Vertebral Tortuosity Is Associated With Increased Rate of Cardiovascular Events in Vascular Ehlers-Danlos Syndrome.

Background Arterial tortuosity is associated with adverse events in Marfan and Loeys-Dietz syndromes but remains understudied in Vascular Ehlers-Danlos syndrome. Methods and Results Subjects with a pathogenic COL3A1 variant diagnosed at age <50 years were included from 2 institutions and the GenTAC Registry (National Registry of Genetically Triggered Thoracic Aortic Aneurysms and Cardiovascular Conditions). Height-adjusted vertebral artery tortuosity index (VTI-h) using magnetic resonance or computed tomography angiography was calculated. Associations between VTI-h and outcomes of (1) cardiovascular events (arterial dissection/rupture, aneurysm requiring intervention, stroke), or (2) hollow organ collapse/rupture at age <50 years were evaluated using receiver operator curve analysis (using outcome by age 30 years) and mixed-effects Poisson regression for incidence rate ratios. Of 65 subjects (54% male), median VTI-h was 12 (interquartile range, 8-16). Variants were missense in 46%, splice site in 31%, and null/gene deletion in 14%. Thirty-two subjects (49%) had 59 events, including 28 dissections, 5 arterial ruptures, 4 aneurysms requiring intervention, 4 strokes, 11 hollow organ ruptures, and 7 pneumothoraces. Receiver operator curve analysis suggested optimal discrimination at VTI-h ≥15.5 for cardiovascular events (sensitivity 70%, specificity 76%) and no association with noncardiovascular events (area under the curve, 0.49 [95% CI, 0.22-0.78]). By multivariable analysis, older age was associated with increased cardiovascular event rate while VTI-h ≥15.5 was not (incidence rate ratios, 1.79 [95% CI, 0.76-4.24], P=0.185). However, VTI-h ≥15.5 was associated with events among those with high-risk variants <40 years (incidence rate ratios, 4.14 [95% CI, 1.13-15.10], P=0.032), suggesting effect modification by genotype and age. Conclusions Increased arterial tortuosity is associated with a higher incidence rate of cardiovascular events in Vascular Ehlers-Danlos syndrome. Vertebral tortuosity index may be a useful biomarker for prognosis when evaluated in conjunction with genotype and age.

Spheroid Size Does not Impact Metabolism of the ß-blocker Propranolol in 3D Intestinal Fish Model.

Compared to two-dimensional (2D) cell culture, cellular aggregates or spheroids (3D) offer a more appropriate alternative in vitro system where individual cell-cell communication and micro-environment more closely represent the in vivo organ; yet we understand little of the physiological conditions at this scale. The relationship between spheroid size and oxygen microenvironment, an important factor influencing the metabolic capacity of cells, was first established using the fish intestine derived RTgutGC cell line. Subsequently, pharmaceutical metabolism (Propranolol), as determined by high performance liquid chromatography, in this intestinal model was examined as a function of spheroid size. Co-efficient of variation between spheroid size was below 12% using the gyratory platform method, with the least variation observed in the highest cell seeding density. The viable, high oxygen micro-environment of the outer rim of the spheroid, as determined by electron paramagnetic resonance (EPR) oximetry, decreased over time, and the hypoxic zone increased as a function of spheroid size. Despite a trend of higher metabolism in smaller spheroids, the formation of micro-environments (quiescent, hypoxic or anoxic) did not significantly affect metabolism or function of an environmentally relevant pharmaceutical in this spheroid model.

Altered cellular redox homeostasis and redox responses under standard oxygen cell culture conditions versus physioxia.

In vivo, mammalian cells reside in an environment of 0.5-10% O2 (depending on the tissue location within the body), whilst standard in vitro cell culture is carried out under room air. Little is known about the effects of this hyperoxic environment on treatment-induced oxidative stress, relative to a physiological oxygen environment. In the present study we investigated the effects of long-term culture under hyperoxia (air) on photodynamic treatment. Upon photodynamic irradiation, cells which had been cultured long-term under hyperoxia generated higher concentrations of mitochondrial reactive oxygen species, compared with cells in a physioxic (2% O2) environment. However, there was no significant difference in viability between hyperoxic and physioxic cells. The expression of genes encoding key redox homeostasis proteins and the activity of key antioxidant enzymes was significantly higher after the long-term culture of hyperoxic cells compared with physioxic cells. The induction of antioxidant genes and increased antioxidant enzyme activity appear to contribute to the development of a phenotype that is resistant to oxidative stress-induced cellular damage and death when using standard cell culture conditions. The results from experiments using selective inhibitors suggested that the thioredoxin antioxidant system contributes to this phenotype. To avoid artefactual results, in vitro cellular responses should be studied in mammalian cells that have been cultured under physioxia. This investigation provides new insights into the effects of physioxic cell culture on a model of a clinically relevant photodynamic treatment and the associated cellular pathways.

Linking genotoxicity and cytotoxicity with membrane fluidity: A comparative study in ovarian cancer cell lines following exposure to auranofin.

Auranofin, an organogold compound classified as an anti-rheumatic agent is under phase 2 clinical trials for re-purposing to treat recurrent epithelial ovarian cancer. We have reported earlier that Breast cancer 1, early onset (BRCA1) mutant ovarian cancer cells exhibit increased sensitivity to auranofin. BRCA1 is a DNA repair protein whose functional status is critical in the prognosis of ovarian cancer. Apart from DNA repair capability of cancer cells, membrane fluidity is also implicated in modulating resistance to chemotherapeutics. We report here that membrane fluidity influences the sensitivity of ovarian cancer cell lines, OVCAR5 and IGROV1, to auranofin. Electron spin resonance (ESR) analysis revealed a more fluidized membrane in IGROV1 compared to OVCAR5. Interestingly, IGROV1 cells were more sensitive to auranofin induced cytotoxicity than OVCAR5. In comparison to OVCAR5, IGROV1 cells also exhibited an increased number of DNA double strand breaks (DSBs) upon auranofin treatment as assessed by 53BP1 immunostaining. Furthermore, correlation analysis demonstrated a strong positive correlation (r=0.856) between membrane fluidity and auranofin sensitivity in these cell lines. Auranofin-treated IGROV1 cells also exhibited increased cellular oxidation and apoptosis. Anti-oxidant, N-acetyl cysteine (NAC) inhibited the cellular oxidation and apoptosis in auranofin-treated ovarian cancer cells suggesting reactive oxygen species (ROS) mediate the anti-cancer properties of auranofin. Overall, our study suggests that auranofin mediates its cytotoxicity via ROS production in ovarian cancer cells which correlates positively with membrane fluidity.

Correction: Direct Measurements of Oxygen Gradients in Spheroid Culture System Using Electron Parametric Resonance Oximetry.

[This corrects the article DOI: 10.1371/journal.pone.0149492.].

The hydroxypyridinone iron chelator CP94 increases methyl-aminolevulinate-based photodynamic cell killing by increasing the generation of reactive oxygen species.

Methyl-aminolevulinate-based photodynamic therapy (MAL-PDT) is utilised clinically for the treatment of non-melanoma skin cancers and pre-cancers and the hydroxypyridinone iron chelator, CP94, has successfully been demonstrated to increase MAL-PDT efficacy in an initial clinical pilot study. However, the biochemical and photochemical processes leading to CP94-enhanced photodynamic cell death, beyond the well-documented increases in accumulation of the photosensitiser protoporphyrin IX (PpIX), have not yet been fully elucidated. This investigation demonstrated that MAL-based photodynamic cell killing of cultured human squamous carcinoma cells (A431) occurred in a predominantly necrotic manner following the generation of singlet oxygen and ROS. Augmenting MAL-based photodynamic cell killing with CP94 co-treatment resulted in increased PpIX accumulation, MitoSOX-detectable ROS generation (probably of mitochondrial origin) and necrotic cell death, but did not affect singlet oxygen generation. We also report (to our knowledge, for the first time) the detection of intracellular PpIX-generated singlet oxygen in whole cells via electron paramagnetic resonance spectroscopy in conjunction with a spin trap.

Body MR angiography in children: how we do it.

Vascular pathology is ubiquitous in children. Common indications for angiographic imaging in the body include congenital anomalies, portal hypertension, assessing resectability of neoplasms, renovascular hypertension, vascular malformations, vasculitis, systemic vein thrombosis, and trauma. MR angiography, with or without the use of intravenous contrast agents, is therefore a mainstay in the repertoire of MR imaging in children. Pediatric contrast-enhanced MR angiography has benefited from several innovations in recent years, including improved hardware options like high-field-strength scanners and integrated high-density coil arrays, new sequences that combine parallel imaging, innovative k-space sampling and Dixon fat suppression with time-resolved imaging, new contrast agents with longer blood-pool residence time, and advanced post-processing solutions like image fusion. This article focuses on the principles of contrast-enhanced MR angiography of the body as it pertains to the physiologies and pathologies encountered in children. It also discusses tools to adapt the MR angiographic technique to the clinical indication, as well as pitfalls of post-processing and interpretation in commonly encountered vascular imaging scenarios in the pediatric body.

Comparison of two single-breath-held 3-D acquisitions with multi-breath-held 2-D cine steady-state free precession MRI acquisition in children with single ventricles.

BACKGROUND: Breath-held two-dimensional balanced steady--state free precession cine acquisition (2-D breath-held SSFP), accelerated with parallel imaging, is the method of choice for evaluating ventricular function due to its superior blood-to-myocardial contrast, edge definition and high intrinsic signal-to-noise ratio throughout the cardiac cycle. OBJECTIVE: The purpose of this study is to qualitatively and quantitatively compare the two different single-breath-hold 3-D cine SSFP acquisitions using 1) multidirectional sensitivity encoding (SENSE) acceleration factors (3-D multiple SENSE SSFP), and 2) k-t broad-use linear acceleration speed-up technique (3-D k-t SSFP) with the conventional 2-D breath-held SSFP in non-sedated asymptomatic volunteers and children with single ventricle congenital heart disease. MATERIALS AND METHODS: Our prospective study was performed on 30 non-sedated subjects (9 healthy volunteers and 21 functional single ventricle patients), ages 12.5 +/- 2.8 years. Two-dimensional breath-held SSFP with SENSE acceleration factor of 2, eight-fold accelerated 3-D k-t SSFP, and 3-D multiple SENSE SSFP with total parallel imaging factor of 4 were performed to evaluate ventricular volumes and mass in the short-axis orientation. Image quality scores (blood myocardial contrast, edge definition and interslice alignment) and volumetric analysis (end systolic volume, end diastolic volume and ejection fraction) were performed on the data sets by experienced users. Paired t-test was performed to compare each of the 3-D k-t SSFP and 3-D multiple SENSE SSFP clinical scores against 2-D breath-held SSFP. Bland-Altman analysis was performed on left ventricle (LV) and single ventricle volumetry. Interobserver and intraobserver variability in volumetric measurements were determined using intraclass coefficients. RESULTS: The clinical scores were highest for the 2-D breath-held SSFP images. Between the two 3-D sequences, 3-D multiple SENSE SSFP performed better than 3-D k-t SSFP. Bland-Altman analysis for volumes indicated that variability was more between 3-D k-t SSFP and 2-D breath-held SSFP acquisitions than between 3-D multiple SENSE SSFP and 2-D breath-held SSFP acquisitions. In the non-sedated population, interslice alignment scores were better for 3-D k-t SSFP and 3-D multiple SENSE SSFP than 2-D breath-held SSFP. The blood-myocardial contrast and edge definition scores were better for 2-D breath-held SSFP than 3-D k-t SSFP and 3-D multiple SENSE SSFP. Scan duration was shorter for 3-D acquisition sequences compared to the 2-D breath-held stack. CONCLUSION: Three-dimensional k-t SSFP and 3-D multiple SENSE for ventricular volumetry release the constraints of multiple breath-holds in children and overcome problems related to interslice misalignment caused by inconsistent amplitude of breathing. Three-dimensional multiple SENSE SSFP performed better in our pediatric population than 3-D k-t SSFP. However, these 3-D sequences produce lower-quality diagnostic images than the gold standard 2-D breath-held SSFP sequence.

Direct Measurements of Oxygen Gradients in Spheroid Culture System Using Electron Parametric Resonance Oximetry.

Advanced in vitro culture from tissues of different origin includes three-dimensional (3D) organoid micro structures that may mimic conditions in vivo. One example of simple 3D culture is spheroids; ball shaped structures typically used as liver and tumour models. Oxygen is critically important in physiological processes, but is difficult to quantify in 3D culture: and the question arises, how small does a spheroid have to be to have minimal micro-environment formation? This question is of particular importance in the growing field of 3D based models for toxicological assessment. Here, we describe a simple non-invasive approach modified for the quantitative measurement and subsequent evaluation of oxygen gradients in spheroids developed from a non-malignant fish cell line (i.e. RTG-2 cells) using Electron Paramagnetic Resonance (EPR) oximetry. Sonication of the paramagnetic probe Lithium phthalocyanine (LiPc) allows for incorporation of probe particulates into spheroid during its formation. Spectra signal strength after incorporation of probe into spheroid indicated that a volume of 20 µl of probe (stock solution: 0.10 mg/mL) is sufficient to provide a strong spectra across a range of spheroid sizes. The addition of non-toxic probes (that do not produce or consume oxygen) report on oxygen diffusion throughout the spheroid as a function of size. We provide evidence supporting the use of this model over a range of initial cell seeding densities and spheroid sizes with the production of oxygen distribution as a function of these parameters. In our spheroid model, lower cell seeding densities (∼2,500 cells/spheroid) and absolute size (118±32 µm) allow control of factors such as pre-existing stresses (e.g. ∼ 2% normoxic/hypoxic interface) for more accurate measurement of treatment response. The applied methodology provides an elegant, widely applicable approach to directly characterize spheroid (and other organoid) cultures in biomedical and toxicological research.

Fast, Ultrasensitive Detection of Reactive Oxygen Species Using a Carbon Nanotube Based-Electrocatalytic Intracellular Sensor.

Herein, we report a highly sensitive electrocatalytic sensor-cell construct that can electrochemically communicate with the internal environment of immune cells (e.g., macrophages) via the selective monitoring of a particular reactive oxygen species (ROS), hydrogen peroxide. The sensor, which is based on vertically aligned single-walled carbon nanotubes functionalized with an osmium electrocatalyst, enabled the unprecedented detection of a local intracellular "pulse" of ROS on a short second time scale in response to bacterial endotoxin (lipopolysaccharide-LPS) stimulation. Our studies have shown that this initial pulse of ROS is dependent on NADPH oxidase (NOX) and toll like receptor 4 (TLR4). The results suggest that bacteria can induce a rapid intracellular pulse of ROS in macrophages that initiates the classical innate immune response of these cells to infection.

Computed tomographic colonography (CTC); colorectal cancer diagnosis with CTC in an Auckland population.

AIM: To determine the sensitivity of computed tomographic colonography (CTC) in the detection of colorectal cancer in our population and evaluate the reasons why these lesions may be missed on CTC. METHODS: All patients who underwent CTC in the 65-month period from 1 January 2004 to 1 July 2009 were included in the analysis. Demographic data and CTC findings were recorded, according to the CT Colonography Reporting and Data System. Data were cross-matched with the National Cancer Registry results for colorectal cancer cases between 1 January 2004 and 1 October 2009, 3 months longer to include any delayed diagnoses. RESULTS: There were 2026 consecutive CTC patients, comprising 52.6% female, average age of 60 years; range 19-87. Approximately 84% were symptomatic. There were 45 confirmed colorectal cancers among this patient group in the National Cancer Registry during the relevant time period compared with 43 suspected cancers on CTC, giving a miss rate of 2 of 45, or 4.4%. CONCLUSION: The sensitivity of 95% for CTC in the detection of colorectal cancer compares favourably with the published national and international data.

Effect of variation of portal venous blood flow on radiofrequency and microwave ablations in a blood-perfused bovine liver model.

PURPOSE: To assess whether there is a significant difference in the effect of incremental changes of portal venous blood flow rates on the size of radiofrequency (RF) versus microwave (MW) ablation lesions in an ex vivo blood-perfused bovine liver model. MATERIALS AND METHODS: This study was exempt from approval by the Institutional Animal Care and Use Committee. Sixty ablations (30 MW and 30 RF ablations) were performed ex vivo in 15 bovine livers perfused with autologous blood via the portal vein at 60, 70, 80, 90, and 100 mL/min per 100 g of liver tissue (three livers were used for each flow rate). Long-axis diameter (LAD), short-axis diameter (SAD), and volume were measured for each ablation lesion. A general linear mixed model was used to examine the effect of location, ablation device, and flow rate on LAD, SAD, and volume. Results were considered to indicate a significant difference at P less than .05. RESULTS: Location was not a significant predictor of LAD, SAD, or volume (P ≥ .4). The slope of the relationship between flow rate and LAD, SAD, and volume was significantly different according to ablation device (P < .0001). For RF ablation lesions, the mean LAD, SAD, and volume demonstrated a significant inverse relationship with flow rate, while the measurements for MW ablation did not change with variation in flow rates. CONCLUSION: The size of RF ablation lesions is highly variable, with a significant inverse relationship to the rate of portal venous blood flow. Conversely, the size of MW ablation lesions is unaffected by changes in portal venous blood flow. The consistency of the size of MW ablation lesions could translate into a higher local tumor eradication rate than that reported with RF ablation.

Computed tomographic colonography: colonic and extracolonic findings in an Auckland population.

AIM: To determine the nature and prevalence of colonic and extracolonic findings in our population. METHODS: All patients who underwent computed tomographic colonography (CTC) in the 72-month period from 1 January 2004 to 1 January 2010 were included in the analysis. Demographic data and CTC findings were recorded, according to the CT colonography reporting and data system (CRADS). RESULTS: There were 2152 consecutive CTC patients; comprising 52.6% female, average age of 60 years; range 19-87. Approximately 84% were symptomatic. CRADS: Colonic findings: 99/2152 patients (4.6%) were C2 category (had 1 or 2 polyps of 6-9 mm). 77/2152 (3.6%) patients were C3 category (>9 mm polyp or >2 polyps of 6-9 mm). 55/2152 (2.5%) were C4 category (possible cancer). This comprises a total potential colonoscopy/surgery referral rate of 10.7%. Extracolonic findings: The majority were normal or clinically unimportant findings. 178/2152 (8.3%) had potentially significant extracolonic findings. CONCLUSION: Our CTC population is largely symptomatic, and there is a referral rate from CTC to colonoscopy, surgery or surveillance of 10.7%. This is similar to other NZ data and international studies. The 8.3% rate of potentially significant extracolonic findings is at the lower end of the reported range.

Photoexcitation of aqueous suspensions of titanium dioxide nanoparticles: an electron spin resonance spin trapping study of potentially oxidative reactions.

It is well-established that exposure of aqueous suspensions of titanium dioxide (TiO(2)) nanoparticles to ultraviolet A (UVA) light produces reactive oxygen species which leads to biological damage. However, there is disagreement in the literature as to the exact nature of these species and how they are formed. Using a number of different spin traps (i.e. PBN, POBN, DMPO, DEPMPO), we have shown that the primary damaging species produced on irradiation of an aqueous suspension of TiO(2) is the hydroxyl radical, which is formed at the valence band hole under both aerobic and hypoxic conditions. Hydroxyl radical production is enhanced by the presence of oxygen which probably reacts with the conduction band electrons or resultant Ti(3+), inhibiting hole-electron recombination, although we find no evidence of reaction of oxygen to form free superoxide radical anions or of the formation of any other radical at that site. The present results suggest that the resulting O(2)(•-) species may not be as labile as previously thought and may possibly undergo further reduction to the O(2)(2-) dianion. Hydroxyl radicals formed at the surface of the TiO(2) readily react with substrates containing an abstractable hydrogen to produce secondary radicals that, in biological systems, could lead to cell damage.

Titanium dioxide induced cell damage: a proposed role of the carboxyl radical.

Titanium dioxide (TiO(2)) nanoparticles have been shown to be genotoxic to cells exposed to ultraviolet A (UVA) radiation. Using the technique of electron spin resonance (ESR) spin trapping, we have confirmed that the primary damaging species produced on irradiation of TiO(2) nanoparticles is the hydroxyl (OH) radical. We have applied this technique to TiO(2)-treated fish and mammalian cells under in vitro conditions and observed the additional formation of carboxyl radical anions (CO(2)(-)) and superoxide radical anions (O(2)(-)). This novel finding suggests a hitherto unreported pathway for damage, involving primary generation of OH radicals in the cytoplasm, which react to give CO(2)(-) radicals. The latter may then react with cellular oxygen to form O(2)(-) and genotoxic hydrogen peroxide (H(2)O(2)).

Hydroxyl radicals (*OH) are associated with titanium dioxide (TiO(2)) nanoparticle-induced cytotoxicity and oxidative DNA damage in fish cells.

TiO(2) nanoparticles (< 100 nm diameter) have been reported to cause oxidative stress related effects, including inflammation, cytotoxicity and genomic instability, either alone or in the presence of UVA irradiation in mammalian studies. Despite the fact that the aquatic environment is often the ultimate recipient of all contaminants there is a paucity of data pertaining to the potential detrimental effects of nanoparticles on aquatic organisms. Therefore, these investigations aimed to evaluate the potential cytotoxic and genotoxic effects of TiO(2) nanoparticles on goldfish skin cells (GFSk-S1), either alone or in combination with UVA. Whilst neutral red retention (NRR) assay (a measure of lysosomal membrane integrity) was used to evaluate cell viability, a modified Comet assay using bacterial lesion-specific repair endonucleases (Endo-III, Fpg) was employed to specifically target oxidative DNA damage. Additionally, electron spin resonance (ESR) studies with different spin traps were carried out for qualitative analysis of free radical generation. For cell viability, TiO(2) alone (0.1-1000 microg ml(-1)) had little effect whereas co-exposure with UVA (0.5-2.0 kJm(-2)) caused a significant dose-dependent decrease which was dependent on both the concentration of TiO(2) and the dose of UVA administered. For the Comet assay, doses of 1, 10 and 100 microg ml(-1) in the absence of UVA caused elevated levels of Fpg-sensitive sites, indicating the oxidation of purine DNA bases (i.e. guanine) by TiO(2). UVA irradiation of TiO(2)-treated cells caused further increases in DNA damage. ESR studies revealed that the observed toxic effects of nanoparticulate TiO(2) were most likely due to hydroxyl radical (OH) formation.