Real-time Ratiometric Fluorescent Assay for Alkaline Phosphatase Activity with Stimulus Responsive Infinite Coordination Polymer Nanoparticles

This study demonstrates a novel ratiometric fluorescent method for real-time alkaline phosphatase (ALP) activity assay with stimulus responsive infinite coordination polymer (ICP) nanoparticles as the probe. The ICP nanoparticles used in this study are composed of two components; one is the supramolecular ICP network formed with guanine monophosphate (GMP) as the ligand and Tb³⁺ as the central metal ion, and the other is a fluorescent dye, i.e., 7-amino-4-methyl coumarin (coumarin) encapsulated into the ICP network. Upon being excited at 315 nm, the ICP network itself emits green fluorescence at 552 nm. Coumarin dye encapsulated in the ICP network emits weak fluorescence at 450 nm upon excitation at the same wavelength (315 nm), and this fluorescence emission becomes strong when the encapsulated dye is released from the network into the solution phase. Hence, we develop a ratiometric fluorescent assay based on the ALP-induced destruction of the supramolecular ICP network and the release of coumarin. This mechanism can be used for real-time ratiometric fluorescent monitoring of ALP activity by continuously measuring the ratio of fluorescent intensity at the wavelength of 552 nm (<i>F</i>₅₅₂) to that at 450 nm (<i>F</i>₄₅₀) (<i>F</i>₅₅₂/<i>F</i>₄₅₀) in the time-dependent fluorescent spectra of the coumarin@Tb-GMP suspension containing ALP with different activities. Under the experimental conditions employed here, the <i>F</i>₅₅₂/<i>F</i>₄₅₀ value is linear with the ALP activity within a range from 0.025 U/mL to 0.2 U/mL. The detection limit is down to 0.010 U/mL (<i>S</i>/<i>N</i> = 3). Moreover, the assay developed here is employed for ALP inhibitor evaluation. This study offers a simple yet sensitive method for real-time ALP activity assay

Stimulus Response of Au-NPs@GMP-Tb Core–Shell Nanoparticles: Toward Colorimetric and Fluorescent Dual-Mode Sensing of Alkaline Phosphatase Activity in Algal Blooms of a Freshwater Lake

In this study, we demonstrate a colorimetric and fluorescent dual-mode method for alkaline phosphatase activity (APA) sensing in freshwater lake with stimuli-responsive gold nanoparticles@terbium-guanosine monophosphate (Au-NPs@GMP-Tb) core–shell nanoparticles. Initially, the core–shell nanoparticles were fabricated based on Au-NPs decorated with a fluorescent GMP-Tb shell. Upon being excited at 290 nm, the as-formed Au-NPs@GMP-Tb core–shell nanoparticles emit green fluorescence, and the decorated GMP-Tb shell causes the aggregation of Au-NPs. However, the addition of ALP destroys GMP-Tb shell, resulting in the release of Au-NPs from the shell into the solvent. As a consequence, the aggregated Au-NPs solubilizes with the changes in the UV–vis spectrum of the dispersion, and in the meantime, the fluorescence of GMP-Tb shell turns off, which constitutes a new mechanism for colorimetric and fluorescent dual-mode sensing of APA. With the method developed here, we could monitor the dynamic change of APA during an algal bloom of a freshwater lake, both by the naked eye and further confirmed by fluorometric determination. This study not only offers a new method for on-site visible detection of APA but also provides a strategy for dual-mode sensing mechanisms by the rational design of the excellent optical properties of Au-NPs and the adaptive inclusion properties of the luminescent infinite coordination polymers

The Loss of miR-26a-Mediated Post-Transcriptional Regulation of Cyclin E2 in Pancreatic Cancer Cell Proliferation and Decreased Patient Survival

<div><p>Background</p><p>miR-26a plays a critical role in tumorigenesis, either as a tumor suppressor or as an oncogenic miRNA, depending on different tumor types. However, the function of miR-26a in pancreatic cancer has not been clearly elucidated. The present study was designed to determine the roles of miR-26a in pancreatic cancer and its association with the survival of patients with pancreatic cancer.</p><p>Methods</p><p>The expression of miR-26a was examined in 15 pairs of pancreatic duct adenocarcinoma (PDAC) and their adjacent benign pancreatic tissues (ABPT), by qRT-PCR. The results were confirmed by <i>in situ</i> hybridization using two panels of 106 PDACs and their ABPT microarray. The association of miR-26a expression with overall survival was determined. The proliferation and cell cycle distribution of Capan-2, SW-1990, and Panc-1 cells, transfected with miR-26a mimics or a miR-26a inhibitor, were assessed using the Cell Counting Kit-8 assay and flow cytometry, respectively. The cell tumorigenicity was evaluated via murine xenograft experiments. Cyclin D2, E2, EZH2, and PCNA levels were analyzed by Western blot and immunohistochemistry.</p><p>Results</p><p>miR-26a was expressed in the cytoplasm of pancreatic ductal epithelial cells, whereas its expression was significantly downregulated in PDAC tissues compared with that of ABPT. Patients with low miR-26a expression had a significantly shorter survival than those with high miR-26a expression. The <i>in vitro</i> and <i>in vivo</i> assays showed that overexpression of miR-26a resulted in cell cycle arrest, inhibited cell proliferation, and decreased tumor growth, which was associated with cyclin E2 downregulation.</p><p>Conclusions</p><p>miR-26a is an important suppressor of pancreatic ductal carcinoma, and can prove to be a novel prognostic factor and therapeutic target for pancreatic cancer treatment.</p></div

Ectopic expression of miR-26a inhibits pancreatic cancer growth in nude mice.

<p>(A) Nude mice were subcutaneously inoculated with SW-1990 cells transfected with pTM (pTM: hTERT–miR-26a plasmid) or pT (pT: hTERT control plasmid), in their flanks. The image is representative of tumors formed in 8 mice. (B) Growth curves of tumor volumes. The graph is representative of tumor growth, 5 weeks after inoculation. Tumor volume was calculated and all data are shown as the mean ± S.D. (<i>n</i> = 8). (C–H) Expression of cyclin D2, cyclin E2, and PCNA was measured by immunohistochemistry in the tissues of mice inoculated with pTM- transfected SW-1990 cells or the control cells. The figure insets in C, E, and G indicate a negative control field. Cells colored brown indicate positive staining. Original magnification, 200×.</p

miR-26a expression in pancreatic cancer specimens and overall survival.

<p>(A) Average expression level of miR-26a in human PDAC specimens (<i>n</i> = 15) and normal pancreatic tissues (<i>n</i> = 15). miRNA abundance was assessed by qRT-PCR and normalized to U6 RNA. Values are presented as the mean ± S.D. (B) Overall survival following resection of pancreatic cancer with the miR-26a-negative versus miR-26a-positive groups. The miR-26a-negative group had significantly shorter survival than the miR-26a-positive group (<i>P</i> = 0.029). (C,D) <i>In situ</i> hybridization for miR-26a in pancreatic lesions. <i>In situ</i> hybridization showed much lower miR-26a expression in PDAC tissues (C) than in ABPT (D). The inset shows the negative control (scrambled sequence probe). Cytoplasmic staining in the ductal epithelial cells stands in contrast with the negative staining with the scrambled probe. (E,F) <i>In situ</i> hybridization for miR-21 (positive control) in pancreatic lesions. <i>In situ</i> hybridization showed much stronger miR-21 expression in PDAC tissues (E) than in ABPT (F). The inset shows the negative control (scrambled sequence probe). Cytoplasmic staining in tumor cells stands in contrast with the negative staining of the scrambled probe. Original magnification, 100×.</p

Clinicopathological characteristics of 106 cases of pancreatic cancer.

<p>Clinicopathological characteristics of 106 cases of pancreatic cancer.</p

Real-Time Colorimetric Assay of Inorganic Pyrophosphatase Activity Based on Reversibly Competitive Coordination of Cu²⁺ between Cysteine and Pyrophosphate Ion

In this study we demonstrate a new colorimetric method for real-time pyrophosphatase (PPase) activity assay based on reversible tuning of the dispersion/aggregation states of gold nanoparticles (Au-NPs) by controlling the coordination of Cu²⁺ between cysteine and pyrophosphate ion (PPi) with PPase. The addition of Cu²⁺ to the cysteine-stabilized Au-NP dispersion results in the aggregation of Au-NPs, while the further addition of PPi to this aggregation turns the aggregated Au-NPs into their dispersed state because of the higher coordination reactivity between Cu²⁺ and PPi than that between Cu²⁺ and cysteine. The subsequent addition of PPase to the PPi-triggered dispersed Au-NPs restores the aggregation state of Au-NPs because PPase catalyzes the hydrolysis of PPi into orthophosphate and thus consumes PPi in the reaction system. In this study, we utilize this reversibility of the change between the aggregation/dispersion states of Au-NPs for real-time colorimetric monitoring of PPase activity by continuously measuring the ratio of absorbance at the wavelength of 650 nm (<i>A</i>₆₅₀) to that at 522 nm (<i>A</i>₅₂₂) in the time-dependent UV–vis spectra of Au-NP dispersions containing different activities of PPase. To calculate the kinetics of the PPase-catalyzed hydrolysis of PPi, the <i>A</i>₆₅₀/<i>A</i>₅₂₂ values are converted into PPi concentrations to obtain the time-dependent changes of PPi concentrations in the dispersions containing different activities of PPase. The initial reaction rates (<i>v</i>₀) are thus achieved from the time-dependent logarithm of PPi concentrations with the presence of different PPase activities. Under the experimental conditions employed here, the <i>v</i>₀ values are linear with the PPase activity within a range from 0.025 to 0.4 U with a detection limit down to 0.010 U (S/N = 3). Moreover, the colorimetric method developed here is also employed for PPase inhibitor evaluation. This study offers a simple yet effective method for real-time PPase activity assay

Immunohistochemical expression of cyclin D2, cyclin E2, and PCNA in PDAC tissues as well as overall survival.

<p>The PDAC tumor tissues (PDAC) were negative for cyclin D2 (A) and strongly positive for cyclin E2 (B), with a high PCNA proliferative index (C). The adjacent benign pancreatic tissues (ABPT) were negative for cyclin D2 was (D), and positive for cyclin E2, as observed in the ductal epithelial cells of ABPT (E). PCNA was very low in normal pancreatic tissues (F). The insets of A, B, and C show the negative controls. The inset in D indicates that cyclin D2 is a positive control of lung adenocarcinoma. Original magnification, 200×. Overall survival after resection of pancreatic cancer with the cyclinE2-positive versus cyclinE2-negative groups was not significant (<i>P</i> = 0.676) (G), whereas overall survival after resection of pancreatic cancer with the PCNA high proliferative index versus PCNA low proliferative index groups had a significantly shorter survival (<i>P</i> = 0.007) (H).</p

miR-26a overexpression inhibited pancreatic cancer cell growth by the downregulation of cyclin E2 expression.

<p>The qRT-PCR analysis demonstrated the transcription of miR-26a in mimics, inhibitor, and control groups (A,B,C), and the expression of cyclin E2 in cyclin E2 siRNA, control siRNA, and control groups (D,E,F). The proliferation of PDAC cell lines transiently transfected with miR-26a mimics, miR26a inhibitor or cyclin E2 siRNA was analyzed by the CCK-8 proliferation assay (G,H,I). The regulation of cyclin E2 or EZH2 expression by miR-26a was analyzed by Western blot (J,K,L), and Western blot analysis also confirmed the expected efficiency of cyclin E2 siRNA in three PDAC cell lines (M,N,O).</p