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.

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.

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.

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.

Gut microbiome for predicting immune checkpoint blockade-associated adverse events.

BACKGROUND: The impact of the gut microbiome on the initiation and intensity of immune-related adverse events (irAEs) prompted by immune checkpoint inhibitors (ICIs) is widely acknowledged. Nevertheless, there is inconsistency in the gut microbial associations with irAEs reported across various studies. METHODS: We performed a comprehensive analysis leveraging a dataset that included published microbiome data (n = 317) and in-house generated data from 16S rRNA and shotgun metagenome samples of irAEs (n = 115). We utilized a machine learning-based approach, specifically the Random Forest (RF) algorithm, to construct a microbiome-based classifier capable of distinguishing between non-irAEs and irAEs. Additionally, we conducted a comprehensive analysis, integrating transcriptome and metagenome profiling, to explore potential underlying mechanisms. RESULTS: We identified specific microbial species capable of distinguishing between patients experiencing irAEs and non-irAEs. The RF classifier, developed using 14 microbial features, demonstrated robust discriminatory power between non-irAEs and irAEs (AUC = 0.88). Moreover, the predictive score from our classifier exhibited significant discriminative capability for identifying non-irAEs in two independent cohorts. Our functional analysis revealed that the altered microbiome in non-irAEs was characterized by an increased menaquinone biosynthesis, accompanied by elevated expression of rate-limiting enzymes menH and menC. Targeted metabolomics analysis further highlighted a notably higher abundance of menaquinone in the serum of patients who did not develop irAEs compared to the irAEs group. CONCLUSIONS: Our study underscores the potential of microbial biomarkers for predicting the onset of irAEs and highlights menaquinone, a metabolite derived from the microbiome community, as a possible selective therapeutic agent for modulating the occurrence of irAEs.

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.

Tunable poly(lauryl methacrylate) surface grafting via SI-ATRP on a one-pot synthesized cellulose nanofibril macroinitiator core as a shear-thinning rheology modifier and drag reducer.

The optimally one-pot synthesized 2-bromoproponyl esterified cellulose nanofibril (Br-CNF) has been validated as a robust macroinitiator for self-surface-initiated atom transfer radical polymerization (SI-ATRP) of lauryl methacrylate (LMA) in tunable graft lengths and high conversions of up to 92.7%. SI-ATRP of LMA surface brushes on Br-CNF followed first order kinetics in lengths at up to 46 degree of polymerization (DP) based on mass balance or 31 DP by solution-state 1H NMR in DMSO-d6. With increasing PLMA graft lengths, Br-CNF-g-PLMA cast films exhibited increasing hydrophobicity with water contact angles from 80.9° to 110.6°. The novel Br-CNF-g-PLMA exhibited dual shear thinning behavior of the Br-CNF core as evident by n < 1 flow behavior index and drag reducing properties of PLMA grafts with increased viscosity at up to 21 071×. Br-CNF-g-PLMA with 46 DP could be fully dispersed in silicon pump oil to function as a drag reducer to enhance viscosity up to 5× at 25, 40, and 55 °C. The novel macroinitiator capability of Br-CNF in SI-ATRP of vinyl monomers and the bottlebrush-like LMA surface grafted Br-CNF as highly effective viscosity modifier and drag reducer further demonstrate the versatile functionality of Br-CNF beyond hydrophobic coatings and reactive polyols previously reported.

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.

One-pot synthesis of 2-bromopropionyl esterified cellulose nanofibrils as hydrophobic coating and film.

Hydrophobic 2-bromopropionyl esterified cellulose nanofibrils (Br-CNFs) have been facilely produced via one-pot esterification of cellulose with 2-bromopropionyl bromide (BPB) then in situ disintegrated by ultrasonication in the same reaction media. Br-CNFs optimally produced by this robust esterification-ultrasonication approach, i.e., 5 : 1 BPB to anhydroglucose (AGU) molar ratio, 23 °C, 6 h and ultrasonication (50% amplitude, 30 min), were 4.6 nm thick, 29.3 nm wide, and 1 µm long in 71% yield and 48% crystallinity. Successful 2-bromopropionyl esterification of cellulose was confirmed by FTIR and 1H NMR. The degree of substitution (DS) of surface hydroxyl to 2-bromopropanoate was determined to be between 0.53 (DSs) based on XRD and Br-CNF dimensions and 0.56 (DSNMR) from solution-state 1H NMR. Br-CNF dispersions in DMF exhibited Newtonian behaviors at concentrations below and shear thinning behaviors above 0.5%, enabling homogeneous deposition at dilute concentrations up to 0.01% into a few nm ultra-thin layers as well as blade coating of gel into ca. 100 µm thick film, all similarly hydrophobic with surface water contact angles (WCAs) in the range of 70-75°. The ultra-high modulus and strength film from gel coating further showed the potential for dual high-strength and hydrophobic applications of Br-CNFs.

Analysis of plasma free amino acids in diabetic rat and the intervention of Ginkgo biloba leaves extract using hydrophilic interaction liquid chromatography coupled with tandem mass-spectrometry.

Amino acids (AAs) are important metabolites that are related with diabetes. However, their roles in the initiation and development of diabetes mellitus (DM), especially in the treatment of Ginkgo biloba leaves extract (GBE) have not been fully explored. Thus, we investigated the roles that AAs played in the progression and GBE supplementation of DM rat induced by streptozotocin. The rats were randomly divided into a normal control group treated with drug-free solution, a normal control group treated with GBE, a DM group treated with drug-free solution, and DM group treated with GBE; and maintained on this protocol for 9 weeks. Rat plasma was collected from the sixth week to the ninth week and then analyzed with the optimized hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry method. A total of 17 AAs with differential levels were monitored to indicate dysfunction of AAs metabolism to confirm the occurrence and development of DM. Treatment with GBE partially reversed the changes seen in seven AAs including leucine, isoleucine, tyrosine, glutamic acid, asparagines, lysine and alanine in DM rats, indicating that GBE could prevent the occurrence and development of DM by acting on AAs metabolism. The improvement of those AAs metabolism disorders may play a considerable role in the treatment of GBE on the occurrence and development of DM. Those findings potentially promote the understanding of the pathogenic progression of DM and reveal the therapeutic mechanism of GBE against DM.

6-Glycosylaminoquinoline-assisted LDI MS for detection and imaging of small molecules with enhanced detection selectivity and sensitivity.

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) is a powerful tool in the analysis and imaging of small molecules. However, MALDI MS analysis is easily subjected to poor signal reproducibility and selectivity, especially for complex samples. In this study, a matrix glycosylation strategy was proposed to synthesize glycosylated matrices with excellent performances by enhancing the interaction of the matrix with small molecules. A series of glycosylated matrices including 3-glycosylaminoquinoline (3-GAQ), 6-glycosylaminoquinoline (6-GAQ), and 1-amino-5-glycosylaminoquinoline (GDAN) were synthesized by connecting glucose with the existing amine matrices. Compared with their parent matrices and the existing matrix (1,5-diaminonaphathelene, 1,5-DAN), the glycosylated matrices exhibited remarkably-improved sensitivity, higher signal reproducibility (RSD < 9%) in detecting metabolites, demonstrating the effectiveness of the glycosylation strategy. Among them, 6-GAQ exhibited the best performance. Using 6-GAQ, the detection limit of citric acid reached the low fmol range, and the calibration curve of citric acid had ideal linearity (R2 > 0.99), proving that 6-GAQ was capable of accurate quantitative analysis of metabolites. Furthermore, 6-GAQ was used for the imaging of metabolites in the mouse kidney section, showing higher sensitivity and lower background noise than the commonly-used matrices, 9-aminoquinoline (9-AA), and 1,5-DAN. More importantly, 6-GAQ can selectively detect the hydrophilic metabolites, especially the hydrophilic lipids in the mouse kidney. Overall, 6-GAQ is an ideal matrix potentially applied in the imaging and quantitative analysis of hydrophilic small molecules in complex samples.

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.

Analysis of eight bile acids in urine of gastric cancer patients based on covalent organic framework enrichment coupled with liquid chromatography-tandem mass spectrometry.

Gastric carcinoma is one of the most common and deadly forms of cancer. Early detection is critical for successful treatment of gastric cancer, and examination of BAs in urine may provide a critical diagnostic tool for identifying gastric cancer at stages when it can still be cured. Bile acids (BAs) are a crucial toxic factor correlated with the injury of gastric mucosa and as such, quantifying the amount of BA in patient's urine could provide a new means to quickly and non-invasively identify the presence of gastric cancer in the early stages. Here, a covalent organic framework (COF) material synthesized on the basis of 1,3,5-tris(4-nitrophenyl)benzene (TAPB) and pyromellitic dianhydride (PMDA) was used as stationary phase for SPE column that was coupled to LC-MS/MS for quantitative analysis of eight BAs in human urine, including cholic acid (CA), deoxycholic acid (DCA), glycochenodeoxycholic acid (GCDCA), glycocholic acid (GCA), taurochenodeoxycholic acid (TCDCA), lithocholic acid (LCA), hyodeoxycholic acid (HDCA), and chenodeoxycholic acid (CDCA). The enrichment effect of synthesized COF material was better than commercial SPE and HLB column. The sensitivity can increase 9.37- to 54.30- fold (calculated by the ratio of peak area between before and after enrichment). The probable mechanism is due to the great porosity and the similar polarity with BAs of the COF material. By compared with previous literatures, our method had the minimum limit of detection, which achieved 46.40, 25.75, 47.40, 47.37, 30.42, and 33.92 pg /mL, respectively, for GCA, GCDCA, CA, CDCA, HDCA and DCA after enrichment. These eight BAs also accomplished excellent linearity from 0.34 to 10,000 ng/mL. This material was successfully applied in the measurements of these six BAs in human urine from 76 gastric cancer patients and 32 healthy people. Compared to healthy people, levels of CA, CDCA, DCA, and HDCA were significantly elevated and levels of GCDCA were depressed, respectively, in gastric cancer patients. Our work suggests that these acids may act as potential biomarkers for gastric cancer and our framework provides a method for "non-invasive" diagnosis of gastric cancer.

Enrichment and Quantitative Determination of Free 3,5- Diiodothyronine, 3',5'-Diiodothyronine, and 3,5-Diiodothyronamine in Human Serum of Thyroid Cancer by Covalent Organic Hyper Cross-linked Poly-ionic Liquid.

The incidence of thyroid cancer is increasing worldwide. So far, still no non-invasive clinical test biomarkers were developed for the diagnosis of thyroid cancer. The diiodothyronines (T2s) are precursors and metabolites of thyroid hormone (T4). Some reports predict that T2s may be associated with several thyroid diseases, especially the thyroid cancer. Detecting free T2s in human serum may help the diagnosis of thyroid cancer. However, few works have reported the detection of T2s due to their trace amounts. Here we developed a novel hyper organic cross-linked poly ionic liquid (PIL) material for the enrichment of three main compounds in T2s family, including 3,5- diiodothyronine (3,5-T2), 3',5'-diiodothyronine (3',5'-T2), and 3,5-diiodothyronamine (3,5-T2AM). This PIL material provided specific enrichment superiority for three T2s. After enrichment, the signal intensity of 3,5-T2, 3',5'-T2, and 3,5-T2AM increased 14, 132 and 1.6 folds, respectively, with LOQ of 76, 87, and 107 fM, respectively. Finally, we successfully applied PIL material coupled with HPLC-ESI-MS/MS in enrichment and quantitative determination of free 3,5-T2, 3',5'-T2, and 3,5-T2AM in human serum of 45 thyroid cancer patients and 15 healthy people. We also used free thyroid hormone (FT4) as the calibration reference to eliminate individual differences. We found that the levels of 3,5-T2 (P < 0.001), and 3',5'-T2 (P = 0.001) in patients with thyroid cancer were significantly higher than those in healthy people. Additionally, we further investigated the power of different T2 thyroid hormones divided FT4 to classify thyroid cancer patients and healthy people. And 3,5-T2/FT4 had the highest classification performance for discriminating thyroid cancer patients from healthy people at certain threshold, indicating that 3,5-T2/FT4 in human serum can act as potential biomarkers for "non-invasive" clinical diagnosis of thyroid cancer.

MALDI-TOF MS Based Bacterial Antibiotics Resistance Finger Print for Diabetic Pedopathy.

Diabetes mellitus has become a major global health issue. Currently, the use of antibiotics remains the best foundational strategy in the control of diabetic foot infections. However, the lack of accurate identification of pathogens and the empirical use of antibiotics at early stages of infection represents a non-targeted treatment approach with a poor curative effect that may increase the of bacterial drug resistance. Therefore, the timely identification of drug resistant bacteria is the key to increasing the efficacy of treatments for diabetic foot infections. The traditional identification method is based on bacterial morphology, cell physiology, and biochemistry. Despite the simplicity and low costs associated with this method, it is time-consuming and has limited clinical value, which delays early diagnosis and treatment. In the recent years, MALDI-TOF MS has emerged as a promising new technology in the field of clinical microbial identification. In this study, we developed a strategy for the identification of drug resistance in the diagnosis of diabetic foot infections using a combination of macro-proteomics and MALDI MS analysis. The macro-proteomics result was utilized to determine the differential proteins in the resistance group and the corresponding peptide fragments were used as the finger print in a MALDI MS analysis. This strategy was successfully used in the research of drug resistance in patients with diabetic foot infections and achieved several biomarkers that could be used as a finger print for 4 different drugs, including ceftazidime, piperacillin, levofloxacin, and tetracycline. This method can quickly confirm the drug resistance of clinical diabetic foot infections, which can help aid in the early treatment of patients.

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.

Multi-Omics Analysis of Diabetic Nephropathy Reveals Potential New Mechanisms and Drug Targets.

Diabetic nephropathy (DN) is one of the most common diabetic complications, which is the major course of end-stage renal disease (ESRD). However, the systematical molecular characterizations during DN pathogenesis and progression has not been not well understood. To identify the fundamental mediators of the pathogenesis and progression of DN. we performed a combination RNASeq, proteomics, and metabolomics analyses of both patients' derived kidney biopsy samples and kidneys from in vivo DN model. As a result, molecular changes of DN contain extracellular matrix accumulation, abnormal activated inflamed microenvironment, and metabolism disorders, bringing about glomerular sclerosis and tubular interstitial fibrosis. Specificity, Further integration analyses have identified that the linoleic acid metabolism and fatty-acids ß-oxidation are significantly inhibited during DN pathogenesis and progression, the transporter protein ABCD3, the fatty acyl-CoA activated enzymes ACOX1, ACOX2, and ACOX3, and some corresponding metabolites such as 13'-HODE, stearidonic acid, docosahexaenoic acid, (±)10(11)-EpDPA were also significantly reduced. Our study thus provides potential molecular mechanisms for DN progression and suggests that targeting the key enzymes or supplying some lipids may be a promising avenue in the treatment of DN, especially advanced-stage DN.