GutUDB: A comprehensive multiomics database for intestinal diseases.

Gut Universe Database (GutUDB) provides a comprehensive, systematic, and practical platform for researchers, and is dedicated to the management, analysis, and visualization of knowledge related to intestinal diseases. Based on this database, eight major categories of omics data analyses are carried out to explore the genotype-phenotype characteristics of a certain intestinal disease. The first tool for comprehensive omics data research on intestinal diseases will help each researcher better understand intestinal diseases.

Dynamic m6 A profiles reveal the role of YTHDC2-TLR2 signaling axis in Talaromyces marneffei infection.

Talaromyces marneffei (TM) immune evasion is an important factor leading to the high mortality rate of Penicilliosis marneffei. N6 -methyladenosine (m6 A) plays important roles in host immune response to various pathogen infections, yet its role in TM and HIV/TM coinfection remains largely unexplored. Here we reported genome-wide transcriptional m6 A profiles of TM mono-infection and HIV/TM coinfection. Our finding revealed dynamic alterations in global m6 A levels and upregulation of the m6 A reader YTH N6 -methyladenosine RNA binding protein C2 (YTHDC2) in TM-infected macrophages. Knockdown of YTHDC2 in TM-infected cells showed an elevated expression of TLR2 through m6 A-dependence, along with upregulation of TNF-α and IL1-ß. Overall, we characterized the m6 A profiles of the host and fungus before and after TM infection, and demonstrated that YTHDC2 mediates the key m6 A site of TLR2 to exert its function. These findings provide new insights into the underlying mechanisms and novel therapeutic approaches for TM diseases.

Downregulation of NEBL promotes migration and invasion of clear cell renal cell carcinoma by inducing epithelial-mesenchymal transition.

As a member of the nebulin protein family and a structural protein of cytoskeleton, NEBL plays an important role in cardiac diseases. Recently, literature have reported the involvement of NEBL in the occurrence and development of various cancers except clear cell renal cell carcinoma (ccRCC). In this study, we found that mRNA and protein of NEBL are downregulated remarkably in ccRCC tissues based on both the TCGA database and clinical samples we collected. The areas under curve values of NEBL analyzed based on the TCGA database, qRT-PCR and IHC results were 0.9376, 0.9733 and 0.9807, respectively. The lower mRNA level of NEBL was associated with worse outcomes in ccRCC patients. When overexpressing NEBL in ccRCC cell lines, the proliferation, migration and invasion of ccRCC cells were suppressed significantly, suggesting a tumor suppressor role of NEBL. In addition, we identified that NEBL is closely related to epithelial-mesenchymal transition (EMT), thereby reducing the motility of ccRCC cells. Furthermore, the lower expression of NEBL was correlated with ccRCC patients with distant organ metastasis. In summary, we firstly described the aberrant expression of NEBL and revealed its tumor suppressor role in ccRCC. Our data support that NEBL could serve as a valuable diagnostic and prognostic biomarker in ccRCC, as well as a promising therapeutic target.

Novosphingobium beihaiensis sp. nov., a novel pesticide-tolerant bacterium isolated from mangrove sediments.

A novel Novosphingobium species, designated strain B2638T, was isolated from mangrove sediments which was collected from Beibu Gulf, Beihai, P. R. China. The isolate could grow in the presence of chlorpyrifos. Phylogenetic analysis based on 16S rRNA gene sequence revealed that the isolate belonged to the genus Novosphingobium, showing 99.9% sequence similarity with N. decloroationis 502str22T and less than 98% similarity with other type strain of species of this genus. Molecular typing by BOX-PCR divided strain B2638T and N. declorationis 502str22T into two clusters and indicated that they were not identical. Genomic comparison referenced by values of the DNA-DNA hybridization (dDDH) and the average nucleotide identity (ANI) between strain B2638T and its close phylogenetic neighbors were 20.0-29.5% and 75.3-85.3%, respectively, that were lower than proposed thresholds for bacterial species delineation. The major fatty acids (> 10%) were identified as C18:1 ω7c, C17:1 iso ω9c and C16:0. The main polar lipids contained diphosphatidylglycerol, phosphatidylethanolamine, sphingoglycolipid, phosphatidyl glycerol, unidentified lipid and unidentified aminolipid. Results from phenotypic, chemotaxonomic and genotypic analyses proposed that strain B2638T (= MCCC 1K07406T = KCTC 72968 T) is represented a novel species in the genus Novosphingobium, for which the names Novosphingobium beihaiensis sp. nov. is proposed.

Loss of ACOX1 in clear cell renal cell carcinoma and its correlation with clinical features.

Clear cell renal cell carcinoma (ccRCC) is a major pathological type of kidney cancer with a poor prognosis due to a lack of biomarkers for early diagnosis and prognosis prediction of ccRCC. In this study, we investigated the aberrant expression of Acyl-coenzyme A oxidase 1 (ACOX1) in ccRCC and evaluated its potential in diagnosis and prognosis. ACOX1 is the first rate-limiting enzyme in the peroxidation ß-oxidation pathway and is involved in the regulation of fatty acid oxidative catabolism. The mRNA and protein levels of ACOX1 were significantly downregulated in ccRCC, and its downregulation was closely associated with the tumor-node-metastasis stage of patients. The ROC curves showed that ACOX1 possesses a high diagnostic value for ccRCC. The OS analysis suggested that lower expression of ACOX1 was closely related to the worse outcome of patients. In addition, gene set enrichment analysis suggested that expression of ACOX1 was positively correlated with CDH1, CDH2, CDKL2, and EPCAM, while negatively correlated with MMP9 and VIM, which strongly indicated that ACOX1 may inhibit the invasion and migration of ccRCC by reversing epithelial-mesenchymal transition. Furthermore, we screened out that miR-16-5p is upregulated at the mRNA transcript level in ccRCC and negatively correlated with ACOX1. In conclusion, our results showed that ACOX1 is abnormally low expressed in ccRCC, suggesting that it could serve as a diagnostic and prognostic biomarker for ccRCC. Overexpression of miR-16-5p may be responsible for the inactivation of ACOX1.

Taxonomy and anticancer potential of Streptomyces niphimycinicus sp. nov. against nasopharyngeal carcinoma cells.

Streptomyces species are ubiquitous, Gram-positive, spore-forming bacteria with the ability to produce various clinically relevant compounds. The strain 4503 T was isolated from mangrove sediments, showing morphological and chemical properties which were consistent with those of members of the genus Streptomyces. Phylogenetic analysis based on 16S rRNA gene sequences revealed that the isolate was primarily identified as members of the genus Streptomyces, sharing more than 99% sequence identity to Streptomyces yatensis DSM 41771 T, S. antimycoticus NBRC 12839 T, and S. melanosporofaciens NBRC 13061 T. Average nucleotide identities (ANI) and digital DNA-DNA hybridization (dDDH) values between strain 4503 T and its close relatives were all below 95-96% and 75% of the novel species threshold, respectively. Results from phylogenetic, genomic, phenotypic, and chemotaxonomic characteristics analyses confirmed that the isolate represented a novel species of the genus Streptomyces, for which the name Streptomyces niphimycinicus sp. nov. 4503 T (= MCCC 1K04557T = JCM 34996 T) is proposed. The bioassay-guided fractionation of the extract of strain 4503 T resulted in the isolation of a known compound niphimycin C, which showed cytotoxic activity against nasopharyngeal carcinoma (NPC) cell lines TW03 and 5-8F with half maximal inhibitory concentration (IC50) values of 12.24 µg/mL and 9.44 µg/mL, respectively. Further experiments revealed that niphimycin C not only exhibited the capacity of anti-proliferation, anti-metastasis, induction of cell cycle arrest, and apoptosis, but was also able to increase the reactive oxygen species (ROS) production and regulate several signaling pathways in NPC cells. KEY POINTS: • Strain 4503 T was classified as a novel species of Streptomyces. • Niphimycin C correlates with the cytotoxic effect of strain 4503 T against NPC cells. • Niphimycin C induces apoptosis, autophagic flux disruption and cell cycle arrest.

HIGD1A inactivated by DNA hypermethylation promotes invasion of kidney renal clear cell carcinoma.

Hypoxia contributes to the tumorigenesis and metastasis of the tumor. However, the detailed mechanisms underlying hypoxia and kidney renal clear cell carcinoma (KIRC) development and progression remain unclear. Here, we investigated the role of the system HIG1 hypoxia inducible domain family member 1 A (HIGD1A) in the proliferation and metastasis of KIRC and elucidated the underlying molecular mechanisms. The expression of HIGD1A is significantly downregulated in KIRC due to promoter hypermethylation. HIGD1A could serve as a valuable diagnostic biomarker in KIRC. In addition, ectopic overexpression of HIGD1A significantly suppressed the growth and invasive capacity of KIRC cells in vitro under normal glucose conditions. Interestingly, the suppressive efficacy in invasion is much more significant when depleted glucose, but not in proliferation. Furthermore, mRNA expression of HIGD1A positively correlates with CDH1 and EPCAM, while negatively correlated with VIM and SPARC, indicating that HIGD1A impedes invasion of KIRC by regulating epithelial-mesenchymal transition (EMT). Our data suggest that HIGD1A is a potential diagnostic biomarker and tumor suppressor in KIRC.

Weighted gene co-expression network analysis revealed T cell differentiation associated with the age-related phenotypes in COVID-19 patients.

The risk of severe condition caused by Corona Virus Disease 2019 (COVID-19) increases with age. However, the underlying mechanisms have not been clearly understood. The dataset GSE157103 was used to perform weighted gene co-expression network analysis on 100 COVID-19 patients in our analysis. Through weighted gene co-expression network analysis, we identified a key module which was significantly related with age. This age-related module could predict Intensive Care Unit status and mechanical-ventilation usage, and enriched with positive regulation of T cell receptor signaling pathway biological progress. Moreover, 10 hub genes were identified as crucial gene of the age-related module. Protein-protein interaction network and transcription factors-gene interactions were established. Lastly, independent data sets and RT-qPCR were used to validate the key module and hub genes. Our conclusion revealed that key genes were associated with the age-related phenotypes in COVID-19 patients, and it would be beneficial for clinical doctors to develop reasonable therapeutic strategies in elderly COVID-19 patients.

Potential Inhibitors of Monkeypox Virus Revealed by Molecular Modeling Approach to Viral DNA Topoisomerase I.

The monkeypox outbreak has become a global public health emergency. The lack of valid and safe medicine is a crucial obstacle hindering the extermination of orthopoxvirus infections. The identification of potential inhibitors from natural products, including Traditional Chinese Medicine (TCM), by molecular modeling could expand the arsenal of antiviral chemotherapeutic agents. Monkeypox DNA topoisomerase I (TOP1) is a highly conserved viral DNA repair enzyme with a small size and low homology to human proteins. The protein model of viral DNA TOP1 was obtained by homology modeling. The reliability of the TOP1 model was validated by analyzing its Ramachandran plot and by determining the compatibility of the 3D model with its sequence using the Verify 3D and PROCHECK services. In order to identify potential inhibitors of TOP1, an integrated library of 4103 natural products was screened via Glide docking. Surface Plasmon Resonance (SPR) was further implemented to assay the complex binding affinity. Molecular dynamics simulations (100 ns) were combined with molecular mechanics Poisson-Boltzmann surface area (MM/PBSA) computations to reveal the binding mechanisms of the complex. As a result, three natural compounds were highlighted as potential inhibitors via docking-based virtual screening. Rosmarinic acid, myricitrin, quercitrin, and ofloxacin can bind TOP1 with KD values of 2.16 µM, 3.54 µM, 4.77 µM, and 5.46 µM, respectively, indicating a good inhibitory effect against MPXV. The MM/PBSA calculations revealed that rosmarinic acid had the lowest binding free energy at -16.18 kcal/mol. Myricitrin had a binding free energy of -13.87 kcal/mol, quercitrin had a binding free energy of -9.40 kcal/mol, and ofloxacin had a binding free energy of -9.64 kcal/mol. The outputs (RMSD/RMSF/Rg/SASA) also indicated that the systems were well-behaved towards the complex. The selected compounds formed several key hydrogen bonds with TOP1 residues (TYR274, LYS167, GLY132, LYS133, etc.) via the binding mode analysis. TYR274 was predicted to be a pivotal residue for compound interactions in the binding pocket of TOP1. The results of the enrichment analyses illustrated the potential pharmacological networks of rosmarinic acid. The molecular modeling approach may be acceptable for the identification and design of novel poxvirus inhibitors; however, further studies are warranted to evaluate their therapeutic potential.

CASA: a comprehensive database resource for the COVID-19 Alternative Splicing Atlas.

BACKGROUND: As a key process in transcriptional regulatory mechanisms, alternative splicing (AS) plays a crucial role in maintaining the diversity of RNA and protein expression, and mediates the immune response in infectious diseases, especially for the COVID-19. Therefore, urgent data gathering and more research of AS profiles in microbe-infected human cells are needed to improve understanding of COVID-19 and related infectious diseases. Herein, we have created CASA, the COVID-19 Alternative Splicing Atlas to provide a convenient computing platform for studies of AS in COVID-19 and COVID-19-related infectious diseases. METHODS: In CASA, we reanalyzed thousands of RNA-seq datasets generated from 65 different tissues, organoids and cell lines to systematically obtain quantitative data on AS events under different conditions. A total of 262,994 AS events from various infectious diseases with differing severity were detected and visualized in this database. In order to explore the potential function of dynamics AS events, we performed analysis of functional annotations and drug-target interactions affected by AS in each dataset. RNA-binding proteins (RBPs), which may regulate these dynamic AS events are also provided for users in this database. RESULTS: CASA displays microbe-induced alterations of the host cell splicing landscape across different virus families and helps users identify condition-specific splicing patterns, as well as their potential regulators. CASA may greatly facilitate the exploration of AS profiles and novel mechanisms of host cell splicing by viral manipulation. CASA is freely available at http://www.splicedb.net/casa/ .

Genome-Wide Profiling Reveals Alternative Polyadenylation of Innate Immune-Related mRNA in Patients With COVID-19.

The coronavirus disease 2019 (COVID-19) pandemic has caused many deaths worldwide. To date, the mechanism of viral immune escape remains unclear, which is a great obstacle to developing effective clinical treatment. RNA processing mechanisms, including alternative polyadenylation (APA) and alternative splicing (AS), are crucial in the regulation of most human genes in many types of infectious diseases. Because the role of APA and AS in response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection remains unknown, we performed de novo identification of dynamic APA sites using a public dataset of human peripheral blood mononuclear cell (PBMC) RNA-Seq data in COVID-19 patients. We found that genes with APA were enriched in innate immunity -related gene ontology categories such as neutrophil activation, regulation of the MAPK cascade and cytokine production, response to interferon-gamma and the innate immune response. We also reported genome-wide AS events and enriched viral transcription-related categories upon SARS-CoV-2 infection. Interestingly, we found that APA events may give better predictions than AS in COVID-19 patients, suggesting that APA could act as a potential therapeutic target and novel biomarker in those patients. Our study is the first to annotate genes with APA and AS in COVID-19 patients and highlights the roles of APA variation in SARS-CoV-2 infection.

Enhanced Signaling Through the TLR9 Pathway Is Associated With Resistance to HIV-1 Infection in Chinese HIV-1-Exposed Seronegative Individuals.

Innate immunity is the first line of defense against invading pathogens and may mediate HIV-1 resistance in HIV-1-exposed seronegative (HESN) individuals. This study aims to identify components of innate immunity that confer natural HIV-1 resistance in Chinese HESN individuals. Specifically, we compared the expression levels of Toll-like receptors (TLRs) and associated pathway molecules in peripheral blood mononuclear cells (PBMCs), monocytes/macrophages, and plasma obtained from HESN and control individuals. HESN individuals had higher expression of TLR9, IRF7, IFN-α/ß, RANTES, and MIP-1α/1ß in PBMCs and plasma than control subjects. Upon TLR9 stimulation, significantly higher expression of TLR9 and IRF7, as well as higher production of IFN-α/ß, RANTES, and MIP-1α/1ß, was observed in PBMCs and monocytes/macrophages from HESN individuals than in the corresponding cells from control individuals. More importantly, both with and without TLR9 stimulation, the levels of HIV-1 replication in monocyte-derived macrophages (MDMs) from HESN individuals were significantly lower than those in MDMs from control individuals. These data suggest that increased TLR9 activity and subsequent release of antiviral factors contribute to protection against HIV-1 in HESN individuals.

Identification of a Novel HIV-1 CRF55_01B/B Recombinant Isolate in Guangxi, China.

Dynamic recombination is the driving force in the genetic diversity of human immunodeficiency virus type 1 (HIV-1). When multiple subtypes are circulating in the same area of a population, new HIV-1 strains are likely to be generated through recombination. In this study, we report a novel recombinant strain (2018GXQZLSHET001) of HIV-1, isolated from a HIV-1-positive heterosexual individual infected in Guangdong province, who recently lived in Guangxi province, China. Phylogenetic analysis of the near full-length genome suggested that 2018GXQZLSHET001 was a recombinant of strains CRF55_01B and subtype B. Similarity plotting and bootscaning showed that a subtype B segment was inserted into the CRF55_01B genome with one breakpoint in the nef and 3' long terminal repeat regions. Further subregion phylogenetic analysis demonstrated that the CRF55_01B segment originated from Guangdong. The subtype B segment was similar to a Thai B lineage. This indicated that the strain might be a novel recombinant, comprising sequences of both CRF55_01B and B. The emergence of this unique recombinant strain illustrated the complexity of the HIV-1 epidemic, and the need to strengthen molecular epidemiological surveillance and measures to reduce its spread.

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.

Metal oxide-based dispersive solid-phase extraction coupled with mass spectrometry analysis for determination of ribose conjugates in human follicular fluid.

Polycystic ovary syndrome (PCOS) is the most common cause of anovulatory infertility. The pathogenesis of PCOS remains unclear and early diagnosis of PCOS is challenging. Follicular fluid provides a unique window in the critical processes during oocyte and follicular maturation, and the metabolic level of follicular fluid has important impact on the developmental potential of oocytes and subsequent embryos. Previous studies demonstrated some modified ribonucleosides in biological fluids were diseases related metabolites. In this respect, analysis of endogenous modified ribonucleosides in follicular fluids will facilitate the investigation of follicular development. Here, we developed a strategy for determination of ribose conjugates from follicular fluid using metal oxide-based dispersive solid-phase extraction (DSPE) coupled with liquid chromatography-multiple reaction monitoring-mass spectrometry analysis (DSPE-LC-MRM-MS/MS). Cerium dioxide (CeO2) was used to selectively recognize and capture cis-diol containing ribose conjugates from complex biological samples under basic environment. The trapped ribose conjugates were then easily released under acidic environment. The results showed that 50 potential ribose conjugates were detected in follicular fluid by the developed DSPE-LC-MRM-MS/MS method. We then further investigated the contents change of the detected ribose conjugates in follicular fluid from PCOS patients. The results indicated that the follicular fluid from healthy controls and PCOS patients can be clearly differentiated with the partial least squares-discriminate analysis (PLS-DA) based on the detected ribose conjugates. In addition, the contents of 8 ribose conjugates were significantly different between PCOS patients and healthy controls, which could potentially serve as the indicator of PCOS.

Comprehensive profiling of ribonucleosides modification by affinity zirconium oxide-silica composite monolithic column online solid-phase microextraction - Mass spectrometry analysis.

More than 140 modified ribonucleosides have been identified in RNA. Determination of endogenous modified ribonucleosides in biological fluids may serve as non-invasive disease diagnostic strategy. However, detection of the modified ribonucleosides in biological fluids is challenging, especially for the low abundant modified ribonucleosides due to the serious matrix interferences of biological fluids. Here, we developed a facile preparation strategy and successfully synthesized zirconium oxide-silica (ZrO2/SiO2) composite capillary monolithic column that exhibited excellent performance for the selective enrichment of cis-diol-containing compounds. Compared with the boronate-based affinity monolith, the ZrO2/SiO2 monolith showed ∼2 orders of magnitude higher extraction capacity and can be used under physiological pH (pH 6.5-7.5). Using the prepared ZrO2/SiO2 composite monolith as the trapping column and reversed-phase C18 column as the analytical column, we further established an online solid-phase microextraction (SPME) in combination with liquid chromatography-mass spectrometry (online SPME-LC-MS/MS) analysis for the comprehensive profiling of ribonucleosides modification in human urine. Our results showed that 68 cis-diol-containing ribosylated compounds were identified in human urine, which is, to the best of our knowledge, the highest numbers of cis-diol-containing compounds were determined in a single analysis. It is worth noting that four modified ribonucleosides were discovered in the human urine for the first time. In addition, the quantification results from the pooled urine samples showed that compared to healthy controls, the contents of sixteen ribose conjugates in the urine of gastric cancer, eleven in esophagus cancer and seven in lymphoma increased more than two folds. Among these ribose conjugates, four ribose conjugates increased more than two folds in both gastric cancer and esophagus cancer; three ribose conjugates increased more than two folds in both gastric cancer and lymphoma; one ribose conjugate increased more than two folds in both esophagus cancer and lymphoma. The developed analytical method provides a good platform to study the modified ribonucleosides in human body fluids.

A highly sensitive fluorescence assay for methyltransferase activity by exonuclease-aided signal amplification.

DNA methylation, catalyzed by methyltransferases, plays critical roles in various biological processes in both prokaryotes and eukaryotes. Bacterial DNA adenine methyltransferases (DAM) are associated with bacterial pathogenesis and essential for bacterial virulence and viability. Since mammals do not methylate DNA at adenine, bacterial DAM is considered to be a great candidate target for developing new therapeutics for diseases. In the current study, we developed a simple, rapid and highly sensitive fluorescence method for the detection of DAM based on exonuclease-aided signal amplification. In the proposed strategy, a liberated amplifier upon DAM methylation and Dpn I digestion of the substrate can hybridize with a reporter (FT) that contains a quencher (TAMRA) at the second base of the 3' end and a fluorophore (FAM) at the fifth base. Upon hybridization, exonuclease III degrades the reporter in the formed duplex DNA from the 3' end successively, releasing the fluorophore from the quencher and resulting in an intensive appearance of the fluorescent signal. The amplifier will hybridize with another reporter and enter a new cycle, which therefore can amplify the signal and dramatically increase the detection sensitivity even with an extremely low amount of amplifier. Using this strategy, the detection limit down to 0.0025 U mL(-1) of DAM was achieved within a short assay time of 30 min. Furthermore, the assay was applied to evaluate endogenous DAM activity in E. coli cell at different growth stages as well as the effects of inhibitors on DAM activity. Given the attractive analytical performance, the sensing strategy may find many important applications in biomedical research and clinical diagnosis.

Metal Oxide-Based Selective Enrichment Combined with Stable Isotope Labeling-Mass Spectrometry Analysis for Profiling of Ribose Conjugates.

Some modified ribonucleosides in biological fluids have been evaluated as cancer-related metabolites. Detection of endogenous modified ribonucleosides in biological fluids may serve as a noninvasive cancers diagnostic method. However, determination of modified ribonucleosides is still challenging because of their low abundance and serious matrix interferences in biological fluids. Here, we developed a novel strategy for comprehensive profiling of ribose conjugates from biological fluids using metal oxide-based dispersive solid-phase extraction (DSPE) followed with in vitro stable isotope labeling and double neutral loss scan-mass spectrometry analysis (DSPE-SIL-LC-DNLS-MS). Cerium dioxide (CeO2) was used to selectively recognize and capture ribose conjugates from complex biological samples under basic environment. The enriched ribose conjugates were subsequently labeled with a pair of isotope labeling reagents (acetone and acetone-d6). The glucosidic bond of acetone labeled ribose conjugates is readily ruptured, and the generated ribose that carries an isotope tag can be lost as a neutral fragment under collision induced dissociation (CID). Since the light (acetone) and heavy (acetone-d6) labeled compounds have the same chemical structures and can generate different neutral loss fragments (NL 172 and 178 Da), it is therefore highly convenient to profile ribose conjugates by double neutral loss scan mode in mass spectrometry analysis. In this respect, the light and heavy labeled compounds were ionized at the same condition but recorded separately on MS spectra, which can significantly improve the detection specificity and facilitate the identification of ribose conjugates. Using the developed DSPE-SIL-LC-DNLS-MS strategy, we profiled the ribose conjugates in human urine, and 49 ribose conjugates were readily identified, among which 7 ribose conjugates exhibited significant contents change between healthy controls and lymphoma patients. The DSPE-SIL-LC-DNLS-MS strategy combines the selective enrichment, stable isotope labeling, and double neutral loss scan - MS analysis, which therefore can efficiently minimize false positive results, facilitate the relative quantification, and notably increase the numbers of identified ribose conjugates in biological fluids samples. Taken together, this study established a promising strategy for the effective profiling of urinary modified ribonucleosides, and simultaneous evaluation of the contents change of multiple modified ribonucleosides should provide more accurate and conclusive results for the use of urinary modified ribonucleosides as indicators of cancers.

Profiling of cis-diol-containing nucleosides and ribosylated metabolites by boronate-affinity organic-silica hybrid monolithic capillary liquid chromatography/mass spectrometry.

RNA contains a large number of modified nucleosides. In the metabolic re-exchange of RNA, modified nucleosides cannot be recycled and are thus excreted from cells into biological fluids. Determination of endogenous modified nucleosides in biological fluids may serve as non-invasive cancers diagnostic methods. Here we prepared boronate-affinity organic-silica hybrid capillary monolithic column (BOHCMC) that exhibited excellent selectivity toward the cis-diol-containing compounds. We then used the prepared BOHCMC as the on-line solid-phase microextraction (SPME) column and developed an on-line SPME-LC-MS/MS method to comprehensively profile cis-diol-containing nucleosides and ribosylated metabolites in human urine. Forty-five cis-diol-containing nucleosides and ribosylated metabolites were successfully identified in human urine. And five ribose conjugates, for the first time, were identified existence in human urine in the current study. Furthermore, the relative quantification suggested 4 cis-diol-containing compounds (5'-deoxy-5'-methylthioadensine, N(4)-acetylcytidine, 1-ribosyl-N-propionylhistamine and N(2),N(2),7-trimethylguanosine) increased more than 1.5 folds in all the 3 types of examined cancers (lung cancer, colorectal cancer, and nasopharyngeal cancer) compared to healthy controls. The on-line SPME-LC-MS/MS method demonstrates a promising method for the comprehensive profiling of cis-diol-containing ribose conjugates in human urines, which provides an efficient strategy for the identification and discovery of biomarkers and may be used for the screening of cancers.

Sensitive detection of DNA methyltransferase activity based on exonuclease-mediated target recycling.

DNA methylation plays vital roles in various biological processes in both prokaryotes and eukaryotes. In bacteria, modification of adenine at N6 can protect bacterial DNA against cleavage by restriction enzymes, and bacterial DNA adenine methyltransferases are essential for bacterial virulence and viability. DNA adenine methyltransferase (DAM) targets the sequence of 5'-GATC-3' and can convert adenine into N(6)-methyladenine (m(6)A). Because mammals do not methylate DNA at adenine, bacterial DAM represents an excellent candidate for antibiotic development. Here, we developed an exonuclease III-aided target recycling strategy to sensitively assay activity of DAM. In this method, a hairpin probe labeled with a donor fluorophore (FAM) at the 5' end and a quencher (BHQ) close to the 3' end (FQ probe) was employed as reporter. Another hairpin substrate containing sequence of GATC was used as the methylation substrate of DAM. Once the hairpin substrate was methylated by DAM, it could be recognized and cleaved by Dpn I, which allows the release of a single-stranded oligodeoxynucleotide (ssODN). The ssODN can then hybridize to the 3' protruding terminus of FQ probe, which subsequently triggers the exonuclease III-mediated target recycling reaction and therefore can significantly improve the detection sensitivity of DAM. The exonuclease-mediated target recycling strategy is extremely sensitive and as low as 0.01 U/mL DAM can be distinctly determined. Using this developed method, we evaluated DAM activity in different growth stages of E. coli cells, and we also demonstrated that the assay has the potential to screen suitable inhibitor drugs for DAM for disease(s) treatment.