Memories of studying life in Emory University

<p>Gestational-age specific risks^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155692#t004fn001" target="_blank">*</a>} of adverse perinatal outcomes comparing cesarean-section (130,808 infants) vs. vaginal (n = 232,812 infants) deliveries in twin pregnancies.</p

Mitochondria-Targeted Ratiometric Fluorescent Nanosensor for Simultaneous Biosensing and Imaging of O₂^•– and pH in Live Cells

Intracellular pH undertakes critical functions in the formation of a proton gradient and electrochemical potential that drives the adenosine triphosphate synthesis. It is also involved in various metabolic processes occurring in mitochondria, such as the generation of reactive oxygen species, calcium regulation, as well as the triggering of cell proliferation and apoptosis. Meanwhile, the aberrant accumulation of O₂^•– within mitochondria is frequently intertwined with mitochondrial dysfunction and disease development. To disentangle the complicated inter-relationship between pH and O₂^•– in the signal transduction and homeostasis in mitochondria, herein we developed a mitochondria-targeted single fluorescent probe for simultaneous sensing and imaging of pH and O₂^•– in mitochondria. CdSe/ZnS quantum dots encapsulated in silica shell was designed as an inner reference element for providing a built-in correction, as well as employed as a carrier to assemble the responsive elements for O₂^•– and pH, together with mitochondria-targeted molecule. The developed nanosensor demonstrated high accuracy and selectivity for pH and O₂^•– sensing, against other ROS, metal ions, and amino acids. The remarkable analytical performance of the present nanosensor, as well as good biocompatibility, established an accurate and selective approach for real-time imaging and biosensing of O₂^•– and pH in mitochondria of live cells

OATP1B1 is glycosylated in HEK293 cells.

<p>A. The effect of tunicamycin on protein size of OATP1B1. B. Transport of E-3-S (0.1 μM) in OATP1B1-expressed HEK293 cells with or without tunicamycin treatment. C. Plasma membrane proteins from cells expressing OATP1B1 treated with N-glycosidase F. Cells expressing OATP1B1 were treated with 0.5 μg/ml of tunicamycin for 42 h before analysis. For protein expression, cells were lysed with RIPA buffer, separated by SDS-PAGE, followed by Western blotting with anti-HA antibody. Fifty micrograms of protein was loaded for each lane. Transport function of tunicamycin treated cells was expressed as a percentage of the uptake measured in the untreated control. The results represent data from three experiments, with triplicate measurements for each sample. The results shown are means ± S.E. (<i>n  = </i>3). For glycosidase treatment, cell surface proteins were biotinylated and precipitated with streptavidin beads. Proteins were then denatured with 0.5%SDS and 1% β-mercaptoethanol at 75°C for 15 min. The denatured proteins were incubated with or without N-glycosidase F overnight at 37°C before subjected to SDS-PAGE.</p

Functional analysis of triple glycosylation site mutants.

<p>Cells were incubated with 0.1 μM E-3-S for 2 min at 37°C. Transport function of mutants was expressed as a percentage of the uptake measured in wild-type OATP1B1. The results represent data from three experiments, with triplicate measurements for each mutant. The results shown are means ± S.E. (<i>n  = </i>3). Asterisks indicate values significantly different (<i>p</i><0.05) from that of wild-type OATP1B1.</p

Effect of multiple disruption of OATP1B1 glycosylation sites.

<p>A. Western blot analysis for plasma membrane protein expression of double mutants. B. Transport of E-3-S (0.1 μM) in OATP1B1 and double mutants. C. Western blot analysis for plasma membrane protein expression of triple mutants. D. Plasma membrane proteins of wild-type OATP1B1 and N134/503/516Q treated with N-glycosidase F. E. Total protein expression of OATP1B1 and triple mutants. Same blot was probed with actin antibody as loading control. F. Western blot analysis of wild-type OATP1B1 and triple mutant N134/503/516Q treated with proteasome inhibitor MG132. Cells were treated with 10 μM MG132 for 6 h before being lysed with RIPA buffer and subjected to Western blotting. Fifty micrograms of protein was loaded for each lane.</p

Secondary structure model of OATP1B1.

<p>Putative glycosylation sites were identified with NetNGlyc 1.0 Server and compared with membrane protein topology prediction analysis TopPred (Kyte-Doolittle hydrophobicity scale), only asparagines located extracellularly were considered as candidates for glycosylation sites. Putative sites were marked as black diamonds and each position was indicated with arrows.</p

Time course of N400 effects in Experiment 1 (music major group).

<p>Difference-wave topography is shown in maps of 50ms duration from 260ms to 510ms.</p

Immunofluorescence study of triple mutant N134/503/516Q.

<p>Cells expressing OATP1B1 wild-type and N134/503/516Q were stained with anti-HA antibody (1∶100) and anti-calnexin antibody (1∶100) and reacted with Alexa Fluor 488 goat anti-mouse IgG or Alexa Fluor 555 goat anti-rabbit IgG antibody. Specific immunostaining shown as green (OATP1B1) or red (calnexin) fluorescence.</p

Comparison of TM2 sequences within OATP family.

<p>Full length sequences of 11 OATP family members were aligned with ClustalW. Only partial sequences were shown here. The corresponding sequences of TM2 were in bold.</p

Time course of N400 effects in Experiment 2 (non-music major group).

<p>Difference-wave topography is shown in maps of 50ms duration from 260ms to 510ms.</p