Heterologous production and characterisation of cation diffusion facilitator proteins from the marine bacterium Maricaulis maris MCS10

Cation diffusion facilitators are a diverse family of transporters responsible for the efflux of zinc and other divalent metals from cells. Several putative members of this family were selected as potential targets for research with the intent of removing difficult targets as problems arose. Of particular interest was the smallest target, MmCDF3, from Maricaulis maris, due to having no apparent C-terminal cytoplasmic domain, yet seemingly maintaining the tetrahedral zinc-coordinating active site and six transmembrane helices found in all known CDFs. This study focused on the expression, purification, characterisation and crystallisation of members of the CDF family, specifically targets from M. maris. High level expression (6-7 mg/L) and purity has been obtained for the MmCDF1 and MmCDF3 proteins, although both could only be obtained in a heavily aggregated state when purified in DDM. Acquiring CDF proteins in a non-aggregated stable form was necessary for repeatable crystallisation experiments and was accomplished by means of a detergent exchange in the presence of high salt concentrations. MmCDF3 was determined to be stable after an exchange with the detergent FC12, but only into a mixed micelle consisting of both FC12 and DDM. Investigations into the exact nature of the micelle containing stable MmCDF3 were performed by constructing mixed micelles of specific detergent ratios. Determination of function was performed by isothermal titration calorimetry, which showed that MmCDF3 was capable of binding zinc and cadmium ions, although in a much reduced fashion to the larger CDF proteins. This study did highlight a potential mechanism through which MmCDF3 and other smaller CDFs might bind their substrate ions in the cytoplasm. Following successful recovery of the stable homogenous protein, crystallisation was attempted with little success. However, now that a method has been established for obtaining a pure, homogenous and stable CDF protein, further crystallisation studies can be attempted

Synthesis of Indole-2-carboxylate Derivatives via Palladium-Catalyzed Aerobic Amination of Aryl C–H Bonds

A direct oxidative C–H amination affording 1-acetyl indolecarboxylates starting from 2-acetamido-3-arylacrylates has been achieved. Indole-2-carboxylates can be targeted with a straightforward deacetylation of the initial reaction products. The C–H amination reaction is carried out using a catalytic Pd­(II) source with oxygen as the terminal oxidant. The scope and application of this chemistry is demonstrated with good to high yields for numerous electron-rich and electron-poor substrates. Further reaction of selected products via Suzuki arylation and deacetylation provides access to highly functionalized indole structures

Effect of osmotic shock on the keratin cytoskeleton in the NEB-1 cell line (A) and the EBS derived cell line KEB-7 (B).

<p>Both cell lines were subjected to hypo-osmotic shock, then fixed and permeabilized and stained for K14 intermediate filaments. Clear peripheral aggregates were seen in KEB-7 cells 30 min after osmotic shock. No filament fragmentation or aggregates were observed for NEB-1 cells. Scale bar = 22 µm.</p

Fluorescent images of (A) NEB-1 K14wt-GFP and (B) NEB-1 K14R125P-GFP keratinocytes undergoing incremental uniaxial strain.

<p>The average cell strain is depicted in the top left corner of each image. The cytokeratin networks of the NEB-1 K14R125P-GFP cells withstood extreme cellular strains of 133% and there was no evidence of intermediate filament bundle rupture or the development of keratin aggregates (n = 10). Scale bar = 20 µm.</p

The green vital inclusion dye FDA and blue vital exclusion dye DAPI were used to test for necrosis after extreme strain in NEB-1 K14wt-GFP and NEB-1 K14R125P-GFP cells.

<p>The keratinocytes were stretched to a specific strain and then returned to the relaxed state for viability staining. Necrosis increased significantly with increasing cell strain (* = p<0.05; ** = p<0.001). No significant difference in viability was found between the NEB-1 K14wt-GFP and NEB-1 K14R125P-GFP keratinocytes after undergoing extreme uniaxial strain of 133% (p>0.1). Error bars are standard error. Scale bar = 20 µm.</p

Effect of osmotic shock on the keratin cytoskeleton in the two cell lines studied.

<p>A. NEB-1 K14wt-GFP (upper panel) and NEB-1 K14R125P-GFP (lower panel). Both cell lines were subjected to hypo-osmotic shock. Clear peripheral aggregates were seen in R125P cells 20 and 30 min after osmotic shock. No filament fragmentation or aggregates were observed for NEB-1 K14wt-GFP cells. Scale bar = 15 µm. B. Insets from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031320#pone-0031320-g003" target="_blank">Figure 3A</a>, 20 min after osmotic shock, showing K14 aggregates in the NEB-1 K14R125P-GFP cells, and reconstituted K14 filaments in the NEB-1 K14wt-GFP cells.</p

Fluorescent images of the K5/K14, F-actin and microtubule networks in NEB-1 K14wt-GFP and NEB-1 K14R125P-GFP keratinocytes fixed at 0% or 133% strain.

<p>Cells were treated with 1 µg/mL nocodazole (Nc) to disturb the F-actin network, or were untreated (control). The F-actin network was visualized with rhodamine-phalloidin (100 nM), and α-tubulin with immunofluorescence. K14-GFP proteins were expressed and visualized by fluorescence microscopy. Scale bar = 20 µm.</p

The viability of NEB-1 K14wt-GFP and NEB-1 K14R125P-GFP keratinocytes before (0%) and after (133%) extreme uniaxial strain.

<p>The treatments consisted of 1 µg/mL nocodazole (Nc) to disrupt the microtubule network and 0.5 µM latrunculin A (Lat A) to disrupt the F-actin network (n = 6). There was no significant difference in viability between the control cells and cells treated with nocodazole at extreme strain (p>0.1). Cells treated with Lat A exhibited significantly lower necrosis in both cell lines compared to the control (p<0.01).</p

Fluorescent images of (A) WT NEB-1 cells and (B) EBS mutant (R125P) K14 KEB-7 keratinocytes undergoing incremental uniaxial strain.

<p>The average cell strain is depicted in the top (A) or the bottom (B) of each panel. Both cell lines were subjected to stretch, then fixed and permeabilized and stained with the anti-K14 (LL001, Santa Cruz) monoclonal antibody. The cytokeratin networks of the EBS cells withstood extreme cellular strains of 133% and there was no evidence of intermediate filament bundle rupture or the development of keratin aggregates. Arrowheads indicate wavy K14 intermediate filaments. Scale bar = 20 µm.</p

Supplemental Material - A Qualitative Study of Home Health Aides’ Perspectives towards COVID-19 Vaccination

Supplemental Material for A Qualitative Study of Home Health Aides’ Perspectives towards COVID-19 Vaccination by David Russell, Nicole Onorato, Alexis Stern, Sasha Vergez, Mia Oberlink, Matthew Luebke, Penny H. Feldman, Margaret V. McDonald, and Madeline R. Sterling in Journal of Applied Gerontology</p