Differences in Oxygen-Induced Retinopathy Susceptibility Between Two Sprague Dawley Rat Vendors: A Comparison of Retinal Transcriptomes.

PURPOSE: To determine the retinal transcriptomic differences underlying the oxygen-induced retinopathy phenotypes between Sprague Dawley rat pups from two commonly used commercial vendors. This will allow us to discover genes and pathways that may be related to differences in disease severity in similarly aged premature babies and suggest possible new treatment approaches. METHODS: We analyzed retinal vascular morphometry and transcriptomes from Sprague Dawley rat pups from Charles River Laboratories and Envigo (previously Harlan). Room air control and oxygen-induced retinopathy groups were compared. Oxygen-induced retinopathy was induced with the rat 50/10 model. RESULTS: Pups from Charles River Laboratories developed a more severe oxygen-induced retinopathy phenotype, with 3.6-fold larger percent avascular area at P15 and twofold larger % neovascular area at P20 than pups from Envigo. Changes in retinal transcriptomes of rat pups from both vendors were substantial at baseline and in response to oxygen-induced retinopathy. Baseline differences centered on activated pathways of neuronal development in Charles River Laboratories pups. In response to oxygen-induced retinopathy, during the neovascular phase, retinas from Charles River Laboratories pups exhibited activation of pathways regulating necrosis, neuroinflammation, and interferon signaling, supporting the observed increase of neovascularization. Conversely, retinas from Envigo pups showed decreased necrosis and increased focal adhesion kinase signaling, supporting more normal vascular development. Comparing oxygen-induced retinopathy transcriptomes at P15 to those at P20, canonical pathways such as phosphate and tensin homolog, interferon, and coordinated lysosomal expression and regulation element signaling were identified, highlighting potential novel mechanistic targets for future research. CONCLUSION: Transcriptomic profiles differ substantially between rat pup retinas from Charles River Laboratories and Envigo at baseline and in response to oxygen-induced retinopathy, providing insight into vascular morphologic differences. Comparing transcriptomes identified new pathways for further research in oxygen-induced retinopathy pathogenesis and increased scientific rigor of this model.

Identification of reference genes for the normalization of retinal mRNA expression by RT-qPCR in oxygen induced retinopathy, anemia, and erythropoietin administration.

BACKGROUND: Anemia and retinopathy of prematurity (ROP) are common comorbidities experienced by preterm infants, yet the role of anemia on the pathogenesis of ROP remains unclear. Reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR) is a sensitive technique for estimating the gene expression changes at the transcript level but requires identification of stably expressed reference genes for accurate data interpretation. This is particularly important for oxygen induced retinopathy studies given that some commonly used reference genes are sensitive to oxygen. This study aimed to identify stably expressed reference genes among eight commonly used reference genes in the neonatal rat pups' retina upon exposure to cyclic hyperoxia-hypoxia, anemia, and erythropoietin administration at two age groups (P14.5 and P20) using Bestkeeper, geNorm, and Normfinder, three publicly available, free algorithms, and comparing their results to the in-silico prediction program, RefFinder. RESULTS: The most stable reference gene across both developmental stages was Rpp30, as predicted by Genorm, Bestkeeper, and Normfinder. RefFinder predicted Tbp to be the most stable across both developmental stages. At P14.5, stability varied by prediction program; at P20, RPP30 and MAPK1 were the most stable reference genes. Gapdh, 18S, Rplp0, and HPRT were predicted as the least stable reference genes by at least one of the prediction algorithms. CONCLUSION: Expression of Rpp30 is the least affected by experimental conditions of oxygen induced retinopathy, phlebotomy induced anemia and erythropoietin administration at both timepoints of P14.5 and P20.

In Vivo Assay Reveals Microbial OleA Thiolases Initiating Hydrocarbon and ß-Lactone Biosynthesis.

OleA, a member of the thiolase superfamily, is known to catalyze the Claisen condensation of long-chain acyl coenzyme A (acyl-CoA) substrates, initiating metabolic pathways in bacteria for the production of membrane lipids and ß-lactone natural products. OleA homologs are found in diverse bacterial phyla, but to date, only one homodimeric OleA has been successfully purified to homogeneity and characterized in vitro A major impediment for the identification of new OleA enzymes has been protein instability and time-consuming in vitro assays. Here, we developed a bioinformatic pipeline to identify OleA homologs and a new rapid assay to screen OleA enzyme activity in vivo and map their taxonomic diversity. The screen is based on the discovery that OleA displayed surprisingly high rates of p-nitrophenyl ester hydrolysis, an activity not shared by other thiolases, including FabH. The high rates allowed activity to be determined in vitro and with heterologously expressed OleA in vivo via the release of the yellow p-nitrophenol product. Seventy-four putative oleA genes identified in the genomes of diverse bacteria were heterologously expressed in Escherichia coli, and 25 showed activity with p-nitrophenyl esters. The OleA proteins tested were encoded in variable genomic contexts from seven different phyla and are predicted to function in distinct membrane lipid and ß-lactone natural product metabolic pathways. This study highlights the diversity of unstudied OleA proteins and presents a rapid method for their identification and characterization.IMPORTANCE Microbially produced ß-lactones are found in antibiotic, antitumor, and antiobesity drugs. Long-chain olefinic membrane hydrocarbons have potential utility as fuels and specialty chemicals. The metabolic pathway to both end products share bacterial enzymes denoted as OleA, OleC, and OleD that transform acyl-CoA cellular intermediates into ß-lactones. Bacteria producing membrane hydrocarbons via the Ole pathway additionally express a ß-lactone decarboxylase, OleB. Both ß-lactone and olefin biosynthesis pathways are initiated by OleA enzymes that define the overall structure of the final product. There is currently very limited information on OleA enzymes apart from the single representative from Xanthomonas campestris In this study, bioinformatic analysis identified hundreds of new, putative OleA proteins, 74 proteins were screened via a rapid whole-cell method, leading to the identification of 25 stably expressed OleA proteins representing seven bacteria phyla.