Cytotoxicity Induced by Engineered Silver Nanocrystallites Is Dependent on Surface Coatings and Cell Types

Due to their unique antimicrobial properties silver nanocrystallites have garnered substantial attention and are used extensively for biomedical applications as an additive to wound dressings, surgical instruments and bone substitute materials. They are also released into unintended locations such as the environment or biosphere. Therefore it is imperative to understand the potential interactions, fate and transport of nanoparticles with environmental biotic systems. Numerous factors including the composition, size, shape, surface charge, and capping molecule of nanoparticles are known to influence cell cytotoxicity. Our results demonstrate that the physical/chemical properties of the silver nanoparticles including surface charge, differential binding and aggregation potential, which are influenced by the surface coatings, are a major determining factor in eliciting cytotoxicity and in dictating potential cellular interactions. In the present investigation, silver nanocrystallites with nearly uniform size and shape distribution but with different surface coatings, imparting overall high negativity to high positivity, were synthesized. These nanoparticles included poly­(diallyldimethylammonium) chloride-Ag, biogenic-Ag, colloidal-Ag (uncoated), and oleate-Ag with zeta potentials +45 ± 5, −12 ± 2, −42 ± 5, and −45 ± 5 mV, respectively; the particles were purified and thoroughly characterized so as to avoid false cytotoxicity interpretations. A systematic investigation on the cytotoxic effects, cellular response, and membrane damage caused by these four different silver nanoparticles was carried out using multiple toxicity measurements on mouse macrophage (RAW-264.7) and lung epithelial (C-10) cell lines. Our results clearly indicate that the cytotoxicity was dependent on various factors such as surface charge and coating materials used in the synthesis, particle aggregation, and the cell-type for the different silver nanoparticles that were investigated. Poly­(diallyldimethylammonium)-coated Ag nanoparticles were found to be the most toxic, followed by biogenic-Ag and oleate-Ag nanoparticles, whereas uncoated or colloidal silver nanoparticles were found to be the least toxic to both macrophage and lung epithelial cells. Also, based on our cytotoxicity interpretations, lung epithelial cells were found to be more resistant to the silver nanoparticles than the macrophage cells, regardless of the surface coating

Highly Efficient Isolation of <em>Populus</em> Mesophyll Protoplasts and Its Application in Transient Expression Assays

<div><h3>Background</h3><p><em>Populus</em> is a model woody plant and a promising feedstock for lignocellulosic biofuel production. However, its lengthy life cycle impedes rapid characterization of gene function.</p> <h3>Methodology/Principal Findings</h3><p>We optimized a <em>Populus</em> leaf mesophyll protoplast isolation protocol and established a <em>Populus</em> protoplast transient expression system. We demonstrated that <em>Populus</em> protoplasts are able to respond to hormonal stimuli and that a series of organelle markers are correctly localized in the <em>Populus</em> protoplasts. Furthermore, we showed that the <em>Populus</em> protoplast transient expression system is suitable for studying protein-protein interaction, gene activation, and cellular signaling events.</p> <h3>Conclusions/Significance</h3><p>This study established a method for efficient isolation of protoplasts from <em>Populus</em> leaf and demonstrated the efficacy of using <em>Populus</em> protoplast transient expression assays as an <em>in vivo</em> system to characterize genes and pathways.</p> </div

<i>Populus</i> leaf mesophyll protoplasts.

<p>(<b>A</b>) Optimal yield and quality of protoplasts can be isolated from one month-old <i>Populus</i> plants grown on MS medium in a magenta box. (<b>B</b>) High transfection efficiency is indicated with GFP signal.</p

Subcellular localization of various organelle markers in <i>Populus</i> protoplasts.

<p>(<b>A</b>) Plasma membrane; (<b>B</b>) Golgi apparatus; (<b>C</b>) Nucleus; (<b>G</b>) Peroxisome; (<b>H</b>) endoplasmic reticulum (ER); (<b>I</b>) An ubiquitously-localized protein (RACK1, Receptor for Activated C-protein Kinase 1). Shown in (<b>D</b>), (<b>E</b>), (<b>F</b>), (<b>J</b>), (<b>K</b>) and (<b>L</b>) are bright field images for fluorescent images of (<b>A</b>), (<b>B</b>), (<b>C</b>), (<b>G</b>), (<b>H</b>) and (<b>I</b>), respectively. The organelle markers were fused with mCherry fluorescent protein, and RACK1 was fused with YFP fluorescent protein. The mCherry signal was separated from chloroplast autofluorescence using spectral imaging and linear unmixing. The mCherry and YFP signals are false-colored green and the chloroplast autofluorescence is shown in red. Scale bar, 1 µm.</p

The response of <i>Populus</i> protoplasts to various plant hormone treatments.

<p>Shown are the change <i>POPTR_01s30560</i> transcript in response to different concentrations of NAA in protoplasts (A) and intact leaves (E), the change of <i>POPTR_14s08030</i> transcript in response to different concentrations of GA₃ in protoplasts (B) and intact leaves (F), the change of <i>POPTR_10s00320</i> transcript in response to different concentrations of BAP in protoplasts (C) and intact leaves (G), and the change of <i>POPTR_10s08300</i> transcript in response to different concentrations of ACC in protoplasts (D) and intact leaves (H). The protoplasts or intact leaves were incubated with various concentrations of plant hormones for 3h before being harvested for qRT-PCR analysis. The experiments were repeated three times with similar results. The averages of three technical replicates ± standard errors are shown. * indicates a significant difference (at P≤0.01, student’s t-test) between each treatment and the untreated control.</p

Energy sensing signaling in <i>Populus</i> protoplasts.

<p>(<b>A</b>) Semi-quantitative RT-PCR analysis of <i>PtrDIN6</i> transcripts in response to dark and hypoxia treatments. L-Light; D-Dark; H-Hypoxia. The <i>PtrUBQ10</i> gene was used as a control. (<b>B</b>) The change of <i>PtrDIN6</i> transcripts in response to overexpression of AthKIN10 protein. After transfection, protoplasts were incubated overnight to allow the expression of AthKIN10 before samples were harvested for qRT-PCR and western blot analysis. Western blot was used to detect the presence of the introduced HA-tagged AthKIN10 protein. The experiments were repeated three times with similar results. The averages of three technical replicates ± standard errors are presented in the graph. * indicates a significant difference (at P≤0.01, student’s t-test) between protoplasts expressing AthKIN10 and the control (ctrl). (<b>C</b>) The expression of three <i>Populus KIN10</i> homologues in transfected protoplasts examined by semi-quantitative RT-PCR. The expression of <i>PtrUBQ10</i> was used as an internal control. (<b>D</b>) The response of <i>PtrDIN6</i> transcript to the overexpression of three <i>Populus KIN10</i> homologues. The experiments were repeated three times with similar results. The averages of three technical replicates ± standard errors are shown. Protoplasts transfected with an empty vector was used as control (ctrl) for each comparison. (<b>E</b>) The activation of <i>PtrDIN6</i> by PtrKIN10 in a GUS reporter assay. For each co-transfection, a 35S::LUC (Luciferase) was included and the LUC activity was used to normalize GUS activity to account for the potential variations in the transfection efficiency. The averages of three technical replicates ± standard errors are shown. * indicates a significant difference (at P≤0.01, student’s t-test) between each treatment and the control (ctrl).</p

Unexpected Effects of Gene Deletion on Interactions of Mercury with the Methylation-Deficient Mutant Δ<i>hgcAB</i>

The <i>hgcA</i> and <i>hgcB</i> gene pair is essential for mercury (Hg) methylation by certain anaerobic bacteria, but little is known about how deletion of <i>hgcAB</i> affects the cell surface interactions and intracellular uptake of Hg. Here, we compare Δ<i>hgcAB</i> mutants with the wild-type (WT) strains of both <i>Geobacter sulfurreducens</i> PCA and <i>Desulfovibrio desulfuricans</i> ND132 and observe differences in Hg redox transformations, adsorption, and uptake in laboratory incubation studies. In both strains, deletion of <i>hgcAB</i> increased the rate of reduction of Hg­(II) but decreased the rate of oxidation of Hg(0) under anaerobic conditions. The measured cellular thiol content in Δ<i>hgcAB</i> mutants was lower than that in the WT, accounting for the decreased rates of adsorption and uptake of Hg. Despite the lack of methylation activity, uptake of Hg by the Δ<i>hgcAB</i> mutant continued, albeit at a rate slower than that of the WT. These findings demonstrate that deletion of the <i>hgcAB</i> gene pair not only eliminates Hg methylation but also alters cell physiology, resulting in changes to Hg redox reactions, sorption, and uptake by cells

Characterization of Indole-3-acetic Acid Biosynthesis and the Effects of This Phytohormone on the Proteome of the Plant-Associated Microbe <i>Pantoea</i> sp. YR343

Indole-3-acetic acid (IAA) plays a central role in plant growth and development, and many plant-associated microbes produce IAA using tryptophan as the precursor. Using genomic analyses, we predicted that <i>Pantoea</i> sp. YR343, a microbe isolated from <i>Populus deltoides</i>, synthesizes IAA using the indole-3-pyruvate (IPA) pathway. To better understand IAA biosynthesis and the effects of IAA exposure on cell physiology, we characterized proteomes of <i>Pantoea</i> sp. YR343 grown in the presence of tryptophan or IAA. Exposure to IAA resulted in upregulation of proteins predicted to function in carbohydrate and amino acid transport and exopolysaccharide (EPS) biosynthesis. Metabolite profiles of wild-type cells showed the production of IPA, IAA, and tryptophol, consistent with an active IPA pathway. Finally, we constructed an Δ<i>ipdC</i> mutant that showed the elimination of tryptophol, consistent with a loss of IpdC activity, but was still able to produce IAA (20% of wild-type levels). Although we failed to detect intermediates from other known IAA biosynthetic pathways, this result suggests the possibility of an alternate pathway or the production of IAA by a nonenzymatic route in <i>Pantoea</i> sp. YR343. The Δ<i>ipdC</i> mutant was able to efficiently colonize poplar, suggesting that an active IPA pathway is not required for plant association

Characterization of Indole-3-acetic Acid Biosynthesis and the Effects of This Phytohormone on the Proteome of the Plant-Associated Microbe <i>Pantoea</i> sp. YR343

Indole-3-acetic acid (IAA) plays a central role in plant growth and development, and many plant-associated microbes produce IAA using tryptophan as the precursor. Using genomic analyses, we predicted that <i>Pantoea</i> sp. YR343, a microbe isolated from <i>Populus deltoides</i>, synthesizes IAA using the indole-3-pyruvate (IPA) pathway. To better understand IAA biosynthesis and the effects of IAA exposure on cell physiology, we characterized proteomes of <i>Pantoea</i> sp. YR343 grown in the presence of tryptophan or IAA. Exposure to IAA resulted in upregulation of proteins predicted to function in carbohydrate and amino acid transport and exopolysaccharide (EPS) biosynthesis. Metabolite profiles of wild-type cells showed the production of IPA, IAA, and tryptophol, consistent with an active IPA pathway. Finally, we constructed an Δ<i>ipdC</i> mutant that showed the elimination of tryptophol, consistent with a loss of IpdC activity, but was still able to produce IAA (20% of wild-type levels). Although we failed to detect intermediates from other known IAA biosynthetic pathways, this result suggests the possibility of an alternate pathway or the production of IAA by a nonenzymatic route in <i>Pantoea</i> sp. YR343. The Δ<i>ipdC</i> mutant was able to efficiently colonize poplar, suggesting that an active IPA pathway is not required for plant association

Characterization of Indole-3-acetic Acid Biosynthesis and the Effects of This Phytohormone on the Proteome of the Plant-Associated Microbe <i>Pantoea</i> sp. YR343

Indole-3-acetic acid (IAA) plays a central role in plant growth and development, and many plant-associated microbes produce IAA using tryptophan as the precursor. Using genomic analyses, we predicted that <i>Pantoea</i> sp. YR343, a microbe isolated from <i>Populus deltoides</i>, synthesizes IAA using the indole-3-pyruvate (IPA) pathway. To better understand IAA biosynthesis and the effects of IAA exposure on cell physiology, we characterized proteomes of <i>Pantoea</i> sp. YR343 grown in the presence of tryptophan or IAA. Exposure to IAA resulted in upregulation of proteins predicted to function in carbohydrate and amino acid transport and exopolysaccharide (EPS) biosynthesis. Metabolite profiles of wild-type cells showed the production of IPA, IAA, and tryptophol, consistent with an active IPA pathway. Finally, we constructed an Δ<i>ipdC</i> mutant that showed the elimination of tryptophol, consistent with a loss of IpdC activity, but was still able to produce IAA (20% of wild-type levels). Although we failed to detect intermediates from other known IAA biosynthetic pathways, this result suggests the possibility of an alternate pathway or the production of IAA by a nonenzymatic route in <i>Pantoea</i> sp. YR343. The Δ<i>ipdC</i> mutant was able to efficiently colonize poplar, suggesting that an active IPA pathway is not required for plant association