Implementation of FRET Spectrometry Using Temporally Resolved Fluorescence: A Feasibility Study.

Förster resonance energy transfer (FRET) spectrometry is a method for determining the quaternary structure of protein oligomers from distributions of FRET efficiencies that are drawn from pixels of fluorescence images of cells expressing the proteins of interest. FRET spectrometry protocols currently rely on obtaining spectrally resolved fluorescence data from intensity-based experiments. Another imaging method, fluorescence lifetime imaging microscopy (FLIM), is a widely used alternative to compute FRET efficiencies for each pixel in an image from the reduction of the fluorescence lifetime of the donors caused by FRET. In FLIM studies of oligomers with different proportions of donors and acceptors, the donor lifetimes may be obtained by fitting the temporally resolved fluorescence decay data with a predetermined number of exponential decay curves. However, this requires knowledge of the number and the relative arrangement of the fluorescent proteins in the sample, which is precisely the goal of FRET spectrometry, thus creating a conundrum that has prevented users of FLIM instruments from performing FRET spectrometry. Here, we describe an attempt to implement FRET spectrometry on temporally resolved fluorescence microscopes by using an integration-based method of computing the FRET efficiency from fluorescence decay curves. This method, which we dubbed time-integrated FRET (or tiFRET), was tested on oligomeric fluorescent protein constructs expressed in the cytoplasm of living cells. The present results show that tiFRET is a promising way of implementing FRET spectrometry and suggest potential instrument adjustments for increasing accuracy and resolution in this kind of study.

An Analysis of the Protein and Lipid concentrations in the Tear Layer of Contact and Non-contact lens wearers

Purpose: The main reason for embarking upon this specif aspect of the study is to obtain local population statistics in Trinidad, W. I. Most of the research pertaining to the tear layer has been done internationally with statistical analysis stemming from the Wester World. The clinical significance of this study is to provide early means of detection of primary and secondary diseases such as hpyerliproteninemia, dyslipdemia, and triglyceridemia in human population. The significance is to determine the relationship between a contact lens wearer and individual components (total proteins and total lipids) of their tears. This is to examine the tear-lens relationship between the disposition of components of the tear film on soft (hydrogel and silicone hydrogel) contact lenses in relation to lens intolerance, patient discomfort and dry eye syndrome. Method: A total of 93 subjects were interviewd and tear samples were obtained from 81 participants. The capillary tube technique was used to extract tear fluid samples and were stored at -80 degrees celsius. Total Protein and Triglyceride Liquicolour kits were used to analyse tears. Using a mass spectrometer, absorbance values were used to calculate the total proteim and total triglycerid concentrations for non-contact lens and contact lens wearers. Results: It was formulated that the total concentrations of proteins and lipids in the tear film of non-contact lens patients were 7.356 1.31 and 3.76 3.26 mg/dl. The protein and lipid concentrations of contact lens patients were determined; these results were 5.88 0.91 and 2.54 0.72mg/dL. Conclusion: Based on independent statistical analysis, there was no sigificant difference between the protein and lipid concentrations of non-contact lens patients. There was also found to be no significant difference between contact lens and no-contact lens wearers' protein and lipid concentrations.

Effect of secondhand smoke on occupancy of nicotinic acetylcholine receptors in brain.

CONTEXT: Despite progress in tobacco control, secondhand smoke (SHS) exposure remains prevalent worldwide and is implicated in the initiation and maintenance of cigarette smoking. OBJECTIVE: To determine whether moderate SHS exposure results in brain α(4)ß(2)* nicotinic acetylcholine receptor (nAChR) occupancy. DESIGN, SETTING, AND PARTICIPANTS: Positron emission tomography scanning and the radiotracer 2-[18F]fluoro-3-(2(S)azetidinylmethoxy) pyridine (also known as 2-[(18)F]fluoro-A-85380, or 2-FA) were used to determine α(4)ß(2)* nAChR occupancy from SHS exposure in 24 young adult participants (11 moderately dependent cigarette smokers and 13 nonsmokers). Participants underwent two bolus-plus-continuous-infusion 2-FA positron emission tomography scanning sessions during which they sat in the passenger's seat of a car for 1 hour and either were exposed to moderate SHS or had no SHS exposure. The study took place at an academic positron emission tomography center. Main Outcome Measure  Changes induced by SHS in 2-FA specific binding volume of distribution as a measure of α(4)ß(2)* nAChR occupancy. RESULTS: An overall multivariate analysis of variance using specific binding volume of distribution values revealed a significant main effect of condition (SHS vs control) (F(1,22) = 42.5, P < .001) but no between-group (smoker vs nonsmoker) effect. Exposure to SHS led to a mean 19% occupancy of brain α(4)ß(2)* nAChRs (1-sample t test, 2-tailed, P < .001). Smokers had both a mean 23% increase in craving with SHS exposure and a correlation between thalamic α(4)ß(2)* nAChR occupancy and craving alleviation with subsequent cigarette smoking (Spearman ρ = -0.74, P = .01). CONCLUSIONS: Nicotine from SHS exposure results in substantial brain α(4)ß(2)* nAChR occupancy in smokers and nonsmokers. Study findings suggest that such exposure delivers a priming dose of nicotine to the brain that contributes to continued cigarette use in smokers. This study has implications for both biological research into the link between SHS exposure and cigarette use and public policy regarding the need to limit SHS exposure in cars and other enclosed spaces.

Effect of a history of major depressive disorder on smoking-induced dopamine release.

BACKGROUND: Dopamine (DA) system dysfunction is implicated in the pathophysiology of major depressive disorder (MDD). We sought to determine if cigarette smokers with a history of MDD and current mild depressive symptoms have abnormal smoking-induced DA release (measured indirectly as change in (11)C-raclopride binding potential [BP(ND)]). METHODS: Fifty-six cigarette smokers either with (n = 10) or without (n = 46) a history of MDD (MDD+ and MDD-, respectively) underwent bolus-plus-continuous-infusion (11)C-raclopride positron emission tomography, during which they smoked a regular cigarette. Presmoking to postsmoking changes in (11)C-raclopride BP(ND) were compared between groups. Also, correlations were determined between change in BP(ND) and depression, anxiety, and withdrawal rating scale scores for the MDD+ group. RESULTS: The MDD+ group had a significantly greater reduction in (11)C-raclopride BP(ND) (-16.3%) than the MDD- group (-8.4%) (analysis of covariance [ANCOVA], p = .03). Significant negative correlations were found between depression/anxiety and change in (11)C-raclopride BP(ND) (r = -.77, p < .01 and r = -.74, p = .01, respectively). CONCLUSIONS: MDD+ smokers have greater smoking-induced DA release than MDD- smokers, and higher depression/anxiety levels are associated with greater smoking-induced DA release. These findings support the theory that MDD+ smokers have DA system dysfunction, including heightened smoking-induced DA release.