MTH1 suppression enhances the stemness of MCF7 through upregulation of STAT3.

MTH1 protein can sanitize the damaged (d)NTP pool and MTH1 inhibitors have been developed to impede the growth of rapidly proliferating tumor cells; however, the effect of MTH1 inhibition on breast cancer stemness has not been reported yet. Here, we constructed breast cancer cell lines with the stable depletion of MTH1. MTH1 suppression clearly increased the ratio of CD44+CD24-/low subpopulations and promoted the formation of tumorspheres in MCF7 and T47D cells. RNA expression profiling, RT-qPCR and Western blotting showed the upregulation of master stem cell transcription factors Sox2, Oct4 and Nanog in MTH1 knockdown cells. GSEA suggested and Western blotting verified that MTH1 knockdown increased the expression of phosphorylated STAT3 (Tyr705). Furthermore, we indirectly demonstrated that the increased concentration of 8-oxo-dGTP and 8-oxo-GTP in MTH1-knockdown cells and exogenous 8-oxoGTP, rather than 8-oxo-dGTP, could significantly increase the phosphorylation of STAT3. In conclusion, this work indicates that MTH1 inhibition increased the proportion of breast cancer stem cells (BCSCs) and promoted stemness properties in MCF7 cells.

Specific Metabolites Involved in Antioxidation and Mitochondrial Function Are Correlated With Frailty in Elderly Men.

BACKGROUND: As an age-related syndrome, frailty may play a central role in poor health among older adults. Sarcopenia overlaps with the physical domain of frailty, and most existing studies have analyzed the associated factors of frailty and sarcopenia as an isolated state. Perturbations in metabolism may play an important role in the presence of frailty or sarcopenia; however, the metabolites associated with frailty, especially overlapping with sarcopenia remain unclear. In this study, we aimed to explore whether amino acids, carnitines, acylcarnitines and lysophosphatidylcholines, as specific panels, are significantly correlated with frailty, especially overlapping with sarcopenia, to gain insight into potential biomarkers and possible biological mechanisms and to facilitate their management. METHODS: We applied a targeted high-performance liquid chromatography-tandem mass spectrometry approach in serum samples from 246 Chinese older men (age 79.2 ± 7.8 years) with frailty (n = 150), non-frailty (n = 96), frailty and sarcopenia (n = 52), non-frail and non-sarcopenic control (n = 85). Frailty was evaluated using Freid phenotype criteria, sarcopenia was defined by diagnostic algorithm of Asian Working Group on Sarcopenia, and the participants were diagnosed as frailty and sarcopenia when they met the evaluation criteria of both frailty and sarcopenia. A panel of 29 metabolomic profiles was assayed and included different classes of amino acids, carnitines, acylcarnitines, and lysophosphatidylcholines (LPCs). Multivariate logistic regression was used to screen the metabolic factors contributing to frailty status, and orthogonal partial least squares discriminant analysis was used to explore important factors and distinguish different groups. RESULTS: In older men demonstrating the frail phenotype, amino acid perturbations included lower tryptophan and higher glycine levels. With regard to lipid metabolism, the frailty phenotype was characterized by lower concentrations of isovalerylcarnitine (C5), LPC16:0 and LPC18:2, while higher levels of octanoyl-L-carnitine (C8), decanoyl-L-carnitine (C10), dodecanoyl-L-carnitine (C12) and tetradecanoyl-L-carnitine (C14). After adjusting for several clinical confounders, tryptophan, LPC18:2, LPC 16:0 and C5 were negatively correlated with frailty, and C8 and C12 were positively related to frailty. We preliminarily identified metabolic profiles (LPC16:0, LPC18:2, glycine and tryptophan) that may distinguish older men with frailty from those without frailty. Importantly, a set of serum amino acids and LPCs (LPC16:0, LPC18:2, and tryptophan) was characterized in the metabotype of older adults with an overlap of frailty and sarcopenia. The metabolites that were most discriminating of frailty status implied that the underlying mechanism might be involved in antioxidation and mitochondrial dysfunction. CONCLUSIONS: These present metabolic analyses may provide valuable information on the potential biomarkers and possible biological mechanisms of frailty, and overlapping sarcopenia. The findings obtained may offer insight into their management in older adults.

Urinary 8-oxoGuo as a potential novel evaluation index for patients with nephrotic syndrome.

Urinary 8-oxo-7,8-dihydroguanosine (8-oxoGuo) and 8-oxo-7,8-dihydro-2'- deoxyguanosine (8-oxodGuo) are considered biomarkers of oxidative stress, and patients with nephrotic syndrome have been reported to have increased oxidative stress levels. In this study, we aimed to assess the value of 8-oxoGuo and 8-oxodGuo as novel biomarkers to evaluate the severity of nephrotic syndrome. In total, 107 patients with nephrotic syndrome and 116 healthy controls were recruited for this study. The concentrations of urinary 8-oxoGuo and 8-oxodGuo were measured using isotope-labeled liquid chromatography with tandem mass spectrometry. Urinary creatinine was used to regulate 8-oxoGuo and 8-oxodGuo concentrations. Urinary 8-oxoGuo and 8-oxoGuo/Cr levels in patients with nephrotic syndrome were significantly higher than those in healthy control participants. 8-oxoGuo/Cr showed a positive correlation with the 24 h urinary total protein (UTP) and UTP levels and negative correlations with serum total protein and albumin levels. After treatment, urinary 8-oxoGuo and 8-oxoGuo/Cr levels were significantly lower in the group with a low 24 h-UTP value (<3.5 g/d) than in the high value group. 8-oxoGuo can be used as a feasible and reliable biomarker for the assessment of nephrotic syndrome.HighlightsUrinary 8-oxoGuo level was significantly increased in patients with nephrotic syndrome.Urinary 8-oxoGuo level increased with an increase in plasma protein and a decrease in urine protein.Urinary 8-oxoGuo level decreased with nephrotic syndrome remission when urinary microalbumin showed no significant change.Urinary 8-oxoGuo level can be used as novel biomarkers of nephrotic syndrome.

Effects of 31 recombinant CYP2C19 variants on clomipramine metabolism in vitro.

BACKGROUND: CYP2C19 is an important member of the cytochrome P450 enzyme superfamily. We recently identified 31CYP2C19 alleles in the Han Chinese population; studying the effects of CYP2C19 on drug metabolism can help reduce adverse drug reactions and therapeutic failure. AIM: The aim of this study was to assess the catalytic activities of 31 allelic isoforms and their effects on the metabolism of clomipramine in vitro. METHODS: The wild-type and 30 CYP2C19 variants were expressed in insect cells, and each variant was characterized using clomipramine as the substrate. Reactions were performed at 37°C with 5-150 µmol/L substrate for 30 min. By using ultra-high-performance liquid chromatography-mass spectrometry to detect the products, the kinetic parameters Km, Vmax, and intrinsic clearance (Vmax/Km) of N-desmethyl clomipramine were determined. RESULTS: Among the CYP2C19 variants tested, CYP2C19*29, L16F, and T130M showed extremely increased intrinsic clearance of clomipramine, CYP2C19*3C, and N277K showed similar intrinsic clearance (Vmax/Km) values with CYP2C19*1, while the intrinsic clearance values of other variants were significantly decreased (from 0.65% to 63.28%). In addition, CYP2C19*3 and 35FS could not be detected because they have no detectable enzyme activity. CONCLUSIONS: As the first report of 31 CYP2C19 alleles for clomipramine metabolism, our study could provide corresponding reference for clomipramine for further studies in vivo and offer valuable information relevant to the personalized medicine for CYP2C19-metabolized drug.

Systemic RNA oxidation can be used as a biomarker of infection in challenged with Vibrio parahaemolyticus.

More and more evidence support the concept that RNA oxidation plays a substantial role in the progress of multiple diseases; however, only a few studies have reported RNA oxidation caused by microbial pathogens. Urinary 8-oxo-7,8-dihydroguanosine (8-oxo-Gsn) and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dGsn), which are broadly used as indicators of oxidative damage of RNA and DNA, were analyzed in this study to determine which can be used as a biomarker of infection in challenged with Vibrio parahaemolyticus (V. parahaemolyticus). In this work, 24 specific-pathogen-free (SPF) male SD rats were randomly divided into two groups: an infection group and a phosphate-buffered saline (PBS) control group. Our results proved that 8-oxo-Gsn rather than 8-oxo-dGsn was significantly increased after challenged with V. parahaemolyticus in urine and tissue samples of SD rats compared with the PBS control group. Simultaneously, white blood cells (WBCs) counts, intestinal inflammation and inflammatory factors (including CRP, IL-6, IL-1ß, TNF-α, IL-10, and IL-17A) were also increased sharply. Which has more clinical value is that the trend of urinary 8-oxo-Gsn was consistent with WBCs, intestinal inflammation and all kinds of inflammatory factors. More importantly is that urinary 8-oxo-Gsn of infection group was positively correlated with WBCs and various inflammatory cytokines. In a word, our results demonstrated that as a systemic RNA oxidation biomarker, we hope 8-oxo-Gsn can be used as a biomarker of the severity of microbial pathogens infection, rather than a specific biomarker of microbial pathogens infection.

New approach for the detection of peptide- and protein-based radicals using a pre-fluorescent probe.

A novel application for pre-fluorescent probes in the detection of peptide- and protein-based radicals is proposed. Pre-fluorescent probes combine a fluorescent moiety labeled with a paramagnetic nitroxide that acts as a fluorescence quencher. Trapping of a radical by the nitroxide group restores the fluorescence properties. The increase in fluorescence intensity with time reflects the formation and quenching of free radicals and can be employed for the quantitative evaluation of yields and kinetics. In this test system, the pre-fluorescent probe 4-(9-acridinecarbonate)-2,2,6,6-tetramethylpiperidinyl-1-oxyl radical (Ac-Tempo), in which an acridine moiety was labeled with 2,2,6,6-tetramethylpiperidinyl-1-oxy (Tempo), was employed to probe peptide- and protein-based radicals. Peptide-based radicals were generated through the reaction between horseradish peroxidase (HRP)/H(2)O(2) and a derivative of tyrosine. Protein-based radicals were generated through the reaction between myoglobin (Mb) and H(2)O(2). In both cases the Ac-Tempo was found, using a combination of high-performance liquid chromatography (HPLC) and mass spectrometry, to be converted into fluorescent acridine (Ac)-piperidine (4-(9-acridinecarbonate)-2,2,6,6-tetramethylpiperidine).