Mass-Spectrometry-Based Assay at Single-Base Resolution for Simultaneously Detecting m6A and m6Am in RNA.

The methylation modifications of adenosine, especially N6-methyladenosine (m6A) and N6, 2'-odimethyladenosine (m6Am), play vital roles in various biological, physiological, and pathological processes. However, current methods for detecting these modifications at single-base resolution have limitations. Mass spectrometry (MS), a highly accurate and sensitive technique, can be utilized to differentiate between m6A and m6Am by analyzing the molecular weight differences in their fragments during tandem MS analysis. In this study, we present an MS-based method that allows for the simultaneous determination of m6A and m6Am sites in targeted RNA fragments at single-nucleotide resolution. The approach involves the utilization of tandem MS in conjunction with targeted RNA enrichment and enzymatic digestion, eliminating the need for PCR amplification. By employing this strategy, we can accurately identify m6A and m6Am sites in targeted RNA fragments with high confidence. To evaluate the effectiveness of our method, we applied it to detect m6A and m6Am sites in cell and tissue samples. Furthermore, we verified the accuracy of our approach by performing CRISPR/Cas9-mediated knockout of the corresponding methyltransferases. Overall, our MS-based method offers a reliable and precise means for the simultaneous detection of m6A and m6Am modifications in targeted RNA fragments, providing valuable insights into the functional characterization of these modifications in various biological contexts.

Mulberry leaf water extract alleviates type 2 diabetes in mice via modulating gut microbiota-host co-metabolism of branched-chain amino acid.

Elevations in circling branched-chain amino acids (BCAAs) levels associated with insulin resistance and type 2 diabetes mellitus (T2DM). Morus alba L. water extracts (MLE) show hypoglycemic function, but the precise mechanism remains obscure. This study is designed to investigate the association of the antidiabetes effect of MLE with the BCAAs co-metabolism modulated by host and gut microbiota. Tissue-specific expressions of BCAA-catabolizing enzymes were detected by RT-PCR and western blot, respectively. The components of the intestinal microflora were analyzed by high-throughput 16S rRNA gene sequencing. The results showed that MLE administration improved blood glucose and insulin level, decreased inflammatory cytokines expression, and lowered serum and feces BCAAs levels. Furthermore, MLE reversed the abundance changes of the bacterial genera correlated with serum and feces BCAAs, such as Anaerovorax, Bilophila, Blautia, Colidextribacter, Dubosiella, Intestinimonas, Lachnoclostridium, Lachnospiraceae_NK4A136, Oscillibacter, and Roseburia. Functionality prediction indicated that MLE potentially inhibited bacterial BCAAs biosynthesis, and promoted the tissue-specific expression of BCAAs catabolic enzyme. More importantly, MLE had obvious impacts on BCAA catabolism in germ-free-mimic T2DM mice. Those results indicated that MLE improving T2DM-related biochemical abnormalities is associated with not only gut microbiota modification but also the tissue-specific expression of BCAAs catabolic enzyme.

2-Bromopropionyl Esterified Cellulose Nanofibrils as Chain Extenders or Polyols in Stoichiometrically Optimized Syntheses of High-Strength Polyurethanes.

2-Bromopropionyl bromide esterified cellulose nanofibrils (Br-CNFs) facilely synthesized from one-pot esterification of cellulose and in situ ultrasonication exhibited excellent N,N-dimethylformamide (DMF) dispersibility and reactivity to partially replace either chain extender or soft segment diol in the stoichiometrically optimized syntheses of polyurethanes (PUs). PUs polymerized with Br-CNF to replace either 11 mol% 1,4-butadiol chain extender OHs or 1.8 mol% polytetramethylene ether glycol OHs, i.e., 1.5 or 0.3 wt% Br-CNF in PUs, exhibited an over 3 times increased modulus, nearly 4 times higher strength, and a 50% increase in strain. In either role, the experimental modulus exceeding those predicted by the Halpin-Tsai model gave evidence of the stoichiometrically optimized covalent bonding with Br-CNF, while the improved strain was attributed to increased hydrogen-bonding interactions between Br-CNF and the soft segment. These new Br-CNFs not only offer novel synthetic strategies to incorporate nanocelluloses in polyurethanes but also maximize their reinforcing effects via their versatile polyol reactant and cross-linking roles, demonstrating promising applications in the synthesis of other polymers.

Morus alba L. water extract changes gut microbiota and fecal metabolome in mice induced by high-fat and high-sucrose diet plus low-dose streptozotocin.

Gut microbiota plays a key role in the pathophysiology of type 2 diabetes mellitus (T2D). Mulberry leaf has a hypoglycemic effect, but the potential mechanism is not fully understood. This study aimed to explore the influences and potential mechanisms of mulberry leaf water extract (MLWE) intervention on mice with T2D induced through a high-fat and high-sucrose diet combined with streptozotocin by the combination of fecal metabolomics and gut microbiota analysis. Results showed that MLWE could decrease fasting blood glucose and body weight while ameliorating lipid profiles, insulin resistance, liver inflammation, and the accumulation of lipid droplets in T2D mice. MLWE could reverse the abundances of the phyla Actinobacteria and Bacteroidetes and the ratio of Firmicutes/Bacteroidetes, and increase the abundances of the phyla Cyanobacteria and Epsilonbacteraeota in the feces of T2D mice. The abundances of genera Alloprevotella, Parabacteroides, Muribaculaceae, and Romboutsia in the feces of T2D mice could be reversed, while Oscillatoriales_cyanobacterium and Gastranaerophilales could be reinforced by MLWE supplementation. The levels of nine metabolites in the feces of T2D mice were improved, among which glycine, Phe-Pro, urocanic acid, phylloquinone, and lactate were correlated with Romboutsia and Gastranaerophilales. Taken together, we conclude that MLWE can effectively alleviate T2D by mediating the host-microbial metabolic axis.

MS-based metabolite analysis of two licorice chalcones in mice plasma, bile, feces, and urine after oral administration.

Isoliquiritigenin (ILG) and isoliquiritin (ILQ), two kinds of major flavonoids in licorice, are biological active substances with antioxidant, anti-inflammatory, and tumor-suppressive effects. However, their in vivo metabolites, possible material basis of this two licorice chalcones for the treatment of diseases, have not been studied completely. To determine the metabolism of ILG and ILQ, after oral administration of 100 mg/kg/day of these compounds for consecutive 8 days, the metabolites of these two licorice chalcones in mice plasma, urine, feces, and bile were determined using liquid chromatography coupled with quadrupole/time-of-flight mass spectrometry in this study. The structures of those metabolites were tentatively identified according to their fragment pathways, accurate masses, characteristic product ions, metabolism law, and reference standards-matching. As a result, a total of 25 and 29 metabolites of ILG and ILQ were identified, respectively. Seven main metabolic pathways, oxidation and reduction, deglycosylation and glycosylation, dehydroxylation and hydroxylation, demethoxylation and methoxylation, acetylation, glucuronidation, and sulfation, were summarized to tentatively explain how the metabolites were biologically transformed. These results provide the important information on the metabolism of ILG and ILQ, which may be helpful for the further research of their pharmacological mechanism.

Microporous polymer based on the new compound "bi-(4-vinyl phenylquinoline) amide" for enrichment and quantitative determination of lamotrigine in rat and human serum.

Lamotrigine is one of the most widely used antiepileptic drugs in the treatment of epilepsy. This kind of drug needs to be used in the long term and should be quantitatively detected in the blood of patients to avoid drug toxicity caused by individual differences and environmental and pathological changes in the process of taking. The detection of antiepileptic drugs in human blood is challenging because of their low contents and the interference of complex matrices. Thus, the sample enrichment method has been commonly used to improve the sensitivity of detection. In this work, we have synthesized a new "bi-(4-vinyl phenylquinoline) amide" compound and used it as the monomer to produce the hyper-cross-linked microporous polymer for the enrichment of lamotrigine. This material has a high adsorption capacity, specificity, and linearity, which can improve the detection sensitivity of lamotrigine by high-performance liquid chromatography (HPLC). The mechanism of this phenomenon has also been investigated. Finally, we have developed the microporous polymer enrichment coupled with HPLC method for the quantitative determination of lamotrigine in rat and human serum samples. This method has excellent precision, accuracy, and recovery, meeting the test of biological sample. The low limit of quantitation was 0.625 µg/mL. Graphical abstract.

Advances in detection and quantification of methylcytosine and its derivatives.

Methylation of the fifth carbon atom in cytosine is an epigenetic modification of deoxyribonucleic acid that plays important roles in numerous cellular processes and disease pathogenesis. Three additional states of cytosine, that is, 5-hydroxymethylcytosine, 5-formylcytosine and 5-carboxylcytosine, have been identified and associated with the diagnosis and/or prognosis of diseases. However, accurate measurement of those intermediates is a challenge since their global levels are relatively low. A number of innovative methods have been developed to detect and quantify these compounds in biological samples, such as blood, tissue and urine, etc. This review focuses on recent advancement in detection and quantification of four cytosine modifications, based on which, the development, diagnosis, and prognosis of diseases could be monitored through non-invasive procedures.

Enrichment and Quantitative Determination of 5-(Hydroxymethyl)-2'-deoxycytidine, 5-(Formyl)-2'-deoxycytidine, and 5-(Carboxyl)-2'-deoxycytidine in Human Urine of Breast Cancer Patients by Magnetic Hyper-Cross-Linked Microporous Polymers Based on Polyionic Liquid.

5-(Hydroxymethyl)-2'-deoxycytidine (5-hmdC), 5-(formyl)-2'-deoxycytidine (5-fodC), and 5-(carboxyl)-2'-deoxycytidine (5-cadC) are crucial intermediate products of the DNA demethylation pathway, which can also act as potential biomarkers reflecting the diagnosis and prognosis in multiple tumors. Detecting 5-hmdC, 5-fodC, and 5-cadC in human urine has various advantages including readily available samples and being noninvasive to patients. However, few works have reported the detection of 5-fodC and 5-cadC due to their trace amounts. Here we developed a novel magnetic hyper-cross-linked microporous polymer (HMP) material based on polyionic liquid (PIL) for the enrichment of 5-hmdC, 5-fodC, and 5-cadC. These magnetic PIL-HMP materials provided specific enrichment superiority for three modified cytidines. After enrichment, the signal intensity of 5-hmdC, 5-fodC, and 5-cadC increased 10-75-fold with lower limits of quantitation (LLOQ) of 0.049, 0.781, and 0.781 ng/mL, respectively. The recoveries were approximately 86.5-95.2% for 5-hmdC, 95.2-107.8% for 5-fodC, and 99.4-102.4% for 5-cadC under the relative standard deviation (RSD) of 0.2-10.3%. Finally, we successfully applied magnetic PIL-HMP materials coupled with high-performance liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) in enrichment and quantitative determination of 5-hmdC, 5-fodC, and 5-cadC in human urine of 10 breast cancer patients and 10 healthy people. We found that the level of 5-hmdC decreased in breast cancer patients ( p < 0.05), while the levels of 5-fodC and 5-cadC increased ( p < 0.05, p < 0.01). Our results demonstrated that the levels of metabolic 5-hmdC, 5-fodC, and 5-cadC in human urine are closely related to breast cancer, which could contribute to the clinical diagnosis and investigation of breast cancer and its occurrence and development mechanisms.

Investigation of the formation of the [2(iohexol) + Mg]2+ complex and its fragmentation in electrospray ionization tandem mass spectrometry.

RATIONALE: Mass spectrometry has been developed as one of the common tools for the analysis of the organometallic systems in the gas phase over decades. The study of the fragmentation of organics-metal complexes has attracted much attention since the interesting dissociation pathways exhibited by these compounds are usually different from the protonated analogues. METHODS: In this work, iohexol complexed with different dications such as Mg2+ , Cu2+ and Zn2+ have been investigated by electrospray ionization (ESI) tandem mass spectrometry. Additionally, deuterium-labeling experiments and an analogue of iohexol were utilized to confirm the reaction mechanisms. A computational chemistry method was used to identify the coordination conformation between iohexol and metal ions in the gas phase. UV detection was also used to identify the interaction between iohexol and metal ions in the liquid phase. RESULTS: A special gas-phase open-loop reaction of iohexol induced by Mg2+ , leading to the formation of [iohexol + Mg - H - HI - C3 H4 O]+ , was observed in the collision-induced dissociation of [2(iohexol) + Mg]2+ complexes. Moreover, theoretical calculation shows the proposed coordination configuration of iohexol/Mg2+ complexes. The Mg2+ could have tetrahedral coordination with two iohexol molecules. CONCLUSIONS: The percent study is a case for better understanding the formation of a typical organic/metal complex and its gas-phase fragmentation reaction. In addition, it provides useful information for researchers working on analysis or structural elucidation of complicated compounds which contain the iohexol analogues. Copyright © 2016 John Wiley & Sons, Ltd.

Enhanced detection of amino acids in hydrophilic interaction chromatography electrospray tandem mass spectrometry with carboxylic acids as mobile phase additives.

Liquid chromatography coupled with mass spectrometry technique has been widely used in the analysis of biological targets such as amino acids, peptides, and proteins. In this work, eight common single carboxylic acids or diacids, which contain different pKa have been investigated as the additives to the analysis of amino acids. As the results, carboxylic acid additive can improve the signal intensity of acidity amino acids such as Asp and Glu and the chromatographic separation of basic amino acids such as Arg, His, and Lys. In particular, the diacids have better performance than single acids. The proposed mechanism is that the diacid has hydrogen bond interaction with amino acids to reduce their polarity/amphiprotic characteristics. Besides, oxalic acid has been found having better enhancement than phthalic acid by overall consideration. Therefore, we successfully quantified the 15 amino acids in Sepia bulk pharmaceutical chemical by using oxalic acid as the additive.

Development and evaluation of a hydrophilic interaction liquid chromatography-MS/MS method to quantify 19 nucleobases and nucleosides in rat plasma.

As essential endogenous compounds, nucleobases and nucleosides fulfill various functions in living organisms. This study presents the development and validation of a new hydrophilic interaction liquid chromatography tandem mass spectrometry method for simultaneous quantification of 19 nucleobases and nucleosides in rat plasma. For the sample preparation, 15 kinds of protein precipitants were evaluated according to the chromatographic profile and ion response of analytes. The optimization of chromatographic separation was respectively performed using reversed-phase liquid chromatography and hydrophilic interaction liquid chromatography mode; each separation mode included two test columns with different stationary phases. The chromatographic profile and parameters such as half-width (W1/2 ), capacity factor (K') and tailing factor (ft ) were used to evaluate the separation efficiencies. Furthermore, the adopted composition of two mobile phase systems and the concentrations of the additives in the optimum buffer system were also investigated. The developed method was fully validated and successfully applied quantitatively to determine 19 nucleobases and nucleosides in plasma from normal and diabetic nephropathy (DN) rats. Significant differences between normal and DN rats were found in plasma levels of cytosine, xanthine, thymidine, adenosine, guanosine, inosine and 8-hydroxy-2'-deoxyguanosine. This information may provide a useful reference for the discovery of potential biomarkers of DN.

Two-dimensional liquid chromatography and its application in traditional Chinese medicine analysis and metabonomic investigation.

Two-dimensional liquid chromatography has become an attractive analytical tool for the separation of complex samples due to its enhanced selectivity, peak capacity, and resolution compared with one-dimensional liquid chromatography. Recently, more attention has been drawn on the application of this separation technique in studies concerning traditional Chinese medicines, metabonomes, proteomes, and other complex mixtures. In this review, we aim to examine the application of two-dimensional liquid chromatography in traditional Chinese medicine analysis and metabonomic investigation. The classification and evaluation indexes were first introduced. Then, various switching methods were summarized when used in an on-line two-dimensional liquid chromatography system. Finally, the applications of this separation technique in traditional Chinese medicine analysis and metabonomic investigation were discussed on the basis of specific studies.

Phenylboronic acid modified solid-phase extraction column: Preparation, characterization, and application to the analysis of amino acids in sepia capsule by removing the maltose.

Maltose, a common auxiliary material of pharmaceutical preparation, may disturb the analysis of total amino acids in sepia capsule by aldolization. Therefore, it is necessary to remove the maltose through a convenient method. In this work, a phenylboronic acid modified solid-phase extraction column has been synthesized and used to remove the maltose. The materials were synthesized by one step "thiol-ene" reaction and the parameters of the column such as absorption capacity, recovery, and absorption specificity have been investigated. The results showed the column (0.5 cm of length × 0.5 cm of inner diameter) can absorb 4.6 mg maltose with a linear absorption and absorption specificity. Then this technique was applied in the quantification of amino acids in sepia capsule. After the optimization of the method, four kinds of amino acids, which were the most abundant, were quantified by high-performance liquid chromatography with diode array detection. The amounts of the four kinds of amino acids are 1.5∼2 times more than that without the treatment of solid-phase extraction column, which almost overcomes the influence of the maltose. All the results indicate that the phenylboronic acid modified solid-phase extraction column can successfully help to accurately quantify the total amino acids in sepia capsule.

Evaluation of sample preparation and chromatographic separation for the parallel determination of taurine and edaravone in rat tissues using HILIC-MS/MS.

The quantitative analysis of taurine and edaravone in biological sample is critical in pharmaceutical studies. Although each of them can be individually analyzed by different approaches, concurrent quantification is still a highly challenging task with respect to their great polarity variation and the complex composition of tissue sample. In the present study, to simultaneously determine taurine and edaravone in rat tissue, the sample preparation and chromatographic separation conditions were evaluated and discussed in detail. As for the sample preparation, four kinds of solvent and the volume ratio of the optimal solvent to biological sample were both tested and evaluated based on the chromatographic profile, extraction recovery, and matrix effect (ME). The chromatographic separation was performed in a reverse phase (RP) and two hydrophilic interaction liquid chromatography (HILIC) modes, and the corresponding separation efficiencies were assessed using chromatographic parameters like half-width (W 1/2 ), tailing factor (f t), theoretical plates number (N), and ME. Furthermore, adopted composition of two mobile phase systems and the concentrations of the additives in the optimum buffer system were also investigated on an Atlantis HILIC silica column according to the resultant chromatographic profiles and peak areas of the analytes. The optimal results were obtained when the biological samples were deproteined by 4-fold volume of methanol/acetonitrile (1:3, v/v) and separated on a HILIC column with a gradient elution of acetonitrile/water containing 0.2 % formic acid and 10 mM ammonium formate. The proposed approach was validated and successfully applied to the parallel determination of the tissue distribution of edaravone and taurine in rat tissues.

Competitive formation of b(2) and c(2)-H2O ions from b(3) ions containing Asp residue during tandem mass spectrometry: the influence of neighboring Arg.

The fragmentation of b3 ions derived from protonated Arg-Xxx-Asp-Ala-Ala (Xxx = Ala, Asp, Glu, Cys) and Arg-Xxx-Glu-Ala-Ala was investigated by electrospray ionization tandem mass spectrometry (MS (n) ) with collision-induced dissociation. A particular ion, which is 1 Da less than b2 ion, is shown to be the c2-H2O ion. The mechanism for its formation involved the aspartic acid in the third position easily losing anhydride to form a c2 ion, which then lost water to form an eight-membered ring of azacyclooctane derivative under the participation of the guanidine of the N-terminal arginine. However, this phenomenon was not observed when the aspartic acid was replaced by glutamic acid. The Amber program was used to determine the conformation of the original c2 residue from the dynamic energy perspective, and then density functional theory-based calculations and changing N-terminal amino acid from arginine to phenylalanine supported this mechanism.

Regioselectively Carboxylated Cellulose Nanofibril Models from Dissolving Pulp: C6 via TEMPO Oxidation and C2,C3 via Periodate-Chlorite Oxidation.

Regioselective C6 and C2,C3 carboxylated cellulose nanofibrils (CNFs) have been robustly generated from dissolving pulp, a readily available source of unmodified cellulose, via stoichiometrically optimized 2,2,6,6-tetramethylpyperidine-1-oxyl (TEMPO)-mediated and sequential sodium periodate-sodium chlorite (PC) oxidation coupled with high-speed blending. Both regioselectively optimized carboxylated CNF series possess the widest ranges of comparable charges (0.72-1.48 mmol/g for T-CNFs vs. 0.72-1.10 mmol/g for PC-CNFs), but similar ranges of thickness (1.3-2.4 nm for T-CNF, 1.8-2.7 nm PC-CNF), widths (4.6-6.6 nm T-CNF, 5.5-5.9 nm PC-CNF), and lengths (254-481 nm T-CNF, 247-442 nm PC-CNF). TEMPO-mediated oxidation is milder and one-pot, thus more time and process efficient, whereas the sequential periodate-chlorite oxidation produces C2,C3 dialdehyde intermediates that are amenable to further chemical functionalization or post-reactions. These two well-characterized regioselectively carboxylated CNF series represent coherent cellulose nanomaterial models from a single woody source and have served as references for their safety study toward the development of a safer-by-design substance evaluation tool.

Wighteone, a prenylated flavonoid from licorice, inhibits growth of SW480 colorectal cancer cells by allosteric inhibition of Akt.

ETHNOPHARMACOLOGICAL RELEVANCE: Licorice is a frequently used herbal medicine worldwide, and is used to treat cough, hepatitis, cancer and influenza in clinical practice of traditional Chinese medicine. Modern pharmacological studies indicate that prenylated flavonoids play an important role in the anti-tumor activity of licorice, especially the tumors in stomach, lung, colon and liver. Wighteone is one of the main prenylated flavonoids in licorice, and its possible effect and target against colorectal cancer have not been investigated. AIM OF THE STUDY: This study aimed to investigate the anti-colorectal cancer effect and underlying mechanism of wighteone. MATERIALS AND METHODS: SW480 human colorectal cancer cells were used to evaluate the in vitro anti-colorectal cancer activity and Akt regulation effect of wighteone by flow cytometry, phosphoproteomic and Western blot analysis. Surface plasmon resonance (SPR) assay, molecular docking and dynamics simulation, and kinase activity assay were used to investigate the direct interaction between wighteone and Akt. A nude mouse xenograft model with SW480 cells was used to verify the in vivo anti-colorectal cancer activity of wighteone. RESULTS: Wighteone inhibited phosphorylation of Akt and its downstream kinases in SW480 cells, which led to a reduction in cell viability. Wighteone had direct interaction with both PH and kinase domains of Akt, which locked Akt in a "closed" conformation with allosteric inhibition, and Gln79, Tyr272, Arg273 and Lys297 played the most critical role due to their hydrogen bond and hydrophobic interactions with wighteone. Based on Akt overexpression or activation in SW480 cells, further mechanistic studies suggested that wighteone-induced Akt inhibition led to cycle arrest, apoptosis and autophagic death of SW480 cells. Moreover, wighteone exerted in vivo anti-colorectal cancer effect and Akt inhibition activity in the nude mouse xenograft model. CONCLUSION: Wighteone could inhibit growth of SW480 cells through allosteric inhibition of Akt, which led to cell cycle arrest, apoptosis and autophagic death. The results contributed to understanding of the anti-tumor mechanism of licorice, and also provided a rationale to design novel Akt allosteric inhibitors for the treatment of colorectal cancer.

Asymmetric synthesis of P-stereogenic phosphindane oxides via kinetic resolution and their biological activity.

The importance of P-stereogenic heterocycles has been widely recognized with their extensive use as privileged chiral ligands and bioactive compounds. The catalytic asymmetric synthesis of P-stereogenic phosphindane derivatives, however, remains a challenging task. Herein, we report a catalytic kinetic resolution of phosphindole oxides via rhodium-catalyzed diastereo- and enantioselective conjugate addition to access enantiopure P-stereogenic phosphindane and phosphindole derivatives. This kinetic resolution method features high efficiency (s factor up to >1057), excellent stereoselectivities (all >20:1 dr, up to >99% ee), and a broad substrate scope. The obtained chiral phosphindane oxides exhibit promising therapeutic efficacy in autosomal dominant polycystic kidney disease (ADPKD), and compound 3az is found to significantly inhibit renal cyst growth both in vitro and in vivo, thus ushering in a promising scaffold for ADPKD drug discovery. This study will not only advance efforts towards the asymmetric synthesis of challenging P-stereogenic heterocycles, but also surely inspire further development of P-stereogenic entities for bioactive small-molecule discovery.

Mulberry (Morus alba L.) leaf water extract attenuates type 2 diabetes mellitus by regulating gut microbiota dysbiosis, lipopolysaccharide elevation and endocannabinoid system disorder.

ETHNOPHARMACOLOGICAL RELEVANCE: Mulberry (Morus alba L.) leaf is a well-known herbal medicine and has been used to treat diabetes in China for thousands of years. Our previous studies have proven mulberry leaf water extract (MLWE) could improve type 2 diabetes mellitus (T2D). However, it is still unclear whether MLWE could mitigate T2D by regulating gut microbiota dysbiosis and thereof improve intestinal permeability and metabolic dysfunction through modulation of lipopolysaccharide (LPS) and endocannabinoid system (eCBs). AIM OF STUDY: This study aims to explore the potential mechanism of MLWE on the regulation of metabolic function disorder of T2D mice from the aspects of gut microbiota, LPS and eCBs. MATERIALS AND METHODS: Gut microbiota was analyzed by high-throughput 16S rRNA gene sequencing. LPS, N-arachidonoylethanolamine (AEA) and 2-ararchidonylglycerol (2-AG) contents in blood were determined by kits or liquid phase chromatography coupled with triple quadrupole tandem mass spectrometry, respectively. The receptors, enzymes or tight junction protein related to eCBs or gut barrier were detected by RT-PCR or Western blot, respectively. RESULTS: MLWE reduced the serum levels of AEA, 2-AG and LPS, decreased the expressions of N-acylphophatidylethanolamine phospholipase D, diacylglycerol lipase-α and cyclooxygenase 2, and increased the expressions of fatty acid amide hydrolase (FAAH), N-acylethanolamine-hydrolyzing acid amidase (NAAA), alpha/beta hydrolases domain 6/12 in the liver and ileum and occludin, monoacylglycerol lipase and cannabinoid receptor 1 in the ileum of T2D mice. Furthermore, MLWE could change the abundances of the genera including Acetatifactor, Anaerovorax, Bilophila, Colidextribacter, Dubosiella, Gastranaerophilales, Lachnospiraceae_NK4A136_group, Oscillibacter and Rikenella related to LPS, AEA and/or 2-AG. Moreover, obvious improvement of MLWE treatment on serum AEA level, ileum occludin expression, and liver FAAH and NAAA expression could be observed in germ-free-mimic T2D mice. CONCLUSION: MLWE could ameliorate intestinal permeability, inflammation, and glucose and lipid metabolism imbalance of T2D by regulating gut microbiota, LPS and eCBs.

m6Am methyltransferase PCIF1 negatively regulates ciliation by inhibiting BICD2 expression.

N6, 2'-O-dimethyladenosine (m6Am) is a widespread RNA modification catalyzed by the methyltransferase PCIF1 (phosphorylated CTD interacting factor 1). Despite its prevalence, the biological functions of m6Am in RNA remain largely elusive. Here, we report a critical role of PCIF1-dependent m6Am RNA modification in ciliogenesis in RPE-1 cells. Our findings demonstrate that PCIF1 acts as a negative regulator of ciliation through its m6Am methyltransferase activity. A quantitative proteomic analysis identifies BICD2 as a downstream target of PCIF1, with PCIF1 depletion resulting in a significant increase in BICD2 levels. BICD2 depletion leads to a significant reduction in ciliation. Crucially, the ciliary phenotype in PCIF1-depleted cells is reversed upon BICD2 knockdown. Further investigations reveal that PCIF1 regulates BICD2 protein levels through its m6Am catalytic activity, which reduces the stability and translation efficiency of BICD2 mRNA. Single-base resolution LC-MS analysis identifies the m6Am site on BICD2 mRNA modified by PCIF1. These findings establish the essential involvement of PCIF1-dependent m6Am modification in ciliogenesis.