Pressure induced structural changes and gas diffusion pathways in monomeric fluorescent proteins

Fluorescent proteins (FPs) are extremely valuable biochemical markers which have found a wide range of applications in cellular and molecular biology research. The monomeric variants of red fluorescent proteins (RFPs), known as mFruits, have been especially valuable for in vivo applications in mammalian cell imaging. Fluorescent proteins consist of a chromophore caged in the beta-barrel protein scaffold. The photophysical properties of an FP is determined by its chromophore structure and its interactions with the protein barrel. Application of hydrostatic pressure on FPs results in the modification of the chromophore environment which allows a systematic study of the role of the protein-chromophore interactions on photophysical properties of FPs. Using Molecular Dynamics (MD) computer simulations, I investigated the pressure induced structural changes in the monomeric variants mCherry, mStrawberry, and Citrine. The results explain the molecular basis for experimentally observed pressure responses among FP variants. It is found that the barrel flexibility, hydrogen bonding interactions and chromophore planarity of the FPs can be correlated to their contrasting photophysical properties at vaious pressures. I also investigated the oxygen diffusion pathways in mOrange and mOrange2 which exhibit marked differences in oxygen sensitivities as well as photostability. Such computational identifications of structural changes and oxygen diffusion pathways are important in guiding mutagenesis efforts to design fluorescent proteins with improved photophysical properties.

Pressure Induced Structural Changes and Gas Diffusion Pathways in Monomeric Fluorescent Proteins

Fluorescent proteins (FPs) are extremely valuable biochemical markers which have found a wide range of applications in cellular and molecular biology research. The monomeric variants of red fluorescent proteins (RFPs), known as mFruits, have been especially valuable for in vivo applications in mammalian cell imaging. Fluorescent proteins consist of a chromophore caged in the beta-barrel protein scaffold. The photophysical properties of an FP is determined by its chromophore structure and its interactions with the protein barrel. Application of hydrostatic pressure on FPs results in the modification of the chromophore environment which allows a systematic study of the role of the protein-chromophore interactions on photophysical properties of FPs. Using Molecular Dynamics (MD) computer simulations, I investigated the pressure induced structural changes in the monomeric variants mCherry, mStrawberry, and Citrine. The results explain the molecular basis for experimentally observed pressure responses among FP variants. It is found that the barrel flexibility, hydrogen bonding interactions and chromophore planarity of the FPs can be correlated to their contrasting photophysical properties at vaious pressures. I also investigated the oxygen diffusion pathways in mOrange and mOrange2 which exhibit marked differences in oxygen sensitivities as well as photostability. Such computational identifications of structural changes and oxygen diffusion pathways are important in guiding mutagenesis efforts to design fluorescent proteins with improved photophysical properties.

Vegetation dynamics and their implications for the management of wetlands in the lowlands of Nepal

The maintenance of species richness is often a priority in the management of nature reserves, where consumptive use of resources is generally prohibited. The purpose of this research was to improve management by understanding the vegetation dynamics in the lowlands of Nepal. The objectives were to determine vegetation associations in relation to environments and human-induced disturbances that affect vegetation dynamics on floodplains, where upstream barrages had altered flooding patterns, and consumptive use of plant resources was influencing natural processes. Floodplain vegetation in relation to physical environments and disturbances were studied along transects, perpendicular to the course of the Mahakali River in the western Terai, Nepal. Forest structural changes were studied for three years in ten plots. A randomized split-block experiment with nine burning and grazing treatments was performed in seasonally flooded grasslands. A semi-structured questionnaire was used to assess people's socio-economic status, natural resource use patterns and conservation attitudes. ^ Elevation, soil organic matter, nitrogen, percentage of sand and grazing intensity were significant in delineating herbaceous vegetation assemblages, whereas elevation and livestock grazing were significant in defining forest type boundaries. On the floodplain islands, highly grazed Dalbergia sissoo-Acacia catechu forests were devoid of understory woody vegetation, but the lightly grazed D. sissoo-mixed forests had a well-developed second canopy layer, comprising woody species other than D. sissoo and A. catechu. In grasslands, species richness and biomass production were highest at intermediate disturbance level represented by the lightly grazed and ungrazed early-burned treatments. Ethnicity, education and resource use patterns were important in influencing conservation attitudes. A succession towards the mixed forests would occur in D. sissoo-dominated floodplain forests, where dams and barrages reduce flooding and associated fluvial processes, and if livestock grazing is stopped, as occasionally suggested by nature conservationists. In seasonally flooded grasslands, early burning with moderate grazing would enhance the species diversity and productivity. There is a need to implement a participatory integrated wetland management plan, to include community development, education and off farm income generation, to assure participatory conservation and management of wetlands in Nepal. ^

Ecosystem Structure and Function Still Altered Two Decades After Short-Term Fertilization of a Seagrass Meadow

An oligotrophic phosphorus (P) limited seagrass ecosystem in Florida Bay was experimentally fertilized in a unique way. Perches were installed to encourage seabirds to roost and deliver an external source of nutrients via defecation. Two treatments were examined: (1) a chronic 23-year fertilization and (2) an earlier 28-month fertilization that was discontinued when the chronic treatment was initiated. Because of the low mobility of P in carbonate sediments, we hypothesized long-term changes to ecosystem structure and function in both treatments. Structural changes in the chronic treatment included a shift in the dominant seagrass species from Thalassia testudinum to Halodule wrightii, large increases in epiphytic biomass and sediment chlorophyll-a, and a decline in species richness. Functional changes included increased benthic metabolism and quantum efficiency. Initial changes in the 28-month fertilization were similar, but after 23 years of nutrient depuration T. testudinum has reestablished itself as the dominant species. However, P remains elevated in the sediment and H. wrightii has maintained a presence. Functionally the discontinued treatment remains altered. Biomass exceeds that in the chronic treatment and indices of productivity, elevated relative to control, are not different from the chronic fertilization. Cessation of nutrient loading has resulted in a superficial return to the pre-disturbance character of the community, but due to the nature of P cycles functional changes persist.

Guest Editorial: Debunking the Myths

The modem retail travel industry has been plagued by myths created by popular media in an effort to create hype and readership, rather than report an accurate portrayal of the extraordinary health of the travel distribution system. Regardless of the structural changes in the travel industry, the outlook for growth and profitability of travel agencies and travel agents has never been better

Simulating everglades national park hydrology and phosphorus transport under existing and future scenarios using numerical modeling

The Florida Everglades has a long history of anthropogenic changes which have impacted the quantity and quality of water entering the system. Since the construction of Tamiami Trail in the 1920's, overland flow to the Florida Everglades has decreased significantly, impacting ecosystems from the wetlands to the estuary. The MIKE Marsh Model of Everglades National Park (M3ENP) is a numerical model, which simulates Everglades National Park (ENP) hydrology using MIKE SHE/MIKE 11software. This model has been developed to determine the parameters that effect Everglades hydrology and understand the impact of specific flow changes on the hydrology of the system. ^ As part of the effort to return flows to the historical levels, several changes to the existing water management infrastructure have been implemented or are in the design phase. Bridge construction scenarios were programed into the M3ENP model to review the effect of these structural changes and evaluate the potential impacts on water levels and hydroperiods in the receiving Northeast Shark Slough ecosystem. These scenarios have shown critical water level increases in an area which has been in decline due to low water levels. Results from this work may help guide future decisions for restoration designs. ^ Excess phosphorus entering Everglades National Park in South Florida may promote the growth of more phosphorus-opportunistic species and alter the food chain from the bottom up. Two phosphorus transport methods were developed into the M3ENP hydrodynamic model to determine the factors affecting phosphorus transport and the impact of bridge construction on water quality. Results showed that while phosphorus concentrations in surface waters decreased overall, some areas within ENP interior may experience an increase in phosphorus loading which the addition of bridges to Tamiami Trail. Finally, phosphorus data and modeled water level data was used to evaluate the spectral response of Everglades vegetation to increasing phosphorus availability using Landsat imagery.^

Simulating Everglades National Park hydrology and phosphorus transport under existing and future scenarios using numerical modeling

The Florida Everglades has a long history of anthropogenic changes which have impacted the quantity and quality of water entering the system. Since the construction of Tamiami Trail in the 1920's, overland flow to the Florida Everglades has decreased significantly, impacting ecosystems from the wetlands to the estuary. The MIKE Marsh Model of Everglades National Park (M3ENP) is a numerical model, which simulates Everglades National Park (ENP) hydrology using MIKE SHE/MIKE 11software. This model has been developed to determine the parameters that effect Everglades hydrology and understand the impact of specific flow changes on the hydrology of the system. As part of the effort to return flows to the historical levels, several changes to the existing water management infrastructure have been implemented or are in the design phase. Bridge construction scenarios were programed into the M3ENP model to review the effect of these structural changes and evaluate the potential impacts on water levels and hydroperiods in the receiving Northeast Shark Slough ecosystem. These scenarios have shown critical water level increases in an area which has been in decline due to low water levels. Results from this work may help guide future decisions for restoration designs. Excess phosphorus entering Everglades National Park in South Florida may promote the growth of more phosphorus-opportunistic species and alter the food chain from the bottom up. Two phosphorus transport methods were developed into the M3ENP hydrodynamic model to determine the factors affecting phosphorus transport and the impact of bridge construction on water quality. Results showed that while phosphorus concentrations in surface waters decreased overall, some areas within ENP interior may experience an increase in phosphorus loading which the addition of bridges to Tamiami Trail. Finally, phosphorus data and modeled water level data was used to evaluate the spectral response of Everglades vegetation to increasing phosphorus availability using Landsat imagery.