Development of FRET Assay into Quantitative and High-throughput Screening Technology Platforms for Protein–Protein Interactions

Förster resonance energy transfer (FRET) technology has been widely used in biological and biomedical research and is a very powerful tool in elucidating protein interactions in many cellular processes. Ubiquitination and SUMOylation are multi-step cascade reactions, involving multiple enzymes and protein–protein interactions. Here we report the development of dissociation constant (Kd) determination for protein–protein interaction and cell-based high-throughput screening (HTS) assay in SUMOylation cascade using FRET technology. These developments are based on steady state and high efficiency of fluorescent energy transfer between CyPet and YPet fused with SUMO1 and Ubc9, respectively. The developments in theoretical and experimental procedures for protein interaction Kd determination and cell-based HTS provide novel tools in affinity measurement and protein interaction inhibitor screening. The Kd determined by FRET between SUMO1 and Ubc9 is compatible with those determined with other traditional approaches, such as isothermal titration calorimetry (ITC) and surface plasmon resonance (SPR). The FRET-based HTS is pioneer in cell-based HTS. Both Kd determination and cell-based HTS, carried out in 384-well plate format, provide powerful tools for large-scale and high-throughput applications

Global Assessment of Extinction Risk to Populations of Sockeye Salmon Oncorhynchus nerka

BACKGROUND: Concern about the decline of wild salmon has attracted the attention of the International Union for the Conservation of Nature (IUCN). The IUCN applies quantitative criteria to assess risk of extinction and publishes its results on the Red List of Threatened Species. However, the focus is on the species level and thus may fail to show the risk to populations. The IUCN has adapted their criteria to apply to populations but there exist few examples of this type of assessment. We assessed the status of sockeye salmon Oncorhynchus nerka as a model for application of the IUCN population-level assessments and to provide the first global assessment of the status of an anadromous Pacific salmon. METHODS/PRINCIPAL FINDINGS: We found from demographic data that the sockeye salmon species is not presently at risk of extinction. We identified 98 independent populations with varying levels of risk within the species' range. Of these, 5 (5%) are already extinct. We analyzed the risk for 62 out of 93 extant populations (67%) and found that 17 of these (27%) are at risk of extinction. The greatest number and concentration of extinct and threatened populations is in the southern part of the North American range, primarily due to overfishing, freshwater habitat loss, dams, hatcheries, and changing ocean conditions. CONCLUSIONS/SIGNIFICANCE: Although sockeye salmon are not at risk at the species-level, about one-third of the populations that we analyzed are at risk or already extinct. Without an understanding of risk to biodiversity at the level of populations, the biodiversity loss in salmon would be greatly underrepresented on the Red List. We urge government, conservation organizations, scientists and the public to recognize this limitation of the Red List. We also urge recognition that about one-third of sockeye salmon global population diversity is at risk of extinction or already extinct

Indications for free vascularized fibular grafting for the treatment of osteonecrosis of the femoral head

BACKGROUND: The present study aimed to determine the indications for free vascularized fibular grafting for the treatment of osteonecrosis of the femoral head. METHODS: Seventy-one hips (60 patients) were clinically followed for a minimum of 3 years. Average follow-up period was 7 years. Etiologies were alcohol abuse in 31 hips, steroid use in 27, idiopathic in 7 and trauma in 6. Preoperative staging of the necrotic lesion was done using the Steinberg's classification system. The outcomes of free vascularized fibular grafting were determined clinically using the Harris hip-scoring system, radiographically by determining progression, and survivorship by lack of conversion to total hip replacement. RESULTS: The average preoperative Harris hip score was 56 points and the average score at the latest follow-up examination was 78 points. Forty-seven hips (67%) were clinically rated good to excellent, 4 hips (6%) were rated fair, and 20 hips (28%) were rated poor. Thirty-six hips (51%) did not show radiographic progression while 35 hips (49%) did, and with an overall survivorship of 83% at 7 years. Steroid-induced osteonecrosis was significantly associated with poor scores and survival rate (68%). Preoperative collapse was significantly associated with poor scores, radiographic progression and poor survival rate (72%). A large extent of osteonecrosis greater than 300 degrees was significantly associated with poor scores, radiographic progression and poor survival rate (67%). There was no relationship between the distance from the tip of the grafted fibula to the subchondral bone of the femoral head and postoperative radiographic progression. CONCLUSION: In conclusion, small osteonecrosis (less than 300 degrees of the femoral head) without preoperative collapse (Steinberg's stages I and II) is the major indication for free vascularized fibular grafting. Steroid-induced osteonecrosis is a relative contraindication. Large osteonecrosis (greater than 300 degrees) with severe preoperative collapse (greater than 3 mm) is a major contraindication. Hips with 2 negative factors such as severe preoperative collapse and a large extent of osteonecrosis, require hip replacements

Quantitative assessment of fluorescent proteins

The advent of fluorescent proteins (FP) for genetic labeling of molecules and cells has revolutionized fluorescence microscopy. Genetic manipulations have created a vast array of bright and stable FPs spanning the blue to red spectral regions. Common to autofluorescent FPs is their tight β-barrel structure, which provides the rigidity and chemical environment needed for effectual fluorescence. Despite the common structure, each FP has its own unique photophysical properties. Thus, there is no single “best” fluorescent protein for every circumstance, and each FP has advantages and disadvantages. To guide decisions about which FP is right for any given application, we have characterized quantitatively over 40 different FPs for their brightness, photostability, pH stability, and monomeric properties, which permits easy apples-to-apples comparisons between these FPs. We report the values for all of the FPs measured, but focus the discussion on the more popular and/or best performing FPs in each spectral region