acids. It has been demonstrated that many of these methylation are reversible and undergo dynamic demethylation. Previous studies established that the demethylation of the two most important and prevalent modifications of 5-methylcytidine (m5C) and N6-methyladenosine (m6A) in nucleic acids is through the hydroxylation of m5C and m6A, forming 5-hydroxymethylcytidine (hm5C) and N6-hydroxymethyladenosine (hm6A), respectively. This indicates the hydroxylation of the methylated nucleosides may be a general pathway for the demethylation of nucleic acid methylation. However, few other hydroxylmethylation modifications have yet to be reported in existence in mammals. In the current study, we developed a neutral enzymatic digestion method for the mild digestion of nucleic acids, followed by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) analysis. With the established method, we reported the existence of a new hydroxylmethylated nucleosides, N2-hydroxymethylguanosine (hm2G), in mammalian RNA. In addition, we found that the contents of hm2G, as well as N2-methylguanosine (m2G), showed significant differences between thyroid carcinoma tissues and tumor-adjacent normal tissues, indicating that m2G and hm2G in RNA may play certain roles in the carcinogenesis of thyroid carcinoma. Collectively, our study suggests that RNA hydroxylmethylation may be a new prevalent group of modifications existing in RNA, which expands the diversity of nucleic acid modifications and should exert regulatory functions in living organisms.

AlkB Homologue 1 Demethylates N3-Methylcytidine in mRNA of Mammals.

RNA contains diverse modifications that exert important influences in a variety of cellular processes. So far more than 150 modifications have been identified in various RNA species, mainly in rRNA and tRNA. Recent research advances in RNA modifications have been sparked by the discovery of dynamic and reversible modifications in mRNA. Moving beyond the abundant tRNA and rRNA to mRNA is opening new directions in understanding RNA modification-mediated regulation of gene expression. Recently, it was reported that N3-methylcytidine (m3C) existed in mRNA of mammalian cells, and methyltransferase-like 8 (METTL8) was identified to be the writer enzyme of m3C. However, little is known about the eraser enzyme of m3C in mRNA. In the current study, we found that the AlkB homologue 1 (ALKBH1) was capable of demethylating m3C in mRNA of mammalian cells in vitro. Overexpression and knockdown of ALKBH1 in cultured human cells can induce decrease and increase of the level of m3C in mRNA, respectively, revealing the eraser enzyme property of ALKBH1 on m3C in mRNA. In addition, we observed significant decrease of the level of m3C in mRNA in hepatocellular carcinoma (HCC) tissues compared to tumor-adjacent normal tissues, which could be attributed to the increased expression of ALKBH1 as well as the decreased expression of METTL8 in HCC tissues. These results indicated that m3C in mRNA may play certain roles in tumorigenesis. Our study shed light on understanding the demethylation of m3C in mRNA.

N 6-Hydroxymethyladenine: a hydroxylation derivative of N6-methyladenine in genomic DNA of mammals.

In addition to DNA cytosine methylation (5-methyl-2'-deoxycytidine, m5dC), DNA adenine methylation (N6-methyl-2'-deoxyadenosine, m6dA) is another DNA modification that has been discovered in eukaryotes. Recent studies demonstrated that the content and distribution of m6dA in genomic DNA of vertebrates and mammals exhibit dynamic regulation, indicating m6dA may function as a potential epigenetic mark in DNA of eukaryotes besides m5dC. Whether m6dA undergoes the further oxidation in a similar way to m5dC remains elusive. Here, we reported the existence of a new DNA modification, N6-hydroxymethyl-2'-deoxyadenosine (hm6dA), in genomic DNA of mammalian cells and tissues. We found that hm6dA can be formed from the hydroxylation of m6dA by the Fe2+- and 2-oxoglutarate-dependent ALKBH1 protein in genomic DNA of mammals. In addition, the content of hm6dA exhibited significant increase in lung carcinoma tissues. The increased expression of ALKBH1 in lung carcinoma tissues may contribute to the increase of hm6dA in DNA. Taken together, our study reported the existence and formation of hm6dA in genomic DNA of mammals.

Modified nucleoside triphosphates exist in mammals.

DNA and RNA contain diverse chemical modifications that exert important influences in a variety of cellular processes. In addition to enzyme-mediated modifications of DNA and RNA, previous in vitro studies showed that pre-modified nucleoside triphosphates (NTPs) can be incorporated into DNA and RNA during replication and transcription. Herein, we established a chemical labeling method in combination with liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) analysis for the determination of endogenous NTPs in the mammalian cells and tissues. We synthesized 8-(diazomethyl)quinoline (8-DMQ) that could efficiently react with the phosphate group under mild condition to label NTPs. The developed method allowed sensitive detection of NTPs, with the detection limits improved by 56-137 folds. The results showed that 12 types of endogenous modified NTPs were distinctly determined in the mammalian cells and tissues. In addition, the majority of these modified NTPs exhibited significantly decreased contents in human hepatocellular carcinoma (HCC) tissues compared to tumor-adjacent normal tissues. Taken together, our study revealed the widespread existence of various modified NTPs in eukaryotes.

Existence of Internal N7-Methylguanosine Modification in mRNA Determined by Differential Enzyme Treatment Coupled with Mass Spectrometry Analysis.

The recent discovery of reversible chemical modifications on mRNA has opened a new era of post-transcriptional gene regulation in eukaryotes. Among the 15 types of modifications identified in mRNA of eukaryotes, N7-methylguanosine (m7G) is unique owing to its presence in the 5' cap structure. It remains unknown whether m7G is also present internally in mRNA, and this is largely attributed to the lack of an appropriate analytical method to differentiate internal m7G in mRNA from that in the 5' cap. To address this analytical challenge, we developed a novel strategy of combining differential enzymatic digestion with liquid chromatography-tandem mass spectrometry analysis to quantify the levels of these two types of m7G modifications in mRNA. In particular, we found that S1 nuclease and phosphodiesterase I exhibit differential activities toward internal and 5'-terminal m7G. By using this method, we found that internal m7G was present in mRNA of cultured human cells as well as plants and rat tissue. In addition, our results showed that plants contain higher levels of internal m7G in mRNA than mammals. We also observed that exposure of rice to cadmium (Cd) stimulated marked diminution in the levels of m7G at both the 5' cap and internal positions of mRNA, which was correlated with the Cd-induced elevated expression of m7G-decapping enzymes. Taken together, we reported here a strategy to distinguish internal and 5'-terminal m7G in mRNA, and by using this method, we demonstrated the prevalence of internal m7G modification in mRNA, which we believe will stimulate future functional studies of m7G on post-transcriptional gene regulation in eukaryotes.