Activities of α-amylase immobilized on ferromagnetic particles and free chitinase under an ac magnetic field.

<p>(A) Schematic representation of the experiment. We prepare a solution, in which α-amylase/ferromagnetic particle hybrids and free, nonimmobilized chitinase are dispersed, and examine the effect of heat generation from the particles under an ac magnetic field on the activities of both immobilized α-amylase and nonimmobilized chitinase. (B) Activities of α-amylase immobilized on the surface of ferromagnetic particles and free chitinase under the ac magnetic field, the amplitude and frequency of which are, respectively, 30 kA/m and 0.34 MHz. The activity of each enzyme shown in the graph is normalized by that in the absence of a magnetic field. The standard deviations are obtained from 3 independent experiments.</p

Dependence of the activity of α-amylase immobilized on ferromagnetic particles on the temperature.

<p>The ordinate axis represents the activity of α-amylase immobilized on ferromagnetic particles in the absence of a magnetic field, which is normalized by that measured at 25°C. The inset shows the detailed data between 25 and 41°C. The standard deviations are obtained from 3 independent experiments.</p

Activation of α-amylase immobilized on ferromagnetic particles under a high frequency ac magnetic field.

<p>(A) Dependence of the activity of α-amylase immobilized on the surface of ferromagnetic particles under an ac magnetic field of 0.34 MHz on the amplitude of the magnetic field. The ambient temperature is 25°C. The ordinate axis represents the activity of α-amylase immobilized on ferromagnetic particles under an ac magnetic field, which is normalized by that in the absence of a magnetic field. The standard deviations are obtained from 3 independent experiments. (B) Dependence of the surface temperature increase and the amount of heat generation per unit time of a ferromagnetic particle under an ac magnetic field of 0.34 MHz on the amplitude of the magnetic field. The surface temperature is estimated comparing the activity increase of α-amylase immobilized on ferromagnetic particles under the ac magnetic fields [see (A)] and the temperature dependence of the activity in the absence of a magnetic field (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127673#pone.0127673.g003" target="_blank">Fig 3</a>). The heat generation of a particle is calculated from the surface temperature and Eqs (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127673#pone.0127673.e003" target="_blank">3</a>) and (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127673#pone.0127673.e004" target="_blank">4</a>). The inset shows the plot of the heat generation versus the square of the amplitude.</p

Isolation of Spirastrellolides A and B from a Marine Sponge <i>Epipolasis</i> sp. and Their Cytotoxic Activities

Spirastrellolides A (<b>1</b>) and B (<b>3</b>) have been isolated as free acids from a marine sponge <i>Epipolasis</i> sp. collected in the East China Sea. These compounds had been isolated from the Caribbean marine sponge <i>Spirastrella coccinea</i> after conversion to the methyl ester. We examined the cytotoxic activities of <b>1</b> and <b>3</b> and found that the activities of the free acids are comparable to those of the corresponding methyl esters

A Stable Chimeric Fibroblast Growth Factor (FGF) Can Successfully Replace Basic FGF in Human Pluripotent Stem Cell Culture

<div><p>Fibroblast growth factors (FGFs) are essential for maintaining self-renewal in human embryonic stem cells and induced pluripotent stem cells. Recombinant basic FGF (bFGF or FGF2) is conventionally used to culture pluripotent stem cells; however, because of the instability of bFGF, repeated addition of fresh bFGF into the culture medium is required in order to maintain its concentration. In this study, we demonstrate that a heat-stable chimeric variant of FGF, termed FGFC, can be successfully used for maintaining human pluripotent stem cells. FGFC is a chimeric protein composed of human FGF1 and FGF2 domains that exhibits higher thermal stability and protease resistance than do both FGF1 and FGF2. Both human embryonic stem cells and induced pluripotent stem cells were maintained in ordinary culture medium containing FGFC instead of FGF2. Comparison of cells grown in FGFC with those grown in conventional FGF2 media showed no significant differences in terms of the expression of pluripotency markers, global gene expression, karyotype, or differentiation potential in the three germ lineages. We therefore propose that FGFC may be an effective alternative to FGF2, for maintenance of human pluripotent stem cells.</p></div

Replacement of bFGF by FGFC in long-term culture.

<p>(A-C) Pluripotent marker expression in H1 ESCs maintained with E8 Medium containing bFGF (A) or FGFC (B) for 30 days through 10 passages. The results of a single independent experiment are shown. (C) The expression levels of pluripotent marker genes (<i>Nanog</i>, <i>Oct-4</i>, <i>Cripto</i>, <i>Tert</i>, <i>Rex1</i> and <i>Dnmt3b</i>) were analyzed by quantitative RT-PCR. The graphs represent relative gene expression levels when the level of H1 ESCs prior to starting the series of cultures was set as 1. Human dermal fibroblasts (HDFs) were used as a negative control. (D-F) Scatter plots showing log₂ transformed average expression values from gene expression profiles between pre-culture H1 ESCs and H1 ESCs cultured with bFGF (D, corresponding to the sample in A) or FGFC (E, corresponding to the sample in B) for 30 days (30d) or KhES-1 ESCs maintained with bFGF (F) using arrayed 60 k probe sets. A typical data set from duplicate microarray experiments is shown. The full array data set was deposited in the GEO databank (GSE55428). Black lines indicate 2-fold up-regulation or down-regulation. (G-I) G-banded karyotype analyses. The results of each single culture experiment are shown. (G) H1 ESCs, which had been cultured in MEF-conditioned medium with FGFC for 41 days (41d) through 10 passages (46,XY[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118931#pone.0118931.ref020" target="_blank">20</a>]). (H) KhES-1 ESCs, which had been cultured in medium with FGFC, instead of bFGF, on MMC-MEF feeder cells for 30 days (30d) through 10 passages (46,XX[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118931#pone.0118931.ref020" target="_blank">20</a>]). (I) 201B7 iPSCs, which had been cultured in medium with FGFC, instead of bFGF, on MMC-MEF feeder cells for 50 days (50d) through 10 passages (46,XX[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118931#pone.0118931.ref020" target="_blank">20</a>]). The karyotype notation complies with ISCN. Scale bar: 100 μm in (A and B).</p

Multifunctional Carboxymethyl Cellulose-Based Magnetic Nanovector as a Theragnostic System for Folate Receptor Targeted Chemotherapy, Imaging, and Hyperthermia against Cancer

A multifunctional biocompatible nanovector based on magnetic nanoparticle and carboxymethyl cellulose (CMC) was developed. The nanoparticles have been characterized using TEM, SEM, DLS, FT-IR spectra, VSM, and TGA studies. We found that the synthesized carboxymethyl cellulose magnetic nanoparticles (CMC MNPs) were spherical in shape with an average size of 150 nm having low aggregation and superparamagnetic properties. We found that the folate-tagged CMC MNPs were delivered to cancer cells by a folate-receptor-mediated endocytosis mechanism. 5-FU was encapsulated as a model drug for delivering cytotoxicity, and we could demonstrate the sustained release of 5-FU. It was also observed that the FITC-labeled CMC MNPs could effectively enter cells, and the fate of nanoparticles was tracked with Lysotracker. The CMC MNPs could induce significant cell death when an alternating magnetic field was applied. These results indicate that the multifunctional CMC MNPs possess a high drug loading efficiency and high biocompatibility and with low cell cytotoxicity and can be considered to be promising candidates for CMC-based targeted drug delivery, cellular imaging, and magnetic hyperthermia (MHT)

FGFC did not affect the expression of pluripotent markers in human ESCs.

<p>(A-D) Pluripotent marker expression in H1 ESCs cultured in E8 Medium with bFGF (A), FGFC (B), or no added FGF (C) for 4 days. A typical data set from two independent experiments is shown. (D) Expression levels of pluripotent marker genes (<i>Nanog</i>, <i>Oct-4</i>, <i>Cripto</i>, <i>Tert</i>, <i>Rex1</i> and <i>Dnmt3b</i>) were analyzed by quantitative RT-PCR. The graphs represent relative gene expression levels when the level of H1 ESCs prior to starting each series of experiments was set as 1. Error bars indicate the standard deviations of three independent experiments. Human dermal fibroblasts (HDFs) were used as a negative control. *, P < 0.05 compared to ESCs cultured with FGFC by paired t-tests. (E–G) Scatter plots showing log₂ transformed average expression values from gene expression profiles of H1 ESCs prior to starting a series of cultures (pre-culture H1) and H1 ESCs cultured with bFGF (E), FGFC (F), or no FGF (G) using arrayed 60 k probe sets. A typical data set from three independent experiments is shown. The full array data set was deposited in the GEO databank (GSE55428). Black lines indicate 2-fold up-regulation or down-regulation. Scale bar: 100 μm in (A-C).</p

FGFC maintained the pluripotency of human ESCs in long-term culture.

<p>hESCs were first maintained in E8 Medium with either bFGF (A) or FGFC (B) and then prepared for the <i>in vitro</i> differentiation assay. Differentiation into three germ lineages (neural, muscle, and liver) was detected by immunocytochemistry. The result of a single experiment is shown. TUJ1: anti-Class IIIβ-Tubulin, SMA: anti-human smooth muscle actin, AFP: anti-α-fetoprotein. no 1° Ab: negative control cells stained with each secondary antibody alone. Scale bar: 100 μm.</p

FGFC induced phosphorylation of MAPKs in human ESCs.

<p>H1 hESCs were treated with E8 Medium with either 100 ng/mL bFGF (A), 100 ng/mL FGFC (B), or no added FGF (C) for 15 min. Each antibody was spotted in duplicate. A typical data set from two independent experiments is shown. b2: Akt1, b3: Akt2, b4: Akt3, b5: Akt pan, b7: ERK1, b8: ERK2, b9: GSK-3α/β, b10: GSK-3β, d3: p38δ. Array coordinate details are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118931#pone.0118931.s004" target="_blank">S1 Table</a>.</p