Inhibition of endogenous MTF-1 signaling in zebrafish embryos identifies novel roles for MTF-1 in development.

The metal responsive element-binding transcription factor-1 (MTF-1) responds to changes in cellular zinc levels caused by zinc exposure or disruption of endogenous zinc homeostasis by heavy metals or oxygen-related stress. Here we report the functional characterization of a complete zebrafish MTF-1 in comparison with the previously identified isoform lacking the highly conserved cysteine-rich motif (Cys-X-Cys-Cys-X-Cys) found in all other vertebrate MTF-1 orthologs. In an effort to develop novel molecular tools, a constitutively nuclear dominant-negative MTF-1 (dnMTF-1) was generated as tool for inhibiting endogenous MTF-1 signaling. The in vivo efficacy of the dnMTF-1 was determined by microinjecting in vitro transcribed dnMTF-1 mRNA into zebrafish embryos (1-2 cell stage) followed by transcriptomic profiling using an Agilent 4x44K array on 28- and 36-hpf embryos. A total of 594 and 560 probes were identified as differentially expressed at 28hpf and 36hpf, respectively, with interesting overlaps between timepoints. The main categories of genes affected by the inhibition of MTF-1 signaling were: nuclear receptors and genes involved in stress signaling, neurogenesis, muscle development and contraction, eye development, and metal homeostasis, including novel observations in iron and heme homeostasis. Finally, we investigate both the transcriptional activator and transcriptional repressor role of MTF-1 in potential novel target genes identified by transcriptomic profiling during early zebrafish development.

Rate of Endothelial Cell Loss and Graft Survival in Descemet Membrane Endothelial Keratoplasty in Eyes Requiring a Rebubble.

PURPOSE: We aimed to compare the rate of 6-month endothelial cell loss (ECL) and 6-month graft survival in eyes that did not require a postoperative rebubble with eyes that did require a postoperative rebubble after Descemet membrane endothelial keratoplasty (DMEK) surgery. METHODS: A consecutive series of DMEK surgeries performed from September 2013 to March 2020 was retrospectively analyzed. Eyes that did not require a rebubble for graft detachment were compared with eyes with 1 rebubble and eyes with 2 or more rebubbles for 6-month ECL and graft survival. A subanalysis of the rebubble rate for different indications for transplantation was also performed. RESULTS: One thousand two hundred ninety-eight eyes were included in this study. The 6-month ECL for eyes with no rebubbles, 1 rebubble, and ≥2 rebubbles was 29.3% ± 16.2% (n = 793), 36.4% ± 18.6% (n = 97, P = 0.001), and 50.1% ± 19.6% (n = 28, P < 0.001), respectively. The 6-month graft survival rate for eyes with no rebubbles, 1 rebubble, and ≥2 rebubbles was 99.5%, 97.8% ( P = 0.035), and 81.8% ( P < 0.001), respectively. When compared to the rebubble rate for DMEK for Fuchs dystrophy (156/1165 eyes = 13.4%), the rebubble rates were statistically higher for DMEK for failed penetrating keratoplasty (28.5%, P = 0.021) and pseudophakic bullous keratopathy (28.0%, P = 0.036). CONCLUSIONS: Eyes undergoing any rebubble procedure in the postoperative period after DMEK have an increased risk of endothelial cell loss and graft failure at 6 months postoperative. DMEK in eyes for failed penetrating keratoplasty and failed DMEK had the highest rebubble rate, with the former reaching statistical significance.

Transcriptomic evaluation of the American oyster, Crassostrea virginica, deployed during the Deepwater Horizon oil spill: Evidence of an active hydrocarbon response pathway.

Estuarine organisms were impacted by the Deepwater Horizon oil spill which released ∼5 million barrels of crude oil into the Gulf of Mexico in the spring and summer of 2010. Crassostrea virginica, the American oyster, is a keystone species in these coastal estuaries and is routinely used for environmental monitoring purposes. However, very little is known about their cellular and molecular responses to hydrocarbon exposure. In response to the spill, a monitoring program was initiated by deploying hatchery-reared oysters at three sites along the Alabama and Mississippi coast (Grand Bay, MS, Fort Morgan, AL, and Orange Beach, AL). Oysters were deployed for 2-month periods at five different time points from May 2010 to May 2011. Gill and digestive gland tissues were harvested for gene expression analysis and determination of aliphatic and polycyclic aromatic hydrocarbon (PAH) concentrations. To facilitate identification of stress response genes that may be involved in the hydrocarbon response, a nearly complete transcriptome was assembled using Roche 454 and Illumina high-throughput sequencing from RNA samples obtained from the gill and digestive gland tissues of deployed oysters. This effort resulted in the assembly and annotation of 27,227 transcripts comprised of a large assortment of stress response genes, including members of the aryl hydrocarbon receptor (AHR) pathway, Phase I and II biotransformation enzymes, antioxidant enzymes and xenobiotic transporters. From this assembly several potential biomarkers of hydrocarbon exposure were chosen for expression profiling, including the AHR, two cytochrome P450 1A genes (CYP1A-like 1 and CYP1A-like 2), Cu/Zn superoxide dismutase (CuZnSOD), glutathione S-transferase theta (GST theta) and multidrug resistance protein 3 (MRP3). Higher expression levels of GST theta and MRP3 were observed in gill tissues from all three sites during the summer to early fall 2010 deployments. Linear regression analysis indicated a statistically significant relationship between total PAH levels in digestive gland tissue samples with CYP1A-like 2, CuZnSOD, GST theta and MRP3 induction. These observations provide evidence of a potentially conserved AHR pathway in invertebrates and yield new insight into the development of novel biomarkers for use in environmental monitoring activities.

Feasibility and efficacy of diabetic retinopathy screening among youth with diabetes in a pediatric endocrinology clinic: a cross-sectional study.

BACKGROUND: We examined the feasibility and efficacy of using a non-mydriatic camera to screen for diabetic retinopathy (DR) among youth with type 1 or type 2 diabetes seen in a pediatric endocrinology clinic serving Alabama, the state that has the highest diabetes rate in the United States. METHODS: 236 youths with type 1 or type 2 diabetes were screened for DR using a non-mydriatic camera. Visual acuity was also assessed. A questionnaire asked parents about diabetes and eye care history. RESULTS: Mean duration since diabetes diagnosis was 5.5 years. 66 % reported receiving an eye examination within the previous year. 97.5 % had images that were gradable. DR was detected in 3.8 % of participants. 9.1 % were visually impaired. CONCLUSIONS: Use of a non-mydriatic fundus camera is feasible and efficacious for DR screening in youth with diabetes. DR screening at routine endocrinology visits may be beneficial in managing youth with diabetes and preventing irreversible vision loss, particularly for those in regions where diabetes rates are high.