Functionalization of Nanostructured ZnO Films by Copper-Free Click Reaction

The copper-free click reaction was explored as a surface functionalization methodology for ZnO nanorod films grown by metal organic chemical vapor deposition (MOCVD). 11-Azidodecanoic acid was bound to ZnO nanorod films through the carboxylic acid moiety, leaving the azide group available for Cu-free click reaction with alkynes. The azide-functionalized layer was reacted with 1-ethynylpyrene, a fluorescent probe, and with alkynated biotin, a small biomolecule. The immobilization of pyrene on the surface was probed by fluorescence spectroscopy, and the immobilization of biotin was confirmed by binding with streptavidin–fluorescein isothiocyanate (streptavidin–FITC). The functionalized ZnO films were characterized by Fourier transform infrared attenuated total reflectance (FTIR–ATR), steady-state fluorescence emission, fluorescence microscopy, and field emission scanning electron microscopy (FESEM)

Morphology Effects on the Biofunctionalization of Nanostructured ZnO

A stepwise surface functionalization methodology was applied to nanostructured ZnO films grown by metal organic chemical vapor deposition (MOCVD) having three different surface morphologies (i.e., nanorod layers (ZnO films-N), rough surface films (ZnO films-R), and planar surface films (ZnO films-P). The films were grown on glass substrates and on the sensing area of a quartz crystal microbalance (nano-QCM). 16-(2-Pyridyldithiol)-hexadecanoic acid (PDHA) was bound to ZnO films-N, -R, and -P through the carboxylic acid unit, followed by a nucleophilic displacement of the 2-pyridyldithiol moiety by single-stranded DNA capped with a thiol group (SH-ssDNA). The resulting ssDNA-functionalized films were hybridized with complementary ssDNA tagged with fluorescein (ssDNA-Fl). In a selectivity control experiment, no hybridization occurred upon treatment with non complementary DNA. The ZnO films' surface functionalization, characterized by FT-IR-ATR and fluorescence spectroscopy and detected on the nano-QCM, was successful on films-N and -R but was barely detectable on the planar surface of films-P

Neuronal Transcription Factors Induce Conversion of Human Glioma Cells to Neurons and Inhibit Tumorigenesis

<div><p>Recent findings have demonstrated that the overexpression of lineage-specific transcription factors induces cell fate changes among diverse cell types. For example, neurons can be generated from mouse and human fibroblasts. It is well known that neurons are terminally differentiated cells that do not divide. Therefore, we consider how to induce glioma cells to become neurons by introducing transcription factors. Here, we describe the efficient generation of induced neuronal (iN) cells from glioma cells by the infection with three transcription factors: Ascl1, Brn2 and Ngn2 (ABN). iN cells expressed multiple neuronal markers and fired action potentials, similar to the properties of authentic neurons. Importantly, the proliferation of glioma cells following ABN overexpression was dramatically inhibited in both in vitro and in vivo experiments. In addition, iN cells that originated from human glioma cells did not continue to grow when they were sorted and cultured in vitro. The strategies by which glioma cells are induced to become neurons may be used to clinically study methods for inhibiting tumor growth.</p> </div

Characterization of iN cells converted from human glioma cells.

<p><b>A</b>, GBM-1 primary human glioma cells were effectively induced to become neurons and expressed the neuronal proteins Tuj1 and MAP2 thirteen days after infection with ABN. <b>B,C,D</b>, iN cells expressed the mature neuronal proteins Neurofilament, NeuN and Synapsin three weeks after infection with ABN. <b>E,F,G,H</b>, GBM-2 primary human glioma cells expressed the neuronal proteins Tuj1, MAP2, Neurofilament, NeuN and Synapsin. Scale bars: 25 µm.</p

Physiological characterization of iN cells from primary human glioma cells.

<p><b>A</b>, Representative traces of action potentials (left) and voltage dependent membrane currents (right) in control glioma cells. <b>B</b>, Representative traces of action potentials (left) and voltage gated membrane currents (right) in iN cells at 23 days after infection. <b>C</b>, Representative traces of action potentials (left) and voltage gated membrane currents (right) in iN cells at 33 days after infection. <b>D</b>, Quantification of currents density of different groups (control, n = 4; iN, 23 days, n = 6, 33 days, n = 3). <b>E</b>, Representative traces of currents measured by ramp protocol. <b>F,G</b>, Quantification of the input resistant (control, n = 4; iN, 23 days, n = 6, 33 days, n = 3), and membrane potentials (control, n = 4; iN, 23 days, n = 6, 33 days, n = 3). *:p<0.05, **:p<0.01 vs control group. Error bars indicate ±s.d. Scale bars: 25 µm.</p

The ABN neuronal transcription factors inhibit glioma cell proliferation.

<p><b>A</b>, Primary glioma cells were counted 2, 3 or 4 days post-infection by ABN or control virus. Cell numbers were normalized to the number of cells plated at day 0. <b>B</b>, U251 cells were counted 2, 3 or 4 days post-infection by ABN or control virus. <b>C</b>, U87 cells were counted 2, 3 or 4 days post-infection by ABN or control virus. The data represent six independent experiments. <b>D</b>, Synpasin positive cells were isolated from synapsin-mcherry infected primary human glioma cells (GBM-1). Synapsin positive cells were sorted out and cultured for 1, 3, 5 or 7 days. Synapsin positive iN cells did not grow completely. *P<0.05. Data are presented as mean ±s.d.</p

Induction of human glioma cells to neurons.

<p>A,B, Thirteen days after infection with Asc1, Brn2 and Ngn2, primary human glioma cells (GBM-1, -2) expressed neuronal protein Tuj1 and displayed a neuronal morphology. iN cell generation efficiencies estimated from Tuj1-positive cells. <b>C</b>, Induction of U251 human glioma cells to neurons by overexpressing ABN. <b>D</b>, Induction of U87 human glioma cells to neurons by overexpressing ABN. <b>E,F,G,H</b>, Quantification of iN cells induced from primary GBM-1, GBM-2, U251 and U87 cells. Scale bars: 100 µm. Error bars indicate ±s.d.</p

High Cytoplasmic FOXO1 and pFOXO1 Expression in Astrocytomas Are Associated with Worse Surgical Outcome

<div><p>FOXO1 is at a convergence point of receptor tyrosine kinase (RTK) signaling, which is one of the three core pathways implicated in glioblastoma. It was recently shown that FOXO1 can effectively induce glioma cell death and inhibit tumor growth through cell cycle arrest and apoptosis. We therefore evaluated FOXO1 and pFOXO1 protein expression in 181 primary astrocytoma samples and 16 normal brain samples. Astrocytoma samples expressed higher cytoplasmic FOXO1 and pFOXO1 than normal brain samples. Nuclear pFOXO1 level was significantly higher than nuclear FOXO1 in astrocytomas. High cytoplasmic FOXO1 expression was associated with older onset age (P = 0.001) and higher WHO grade (P = 0.001). The trend was also observed between cytoplasmic pFOXO1 expression and WHO grade although not significant. Univariate survival analysis showed that both high cytoplasmic FOXO1 and pFOXO1 expression indicated a significantly shorter median overall survival and progression-free survival. Multivariate survival analysis revealed cytoplasmic FOXO1 expression, cytoplasmic pFOXO1 expression, WHO grade, gender, extent of resection and radiotherapy to be independent prognostic factors for overall survival and progression-free survival. Thus, our data suggested that cytoplasmic FOXO1 and pFOXO1 expression may serve as valuable prognostic variables in astrocytomas and may have significant implications for the development and application of targeted therapy.</p></div

Tissue microarray analysis of FOXO1 expression.

<p>(A, B) Cytoplasmic and nuclear FOXO1 expressions were higher in astrocytomas than in normal brain tissues. High-grade gliomas express more cytoplasmic FOXO1 than low-grade gliomas while nuclear FOXO1 expression was comparable in both. The horizontal line inside the box represents the median. The outliers are cases with the values between 1.5 and 3 box-lengths from the 75th percentile or 25th percentile. Representative images of FOXO1 expression in normal brain tissue (C, ×200); grade II diffuse astrocytoma (D, ×200); grade III anaplastic astrocytoma (E, ×200) and grade IV glioblastoma (F, ×200).</p

Clinio-pathological characteristics of 181 patients of astrocytomas.

<p>Abbreviations: IICP, increased intracranial pressure; MTD, mean tumor diameter.</p>*<p>The survival data of 11 patients, including 5 diffuse astrocytomas (Grade II), 2 anaplastic astrocytomas (Grade III) and 4 glioblastomas (Grade IV) was not available.</p