Photo-Induced Cell Damage Analysis for Single- and Multifocus Coherent Anti-Stokes Raman Scattering Microscopy

In this study, we investigated photo-induced damage to living cells during single-and multifocus excitations for coherent anti-Stokes Raman scattering (CARS) imaging. A near-infrared pulsed laser (709 nm) was used to induce cell damage. We compared the photo-induced cell damage in the single- and the multifocus excitation schemes with the condition to obtain the same CARS signal in the same frame rate. For the evaluation of cell viability, we employed 4', 6-diamidino-2-phenylindole (DAPI) fluorophores that predominantly stained the damaged cells. One-and two-photon fluorescence of DAPI fluorophores were, respectively, excited by an ultraviolet light source and the same near-infrared light source and were monitored to evaluate the cell viability during near-infrared pulsed laser irradiation. We found lower uptake of DAPI fluorophores into HeLa cells during the multifocus excitation compared with the single- focus excitation scheme in both the one- and the two-photon fluorescence examinations. This indicates a reduction of photo-induced cell damage in the multifocus excitation. Our findings suggested that the multifocus excitation scheme is expected to be suitable for CARS microscopy in terms of minimal invasiveness

Photo-Induced Cell Damage Analysis for Single- and Multifocus Coherent Anti-Stokes Raman Scattering Microscopy

In this study, we investigated photo-induced damage to living cells during single- and multifocus excitations for coherent anti-Stokes Raman scattering (CARS) imaging. A near-infrared pulsed laser (709 nm) was used to induce cell damage. We compared the photo-induced cell damage in the single- and the multifocus excitation schemes with the condition to obtain the same CARS signal in the same frame rate. For the evaluation of cell viability, we employed 4',6-diamidino-2-phenylindole (DAPI) fluorophores that predominantly stained the damaged cells. One- and two-photon fluorescence of DAPI fluorophores were, respectively, excited by an ultraviolet light source and the same near-infrared light source and were monitored to evaluate the cell viability during near-infrared pulsed laser irradiation. We found lower uptake of DAPI fluorophores into HeLa cells during the multifocus excitation compared with the single-focus excitation scheme in both the one- and the two-photon fluorescence examinations. This indicates a reduction of photo-induced cell damage in the multifocus excitation. Our findings suggested that the multifocus excitation scheme is expected to be suitable for CARS microscopy in terms of minimal invasiveness

Noninvasive Demonstration of Dual Coronary Artery Fistulas to Main Pulmonary Artery with 64-Slice Multidetector-Computed Tomography: A Case Report

Coronary artery fistulas, including coronary pulmonary fistulas, are usually discovered accidently among the adult population when undergoing invasive coronary angiographies. We report here a 58-year-old woman with dual fistulas originating from the left anterior descending coronary artery and right coronary sinus to the main pulmonary artery, demonstrating noninvasively with multidetector-computed tomography (MDCT) and transthoracic echocardiography (TTE)

Associations between dietary n-6 and n-3 fatty acids and arachidonic acid compositions in plasma and erythrocytes in young and elderly Japanese volunteers

<p>Abstract</p> <p>Background</p> <p>We reported that the compositions of arachidonic acid (ARA) in erythrocytes and plasma phospholipids (PL) in the elderly were lower than those in the young, though the ARA intake was nearly identical.</p> <p>Objective</p> <p>We further analyzed data in four study groups with different ages and sexes, and determined that the blood ARA levels were affected by the kinds of dietary fatty acids ingested.</p> <p>Methods</p> <p>One hundred and four healthy young and elderly volunteers were recruited. Dietary records together with photographic records from 28 consecutive days were reviewed and the fatty acid composition in plasma lipid fractions and erythrocyte PL was analyzed.</p> <p>Results</p> <p>No correlations for ARA between dietary fatty acids and blood lipid fractions were observed. A significant negative correlation between eicosapentaenoic acid (EPA) + docosahexaenoic acid (DHA) intake and ARA composition in erythrocyte PL was observed. ARA composition in erythrocyte PL was significantly lower in elderly subjects than in young subjects, because EPA and DHA intake in elderly subjects was higher than in young subjects. However, after removing the effect of dietary EPA+DHA intake, the ARA composition in erythrocyte PL in elderly subjects was significantly lower than that in young subjects.</p> <p>Conclusions</p> <p>Changes in physical conditions with aging influenced the low ARA composition of erythrocyte in elderly subjects in addition to the effects of dietary EPA and DHA.</p

Oncolytic virus-mediated p53 overexpression promotes immunogenic cell death and efficacy of PD-1 blockade in pancreatic cancer

Immune checkpoint inhibitors, including anti-programmed cell death 1 (PD-1) antibody, provide improved clinical outcome in certain cancers. However, pancreatic ductal adeno-carcinoma (PDAC) is refractory to PD-1 blockade therapy due to poor immune response. Oncolytic virotherapy is a novel approach for inducing immunogenic cell death (ICD). We demonstrated the therapeutic potential of p53-expressing telo-merase-specific oncolytic adenovirus OBP-702 to induce ICD and anti-tumor immune responses in human PDAC cells with different p53 status (Capan-2, PK-59, PK-45H, Capan-1, MIA PaCa-2, BxPC-3) and murine PDAC cells (PAN02). OBP-702 significantly enhanced ICD with secretion of extracel-lular adenosine triphosphate and high-mobility group box pro-tein B1 by inducing p53-mediated apoptosis and autophagy. OBP-702 significantly promoted the tumor infiltration of CD8+ T cells and the anti-tumor efficacy of PD-1 blockade in a subcutaneous PAN02 syngeneic tumor model. Our results suggest that oncolytic adenovirus-mediated p53 overexpres-sion augments ICD and the efficacy of PD-1 blockade therapy against cold PDAC tumors. Further in vivo experiments would be warranted to evaluate the survival benefit of tumor-bearing mice in combination therapy with OBP-702 and PD-1 blockade

CSC with and without steroids

We investigated the rates of the use of steroids in Japanese central serous chorioretinopathy (CSC) cases and differences in the characteristics of CSC with and without steroids. A total of 538 eyes of 477 patients diagnosed with CSC, with 3 months or more of follow-up between April 2013 and June 2017 at 8 institutions. Patients with CSC with more than 3 months of follow-up were identified by OCT and fluorescein angiography at 8 institutions. Data collected included patient demographics, history of corticosteroid medication and smoking, spherical errors, findings of angiography, subfoveal choroidal thickness, and changes through the follow-up period. Differences in these findings were analyzed in cases with and without corticosteroid treatment. Among the 477 patients (344 men,133 women), 74 (15.5%) (39 men, 35 women) underwent current or prior steroid treatment. Cases with steroids were higher age (p = 0.0403) and showed no male prevalence, more bilateral involvement (p < 0.0001), and the affected eyes had multiple pigment epithelial detachment (p <0.0001), more fluorescein leakage sites (p < 0.0001), greater choroidal thickness (p = 0.0287) and a higher recurrence rate (p = 0.0412). Steroids can cause severer CSC through an effect on choroidal vessels and an impairment of retinal pigment epithelium

Vasohibin-1 is identified as a master-regulator of endothelial cell apoptosis using gene network analysis.

BACKGROUND: Apoptosis is a critical process in endothelial cell (EC) biology and pathology, which has been extensively studied at protein level. Numerous gene expression studies of EC apoptosis have also been performed, however few attempts have been made to use gene expression data to identify the molecular relationships and master regulators that underlie EC apoptosis. Therefore, we sought to understand these relationships by generating a Bayesian gene regulatory network (GRN) model. RESULTS: ECs were induced to undergo apoptosis using serum withdrawal and followed over a time course in triplicate, using microarrays. When generating the GRN, this EC time course data was supplemented by a library of microarray data from EC treated with siRNAs targeting over 350 signalling molecules.The GRN model proposed Vasohibin-1 (VASH1) as one of the candidate master-regulators of EC apoptosis with numerous downstream mRNAs. To evaluate the role played by VASH1 in EC, we used siRNA to reduce the expression of VASH1. Of 10 mRNAs downstream of VASH1 in the GRN that were examined, 7 were significantly up- or down-regulated in the direction predicted by the GRN.Further supporting an important biological role of VASH1 in EC, targeted reduction of VASH1 mRNA abundance conferred resistance to serum withdrawal-induced EC death. CONCLUSION: We have utilised Bayesian GRN modelling to identify a novel candidate master regulator of EC apoptosis. This study demonstrates how GRN technology can complement traditional methods to hypothesise the regulatory relationships that underlie important biological processes

Gene network inference and visualization tools for biologists: application to new human transcriptome datasets.

Gene regulatory networks inferred from RNA abundance data have generated significant interest, but despite this, gene network approaches are used infrequently and often require input from bioinformaticians. We have assembled a suite of tools for analysing regulatory networks, and we illustrate their use with microarray datasets generated in human endothelial cells. We infer a range of regulatory networks, and based on this analysis discuss the strengths and limitations of network inference from RNA abundance data. We welcome contact from researchers interested in using our inference and visualization tools to answer biological questions