Status of breast cancer detection in young women and potential of liquid biopsy

Young onset breast cancer (YOBC) is an increasing demographic with unique biology, limited screening, and poor outcomes. Further, women with postpartum breast cancers (PPBCs), cancers occurring up to 10 years after childbirth, have worse outcomes than other young breast cancer patients matched for tumor stage and subtype. Early-stage detection of YOBC is critical for improving outcomes. However, most young women (under 45) do not meet current age guidelines for routine mammographic screening and are thus an underserved population. Other challenges to early detection in this population include reduced performance of standard of care mammography and reduced awareness. Women often face significant barriers in accessing health care during the postpartum period and disadvantaged communities face compounding barriers due to systemic health care inequities. Blood tests and liquid biopsies targeting early detection may provide an attractive option to help address these challenges. Test development in this area includes understanding of the unique biology involved in YOBC and in particular PPBCs that tend to be more aggressive and deadly. In this review, we will present the status of breast cancer screening and detection in young women, provide a summary of some unique biological features of YOBC, and discuss the potential for blood tests and liquid biopsy platforms to address current shortcomings in timely, equitable detection

Standard-Level Herbivory in an Old-Growth Conifer Forest Canopy

Herbivory is an important ecological process in forest canopies but is difficult to measure, especially for whole stands. We used the Wind River Canopy Crane in Washington State to access 101 randomly-located sample points throughout the forest canopy. This provided a relatively quick and convenient way to estimate herbivory for a whole stand. The overall level of herbivory was estimated at 1.6% of leaf area. The distribution was strongly skewed to the lower canopy where broad-leafed species experienced higher levels of herbivory. Herbivory averaged 0.3% in conifers and 13.5% in broad-leafed species. Fully half of the sample points had no detectable herbivory. Herbivory in this old-growth conifer forest is among the lowest levels published for forests around the globe and may reflect the general levels of herbivory in temperate coniferous forests during nonoutbreak conditions. Our whole-stand estimate is the first attempt at measuring herbivory for an entire forest stand in the Pacific Northwest

Methicillin resistant Staphylococcus aureus adhesion to human umbilical vein endothelial cells demonstrates wall shear stress dependent behaviour

<p>Abstract</p> <p>Background</p> <p>Methicillin-resistant <it>Staphylococcus aureus </it>(MRSA) is an increasingly prevalent pathogen capable of causing severe vascular infections. The goal of this work was to investigate the role of shear stress in early adhesion events.</p> <p>Methods</p> <p>Human umbilical vein endothelial cells (HUVEC) were exposed to MRSA for 15-60 minutes and shear stresses of 0-1.2 Pa in a parallel plate flow chamber system. Confocal microscopy stacks were captured and analyzed to assess the number of MRSA. Flow chamber parameters were validated using micro-particle image velocimetry (PIV) and computational fluid dynamics modelling (CFD).</p> <p>Results</p> <p>Under static conditions, MRSA adhered to, and were internalized by, more than 80% of HUVEC at 15 minutes, and almost 100% of the cells at 1 hour. At 30 minutes, there was no change in the percent HUVEC infected between static and low flow (0.24 Pa), but a 15% decrease was seen at 1.2 Pa. The average number of MRSA per HUVEC decreased 22% between static and 0.24 Pa, and 37% between 0.24 Pa and 1.2 Pa. However, when corrected for changes in bacterial concentration near the surface due to flow, bacteria per area was shown to increase at 0.24 Pa compared to static, with a subsequent decline at 1.2 Pa.</p> <p>Conclusions</p> <p>This study demonstrates that MRSA adhesion to endothelial cells is strongly influenced by flow conditions and time, and that MSRA adhere in greater numbers to regions of low shear stress. These areas are common in arterial bifurcations, locations also susceptible to generation of atherosclerosis.</p

RELATING CELLULAR ASSOCIATION WITH LIPOSOME CYTOTOXICITY IN HUMAN ENDOTHELIAL CELLS

INTRODUCTION Interactions with the endothelium play a key role in the behaviour of intravenously administered nanoparticle drug carriers[1]. Hence, quantifying cellular association (membrane adhesion and cell internalization) of liposomes with endothelial cells is an effective screening method of biocompatibility and success of new drug carriers. Current methods are inaccurate as concentration does not necessarily equate to local cellular association. The focus of this experiment is to quantify the cellular association between liposomes and two types of human endothelial cells and compare the associations with cells’ cytotoxic response. Cellular association of liposomes as well as cell viability were quantified on cellular level at different concentrations of liposomes. METHODS Two different types of cells, Human Umbilical Vein Endothelial cell, which is a common cell type used in vitro studies, and Human MicroVascular Cell, which is more accurate representation of in vivo, were used[2]. HUVEC and HMVEC were cultured and passaged onto chamber slides using standard cell culture techniques. The confluent cells were exposed to fluorescent liposomes with hydrodynamic diameter of 90.4 nm at concentrations ranging from 0.08nM to 8nM for 24 hours, membrane stained with CellMask Deep Red and fixed with paraformaldehyde, following same protocols for both types of cells. Cell viability on exposure to the same concentration range of liposomes was determined using Vialight assay using manufacturer protocols. Z-stacks of the treated cells were obtained using Olympus Fluoview FV1000 confocal microscope. Region of interest, limited by cell membranes, was set using the membrane stain channel using ImageJ. The region of interest was superimposed onto the fluorescent liposome channel to determine exclusively the fluorescence of cell adhered and cell internalized liposomes RESULTS Compared to HUVECs, higher cellular association of liposomes was observed for HMVC as shown in Figure 1.While cellular association of liposomes increased with concentration, cell viability was in the range of 85 to nearly 100% for the concentration range of 0.08-4 nM with no significant difference. Only at 8 nM, cell viability decreased significantly to approximately 62 %. DISCUSSION AND CONCLUSIONS Liposome cellular association provide insight into the cytotoxicity and the endothelial cytotoxicity of the liposomes at low concentration of 8nM raises cautions on documented innocuous properties of liposomes. Cytotoxicity and cellular association upon comparison showed exponential relationship. Because the cytotoxicity and cellular association relationship is exponential, slight over-administration can cause severe toxicity. 8nM is lower than concentration of current intravenous liposome-based drug doxorubicin[3]. High toxicity and exponential relationship raise caution on the importance of proper safe dosage

RELATING QUANTUM DOT ASSOCIATION WITH HUMAN ENDOTHELIAL CELLS WITH THEIR CYTOTOXIC EFFECTS

INTRODUCTION Advances in the field of nanotechnology have enabled researchers to pursue biomedical applications of nanoparticles. Quantum dots are commonly used fluorescent probes because they are brighter and less prone to photobleaching than other fluorophores [1]. However, despite the advantages, potential for toxicity must be acknowledged. Quantum dots are commonly made with toxic metal elements, which can cause oxidative stress [2]. Cadmium ions have been shown to disrupt mitochondria activity, leading to cell death [2]. Quantum dots have been shown to attach to the cell membrane as well as be internalized through endocytic mechanisms [3]. In this study, we aim to quantify quantum dot association and compare results from cytotoxicity assays for identical conditions, relating cellular association with cytotoxicity. METHODS Human Umbilical Vein Endothelial Cells (HUVECs) and Human Micro-vascular Endothelial Cells (HMVECs) were cultured in static conditions in 8-well chamber slides then exposed to amino-PEG quantum dots at a concentration of 0.2nM to 200nM. After exposure for 24 hours, the cells were washed, fixed, and stained. Z-stacks were obtained using an Olympus Fluoview FV1000 confocal microscope. Images were analyzed using ImageJ software to quantify mean fluorescence intensity within the defined region of interest, selected from the boundaries of stained cell membranes. Statistical analysis using one-way Analysis of Variance (ANOVA) and post-hoc Tukey HSD test was performed. Finally, Vialight assay was used to test cell viability after exposure to quantum dots under the same experimental conditions used for association experiments. RESULTS Exposure to different concentrations of quantum dots results in significant changes in the observed fluorescence intensity per area. Non-linear dependence of cellular association of quantum dots on exposure concentration was observed. A representative example of mean fluorescence intensity of quantum dots associated with HUVECs is shown in Figure 1.A significant decrease in the viability of HUVECs was observed on exposure to quantum dots (30-50% cell viability relative to 100% for non-exposed cells). However, no significant difference in cell viability was observed between 0.2nM to 200nM concentrations. DISCUSSION AND CONCLUSIONS Nanoparticle association studies play a vital role in predicting cell viability in nanoparticle cytotoxicity studies. The non-linear trend observed suggests that for the range of concentrations examined, cellular association does not increase linearly with exposure concentration, and that cytotoxicity can be related to association, rather than just to exposure concentration. This experiment provides an approach to advance future studies relating cellular association to cytotoxicity

Case Study: Intra-Patient Heterogeneity of Aneurysmal Tissue Properties

Introduction: Current recommendations for surgical treatment of abdominal aortic aneurysms (AAAs) rely on the assessment of aortic diameter as a marker for risk of rupture. The use of aortic size alone may overlook the role that vessel heterogeneity plays in aneurysmal progression and rupture risk. The aim of the current study was to investigate intra-patient heterogeneity of mechanical and fluid mechanical stresses on the aortic wall and wall tissue histopathology from tissue collected at the time of surgical repair.Methods: Finite element analysis (FEA) and computational fluid dynamics (CFD) simulations were used to predict the mechanical wall stress and the wall shear stress fields for a non-ruptured aneurysm 2 weeks prior to scheduled surgery. During open repair surgery one specimen partitioned into different regions was collected from the patient's diseased aorta according to a pre-operative map. Histological analysis and mechanical testing were performed on the aortic samples and the results were compared with the predicted stresses.Results: The preoperative simulations highlighted the presence of altered local hemodynamics particularly at the proximal segment of the left anterior area of the aneurysm. Results from the post-operative assessment on the surgical samples revealed a considerable heterogeneity throughout the aortic wall. There was a positive correlation between elastin fragmentation and collagen content in the media. The tensile tests demonstrated a good prediction of the locally varying constitutive model properties predicted using geometrical variables, i.e., wall thickness and thrombus thickness.Conclusions: The observed large regional differences highlight the local response of the tissue to both mechanical and biological factors. Aortic size alone appears to be insufficient to characterize the large degree of heterogeneity in the aneurysmal wall. Local assessment of wall vulnerability may provide better risk of rupture predictions

EFFECT OF PEG COATING ON NANOPARTICLE DIFFUSION THROUGH TUMOUR EXTRACELLULAR MATRIX

INTRODUCTION Nanoparticle drug delivery systems have the potential to improve current cancer treatments through encapsulating cytotoxic agents and delivering them to specific sites in the body. One such class of particle, liposomes, has already found some commercial success [1]. Liposomes are vesicles composed of a lipid bi-layer surrounding an aqueous solution. Poly(ethylene) glycol (PEG) surface coating is commonly used to improve the hydrophilicity of liposomes, thereby increasing their stability in aqueous solutions. Furthermore, PEG limits the binding of blood antigens, which minimizes opsonisation and phagocytosis, extending circulation time in the blood stream. When applied to the surface of liposomes at lower molecular weights and surface densities, PEG adopts a “mushroom” conformation, in which adjacent chains of PEG do not interact laterally, therefore portions of the bi-layer remain exposed [2]. However, at higher molecular weights and surface densities, the “brush” conformation is adopted; where lateral interactions occur between neighbouring PEG strands and provide complete coverage of the lipid bi-layer [2]. This study will investigate the effect of varying PEG molecular weight and surface density on liposome transport through tumour extracellular matrix. METHODS Seven different formulations of liposomes were synthesized using a modification of the lipid extrusion method described in [1]. Molecular weight and surface density values were chosen to include both PEG conformations. The Type I collagen hydrogel was prepared with a collagen concentration of 2.5mg/mL. Confocal Microscopy was used to track the liposome transport into the gels via the bilayer incorporated Rhodamine dye. While simple collagen hydrogels may not capture all of the complexity of native tumour ECM, they allow for more carefully controlled conditions than in vivo models. Images were taken every 30 minutes until the 900 minute mark. RESULTS As shown in Figure 1, the liposomes with a lower PEG loading (DOPC, 5, 10% PEG 1000, 5, 10% PEG 2000), all accumulated at the interface of the hydrogel, and had identical diffusion coefficients. The 5% and 10% PEG 5000 however, accumulated significantly less and therefore had a much greater diffusion coefficient.DISCUSSION AND CONCLUSIONS The liposomes with low PEG surface density, and DOPC control liposomes shown in Figure 1, are all within the “mushroom” conformation of PEG [2] and therefore would all have exposed bilayer which is not shielded by the PEG strands. The formulations that penetrated deeply were notably only higher PEG surface densities (5 and 10% PEG 5000) which literature suggests would have been in the “brush” conformation [2]. This suggests that the high PEG surface densities sterically shielded the liposomes, and reduced the electrostatic interactions between the hydrogels and the liposomes, allowing increased diffusion

Flow-dependent Smad2 phosphorylation and TGIF nuclear localization in human aortic endothelial cells

Endothelial cells respond to fluid flow stimulation through transient and sustained signal pathway activation. Smad2 is a signaling molecule and transcription factor in the Smad signaling pathway, traditionally associated with TGF-β. Although phosphorylation of Smad2 in the receptor-dependent COOH-terminal region is the most appreciated way Smad2 is activated to affect gene expression, phosphorylation may also occur in the MH1-MH2 linker region (L-psmad2). Here, we show that in human aortic endothelial cells (HAEC), Smad2 was both preferentially phosphorylated in the linker region and localized to the nucleus in a flow-dependent manner. The Smad corepressor transforming growth interacting factor (TGIF) was also found to have flow-dependent nuclear localization. Tissue studies confirmed this L-psmad2 generation trend in rat aorta, indicating likely importance in arterial tissue. HAEC-based inhibitor studies demonstrated that L-psmad2 levels were not related to MAPK phosphorylation, but instead followed the pattern of pAkt473, both with and without the phosphatidylinositol 3-kinase inhibitor PI-103. Akt and Smad species were also shown to directly interact under flow relative to static controls. To further evaluate impacts of PI-103 treatment, expression profiles for two TGF-β and shear stress-dependent genes were determined and showed that mRNAs were lower from untreated 10 dyn/cm2 than 2 dyn/cm2 average shear stress cultures. However, upon exposure to PI-103, this trend was reversed, with a stronger response observed at 10 dyn/cm2. Taken together, the results of this work suggest that fluid flow exposure may influence endothelial gene expression by a novel mechanism involving Akt, L-psmad2, and TGIF