Mechanism and determinants of nanoparticle penetration through human skin

The ability of nanoparticles to penetrate the stratum corneum was the focus of several studies. Yet, there are controversial issues available for particle penetration due to different experimental setups. Meanwhile, there is little known about the mechanism and determinants of their penetration. In this paper the penetration of four model gold nanoparticles of diameter 6 and 15 nm, differing in surface polarity and the nature of the vehicle, through human skin was studied using multiphoton microscopy. This is in an attempt to profoundly investigate the parameters governing particle penetration through human skin. Our results imply that nanoparticles at this size range permeate the stratum corneum in a similar manner to drug molecules, mainly through the intercellular pathways. However, due to their particulate nature, permeation is also dependent on the complex microstructure of the stratum corneum with its tortuous aqueous and lipidic channels, as shown from our experiments performed using skin of different grades of barrier integrity. The vehicle (toluene-versus-water) had a minimal effect on skin penetration of gold nanoparticles. Other considerations in setting up a penetration experiment for nanoparticles were also studied. The results obtained are important for designing a new transdermal carrier and for a basic understanding of skin–nanoparticle interaction

Thrombocytopenia in systemic lupus erythematosus patients and its association with antiphospholipid antibodies

Introduction: Systemic lupus erythematosus (SLE) is a complex systemic autoimmune disease, characterized by immune-mediated inflammation in different organs. The course of the disease is characterized by relapses and remissions, and the degree of severity of the clinical manifestations is greatly affected by the number and nature of the various organ affection. The death rate in patients with SLE is still significant, and it may be due to lupus activity, when vital organs are affected, the complications of treatment especially infections or long-term complications, such as cardiovascular disorders. Objective: To detect the relation between thrombocytopenia in SLE patients and presence antiphospholipid antibodies.Patients and methods: This study was a cross-sectional study included 100 SLE patients who attended to Sohag University Hospitals. Patients included in this study were classified as SLE patients according to either the 2012 SLICC criteria or the new 2017 ACR/EULAR SLE classification criteria. All of the participants were subjected to the following: Full history, full clinical examination, routine investigations, ANA by immunofluorescence, and ANA profile for the most common 19 autoantibodies by immunoblot. All of the participants were subjected to detection of serum titers of all antiphospholipid antibodies (aPLs) including lupus anticoagulant (LA), anti-cardiolipin (aCL) and anti-beta2-glycoprotein I (ab2GPI).Results: In this study, we demonstrated that aPLs are strongly associated with increased risk of thrombocytopenia in SLE patients. We identified aPL profiles, especially LA and IgM isotypes, as biomarkers for the risk stratification of thrombocytopenia in SLE patients.Conclusions: We concluded that aPLs are strongly associated with increased risk of thrombocytopenia in SLE patients

The Measurement Problem and two Dogmas about Quantum Mechanics

According to a nowadays widely discussed analysis by Itamar Pitowsky, the theoretical problems of QT are originated from two ‘dogmas’: the first forbidding the use of the notion of measurement in the fundamental axioms of the theory; the second imposing an interpretation of the quantum state as representing a system’s objectively possessed properties and evolution. In this paper I argue that, contrarily to Pitowsky analysis, depriving the quantum state of its ontological commitment is not sufficient to solve the conceptual issues that affect the foundations of QT. In order to test Pitowsky’s analysis I make use of an argument elaborated by Amit Hagar and Meir Hemmo, showing how some probabilistic interpretations of QT fail at dictating coherent predictions in Wigner’s Friend situations. More specifically, I evaluate three different probabilistic approaches: qBism, as a representative of the epistemic subjective interpretation of the quantum state; Jeff Bub’s information-theoretic interpretation of QT, as an example of the ontic approach to the quantum state; Itamar Pitowsky’s probabilistic interpretation, as an epistemic but objective interpretation. I argue that qBism succeeds in providing a formal solution to the problem that does not lead to a self-contradictory picture, although the resulting interpretation leads to an interpretation where the real subject matter of QT clashes alarmingly with scientific practice. The other two approaches, instead, strictly fail in Wigner’s Friend scenarios, showing in such a way that they don’t provide a genuine solution to the problem

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

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

A comparison of selected parametric and imputation methods for estimating snag density and snag quality attributes

Snags (standing dead trees) are an essential structural component of forests. Because wildlife use of snags depends on size and decay stage, snag density estimation without any information about snag quality attributes is of little value for wildlife management decision makers. Little work has been done to develop models that allow multivariate estimation of snag density by snag quality class. Using climate, topography, Landsat TM data, stand age and forest type collected for 2356 forested Forest Inventory and Analysis plots in western Washington and western Oregon, we evaluated two multivariate techniques for their abilities to estimate density of snags by three decay classes. The density of live trees and snags in three decay classes (D1: recently dead, little decay; D2: decay, without top, some branches and bark missing; D3: extensive decay, missing bark and most branches) with diameter at breast height (DBH) ≥ 12.7 cm was estimated using a nonparametric random forest nearest neighbor imputation technique (RF) and a parametric two-stage model (QPORD), for which the number of trees per hectare was estimated with a Quasipoisson model in the first stage and the probability of belonging to a tree status class (live, D1, D2, D3) was estimated with an ordinal regression model in the second stage. The presence of large snags with DBH ≥ 50 cm was predicted using a logistic regression and RF imputation. Because of the more homogenous conditions on private forest lands, snag density by decay class was predicted with higher accuracies on private forest lands than on public lands, while presence of large snags was more accurately predicted on public lands, owing to the higher prevalence of large snags on public lands. RF outperformed the QPORD model in terms of percent accurate predictions, while QPORD provided smaller root mean square errors in predicting snag density by decay class. The logistic regression model achieved more accurate presence/absence classification of large snags than the RF imputation approach. Adjusting the decision threshold to account for unequal size for presence and absence classes is more straightforward for the logistic regression than for the RF imputation approach. Overall, model accuracies were poor in this study, which can be attributed to the poor predictive quality of the explanatory variables and the large range of forest types and geographic conditions observed in the data.Keywords: Snag size class, Ordinal regression, Snag density, Snag decay class, Nearest neighbor imputatio

A comparison of selected parametric and imputation methods for estimating snag density and snag quality attributes

Snags (standing dead trees) are an essential structural component of forests. Because wildlife use of snags depends on size and decay stage, snag density estimation without any information about snag quality attributes is of little value for wildlife management decision makers. Little work has been done to develop models that allow multivariate estimation of snag density by snag quality class. Using climate, topography, Landsat TM data, stand age and forest type collected for 2356 forested Forest Inventory and Analysis plots in western Washington and western Oregon, we evaluated two multivariate techniques for their abilities to estimate density of snags by three decay classes. The density of live trees and snags in three decay classes (D1: recently dead, little decay; D2: decay, without top, some branches and bark missing; D3: extensive decay, missing bark and most branches) with diameter at breast height (DBH) ≥ 12.7 cm was estimated using a nonparametric random forest nearest neighbor imputation technique (RF) and a parametric two-stage model (QPORD), for which the number of trees per hectare was estimated with a Quasipoisson model in the first stage and the probability of belonging to a tree status class (live, D1, D2, D3) was estimated with an ordinal regression model in the second stage. The presence of large snags with DBH ≥ 50 cm was predicted using a logistic regression and RF imputation. Because of the more homogenous conditions on private forest lands, snag density by decay class was predicted with higher accuracies on private forest lands than on public lands, while presence of large snags was more accurately predicted on public lands, owing to the higher prevalence of large snags on public lands. RF outperformed the QPORD model in terms of percent accurate predictions, while QPORD provided smaller root mean square errors in predicting snag density by decay class. The logistic regression model achieved more accurate presence/absence classification of large snags than the RF imputation approach. Adjusting the decision threshold to account for unequal size for presence and absence classes is more straightforward for the logistic regression than for the RF imputation approach. Overall, model accuracies were poor in this study, which can be attributed to the poor predictive quality of the explanatory variables and the large range of forest types and geographic conditions observed in the data.This is the publisher’s final pdf. The published article is copyrighted by Elsevier and can be found at: http://www.journals.elsevier.com/forest-ecology-and-managemen

A comparison of selected parametric and imputation methods for estimating snag density and snag quality attributes

Snags (standing dead trees) are an essential structural component of forests. Because wildlife use of snags depends on size and decay stage, snag density estimation without any information about snag quality attributes is of little value for wildlife management decision makers. Little work has been done to develop models that allow multivariate estimation of snag density by snag quality class. Using climate, topography, Landsat TM data, stand age and forest type collected for 2356 forested Forest Inventory and Analysis plots in western Washington and western Oregon, we evaluated two multivariate techniques for their abilities to estimate density of snags by three decay classes. The density of live trees and snags in three decay classes (D1: recently dead, little decay; D2: decay, without top, some branches and bark missing; D3: extensive decay, missing bark and most branches) with diameter at breast height (DBH)P12.7 cm was estimated using a nonparametric random forest nearest neighbor imputation technique (RF) and a parametric two-stage model (QPORD), for which the number of trees per hectare was estimated with a Quasipoisson model in the first stage and the probability of belonging to a tree status class (live, D1, D2, D3) was estimated with an ordinal regression model in the second stage. The presence of large snags with DBHP50 cm was predicted using a logistic regression and RF imputation. Because of the more homogenous conditions on private forest lands, snag density by decay class was predicted with higher accuracies on private forest lands than on public lands, while presence of large snags was more accurately predicted on public lands, owing to the higher prevalence of large snags on public lands. RF outperformed the QPORD model in terms of percent accurate predictions, while QPORD provided smaller root mean square errors in predicting snag density by decay class. The logistic regression model achieved more accurate presence/absence classification of large snags than the RF imputation approach. Adjusting the decision threshold to account for unequal size for presence and absence classes is more straightforward for the logistic regression than for the RF imputation approach. Overall, model accuracies were poor in this study, which can be attributed to the poor predictive quality of the explanatory variables and the large range of forest types and geographic conditions observed in the data.Elsevier - Education Use Statement: This article appeared in a journal published by Elsevier. The attached copy is furnished to the author for internal non-commercial research and education use, including for instruction at the authors institution and sharing with colleagues. This is the publisher’s final pdf. The published article is copyrighted by Elsevier and can be found at: http://www.elsevier.com/Keywords: Snag size class, Nearest neighbor imputation, Snag decay class, Ordinal regression, Snag densityKeywords: Snag size class, Nearest neighbor imputation, Snag decay class, Ordinal regression, Snag densit

Canonical PRC1 controls sequence-independent propagation of Polycomb-mediated gene silencing

Polycomb group (PcG) proteins play critical roles in the epigenetic inheritance of cell fate. The Polycomb Repressive Complexes PRC1 and PRC2 catalyse distinct chromatin modifications to enforce gene silencing, but how transcriptional repression is propagated through mitotic cell divisions remains a key unresolved question. Using reversible tethering of PcG proteins to ectopic sites in mouse embryonic stem cells, here we show that PRC1 can trigger transcriptional repression and Polycomb-dependent chromatin modifications. We find that canonical PRC1 (cPRC1), but not variant PRC1, maintains gene silencing through cell division upon reversal of tethering. Propagation of gene repression is sustained by cis-acting histone modifications, PRC2-mediated H3K27me3 and cPRC1-mediated H2AK119ub1, promoting a sequence-independent feedback mechanism for PcG protein recruitment. Thus, the distinct PRC1 complexes present in vertebrates can differentially regulate epigenetic maintenance of gene silencing, potentially enabling dynamic heritable responses to complex stimuli. Our findings reveal how PcG repression is potentially inherited in vertebrates