Light-Scattering Analysis of Native Wood Holocelluloses Totally Dissolved in LiCl–DMI Solutions: High Probability of Branched Structures in Inherent Cellulose

Three holocelluloses (i.e., cellulose and hemicellulose fractions) are prepared from softwood and hardwood by the Wise method. These holocelluloses completely dissolve in 8% lithium chloride/1,3-dimethyl-2-imidazolidinone (LiCl/DMI) after an ethylenediamine (EDA) pretreatment. After diluting the holocellulose solutions to 1% LiCl/DMI, they are subjected to size-exclusion chromatography/multiangle laser-light scattering/photodiode array (SEC-MALLS-PDA) analysis. All holocelluloses exhibit bimodal molecular weight distributions primarily due to high-molecular-weight (HMW) cellulose and low-molecular-weight hemicellulose fractions. Plots of molecular weight vs root-mean-square radius obtained by SEC-MALLS analysis revealed that all the wood celluloses comprise dense conformations in 1% LiCl/DMI. In contrast, bacterial cellulose, which was used as a pure cellulose model, has a random coil conformation as a linear polymer. These results show that both softwood and hardwood HMW celluloses contain branched structures, which are probably present on crystalline cellulose microfibril surfaces. These results are consistent with those obtained by permethylation analysis of wood celluloses

Heterogeneous cell-cycle behavior in response to UVB irradiation by a population of single cancer cells visualized by time-lapse FUCCI imaging

<p>The present study analyzed the heterogeneous cell-cycle dependence and fate of single cancer cells in a population treated with UVB using a fluorescence ubiquitination-based cell-cycle (FUCCI) imaging system. HeLa cells expressing FUCCI were irradiated by 100 or 200 J/m² UVB. Modulation of the cell-cycle and apoptosis were observed by time-lapse confocal microscopy imaging every 30 min for 72 h. Correlation between cell survival and factors including cell-cycle phase at the time of the irradiation of UVB, mitosis and the G₁/S transition were analyzed using the Kaplan–Meier method along with the log rank test. Time-lapse FUCCI imaging of HeLa cells demonstrated that UVB irradiation induced cell-cycle arrest in S/G₂/M phase in the majority of the cells. The cells irradiated by 100 or 200 J/m² UVB during G₀/G₁ phase had a higher survival rate than the cells irradiated during S/G₂/M phase. A minority of cells could escape S/G₂/M arrest and undergo mitosis which significantly correlated with decreased survival of the cells. In contrast, G₁/S transition significantly correlated with increased survival of the cells after UVB irradiation. UVB at 200 J/m² resulted in a greater number of apoptotic cells.</p

<i>Salmonella</i> A1-R targeting and efficacy on the melanoma PDOX model.

<p><b>A)</b> Schematic diagram of the experimental protocol. <b>B)</b> Efficacy of <i>Salmonella</i> A1-R is indicated by the volume ratio of the transplanted tumor at day 28 after injection compared with the tumor at the beginning of the treatment. Tumor size of the <i>Salmonella</i> A1-R-treated group was significantly decreased compared with the untreated control group. The values are mean relative tumor volume ± SEM (bars). There were five mice per group. *<i>p</i> < 0.05 compared to the untreated group. <b>C)</b> Distribution of GFP-labeled <i>Salmonella</i> A1-R in tumor and organs. Representative images of GFP-labeled <i>Salmonella</i> A1-R bacteria isolated and cultured from the tumor and the normal organs (blood, liver and spleen) of the mice treated with <i>Salmonella</i> A1-R. Fluorescence imaging with the iBox small animal imaging system (UVP LLC). Scale bar: 10 mm. <b>D)</b> Colony number of each sample is indicated per mg of harvested tissue. Tissues were collected from three different mice. GFP-labeled <i>Salmonella</i> A1-R was clearly detected in the tumor. A small number of GFP-labeled <i>Salmonella</i> A1-R was detected in the liver and no GFP-labeled <i>Salmonella</i> A1-R was detected in blood and spleen.</p

Effect of a tumor-targeting <i>Salmonella</i> A1-R and chemotherapy on the melanoma PDOX.

<p><b>A)</b> Schematic diagram of the experimental protocol. (1) untreated control (Control); (2) 5-fluorouracil (5-FU; 10 mg/kg, intraperitoneal injection (i.p.), qW×4); (3) cisplatinum (CDDP; 5 mg/kg, i.p., qW×4); (4) <i>Salmonella</i> A1-R (5 × 10⁷ CFU/body, intravenously (i.v.), qW×4) and (5) <i>Salmonella</i> A1-R (3 × 10⁷ CFU/body, i.v., qW×4) + CDDP (CDDP; 3 mg/kg, i.p., qW×4). <b>B)</b> Growth curves of the melanoma PDOX tumor treated with various drugs as described above. B1: Mean change in tumor volume plotted against time; Control, <i>n</i> = 9; 5-FU, <i>n</i> = 4; CDDP, <i>n</i> = 5; <i>Salmonella</i> A1-R, <i>n</i> = 9; <i>Salmonella</i> A1-R + CDDP, <i>n</i> = 8. B-2: Data plotted are linear prediction versus time with adjusted predictions of interaction of treatment group and time with 95% Cis. The treatment-by-time interaction was significant (p = 0.0000) as were the main-effects for treatment and time (p = 0.0000) for <i>Salmonella</i> A1-R, CDDP and <i>Salmonella</i> A1-R and CDDP combined. <b>C)</b> Comparison of body weight of nude mice transplanted PDOX tumors after <i>Salmonella</i> A1-R and/or chemotherapy. All values represent mean ± SEM; Control, <i>n</i> = 8; 5-FU, <i>n</i> = 4; CDDP, <i>n</i> = 4; <i>Salmonella</i> A1-R, <i>n</i> = 7; <i>Salmonella</i> A1-R + CDDP, <i>n</i> = 4. **<i>p</i> < 0.01, compared with the untreated control group.</p

Establishment of a melanoma patient-derived orthotopic xenograft (PDOX) model.

<p><b>A)</b> Schematic diagram of the experimental protocol. <b>B)</b> Representative cross-sections of transplanted tumor 28 days after transplantation obtained from an orthotopically-transplanted patient’s melanoma. Scale bar: 10 mm. <b>C)</b> Immunohistochemical characterization of PDOX melanoma after being grown in nude mice. H&E-stained sections (left column) and immunohistochemistry for human MHC class I (HLA; middle column) and mouse MHC class I (H2 KdtH2 Dd; right column). Strong staining for HLA was observed in the cancer cells (middle column), whereas strong staining for H2 KdtH2 Dd was observed in the stromal cells (right column). Magnified views of boxed region in the upper rows are indicated at the middle rows and magnified views of boxed region in the middle rows are indicated in the lower rows. Black arrowhead indicates necrotic region of the tumor. Scale bars: (top and middle row) 200 μm; (bottom row) 100 μm.</p

Cell-cycle fate-monitoring distinguishes individual chemosensitive and chemoresistant cancer cells in drug-treated heterogeneous populations demonstrated by real-time FUCCI imaging

<p>Essentially every population of cancer cells within a tumor is heterogeneous, especially with regard to chemosensitivity and resistance. In the present study, we utilized the fluorescence ubiquitination-based cell cycle indicator (FUCCI) imaging system to investigate the correlation between cell-cycle behavior and apoptosis after treatment of cancer cells with chemotherapeutic drugs. HeLa cells expressing FUCCI were treated with doxorubicin (DOX) (5 μM) or cisplatinum (CDDP) (5 μM) for 3 h. Cell-cycle progression and apoptosis were monitored by time-lapse FUCCI imaging for 72 h. Time-lapse FUCCI imaging demonstrated that both DOX and CDDP could induce cell cycle arrest in S/G₂/M in almost all the cells, but a subpopulation of the cells could escape the block and undergo mitosis. The subpopulation which went through mitosis subsequently underwent apoptosis, while the cells arrested in S/G₂/M survived. The present results demonstrate that chemoresistant cells can be readily identified in a heterogeneous population of cancer cells by S/G₂/M arrest, which can serve in future studies as a visible target for novel agents that kill cell-cycle-arrested cells.</p

Representative time-course imaging of tumor recurrence after BLS-only and the combination of BLS + FGS.

<p>Fluorescence imaging, using the iBOX Scientia Small Animal Imaging System [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116865#pone.0116865.ref034" target="_blank">34</a>, 52, 53], showed BLS-only mice treated had tumor recurrence. In contrast, mice treated with the combination of BLS + FGS showed little recurrent tumor growth.</p

Fluorescence-Guided Surgery of Retroperitoneal-Implanted Human Fibrosarcoma in Nude Mice Delays or Eliminates Tumor Recurrence and Increases Survival Compared to Bright-Light Surgery

<div><p>The aim of this study is to determine if fluorescence-guided surgery (FGS) can eradicate human fibrosarcoma growing in the retroperitoneum of nude mice. One week after retroperitoneal implantation of human HT1080 fibrosarcoma cells, expressing green fluorescent protein (GFP) (HT-1080-GFP), in nude mice, bright-light surgery (BLS) was performed on all tumor-bearing mice (n = 22). After BLS, mice were randomized into 2 treatment groups; BLS-only (n = 11) or the combination of BLS + FGS (n = 11). The residual tumors remaining after BLS were resected with FGS using a hand-held portable imaging system under fluorescence navigation. The average residual tumor area after BLS + FGS was significantly smaller than after BLS-only (0.4 ± 0.4 mm² and 10.5 ± 2.4 mm², respectively; p = 0.006). Five weeks after surgery, the fluorescent-tumor areas of BLS- and BLS + FGS-treated mice were 379 ± 147 mm² and 11.7 ± 6.9 mm², respectively, indicating that FGS greatly inhibited tumor recurrence compared to BLS. The combination of BLS + FGS significantly decreased fibrosarcoma recurrence compared to BLS-only treated mice (p < 0.001). Mice treated with BLS+FGS had a significantly higher disease-free survival rate than mice treated with BLS-only at five weeks after surgery. These results suggest that combination of BLS + FGS significantly reduced the residual fibrosarcoma volume after BLS and improved disease-free survival.</p></div

Pre-operative and post-operative images of retroperitoneal fibrosarcoma.

<p>A. Pre- and post-operative images of mice in the BLS-only and BLS + FGS groups. Upper panels are bright-field (BF), middle panels show GFP tumor fluorescence, and lower panels show merged images. The residual tumor after BLS-only was clearly detected with the OV100 at a magnification of 0.56×. The residual tumor after BLS + FGS was marginally detected with the OV100. Fluorescent area of residual tumor after BLS-only and BLS + FGS. Fluorescent areas of residual tumors in BLS-only and BLS + FGS group were 10.5 ± 2.4 mm² and 0.4 ± 0.4 mm², respectively. The residual tumor area after BLS + FGS was significantly smaller than after BLS-only. All images were measured for residual tumor areas using ImageJ. * p < 0.01. B. Bar graphs show the percent of the original tumor remaining after either BLS or the combination of BLS + FGS.</p

Extent of tumor recurrence after BLS-only or BLS+FGS. BLS-only treated mice had tumor recurrence.

<p>BLS + FGS-treated mice had only minimal growth of recurrent tumors. Five weeks after surgery, the fluorescent-tumor areas of BLS-only- and BLS+FGS-treated mice were 379 ± 147 mm² and 11.7 ± 6.9 mm², respectively. The combination of BLS + FGS significantly decreased recurrence compared to BLS-only treated mice. * p < 0.001. </p