Shedding light on intracoronary pathophysiology

There is a new player in the field of interventional cardiology. It is optical coherence tomography (OCT), an imaging modality analogous to intravascular ultrasound (IVUS), but using light instead of sound. The paper by Yonetsu et al in this issue of the IJC is therefore of timely interest, as an illustration of where OCT evaluation of coronary pathophysiology is at present, and what the future might hold.     

Shedding light on a painful rash

We present a case of a rare inherited disorder – erythropoietic protoporphyria – with typical clinical manifestations. The diagnosis was suspected on the basis of a history of a light-sensitive rash and characteristic liver biopsy, and confirmed with genetic testing. The patient was followed up for more than 20 years, and ultimately developed the uncommon associated complication of liver cirrhosis. The clinical features, pathogenesis and management of erythropoietic protoporphyria are discussed herein.     

Shedding light on the dark and weakly fluorescent states of green fluorescent proteins

Recent experiments on various similar green fluorescent protein (GFP) mutants at the single-molecule level and in solution provide evidence of previously unknown short- and long-lived "dark" states and of related excited-state decay channels. Here, we present quantum chemical calculations on cis-trans photoisomerization paths of neutral, anionic, and zwitterionic GFP chromophores in their ground and first singlet excited states that explain the observed behaviors from a common perspective. The results suggest that favorable radiationless decay channels can exist for the different protonation states along these isomerizations, which apparently proceed via conical intersections. These channels are suggested to rationalize the observed dramatic reduction of fluorescence in solution. The observed single-molecule fast blinking is attributed to conversions between the fluorescent anionic and the dark zwitterionic forms whereas slow switching is attributed to conversions between the anionic and the neutral forms. The predicted nonadiabatic crossings are seen to rationalize the origins of a variety of experimental observations on a common basis and may have broad implications for photobiophysical mechanisms in GFP.     

Visualization of retrovirus budding with correlated light and electron microscopy

We have used correlated scanning EM (SEM) and multiphoton fluorescence microscopy to visualize budding of virus-like particles (VLPs) of Rous sarcoma virus (RSV) and HIV type 1 (HIV-1). When the Gag structural protein was expressed alone as a GFP fusion, most budding particles appeared morphologically aberrant, but normal assembly could be rescued by coexpression of untagged Gag protein. Imaging of live cells allowed budding to be seen in real time as the disappearance of fluorescent spots from the dorsal cell surface. The disappearance of very bright spots containing clusters of VLPs often occurred in a stepwise fashion. Even after imaging times >1 h, only a minority of the spots disappeared, suggesting that some might be budding-incompetent complexes. On individual cells, we enumerated both the fluorescent puncta and the budding structures visible by SEM and compared these numbers for WT Gag proteins and for Gag proteins that were blocked at the last step in budding by a late domain mutation. For the mutant HIV-1 and RSV proteins, almost all of the fluorescent spots corresponded to budding structures. For WT RSV, the dorsal side of cells showed 3-fold more fluorescent spots than budding structures, suggesting that formation of the polymerized Gag shell precedes bulging out of the membrane. For WT HIV-1, most fluorescent spots corresponded with budding structures, consistent with the slower budding rate of this virus. Combining these two types of microscopy will allow innovative approaches for elucidating the mechanism of retrovirus budding.