Derivation of scenario-specific water quality guidelines for acid mine drainage in South Africa,using a risk-based approach

Acid mine drainage (AMD) continues to threaten water quality in many mining regions globally. Data paucity renders it challenging to inform appropriate water quality management strategies for a succinct scientific understanding of the effects of AMD on freshwater ecosystems. The current study investigated the effects of AMD collected from a defunct coalmine in Mpumalanga, South Africa, on freshwater ecosystems using a risk-based approach on five indigenous species, Adenophlebia auriculata, Burnupia stenochorias, Caridina nilotica, Pseudokirchneriella subcapitata and Oreochromis mossambicus in 2016. Species responded differently to AMD after 96 hours and 240 hours of exposure in static experimental test designs. Burnupia stenochorias was more sensitive to AMD after 96 and 240 hours of exposure, whereas O. mossambicus was tolerant during short-term exposure, but became more sensitive after 240 hours of exposure than the other species tested. The availability of metals in AMD was directly associated with dilution rate. Scenario-specific water quality guidelines for AMD have been derived as 0.122% for short-term and 0.014% for long-term exposure. These may form important indicative dilutions for other AMDs that do not match the scenarios of this study. The toxicity of AMD to a wide range of aquatic species, including field validations, requires further investigation.     

Activation of protein kinase C by phorbol esters modulates alpha2beta1 integrin on MCF-7 breast cancer cells

Cellular adhesions to other cells and to the extracellular matrix play crucial roles in the malignant progression of cancer. In this study, we investigated the role of protein kinase C (PKC) in the regulation of cell-substratum adhesion by the breast adenocarcinoma cell line MCF-7. A PKC activator, 12-O-tetradecanoylphorbol-l, 3-acetate (TPA), stimulated cell adhesion to laminin and collagen I in a dose-dependent manner over a 1- to 4-h interval. This enhanced adhesion was mediated by alpha2beta1 integrin, since both anti-alpha2 and anti-beta1 blocking antibodies each completely abrogated the TPA-induced adhesion. FACS analysis determined that TPA treatment does not change the cell surface expression of alpha2beta1 integrin over a 4-h time interval. However, alpha2beta1 levels were increased after 24 h of TPA treatment. Thus, the enhanced avidity of alpha2beta1-dependent cellular adhesion preceded the induction of alpha2beta1 cell surface expression. Northern blot analysis revealed that mRNA levels of both alpha2 and beta1 subunits were increased after exposure to TPA for 4 h, indicating that the induction of alpha2beta1 mRNA preceded that of its cell surface expression. This further suggested that the TPA-induced avidity of alpha2beta1 was independent of increased expression of alpha2beta1. Pretreatment of cells with the PKC inhibitor calphostin C partially antagonized the TPA-induced increase in expression of alpha2beta1 integrin expression and of alpha2beta1-mediated cellular adhesion. To identify a possible mechanism by which TPA could be acting to promote the rapid induction of alpha2beta1 adhesion, we treated the cells with the Rho-GTPase inhibitor Clostridium botulinumexotoxin C3. C3 inhibited TPA-induced adhesion to laminin and collagen I in a dose-dependant manner, suggesting a likely role for Rho in TPA-induced adhesion. Together, these results suggest that PKC can modulate the alpha2beta1-dependent adhesion of MCF-7 cells by two distinct mechanisms: altering the gene expression of integrins alpha2 and beta1 and altering the avidity of the alpha2beta1 integrin by a Rho-dependant mechanism.     

Evaluation of the novel folate receptor ligand [18F]fluoro-PEG-folate for macrophage targeting in a rat model of arthritis

Introduction

Detection of (subclinical) synovitis is relevant for both early diagnosis and monitoring of therapy of rheumatoid arthritis (RA). Previously, the potential of imaging (sub)clinical arthritis was demonstrated by targeting the translocator protein in activated macrophages using (R)-[¹¹C]PK11195 and positron emission tomography (PET). Images, however, also showed significant peri-articular background activity. The folate receptor (FR)-β is a potential alternative target for imaging activated macrophages. Therefore, the PET tracer [¹⁸F]fluoro-PEG-folate was synthesized and evaluated in both in vitro and ex vivo studies using a methylated BSA induced arthritis model.

Methods

[¹⁸F]fluoro-PEG-folate was synthesized in a two-step procedure. Relative binding affinities of non-radioactive fluoro-PEG-folate, folic acid and naturally circulating 5-methyltetrahydrofolate (5-Me-THF) to FR were determined using KB cells with high expression of FR. Both in vivo [¹⁸F]fluoro-PEG-folate PET and ex vivo tissue distribution studies were performed in arthritic and normal rats and results were compared with those of the established macrophage tracer (R)-[¹¹C]PK11195.

Results

[¹⁸F]fluoro-PEG-folate was synthesized with a purity >97%, a yield of 300 to 1,700 MBq and a specific activity between 40 and 70 GBq/µmol. Relative in vitro binding affinity for FR of F-PEG-folate was 1.8-fold lower than that of folic acid, but 3-fold higher than that of 5-Me-THF. In the rat model, [¹⁸F]fluoro-PEG-folate uptake in arthritic knees was increased compared with both contralateral knees and knees of normal rats. Uptake in arthritic knees could be blocked by an excess of glucosamine-folate, consistent with [¹⁸F]fluoro-PEG-folate being specifically bound to FR. Arthritic knee-to-bone and arthritic knee-to-blood ratios of [¹⁸F]fluoro-PEG-folate were increased compared with those of (R)-[¹¹C]PK11195. Reduction of 5-Me-THF levels in rat plasma to those mimicking human levels increased absolute [¹⁸F]fluoro-PEG-folate uptake in arthritic joints, but without improving target-to-background ratios.

Conclusions

The novel PET tracer [¹⁸F]fluoro-PEG-folate, designed to target FR on activated macrophages provided improved contrast in a rat model of arthritis compared with the accepted macrophage tracer (R)-[¹¹C]PK11195. These results warrant further exploration of [¹⁸F]fluoro-PEG-folate as a putative PET tracer for imaging (sub)clinical arthritis in RA patients.