Ultrasensitive Detection of Aminopeptidase N Activity in Urine and Cells with a Ratiometric Fluorescence Probe

An ultrasensitive ratiometric fluorescent probe (CVN) has been designed and synthesized by incorporating alanine into the cresyl violet fluorophore. The probe shows ratiometric fluorescence response toward aminopeptidase N (APN) through the increase of fluorescent intensity ratio of 626/575 nm. The sensitivity of the probe is ultrahigh with a detection limit of 33 pg/mL, which can quantify the contents of APN in 500-fold diluted human urine samples. Furthermore, by using ratiometric fluorescence imaging, the probe reveals significantly higher contents of APN in HepG2 cells than those in LO2 cells, which has been further used to distinguish these two types of cells in mixed cocultures. The probe could be of great importance for the APN-related disease diagnosis and pathophysiology elucidation

Facile and Sensitive Method for Protein Kinase A Activity Assay Based on Fluorescent Off-On PolyU-peptide Assembly

Phosphorylation mediated by protein kinases plays a pivotal role in metabolic and cell-signaling processes, and the dysfunction of protein kinases such as protein kinase A (PKA) may induce several human diseases. Therefore, it is of great significance to develop a facile and effective method for PKA activity assay and high-throughput screening of inhibitors. Herein, we develop a new fluorescent off-on method for PKA assay based on the assembly of anionic polyuridylic acid (polyU) and cationic fluorescent peptide. The phosphorylation of the peptide disrupts its electrostatic binding with polyU, suppresses the concentration quenching effect of polyU, and thus causes fluorescence recovery. The recovered fluorescence intensity at 585 nm is directly proportional to the PKA activity in the range of 0.1–3.2 U/mL with a detection limit of 0.05 U/mL. Using our method, the PKA activity in HeLa cell lysate is determined to be 58.2 ± 5.1 U/mg protein. The method has also been employed to evaluate the inhibitory effect of PKA inhibitors with satisfactory results and may be expected to be a promising candidate for facile and cost-effective assay of kinase activity and high-throughput inhibitor screening

Ghrelin Inhibits the Differentiation of T Helper 17 Cells through mTOR/STAT3 Signaling Pathway

<div><p>Enhanced activity of interleukin 17 (IL-17) producing T helper 17 (Th17) cells plays an important role in autoimmune and inflammatory diseases. Significant loss of body weight and appetite is associated with chronic inflammation and immune activation, suggesting the cross talk between immune and neuroendocrine systems. Ghrelin has been shown to regulate the organism immune function. However, the effects of ghrelin on the differentiation of Th17 cells remain elusive. In the present study, we observed the enhanced differentiation of Th17 cells in spleens of growth hormone secretagogue receptor 1a (GHSR1a)^-/- mice. Treatment of ghrelin repressed Th17 cell differentiation in a time- and concentration-dependent manner. Phosphorylation of mammalian target of rapamycin (mTOR) and signal transducer and activator of transcription 3 (STAT3) was increased in the spleens of GHSR1a^-/- mice. Activation of mTOR signaling by injection of Cre-expressiong adenovirus into tuberous sclerosis complex 1 (TSC1) ^loxp/loxp mice increased the differentiation of Th17 cells in spleen, which was associated with an increment in the phosphorylation of STAT3. Activation of mTOR signaling by leucine or overexpression of p70 ribosome protein subunit 6 kinase 1 (S6K1) activated mTOR signaling in isolated T cells, while reversed the ghrelin-induced inhibition of iTh17 cell differentiation. In conclusion, mTOR mediates the inhibitory effect of ghrelin on the differentiation of Th17 cells by interacting with STAT3.</p></div

Real-Time Sensing of TET2-Mediated DNA Demethylation In Vitro by Metal–Organic Framework-Based Oxygen Sensor for Mechanism Analysis and Stem-Cell Behavior Prediction

Active DNA demethylation, mediated by O₂-dependent ten–eleven translocation (TET) enzymes, has essential roles in regulating gene expression. TET kinetics assay is vital for revealing mechanisms of demethylation process. Here, by a metal–organic framework (MOF)-based optical O₂ sensor, we present the first demonstration on real-time TET2 kinetics assay in vitro. A series of luminescent Cu­(I) dialkyl-1,2,4-triazolate MOFs were synthesized, which were noble-metal-free and able to intuitively response to dissolved O₂ in a wide range from cellular hypoxia (≤15 μM) to ambient condition (∼257 μM). By further immobilization of the MOFs onto transparent silicon rubber (MOF@SR) to construct O₂ film sensors, and real-time monitoring of O₂ consumption on MOF@SR over the reaction time, the complete TET2-mediated 5-methylcytosine (5mC) oxidation process were achieved. The method overcomes the limitations of the current off-line methods by considerably shortening the analytical time from 0.5–18 h to 10 min, and remarkably reducing the relative standard deviation from 10%–68% to 0.68%–4.2%. As a result, the Michaelis–Menten constant (<i>K</i>_m) values of TET2 for 5mC and O₂ in ascorbic acid-free (AA^–) condition were precisely evaluated to be 24 ± 1 and 43.8 ± 0.3 μM, respectively. By comparative study on AA-containing (AA⁺) conditions, and further establishing kinetics models, the stem-cell behavior of TETs was successfully predicted, and the effects of key factors (AA, O₂, Fe²⁺) on TETs were revealed, which were fully verified in mouse embryonic stem (mES) cells. The method is promising in wide application in kinetics analysis and cell behavior prediction of other important O₂-related enzymes

Overexpression of S6K1 rescued the inhibitory effect of ghrelin on Th17 cells.

<p>Total T cells were isolated from mouse spleens and induced differentiation to Th17 cells. Adenovirus was used to infect activated T cells to overexpress S6K1, the critical downstream molecule in mTOR signaling pathway. (A) The phosphorylation of S6 was analyzed with Western Blot. Relative protein signal intensity was quantified. (B&C) Differentiated Th17 cells were treated with adenovirus expressing S6K1 (Ad-S6K1) or GFP (Ad-GFP) with final titer indicated. The mRNA level of RORγt (B) and IL-17A (C) were analyzed with RT-PCR. (D) The concentration of IL-17A in the supernatant was examined with ELISA. (E&F) The percentage of IL-17A⁺ cells in splenic total T cells (E) and CD4⁺ T cells (F) was analyzed with flow cytometry. Shown is the representative of three independent experiments. *<i>P</i><0.05 versus control; ^#<i>P</i><0.05 versus ghrelin-treated alone.</p

STAT3 was positively regulated by mTOR.

<p>(A) Total T cells were isolated from the spleen of TSC1^loxp/loxp mice injected with Ad-GFP and Ad-Cre. The phosphorylation of STAT3 was analyzed with Western Blot. (B) Differentiated Th17 cell was stimulated with ghrelin and/or leucine. The phosphorylation of STAT3 was analyzed with Western Blot. (C) Adenovirus was used to infect differentiated Th17 cells to overexpress S6K1. The phosphorylation of STAT3 was analyzed with Western Blot. (D) Differentiated Th17 cells were stimulated with ghrelin and/or colivelin. The phosphorylation of S6 was analyzed with Western Blot. Relative protein signal intensity was quantified.</p

Ghrelin inhibited the differentiation of Th17 cells <i>in vitro</i>.

<p>(A&B) Total T cells were isolated from mouse spleens and induced to differentiate into Th17 cells with TGF-β (5 ng/ml) and IL-6 (20 ng/ml), then treated with ghrelin with final concentration (A) and time (B) as indicated. The mRNA level of RORγt was analyzed with RT-PCR. (C) Differentiated Th17 cells were treated with ghrelin (10^–8 M). The mRNA level of IL-17A was analyzed with RT-PCR. (D)The concentration of IL-17A in the supernatant was examined with ELISA. (E&F) Cells were stimulated with PMA, Ionomycin and Brefeldin A for 4–6 hours. The percentage of IL-17A⁺ cells in splenic total T cells (E) and CD4⁺ T cells (F) was analyzed with flow cytometry. Shown is the representative of three independent experiments. *<i>P</i><0.05 versus control; ^#<i>P</i><0.05 versus ghrelin-treated alone.</p

STAT3 signaling pathway was involved in the inhibitory effect of ghrelin on Th17 cells.

<p>(A) Total T cells were isolated from spleens of GHSR1a^WT and GHSR1a^-/- mice. The phosphorylation of STAT3 was analyzed with Western Blot. (B) Total T cells were isolated from mouse spleens and induced differentiation to Th17 cells, then treated with ghrelin (10^–8 M). The phosphorylation of STAT3 was analyzed with Western Blot. (C) Differentiated Th17 cells were pre-treated with or without Colivelin (100 pM), then treated with or without ghrelin (10^-8M). The phosphorylation of STAT3 was analyzed with Western Blot. Relative protein signal intensity was quantified. (D&E) The mRNA level of RORγt (D) and IL-17A (E) was analyzed with RT-PCR. (F) The concentration of IL-17A in the supernatant was examined with ELISA. (G&H) The percentage of IL-17A⁺ cells in splenic total T cells (G) and CD4⁺ T cells (H) was analyzed with flow cytometry. Shown is the representative of three independent experiments. *<i>P</i><0.05 versus control; ^#<i>P</i><0.05 versus ghrelin-treated alone.</p

mTOR signaling pathway mediated the inhibitory effect of ghrelin on Th17 cells.

<p>(A) Total T cells were isolated from spleens of GHSR1a ^WT and GHSR1a^-/- mice. The phosphorylation of S6 was analyzed with Western Blot. Relative protein signal intensity was quantified. (B) 8 to 10-wk-old TSC1^loxp/loxp mice were injected intravenously through caudal vein with GFP or Cre virus for 4 weeks. The location of Ad-GFP in spleen was observed using immunoluorescence microscopy. (C) Total T cells were isolated from the spleens of mice injected with Ad-GFP or Ad-Cre. The expression of TSC1 and the phosphorylation of S6 were analyzed with Western Blot. Relative protein signal intensity was quantified. (D) Total T cells were isolated from the spleens of mice injected with Ad-GFP and Ad-Cre. The mRNA levels of RORγt and IL-17A were analyzed with RT-PCR. (E) The concentration of IL-17A in mice serum was examined with ELISA. *<i>P</i><0.05 versus Ad-GFP injected group.</p

Leucine rescued the inhibitory effect of ghrelin on Th17 cells.

<p>Total T cells were isolated from mouse spleens and induced differentiation to Th17 cells. (A) Differentiated Th17 cells were treated with ghrelin (10^–8 M) and leucine (1 mM). The phosphorylation of S6 was analyzed with Western Blot. Relative protein signal intensity was quantified. (B&C) Differentiated Th17 cells were treated with ghrelin (10^–8 M) and leucine with final concentration as indicated. The mRNA levels of RORγt (B) and IL-17A (C) were analyzed with RT-PCR. (D) Differentiated Th17 cells were treated with ghrelin (10^–8 M) and leucine (1 mM). The concentration of IL-17A in the supernatant was examined with ELISA. (E&F) The percentage of IL-17A⁺ cells in splenic total T cells (E) and CD4⁺ T cells (F) was analyzed with flow cytometry. Shown is the representative of three independent experiments. *<i>P</i><0.05 versus control; ^#<i>P</i><0.05 versus ghrelin-treated alone.</p