IR Imaging of Solid Lubricant Coatings on Concealed/Disjointed Surfaces for Transparent Polymer Delivery Device Applications.

Transparent polymer delivery devices often contain a solid lubricant coating on a stronger bulk polymer. The distribution of lubricant coating must be monitored for device optimisation appraisals and to ensure consistency during mass production. However, coating evaluation is difficult to perform as surfaces are often concealed and/or disjointed. Dye stain analysis, which is destructive and time-consuming, is the current industry standard. We present a prototype IR transmission microscope to evaluate micron-level coating coverage of polyurethane and/or polyvinylpyrrolidone on a poly(propylene)-based delivery device. The device has a common industrial configuration, containing a duct and bevel. Inferred absorption of the coating was used to identify coating coverage and a multivariate analysis was used to remove the effects of absorption and scattering by the bulk. Coverage on concealed and disjointed surfaces was imaged and evaluated from a single camera viewpoint and ≈50 µm defects were detectable. The industrial applicability of the prototype was demonstrated using comparisons with dye stain analysis by estimating water dilution of coating and identifying artifacts in coating, which may indicate machine malfunction. The sensitivity and speed of the IR technique makes it a favourable alternative to the current industry standard.

Identification of a novel K311 ubiquitination site critical for androgen receptor transcriptional activity.

The androgen receptor (AR) is the main driver of prostate cancer (PC) development and progression, and the primary therapeutic target in PC. To date, two functional ubiquitination sites have been identified on AR, both located in its C-terminal ligand binding domain (LBD). Recent reports highlight the emergence of AR splice variants lacking the LBD that can arise during disease progression and contribute to castrate resistance. Here, we report a novel N-terminal ubiquitination site at lysine 311. Ubiquitination of this site plays a role in AR stability and is critical for its transcriptional activity. Inactivation of this site causes AR to accumulate on chromatin and inactivates its transcriptional function as a consequence of inability to bind to p300. Additionally, mutation at lysine 311 affects cellular transcriptome altering the expression of genes involved in chromatin organization, signaling, adhesion, motility, development and metabolism. Even though this site is present in clinically relevant AR-variants it can only be ubiquitinated in cells when AR retains LBD suggesting a role for AR C-terminus in E2/E3 substrate recognition. We report that as a consequence AR variants lacking the LBD cannot be ubiquitinated in the cellular environment and their protein turnover must be regulated via an alternate pathway.

AGE-modified basement membrane cooperates with Endo180 to promote epithelial cell invasiveness and decrease prostate cancer survival.

Biomechanical strain imposed by age-related thickening of the basal lamina and augmented tissue stiffness in the prostate gland coincides with increased cancer risk. Here we hypothesized that the structural alterations in the basal lamina associated with age can induce mechanotransduction pathways in prostate epithelial cells (PECs) to promote invasiveness and cancer progression. To demonstrate this, we developed a 3D model of PEC acini in which thickening and stiffening of basal lamina matrix was induced by advanced glycation end-product (AGE)-dependent non-enzymatic crosslinking of its major components, collagen IV and laminin. We used this model to demonstrate that antibody targeted blockade of CTLD2, the second of eight C-type lectin-like domains in Endo180 (CD280, CLEC13E, KIAA0709, MRC2, TEM9, uPARAP) that can recognize glycosylated collagens, reversed actinomyosin-based contractility [myosin-light chain-2 (MLC2) phosphorylation], loss of cell polarity, loss of cell-cell junctions, luminal infiltration and basal invasion induced by AGE-modified basal lamina matrix in PEC acini. Our in vitro results were concordant with luminal occlusion of acini in the prostate glands of adult Endo180(Δ) (Ex2-6/) (Δ) (Ex2-6) mice, with constitutively exposed CTLD2 and decreased survival of men with early (non-invasive) prostate cancer with high epithelial Endo180 expression and levels of AGE. These findings indicate that AGE-dependent modification of the basal lamina induces invasive behaviour in non-transformed PECs via a molecular mechanism linked to cancer progression. This study provides a rationale for targeting CTLD2 in Endo180 in prostate cancer and other pathologies in which increased basal lamina thickness and tissue stiffness are driving factors. © 2014 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

The lysine demethylase, KDM4B, is a key molecule in androgen receptor signalling and turnover.

The androgen receptor (AR) is a key molecule involved in prostate cancer (PC) development and progression. Post-translational modification of the AR by co-regulator proteins can modulate its transcriptional activity. To identify which demethylases might be involved in AR regulation, an siRNA screen was performed to reveal that the demethylase, KDM4B, may be an important co-regulator protein. KDM4B enzymatic activity is required to enhance AR transcriptional activity; however, independently of this activity, KDM4B can enhance AR protein stability via inhibition of AR ubiquitination. Importantly, knockdown of KDM4B in multiple cell lines results in almost complete depletion of AR protein levels. For the first time, we have identified KDM4B to be an androgen-regulated demethylase enzyme, which can influence AR transcriptional activity not only via demethylation activity but also via modulation of ubiquitination. Together, these findings demonstrate the close functional relationship between AR and KDM4B, which work together to amplify the androgen response. Furthermore, KDM4B expression in clinical PC specimens positively correlates with increasing cancer grade (P < 0.001). Consequently, KDM4B is a viable therapeutic target in PC.

Deubiquitinating enzyme Usp12 is a novel co-activator of the androgen receptor.

The androgen receptor (AR), a member of the nuclear receptor family, is a transcription factor involved in prostate cell growth, homeostasis, and transformation. AR is a key protein in growth and development of both normal and malignant prostate, making it a common therapeutic target in prostate cancer. AR is regulated by an interplay of multiple post-translational modifications including ubiquitination. We and others have shown that the AR is ubiquitinated by a number of E3 ubiquitin ligases, including MDM2, CHIP, and NEDD4, which can result in its proteosomal degradation or enhanced transcriptional activity. As ubiquitination of AR causes a change in AR activity or stability and impacts both survival and growth of prostate cancer cells, deubiquitination of these sites has an equally important role. Hence, deubiquitinating enzymes could offer novel therapeutic targets. We performed an siRNA screen to identify deubiquitinating enzymes that regulate AR; in that screen ubiquitin-specific protease 12 (Usp12) was identified as a novel positive regulator of AR. Usp12 is a poorly characterized protein with few known functions and requires the interaction with two cofactors, Uaf-1 and WDR20, for its enzymatic activity. In this report we demonstrate that Usp12, in complex with Uaf-1 and WDR20, deubiquitinates the AR to enhance receptor stability and transcriptional activity. Our data show that Usp12 acts in a pro-proliferative manner by stabilizing AR and enhancing its cellular function.

Cavity-enhanced absorption using an atomic line source: application to deep-UV measurements.

Optical cavities are commonly used to increase the sensitivity of absorption measurements, but have not been extensively used below 300 nm, mainly owing to the limited light sources at these wavelengths. While some progress has been made using cavity ring-down spectroscopy, these systems rely on complex and expensive lasers. Here we investigate an approach combining Cavity-Enhanced Absorption Spectroscopy (CEAS) with an inexpensive low vapour pressure mercury lamp for sensitive absorption measurements at 253.7 nm. We demonstrate that the CEAS absorption in our system is 50 times greater than the absorption found in a single-pass configuration; using this approach, we obtained limits of detection of 8.1 pptv (66 ng m(-3)) for gaseous elemental mercury and 8.4 ppbv for ozone. We evaluate the performance of the system and discuss potential improvements and applications of this approach.

Coastal iodine emissions. 1. Release of I2 by Laminaria digitata in chamber experiments.

Tidally exposed macroalgae emit large amounts of I(2) and iodocarbons that produce hotspots of iodine chemistry and intense particle nucleation events in the coastal marine boundary layer. Current emission rates are poorly characterized, however, with reported emission rates varying by 3 orders of magnitude. In this study, I(2) emissions from 25 Laminaria digitata samples were investigated in a simulation chamber using incoherent broadband cavity-enhanced absorption spectroscopy (IBBCEAS). The chamber design allowed gradual extraction of seawater to simulate tidal emersion of algae. Samples were exposed to air with or without O(3) and to varying irradiances. Emission of I(2) occurred in four distinct stages: (1) moderate emissions from partially submerged samples; (2) a strong release by fully emerged samples; (3) slowing or stopping of I(2) release; and (4) later pulses of I(2) evident in some samples. Emission rates were highly variable and ranged from 7 to 616 pmol min(-1) gFW(-1) in ozone-free air, with a median value of 55 pmol min(-1) gFW(-1) for 20 samples.

Coastal iodine emissions: part 2. Chamber experiments of particle formation from Laminaria digitata-derived and laboratory-generated I2.

Laboratory studies into particle formation from Laminaria digitata macroalgae were undertaken to elucidate aerosol formation for a range of I(2) (0.3-76 ppb(v)) and O(3) (<3-96 ppb(v)) mixing ratios and light levels (E(PAR) = 15, 100, and 235 µmol photons m(-2) s(-1)). No clear pattern was observed for I(2) or aerosol parameters as a function of light levels. Aerosol mass fluxes and particle number concentrations, were, however, correlated with I(2) mixing ratios for low O(3) mixing ratios of <3 ppb(v) (R(2) = 0.7 and 0.83, respectively for low light levels, and R(2) = 0.95 and 0.98, respectively for medium light levels). Additional experiments into particle production as a function of laboratory-generated I(2), over a mixing ratio range of 1-8 ppb(v), were conducted under moderate O(3) mixing ratios (∼24 ppb(v)) where a clear, 100-fold or greater, increase in the aerosol number concentrations and mass fluxes was observed compared to the low O(3) experiments. A linear relationship between particle concentration and I(2) was found, in reasonable agreement with previous studies. Scaling the laboratory relationship to aerosol concentrations typical of the coastal boundary layer suggests a I(2) mixing ratio range of 6-93 ppt(v) can account for the observed particle production events. Aerosol number concentration produced from I(2) is more than a factor of 10 higher than that produced from CH(2)I(2) for the same mixing ratios.

COVID-19: mask efficacy is dependent on both fabric and fit.

Aim: Face masks are an important addition to our arsenal in the fight against COVID-19. The aim of this study is to present a novel method of measuring mask performance which can simultaneously assess both fabric penetration and leakage due to poor fit. Materials & methods: A synthetic aerosol is introduced into the lung of a medical dummy. A conical laser sheet surrounds the face of the dummy where it illuminates the aerosol emitted during a simulated breath. The system is demonstrated with five mask types. Conclusions: The curved laser sheet highlights both penetration through the mask fabric and leakage around the edges of the mask. A large variation in both material penetration and leakage was observed.

Evaluation of novel cationic gene based liposomes with cyclodextrin prepared by thin film hydration and microfluidic systems.

In gene delivery, non-viral vectors have become the preferred carrier system for DNA delivery. They can overcome major viral issues such as immunogenicity and mutagenicity. Cationic lipid-mediated gene transfer is one of the most commonly used non-viral vectors, which have been shown to be a safe and effective carrier. However, their use in gene delivery often exhibits low transfection efficiency and stability. The aim of this study was to examine the effectiveness of novel non-viral gene delivery systems. This study has investigated the encapsulation and transfection efficiency of cationic liposomes prepared from DOTAP and carboxymethyl-ß-cyclodextrin (CD). The encapsulation efficiency of the CD-lipoplex complexes were also studied with and without the addition of Pluronic-F127, using both microfluidic and thin film hydration methods. In vitro transfection efficiencies of these complexes were determined in COS7 and SH-SY5Y cell lines. Formulation stability was evaluated using liposomes size, zeta potential and polydispersity index. In addition, the external morphology was studied using transmission electron microcopy (TEM). Results revealed that formulations produced by microfluidic method had smaller, more uniform and homogenious size and zeta-potential as well as higher encapsulation efficiency when compared with liposomes manufactured by thin film hydration method. Overall, the results of this study show that carboxymethyl-ß-cyclodextrin increased lipoplexes' encapsulation efficiency using both NanoAssemblr and rotary evaporator manufacturing processes. However, this increase was reduced slightly following the addition of Pluronic-F127. The addition of carboxymethyl-ß-cyclodextrin to cationic liposomes resulted in an increase in transfection efficiency in mammalian cell lines. However, this increase appeared to be cell line specific, COS7 showed higher transfection efficiency compared to SH-SY5Y.

Survival Outcome and EMT Suppression Mediated by a Lectin Domain Interaction of Endo180 and CD147.

UNLABELLED: Epithelial cell-cell contacts maintain normal glandular tissue homeostasis, and their breakage can trigger epithelial-to-mesenchymal transition (EMT), a fundamental step in the development of metastatic cancer. Despite the ability of C-type lectin domains (CTLD) to modulate cell-cell adhesion, it is not known if they modulate epithelial adhesion in EMT and tumor progression. Here, the multi-CTLD mannose receptor, Endo180 (MRC2/uPARAP), was shown using the Kaplan-Meier analysis to be predictive of survival outcome in men with early prostate cancer. A proteomic screen of novel interaction partners with the fourth CTLD (CTLD4) in Endo180 revealed that its complex with CD147 is indispensable for the stability of three-dimensional acini formed by nontransformed prostate epithelial cells (PEC). Mechanistic study using knockdown of Endo180 or CD147, and treatment with an Endo180 mAb targeting CTLD4 (clone 39.10), or a dominant-negative GST-CTLD4 chimeric protein, induced scattering of PECs associated with internalization of Endo180 into endosomes, loss of E-cadherin (CDH1/ECAD), and unzipping of cell-cell junctions. These findings are the first to demonstrate that a CTLD acts as a suppressor and regulatory switch for EMT; thus, positing that stabilization of Endo180-CD147 complex is a viable therapeutic strategy to improve rates of prostate cancer survival. IMPLICATIONS: This study identifies the interaction between CTLD4 in Endo180 and CD147 as an EMT suppressor and indicates that stabilization of this molecular complex improves prostate cancer survival rates.

Dynamic Imaging Analysis of SERS-Active Nanoparticle Clusters in Suspension.

A novel wide-field approach for the real-time Surface Enhanced Raman Scattering (SERS) imaging of multiple silver nanoparticle clusters suspended in solution is described. This method enables direct correlation of the SERS activity of a single nanoparticle aggregate and its size through measurement of the cluster diffusion coefficient and can also be performed in a high-throughput basis. As a first demonstration, we investigate the salt-induced aggregation of silver nanoparticles in the presence of a reporter tag molecule, which has a high affinity for the nanoparticle surface. In addition to tracking individual particles, direct comparison of Rayleigh and SERS videos of the same colloid solution enabled measurement of the fraction of individual clusters that are SERS active and the dependence of this value on the relative concentration of the tag molecule. Furthermore, given the ability to also rapidly profile any nonuniformity in particle size distributions, we expect this approach will not only provide a new tool for the fundamental understanding of SERS but also significantly contribute to the development of an array of emerging nanoparticle-enhanced biomolecule and imaging detection platforms.