Oxidative stress induces mitochondrial dysfunction and a protective unfolded protein response in RPE cells.

How cells degenerate from oxidative stress in aging-related disease is incompletely understood. This study's intent was to identify key cytoprotective pathways activated by oxidative stress and determine the extent of their protection. Using an unbiased strategy with microarray analysis, we found that retinal pigmented epithelial (RPE) cells treated with cigarette smoke extract (CSE) had overrepresented genes involved in the antioxidant and unfolded protein response (UPR). Differentially expressed antioxidant genes were predominantly located in the cytoplasm, with no induction of genes that neutralize superoxide and H2O2 in the mitochondria, resulting in accumulation of superoxide and decreased ATP production. Simultaneously, CSE induced the UPR sensors IRE1α, p-PERK, and ATP6, including CHOP, which was cytoprotective because CHOP knockdown decreased cell viability. In mice given intravitreal CSE, the RPE had increased IRE1α and decreased ATP and developed epithelial-mesenchymal transition, as suggested by decreased LRAT abundance, altered ZO-1 immunolabeling, and dysmorphic cell shape. Mildly degenerated RPE from early age-related macular degeneration (AMD) samples had prominent IRE1α, but minimal mitochondrial TOM20 immunolabeling. Although oxidative stress is thought to induce an antioxidant response with cooperation between the mitochondria and the ER, herein we show that mitochondria become impaired sufficiently to induce epithelial-mesenchymal transition despite a protective UPR. With similar responses in early AMD samples, these results suggest that mitochondria are vulnerable to oxidative stress despite a protective UPR during the early phases of aging-related disease.

Nrf2 signaling modulates cigarette smoke-induced complement activation in retinal pigmented epithelial cells.

Whereas cigarette smoking (CS) and dysregulated complement are thought to play central roles in age-related macular degeneration (AMD), their exact roles are unknown. The aim of this study was to determine if CS activates complement and if the antioxidant transcription factor Nrf2 modulates this response. In AMD specimens, Nrf2 immunolabeling was strong in the cytoplasm, with scattered nuclear labeling of macular retinal pigmented epithelial (RPE) cells that appeared normal, but was decreased and without nuclear labeling in dysmorphic cells overlying drusen, a hallmark AMD lesion. Cigarette smoke extract (CSE) induced Nrf2 nuclear translocation in RPE cells with increased antioxidant and complement gene expression. Whereas CFH protein was not altered by CSE, the cell membrane regulator proteins CD46, CD55, and CD59 were decreased, and C3a and C3b, but not iC3b, were increased compared to controls. C5b-9 was increased by CSE, but at sublytic levels, only after addition of normal human serum. Nrf2 knockdown enhanced the increase in C3a and C3b from CSE, but not iC3b, C5a, or C5b-9. CSE also increased IL-1b expression and secretion after C3a generation and was reduced by a C3aR antagonist. In contrast, the Nrf2 activator CDDO-Im restored complement gene expression in RPE cells exposed to CSE. We provide evidence of altered Nrf2 in human AMD and that CSE induces a proinflammatory environment specifically by generating C3a and C3b, and Nrf2 deficiency magnifies this specific complement response.

Mental fatigue influence on effort-related cardiovascular response: extension across the regulatory (inhibitory)/non-regulatory performance dimension.

Participants first performed a scanning task that was weak (fatigue low) or strong (fatigue high) in self-regulatory (inhibitory) demand. They then were presented a cognitive challenge that had a strong regulatory component (the Stroop color-word conflict task) or a weak regulatory component (single-digit mental multiplication) with instructions that they would avoid noise if they attained a moderate performance standard. Analysis of cardiovascular data collected during the two work periods revealed fatigue main effects for systolic blood pressure, diastolic blood pressure, and mean arterial blood pressure. The effects reflected stronger blood pressure responses for High Fatigue participants across work periods and regardless of the character of the challenge presented in work period 2. Results conceptually replicate previous mental fatigue findings, which have shown extension of fatigue influence across cognitive performance domains. At least as importantly, they also extend those findings by showing extension across a fresh and theoretically significant cognitive performance dimension.

Preparation of pyrrolidine-based PDE4 inhibitors via enantioselective conjugate addition of alpha-substituted malonates to aromatic nitroalkenes.

[reaction: see text] The enantioselective conjugate addition of alpha-substituted malonates to aromatic nitroalkenes generates a stereocenter at the carbon bearing the aromatic group and an adjacent prochiral center from the alpha-substituted malonate. Nitro reduction followed by diastereoselective cyclization provides pyrrolidinones with two contiguous stereocenters, one of which is quaternary. This sequence was used for the preparation of the PDE4 inhibitor IC86518. Additional examples of the enantioselective Michael addition illustrate the scope of the reaction.

Magnetoelectroporation: improved labeling of neural stem cells and leukocytes for cellular magnetic resonance imaging using a single FDA-approved agent.

Cellular magnetic resonance imaging (MRI) relies on the use of intracellular contrast agents, primarily iron oxide compounds. Several techniques have been used to efficiently shuttle iron oxides into nonphagocytic cells, but all methods used until now require a prolonged incubation of cells. We hypothesized that instant magnetic labeling of cells could be achieved using electroporation. Neural stem cells (NSCs) and leukocytes from spleen and lymph nodes were suspended in a ferumoxide labeling solution, loaded into cuvettes, and subjected to electromechanical permeabilization using electroporation. Magnetically labeled cells were assayed for labeling efficiency, as well as for potential toxicity or altered function. To confirm the method's applicability to detect cells, MRI experiments were performed at 11.7 T. Magnetoelectroporation of NSCs, as demonstrated by Prussian blue staining, anti-dextran immunostaining, and a quantitative iron uptake assay, proved to be an efficient intracellular magnetic labeling method. Leukocytes including lymphocytes, which are notoriously difficult to label because of their membrane properties and small cytoplasmic volume, also demonstrated a pronounced uptake of ferumoxide. MRI experiments showed that labeled NSCs could be visualized as single cells and cell clusters in gelatin phantoms, and as proliferating cell masses in mouse brain. We have developed a convenient technique for instant magnetic labeling of cells. Because magnetoelectroporation allows the use of ferumoxides approved by the US Food and Drug Administration without additional agents, it has excellent potential for clinical translation.