Monitoring Biosensor Activity in Living Cells with Fluorescence Lifetime Imaging Microscopy

Live-cell microscopy is now routinely used to monitor the activities of the genetically encoded biosensor proteins that are designed to directly measure specific cell signaling events inside cells, tissues, or organisms. Most fluorescent biosensor proteins rely on Förster resonance energy transfer (FRET) to report conformational changes in the protein that occur in response to signaling events, and this is commonly measured with intensity-based ratiometric imaging methods. An alternative method for monitoring the activities of the FRET-based biosensor proteins is fluorescence lifetime imaging microscopy (FLIM). FLIM measurements are made in the time domain, and are not affected by factors that commonly limit intensity measurements. In this review, we describe the use of the digital frequency domain (FD) FLIM method for the analysis of FRET signals. We illustrate the methods necessary for the calibration of the FD FLIM system, and demonstrate the analysis of data obtained from cells expressing “FRET standard” fusion proteins. We then use the FLIM-FRET approach to monitor the changes in activities of two different biosensor proteins in specific regions of single living cells. Importantly, the factors required for the accurate determination and reproducibility of lifetime measurements are described in detail.     

全世界的设计都是相通的 2008年Andrew martin室内设计大奖

2008年10月29日,由缤纷杂志与ANDREW MARTIN联合主办的"2008 ANDREW MARTIN国际室内设计年奖获奖作品全国巡回展".在北京博洛尼家居体验馆拉开帷幕。并于10月31日在杭州.11月21日在上海、11月25日在深圳、11月26日在广州盛大开展。     

The Computer Aversion, Attitudes, and Familiarity Index (CAAFI): A validity study

This study’s purpose was to provide additional psychometric data on the Computer Aversion, Attitudes, and Familiarity Index (CAAFI). The CAAFI is comprised of 40 items, yielding a total score and four factor scores with 10 items each (one familiarity, one attitudes, two aversion). The measure was administered to a sample of undergraduate psychology students (N = 293) along with the Computer Understanding and Experience Scale, the Computer Attitude Scale, and the Computer Aversion Scale. The factor structure of the CAAFI was supported via confirmatory factor analytic procedures and analysis of internal consistency reliability coefficients. In relation to the other measures of computer-related constructs, the strongest validity support was found for the CAAFI total score and Factors 1 (computer familiarity) and 3 (computer aversion-discomfort), followed by Factor 2 (computer attitudes). With regard to Factor 4 (computer aversion-fear), given the results of the analyses the decision was made to remove these items from the CAAFI in order to enhance the measure’s psychometric properties and practical utility.     

Usability of product X-lessons from a real product

Using the example of a real product, this paper shows how various usability assessments, conducted by different human factors engineers, in several phases of the product's development life-cycle, identified similar potential usability problems. Circumstances dictated that no remedial action was taken, so it was possible to track these potential usability defects to customer sites, where it was found that most of the important problems did indeed occur. Thus, it can be demonstrated that human factors advice was valid and reliable. In simpler terms, early usability evaluation by human factors engineers can save hours of wasted development effort and customer frustration, and can help to ensure that a more usable product is produced.     

Schematic change as a result of an intensive group-therapy day-treatment programs.

Treatment effects of a 12-wk day-treatment program were explored on measures of psychiatric distress (Brief Symptom Inventory) and maladaptive schemas (Schema Questionnaire Short Form; J. Young, 1990). 84 program completers (aged 22–57 yrs) showed significant pre–post change in psychiatric distress and on 3 of 15 schema: vulnerability to harm, social alienation, and defectiveness. Other schema did not change over the 12 wks. 15 waiting-list controls demonstrated no improvement in either distress level or in any of the 15 schema while waiting to be admitted to the program. Only 1 of the 15 schema, abandonment, predicted treatment outcome on changes in psychiatric distress level. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Prospective associations between friendship adjustment and social strategies: Friendship as a context for building social skills.

The proposal that friendships provide a context for the development of social skills is widely accepted. Yet little research exists to support this claim. In the present study, children and adolescents (N = 912) were presented with vignettes in which a friend encountered a social stressor and they could help the friend and vignettes in which they encountered a stressor and could seek help from the friend. Social strategies in response to these vignettes were assessed in the fall and spring of the school year. Different indicators of friendship adjustment had unique effects on youths' strategies in response to helping tasks. Whereas having more friends predicted decreases in avoidant or hostile strategies, having high-quality friendships predicted emotionally engaged strategies that involved talking about the problem. Moreover, whereas having more friends predicted increases in relatively disengaged strategies, like distraction and acting like the problem never happened, having high-quality friendships predicted decreases in these strategies. The present study also tested whether youths' strategies in the fall predicted changes in friendship adjustment by the spring. Only strategies which may be seen as major friendship transgressions (i.e., avoiding or blaming the friend when the friend encounters a problem) predicted changes in friendship over time. Collectively, these results provide important new information on the interplay between social competencies and friendship experiences and suggest that friendships may provide a critical venue for the development of important relationship skills. (PsycINFO Database Record (c) 2011 APA, all rights reserved)     

Hydration-Induced Void-Containing Hydrogels for Encapsulation and Sustained Release of Small Hydrophilic Molecules

Efficient encapsulation and sustained release of small hydrophilic molecules from traditional hydrogel systems are challenging due to the large mesh size of 3D networks and high water content. Furthermore, the encapsulated molecules are prone to early release from the hydrogel prior to use, resulting in a short shelf life of the formulation. Here, a hydration-induced void-containing hydrogel (HVH) based on hyperbranched polyglycerol-poly(propylene oxide)-hyperbranched polyglycerol (HPG-PPG-HPG) as a robust and efficient delivery system is presented for small hydrophilic molecules. Specifically, after the HPG-PPG-HPG is incubated overnight at 4 °C in the drug solution, it is hydrated into a hydrogel containing micron-sized voids, which can encapsulate hydrophilic drugs and achieve 100% drug encapsulation efficiency. In addition, the voids are surrounded by a densely packed polymer matrix, which restricts drug transport to achieve sustained drug release. The hydrogel/drug formulation can be stored for several months without changing the drug encapsulation and release properties. HVH hydrogels are injectable due to shear thinning properties. In rats, a single injection of the HPG-PPG-HPG hydrogel containing 8 µg of tetrodotoxin (TTX) produces sciatic nerve block lasting up to 10 h without any TTX-related systemic toxicity nor local toxicity to nerves and muscles.     

A Membrane-Associated Light-Harvesting Model is Enabled by Functionalized Assemblies of Gene-Doubled TMV Proteins

Photosynthetic light harvesting requires efficient energy transfer within dynamic networks of light-harvesting complexes embedded within phospholipid membranes. Artificial light-harvesting models are valuable tools for understanding the structural features underpinning energy absorption and transfer within chromophore arrays. Here, a method for attaching a protein-based light-harvesting model to a planar, fluid supported lipid bilayer (SLB) is developed.  The protein model consists of the tobacco mosaic viral capsid proteins that are gene-doubled to create a tandem dimer (dTMV). Assemblies of dTMV break the facial symmetry of the double disk to allow for differentiation between the disk faces. A single reactive lysine residue is incorporated into the dTMV assemblies for the site-selective attachment of chromophores for light absorption. On the opposing dTMV face, a cysteine residue is incorporated for the bioconjugation of a peptide containing a polyhistidine tag for association with SLBs. The dual-modified dTMV complexes show significant association with SLBs and exhibit mobility on the bilayer. The techniques used herein offer a new method for protein-surface attachment and provide a platform for evaluating excited state energy transfer events in a dynamic, fully synthetic artificial light-harvesting system.     

Searching for multimedia: analysis of audio,video and image Web queries

The development of digital libraries has enhanced the integration of textual and multimedia information in many document collections. The World Wide Web provides the connectivity for many digital library users. Studies exploring the searching characteristics of Web users are an important and a growing area of research. Most Web user studies have focused on general Web searching, regardless of subject matter or format. Little research has examined how Web users search for multimedia information. Our study examines users' multimedia searching on a major Web search service. The data set examined consisted of 1,025,908 queries from 211,058 users of Excite ®, a major Web search service. From this data set, we identified and analyzed queries for audio, image, and video queries. Our findings were compared to results from general Web searching studies. Implications for the design of Web searching services and interfaces are discussed.