Some Secrets of Fluorescent Proteins: Distinct Bleaching in Various Mounting Fluids and Photoactivation of cyan fluorescent proteins at YFP-Excitation

Background
The use of spectrally distinct variants of green fluorescent protein (GFP) such as cyan or yellow mutants (CFP and YFP, respectively) is very common in all different fields of life sciences, e.g. for marking specific proteins or cells or to determine protein interactions. In the latter case, the quantum physical phenomenon of fluorescence resonance energy transfer (FRET) is exploited by specific microscopy techniques to visualize proximity of proteins.

Methodology/Principal Findings
When we applied a commonly used FRET microscopy technique - the increase in donor (CFP)-fluorescence after bleaching of acceptor fluorophores (YFP), we obtained good signals in live cells, but very weak signals for the same samples after fixation and mounting in commercial microscopy mounting fluids. This observation could be traced back to much faster bleaching of CFP in these mounting media. Strikingly, the opposite effect of the mounting fluid was observed for YFP and also for other proteins such as Cerulean, TFP or Venus. The changes in photostability of CFP and YFP were not caused by the fixation but directly dependent on the mounting fluid. Furthermore we made the interesting observation that the CFP-fluorescence intensity increases by about 10 - 15% after illumination at the YFP-excitation wavelength – a phenomenon, which was also observed for Cerulean. This photoactivation of cyan fluorescent proteins at the YFP-excitation can cause false-positive signals in the FRET-microscopy technique that is based on bleaching of a yellow FRET acceptor.

Conclusions/Significance
Our results show that photostability of fluorescent proteins differs significantly for various media and that CFP bleaches significantly faster in commercial mounting fluids, while the opposite is observed for YFP and some other proteins. Moreover, we show that the FRET microscopy technique that is based on bleaching of the YFP is prone to artifacts due to photoactivation of cyan fluorescent proteins under these conditions

In-hospital safety in field conditions of Nifurtimox Eflornithine Combination Therapy (NECT) for T. B. Gambiense Sleeping Sickness

Trypanosoma brucei (T.b.) gambiense Human African trypanosomiasis (HAT; sleeping sickness) is a fatal disease. Until 2009, available treatments for 2(nd) stage HAT were complicated to use, expensive (eflornithine monotherapy), or toxic, and insufficiently effective in certain areas (melarsoprol). Recently, nifurtimox-eflornithine combination therapy (NECT) demonstrated good safety and efficacy in a randomised controlled trial (RCT) and was added to the World Health Organisation (WHO) essential medicines list (EML). Documentation of its safety profile in field conditions will support its wider use

Floral temperature and optimal foraging: is heat a feasible floral reward for pollinators?

As well as nutritional rewards, some plants also reward ectothermic pollinators with warmth. Bumble bees have some control over their temperature, but have been shown to forage at warmer flowers when given a choice, suggesting that there is some advantage to them of foraging at warm flowers (such as reducing the energy required to raise their body to flight temperature before leaving the flower). We describe a model that considers how a heat reward affects the foraging behaviour in a thermogenic central-place forager (such as a bumble bee). We show that although the pollinator should spend a longer time on individual flowers if they are warm, the increase in total visit time is likely to be small. The pollinator's net rate of energy gain will be increased by landing on warmer flowers. Therefore, if a plant provides a heat reward, it could reduce the amount of nectar it produces, whilst still providing its pollinator with the same net rate of gain. We suggest how heat rewards may link with plant life history strategies

Climatic variability during the last millennium in Western Iceland from lake sediment records

The aim of this research was to create a decadal-scale terrestrial quantitative palaeoclimate record for NW Iceland from lake sediments for the last millennium. Geochemical, stable isotope and chironomid reconstructions were obtained from a lake sequence constrained by tephra deposits on the Snæfellsnes peninsula, western Iceland. Obtaining a quantitative record proved problematic, but the qualitative chironomid record showed clear trends associated with past summer temperatures, and the sedimentological records provided evidence for past changes in precipitation, mediated through catchment soil in-wash. When the full range of chronological uncertainty is considered, four clear phases of climatic conditions were identified: (1) a relatively warm phase between AD 1020 and 1310; (2) a relatively stable period between AD 1310 and 1510, cooler than the preceding period but still notably warmer than the second half of the millennium; (3) a consistent reduction of temperatures between AD 1560 and 1810, with the coolest period between AD 1680 and 1810; and (4) AD 1840–2000 has temperatures mainly warmer than in the preceding two centuries, with a rising trend and increased variability from c. AD 1900 onwards. The reconstructions show clearly that the first half of the millennium experienced warmer climatic conditions than the second half, with a return to the warmer climate only occurring in the last c. 100 years. Much of the variability of the chironomid record can be linked to changes in the North Atlantic Oscillation (NAO). The reconstructions presented can track low-frequency and long-term trends effectively and consistently but high-resolution and calibrated quantitative records remain more of a challenge – not just in finding optimal sedimentary deposits but also in finding the most reliable proxy. It is this that presents the real challenge for Holocene climate reconstruction from this key area of the North Atlantic. Keywords : iceland, palaeolimnology, chironomids, little ice age, medieval climate anomaly, north atlantic oscillatio

Age and environment affect constitutive immune function in Red Knots (Calidris canutus)

We studied subspecies, age and environmental effects on constitutive immune function (natural antibody and complement titres, haptoglobin activity and leukocyte concentrations) in Red Knots (Calidris canutus). We compared C. c. islandica and C. c. canutus in the Wadden Sea and found no difference in immune function between subspecies. However, C. c. canutus on their wintering grounds in Banc d’Arguin had higher natural antibody and lower complement levels than C. c. canutus or C. c. islandica in the Wadden Sea. This suggests that immune function is determined more by the surrounding environment than by subspecies. We also compared age classes in the Wadden Sea and found that first year birds had significantly lower natural antibody levels than adults, but that second year birds no longer differed from adults. Finally, we examined the interaction of age and environment in Banc d’Arguin. We found that first year birds (but not adults) in a low quality habitat had higher leukocyte concentrations than first year birds or adults in a high quality habitat. Differences in available resources and defence needs between environments, and differences among individuals differentially distributed between sites, are likely important contributors to the variation in immune function we report. Future studies, which examine these factors on wild birds, will be important for our understanding of how animals function in their natural environment.

Pharmacokinetic targeting of intravenous busulfan reduces conditioning regimen related toxicity following allogeneic hematopoietic cell transplantation for acute myelogenous leukemia

Optimal conditioning therapy for hematopoietic cell transplantation (HCT) in acute myelogenous leukemia (AML) remains undefined. We retrospectively compared outcomes of a consecutive series of 51 AML patients treated with oral busulfan (1 mg/kg every 6 hours for 4 days) and cyclophosphamide (60 mg/kg IV × 2 days) - (Bu/Cy) with 100 consecutive AML patients treated with pharmacokinetic targeted IV busulfan (AUC < 6000 μM/L*min per day × 4 days) and fludarabine (40 mg/m2 × 4 days) - (t-IV Bu/Flu). The Bu/Cy and t-IV Bu/Flu groups significantly differed according to donor relation, stem cell source, aGVHD prophylaxis, remission status, primary vs. secondary disease, median age, and % blasts prior to HCT (p < 0.01 for each). Conditioning with t-IV Bu/Flu reduced early toxicity including idiopathic pneumonia syndrome (IPS) and hepatic veno-occlusive disease (VOD). Additionally, the trajectory of early NRM (100 day: 16% vs. 3%, and1 year: 25% vs. 15% for Bu/Cy and t-IV Bu/Flu, respectively) favored t-IV Bu/Flu. Grade II-IV aGVHD (48% vs. 82%, p < 0.0001), as well as moderate/severe cGVHD (7% vs. 40%, p < 0.0001) differed between the Bu/Cy and t-IV Bu/Flu groups, due to the predominance of peripheral blood stem cells in the t-IV Bu/Flu group. Pharmacokinetic targeting of intravenous busulfan in combination with fludarabine is associated with reduced conditioning regimen related toxicity compared to oral busulfan and cyclophosphamide. However, multivariable analysis did not demonstrate significant differences in overall survival (p = 0.78) or non-relapse mortality (p = 0.6) according to conditioning regimen delivered

Optimization of the Contrast Mixture Ratio for Simultaneous Direct MR and CT Arthrography: an in Vitro Study

OBJECTIVE: This study was designed to determine the optimal mixture ratio of gadolinium and iodinated contrast agent for simultaneous direct MR arthrography and CT arthrography. MATERIALS AND METHODS: An in vitro study was performed utilizing mixtures of gadolinium at six different concentrations (0.625, 1.25, 2.5, 5.0, 10 and 20 mmol/L) and iodinated contrast agent at seven different concentrations (0, 12.5, 25, 37.5, 50, 75 and 92-99.9%). These mixtures were placed in tissue culture plates, and were then imaged with CT and MR (with T1-weighted sequences, proton-density sequences and T2-weighted sequences). CT numbers and signal intensities were measured. Pearson's correlation coefficients were used to assess the correlations between the gadolinium/iodinated contrast agent mixtures and the CT numbers/MR signal intensities. Scatter diagrams were plotted for all gadolinium/iodinated contrast agent combinations and two radiologists in consensus identified the mixtures that yielded the optimal CT numbers and MR signal intensities. RESULTS: The CT numbers showed significant correlation with iodinated contrast concentrations (r = 0.976, p < 0.001), whereas the signal intensities as measured on MR images showed a significant correlation with both gadolinium and iodinated contrast agent concentrations (r = -484 to -0.719, p < 0.001). A review of the CT and MR images, graphs, and scatter diagram of 42 combinations of the contrast agent showed that a concentration of 1.25 mmol/L gadolinium and 25% iodinated contrast agent was the best combination for simultaneous CT and MR imaging. CONCLUSION: A mixture of 1.25 mmol/L gadolinium and 25% iodinated contrast agent was found to be optimal for simultaneous direct MR arthrography and CT arthrography

Incorporating scale dependence in disease burden estimates:the case of human African trypanosomiasis in Uganda

The WHO has established the disability-adjusted life year (DALY) as a metric for measuring the burden of human disease and injury globally. However, most DALY estimates have been calculated as national totals. We mapped spatial variation in the burden of human African trypanosomiasis (HAT) in Uganda for the years 2000-2009. This represents the first geographically delimited estimation of HAT disease burden at the sub-country scale.Disability-adjusted life-year (DALY) totals for HAT were estimated based on modelled age and mortality distributions, mapped using Geographic Information Systems (GIS) software, and summarised by parish and district. While the national total burden of HAT is low relative to other conditions, high-impact districts in Uganda had DALY rates comparable to the national burden rates for major infectious diseases. The calculated average national DALY rate for 2000-2009 was 486.3 DALYs/100 000 persons/year, whereas three districts afflicted by rhodesiense HAT in southeastern Uganda had burden rates above 5000 DALYs/100 000 persons/year, comparable to national GBD 2004 average burden rates for malaria and HIV/AIDS.These results provide updated and improved estimates of HAT burden across Uganda, taking into account sensitivity to under-reporting. Our results highlight the critical importance of spatial scale in disease burden analyses. National aggregations of disease burden have resulted in an implied bias against highly focal diseases for which geographically targeted interventions may be feasible and cost-effective. This has significant implications for the use of DALY estimates to prioritize disease interventions and inform cost-benefit analyses

Single-Atom Gating of Quantum State Superpositions

The ultimate miniaturization of electronic devices will likely require local and coherent control of single electronic wavefunctions. Wavefunctions exist within both physical real space and an abstract state space with a simple geometric interpretation: this state space--or Hilbert space--is spanned by mutually orthogonal state vectors corresponding to the quantized degrees of freedom of the real-space system. Measurement of superpositions is akin to accessing the direction of a vector in Hilbert space, determining an angle of rotation equivalent to quantum phase. Here we show that an individual atom inside a designed quantum corral can control this angle, producing arbitrary coherent superpositions of spatial quantum states. Using scanning tunnelling microscopy and nanostructures assembled atom-by-atom we demonstrate how single spins and quantum mirages can be harnessed to image the superposition of two electronic states. We also present a straightforward method to determine the atom path enacting phase rotations between any desired state vectors. A single atom thus becomes a real space handle for an abstract Hilbert space, providing a simple technique for coherent quantum state manipulation at the spatial limit of condensed matter.Comment: Published online 6 April 2008 in Nature Physics; 17 page manuscript (including 4 figures) + 3 page supplement (including 2 figures); supplementary movies available at http://mota.stanford.ed

Amplified surface temperature response of cold, deep lakes to inter-annual air temperature variability

Summer lake surface water temperatures (LSWTs) have previously been shown to respond more rapidly to climatic warming compared to local summer surface air temperatures (SATs). In a global- scale analysis, we explore the factors underpinning the observation of an amplified response of summer LSWT to SAT variability using 20 years of satellite-derived temperatures from 144 lakes. We demonstrate that the degree of amplification in inter-annual summer LSWT is variable, and is greater for cold lakes (e.g. high latitude and high altitude), which are characterised by a short warming season, and deep lakes, that exhibit long correlation timescales of temperature anomalies due to increased thermal inertia. Such lakes are more likely to display responses in excess of local inter-annual summer SAT variability. Climatic modification of LSWT has numerous consequences for water quality and lake ecosystems, so quantifying this amplified response at a global scale is important