Class A scavenger receptor expression and function in eight novel tadpole cell lines from the green frog (Lithobates clamitans) and the wood frog (Lithobates sylvatica).

A total of eight tadpole cell lines were established from green frogs (Lithobates clamitans) and wood frogs (Lithobates sylvatica). The five green frog cell lines were named GreenTad-HF1, GreenTad-HF2, GreenTad-HF3, GreenTad-HE4, and GreenTad-gill. The three wood frog cell lines were named WoodTad-HE1, WoodTad-Bone, and WoodTad-rpe. DNA barcoding confirmed the cell lines to be from the correct species and the growth characteristics (optimal temperature and FBS requirement) were elucidated. In order to begin studying the innate immune capacity for each cell line, class A scavenger receptor expression and function were next explored. All cell lines expressed genes for at least 3 of the 5 class A scavenger receptor (SR-A) family members, but the gene expression patterns varied between cell lines. MARCO was only expressed in GreenTad-HE4 and WoodTad-Bone, while only GreenTad-HF3 did not express SCARA5 and only WoodTad-rpe did not express SR-AI. Acetylated low density lipoprotein (AcLDL) is a well-defined ligand for SR-As and WoodTad-rpe was the only cell line to which it was unable to bind. In the other seven tadpole cell lines, the SR-A competitive ligands (dextran sulfate, fucoidan, polyinosinic acid) blocked AcLDL binding whereas the SR-A non-competitive ligand counterparts (chondroitin sulfate, fetuin, polycytidylic acid, respectively) did not. Overall, these new eight cell lines can become important tools in the study of innate immunity in general and SR-A functions in particular in green frogs and wood frogs.

Class A scavenger receptors mediate extracellular dsRNA sensing, leading to downstream antiviral gene expression in a novel American toad cell line, BufoTad.

Viral double-stranded (ds)RNA is a potent pathogen-associated molecular pattern (PAMP), capable of inducing a strong antiviral state within the cell, protecting the cell from virus infection. In mammals and fish, sensing extracellular dsRNA is mediated by cell-surface class A scavenger receptors (SR-As). Currently, very little is known about SR-As in amphibians, including: sequence, expression patterns and function. To this end, SR-A expression and function was studied in a novel American toad (Anaxyrus americanus) tadpole cell line called BufoTad. BufoTad was derived from a whole tadpole. The cell line exhibits a cobblestone morphology and expresses abundant levels of transcripts for cytokeratin 19, vimentin, claudin 3, chemokine receptor CXCR4, and SR-AI, one of the five members of the SR-A family, collectively suggesting that BufoTad could be endothelial-like. BufoTad cells bound acetylated LDL, whereas the Xenopus laevis kidney epithelial A6 cell line did not, suggesting functional SR-A activity in BufoTad cells. Additionally, three SR-A competitive ligands (DxSO4, fucoidan, poly inosine (pI)) completely blocked AcLDL binding in BufoTad cells, whereas their three corresponding non-competitive ligands (ChSO4, fetuin, poly cytosine (pC)) did not. A commercial dsRNA, poly IC, induced robust expression of an Mx-like gene transcript, a possible antiviral protein in BufoTad cells. Employing the same SR-A ligand blocking assay used for AcLDL blocked dsRNA-induced ISG expression. This study is the first demonstration that amphibian SR-As have functional ligand binding activities in a live biological cellular model and that sensing extracellular dsRNA in amphibian cells leads to antiviral gene expression that is mediated by class A scavenger receptors.

Assessing off-target cytotoxicity of the field lampricide 3-trifluoromethyl-4-nitrophenol using novel lake sturgeon cell lines.

Lampricides are currently being applied to streams and rivers to control the population of sea lamprey, an invasive species, in the Great Lakes. The most commonly used lampricide agent used in the field is 3-trifluoromethyl-4-nitrophenol (TFM), which targets larval sea lamprey in lamprey-infested rivers and streams. The specificity of TFM is due to the relative inability of sea lamprey to detoxify the agent relative to non-target fishes. There is increasing concern, however, about non-target effects on fishes, particularly threatened populations of juvenile lake sturgeon (LS; Acipenser fulvescens). There is therefore a need to develop models to better define lake sturgeon's response to TFM. Here we report the establishment of five LS cell lines derived from the liver, gill, skin and intestinal tract of juvenile LS and some of their cellular characteristics. All LS cell lines grew well at 25 °C in Leibovitz's (L)- 15 medium supplemented with 10% FBS. All cell lines demonstrated high senescence-associated ß-galactosidase activity and varying levels of Periodic acid Schiff-positive polysaccharides, indicating substantial production of glycoproteins and mucosubstances by the cells. Comparative toxicity of TFM in the five LS cell lines was assessed by two fluorescent cell viability dyes, Alamar Blue and CFDA-AM, in conditions with and without serum and at 24 or 72 h exposure. Deduced EC50 values were compared between the cell lines and to the reported in vivo LC50s. Tissues sensitive to the effects of TFM in vivo correlated with cell lines from the same tissues being most sensitive to TFM in vitro. EC50 values for the LSliver-e cells was significantly lower than the EC50 for the rainbow trout (RBT) liver cells RTL-W1, reaffirming the in vivo observation that LS was generally more TFM-sensitive than rainbow trout. Our data suggests that whole-fish sensitivity of LS to TFM is likely attributable to sensitivity at the cellular level. Thus, LS cell lines, as well as those of RBT, can be used to screen and evaluate the toxicity of the next generation of lampricides on non-target fish such as lake sturgeon.

Bis-phenylethynyl polyhedral oligomeric silsesquioxanes: new high-temperature, processable thermosetting materials.

Bis-phenylethynyl polyhedral oligomeric silsesquioxane (bis-PE-POSS) compounds were synthesized and thermally cured yielding crosslinked materials. After curing at 370 °C, thermal decomposition occurs near 600 °C under nitrogen. These materials were synthesized by condensation of a new phenylethynyl-functional dichlorosilane onto tetrasilanol phenyl POSS, yielding two geometric isomers.

Three-Dimensional Morphology Control Yielding Enhanced Hole Mobility in Air-Processed Organic Photovoltaics: Demonstration with Grazing-Incidence Wide-Angle X-ray Scattering.

Polymer organic photovoltaic (OPV) device performance is defined by the three-dimensional morphology of the phase-separated domains in the active layer. Here, we determine the evolution of morphology through different stages of tailored solvent vapor and thermal annealing techniques in air-processed poly(3-hexylthiophene-2,5-diyl)/phenyl-C61-butyric acid methyl ester-based OPV blends. A comparative evaluation of the effect of solvent type used for vapor annealing was performed using grazing-incidence wide-angle X-ray scattering, atomic force microscopy, and UV-vis spectroscopy to probe the active-layer morphology. A nonhalogenated orthogonal solvent was found to impart controlled morphological features within the exciton diffusion length scales, enhanced absorbance, greater crystallinity, increased paracrystalline disorder, and improved charge-carrier mobility. Low-boiling, fast-diffusing isopropanol allowed the greatest control over the nanoscale structure of the solvents evaluated and yielded a cocontinuous morphology with narrowed domains and enhanced paths for the charge carrier to reach the anode.