Dual-color fluorescence lifetime correlation spectroscopy to quantify protein-protein interactions in live cell

Dual-color fluorescence correlation spectroscopy is an interesting method to quantify protein interaction in living cells. But, when performing these experiments, one must compensate for a known spectral bleed through artifact that corrupts cross-correlation data. In this article, problems with crosstalk were overcome with an approach based on fluorescence lifetime correlation spectroscopy (FLCS). We show that FLCS applied to dual-color EGFP and mCherry cross-correlation allows the determination of protein-protein interactions in living cells without the need of spectral bleed through calibration. The methodology was validated by using EGFP-mCherry tandem in comparison with coexpressed EGFP and mCherry in live cell. The dual-color FLCS experimental procedure where the different laser intensities do not have to be controlled during experiment is really very helpful to study quantitatively protein interactions in live sample.     

Ketamine effects on the urogenital system--changes in the urinary bladder and sperm motility

Different doses of ketamine (10 mg/kg, 20 mg/kg, 30 mg/kg, 40 mg/kg, 50 mg/kg, and 60 mg/kg) were injected i.p. (I.P.), respectively, to male ICR mice to determine the optimal dosage for chronic administration. At and above 40 mg/kg I.P. injection, mice had almost no hindlimb movement during swimming test. Subsequently, 30 mg/kg was used as the dose for the study in the toxicity of long-term ketamine administration on urinary bladder and sperm motility. The treatment group were subdivided into two (n = 10 each group); one received daily ketamine treatment i.p. for 3 months and another group for 6 months. Corresponding number of mice in control groups (n = 5 each group) received saline injection instead of ketamine. Terminal dUTP nick and labeling (TUNEL) study and Sirius red staining were carried out on the sectioned slides of the urinary bladders to study the degree of apoptosis in both epithelium and muscular layers of the urinary bladder and the relative thickness of the muscular layers in this organ was also computed. Apoptosis in the bladder epithelium was observed initially in the 3-month ketamine treated mice and the number of apoptotic cells was significantly different (P < 0.05) between the 3-month and 6-month ketamine treated mice and the control. The relative thickness of muscular layers in the bladder wall also decreased significantly (P < 0.05) when the 6-month treated mice and the control were compared. Sirius red staining revealed increase of collagen in the urinary bladder of the treated mice, most evidently 6 months after ketamine treatment. In addition, the sperm motility was studied and there was a statistically significant difference between the control and ketamine treated groups in the percentages of sperms which were motile (P < 0.05). This suggested that the chronic administration of ketamine affected the genital system as well.     

Surface treatment of glass fiber and carbon fiber posts: SEM characterization

Morphology, etching patterns, surface modification, and characterization of 2 different fiber posts: Gfp, Glass fiber post; and Cfp, carbon fiber were investigated by SEM analysis, after different surface treatments. Thirty fiber posts, being 15 Gfp and 15 Cfp were divided into a 5 surface treatments (n = 3): C-alcohol 70% (control); HF 4%-immersion in 4% hydrofluoric acid for 1min; H(3) PO(4) 37%-immersion in 37% phosphoric acid for 30s; H(2) O(2) 10%-immersion in 10% hydrogen peroxide for 20 min; H(2) O(2) 24%-immersion in 24% hydrogen peroxide for 10 min. Morphology, etching patterns, surface modification and surface characterization were acessed by SEM analysis. SEM evaluation revealed that the post surface morphology was modified following all treatment when compared with a control group, for both type of reinforced posts. HF seems to penetrate around the fibers of Gfp and promoted surface alterations. The Cfp surface seems to be inert to treatment with HF 4%. Dissolution of epoxy resin and exposure of the superficial fiber was observed in both post groups, regardless the type of reinforcing fiber, H(2) O(2) in both concentrations. Relative smooth surface area was produced by H(3) PO(4) 37% treatment, but with similar features to untreated group. Surface treatment of fiber post is a determinant factor on micromechanical entanglement to resin composite core. Post treatment with hydrogen peroxide resulted strength of carbon and glass/epoxy resin fiber posts to resin composite core.     

Secretory profile of metapleural gland cells of the leaf-cutting ant Acromyrmex coronatus (Formicidae: Attini)

Ants present a pair of metapleural glands located at the posterolateral end of the thorax. Because of its importance in the social organization of ants, the present study was aimed at describing the morphophysiology of this gland in three worker castes of Acromyrmex coronatus, focused on secretory activity using histological and histochemical techniques. Our findings revealed that the secretory and the storage portions of this gland are connected by extracytoplasmic portion of canaliculi that drain the secretion from each secretory cell to the collecting chamber. This secretion contains glycoproteins. In minor workers, the secretion contains higher levels of polysaccharides when compared to that of major workers, supporting the role of the metapleural gland in the maintenance of the fungus garden. The nucleus as well as cytoplasm of secretory cells were strongly positive for RNA indicating that these cells are active in the synthesis of proteins and lipids, compounds found in the final secretion. The variant of the CEC revealed that the secretory activity of the entire gland is synchronous, as all cells exhibit the result.     

Electrostatic force microscopy of self-assembled peptide structures

In this report electrostatic force microscopy (EFM) is used to study different peptide self-assembled structures such as tubes and particles. It is shown that not only geometrical information can be obtained using EFM, but also information about the composition of different structures. In particular we use EFM to investigate the structures of diphenylalanine peptide tubes, particles, and CSGAITIG peptide particles placed on pre-fabricated SiO(2) surfaces with a backgate. We show that the cavity in the peptide tubes could be due to the presence of water residues. Additionally we show that self-assembled amyloid peptides form spherical solid structures containing the same self-assembled peptide in its interior. In both cases transmission electron microscopy is used to verify these structures. Further, the limitations of the EFM technique are discussed, especially when the observed structures become small compared with the radius of the AFM tip used. Finally, an agreement between the detected signal and the structure of the hollow peptide tubes is demonstrated.     

Selective metallization of polymers using laser induced surface activation (LISA)��characterization and optimization of porous surface topography

Laser induced selective activation (LISA) is a molded interconnected devices technique for selective metallization of polymers. On the working piece, only the laser-machined area can be metalized in the subsequent plating. The principle of the technology is introduced. Surface analysis was performed on the laser-machined polymer using an Alicona InfiniteFocus? microscope. Based on previous experiments, bearing area curve and its parameters are chosen to characterize the surface. In this paper, by comparison of plateable and non-plateable surfaces, and two types of plateable surface made by different lasers, it is found that the normalized bearing area curve is an effective method to characterize porous surface for the subsequent plating. The normalized parameters are available to make a quantitative analysis.     

Fizeau interferometer system for fast high resolution studies of spectral line shapes

A monochromator∕Fizeau interferometer∕intensified CCD camera system is described that was developed for the measurement of the shape of spectral lines that are rapidly time varying. The most important operating parameter that determines the performance of the instrument is the size of the entrance aperture as this determines both the light throughput and the effective interferometer wavelength resolution. This paper discusses, both theoretically and experimentally, the effect of the finite source area on the instrumental resolution to assist in optimizing the choice of this parameter. A second effect that often produces a practical limit to the quality of the spectra is drift of the interferometer plates. Measurements of the shapes of spectral lines of ions and atoms ejected from the cathode spot of continuous and pulsed cathodic arcs are presented to demonstrate the utility of this instrument.     

Leucine enkephalin—A mass spectrometry standard

The present article reviews the mass spectrometric fragmentation processes and fragmentation energetics of leucine enkephalin, a commonly used peptide, which has been studied in detail and has often been used as a standard or reference compound to test novel instrumentation, new methodologies, or to tune instruments. The main purpose of the article is to facilitate its use as a reference material; therefore, all available mass spectrometry‐related information on leucine enkephalin has been critically reviewed and summarized. The fragmentation mechanism of leucine enkephalin is typical for a small peptide; but is understood far better than that of most other compounds. Because ion ratios in the MS/MS spectra indicate the degree of excitation, leucine enkephalin is often used as a thermometer molecule in electrospray or matrix‐assisted laser desorption ionization (ESI or MALDI). Other parameters described for leucine enkephalin include collisional cross‐section and energy transfer; proton affinity and gas‐phase basicity; radiative cooling rate; and vibrational frequencies. The lowest‐energy fragmentation channel of leucine enkephalin is the MH⁺ → b₄ process. All available data for this process have been re‐evaluated. It was found that, although the published E_a values were significantly different, the corresponding Gibbs free energy change showed good agreement (1.32 ± 0.07 eV) in various studies. Temperature‐ and energy‐dependent rate constants were re‐evaluated with an Arrhenius plot. The plot showed good linear correlation among all data (R² = 0.97), spanned over a 9 orders of magnitude range in the rate constants and yielded 1.14 eV activation energy and 10^11.0 sec^?1 pre‐exponential factor. Accuracy (including random and systematic errors, with a 95% confidence interval) is ±0.05 eV and 10^±0.5 sec^?1, respectively. The activation entropy at 470 K that corresponds to this reaction is ?38.1 ± 9.6 J mol^?1 K^?1. We believe that these re‐evaluated values are by far the most accurate activation parameters available at present for a protonated peptide and can be considered as “consensus” values; results on other processes might be compared to this reference value. © 2010 Wiley Periodicals, Inc., Mass Spec Rev 30:298–320, 2011     

代码生成器形式化验证技术研究

代码生成器已经广泛应用于嵌入式控制软件的开发.对于安全攸关的软件系统,对软件可靠性有很高的要求,代码生成器作为工具软件被要求与所生成代码有相同的可靠性,形式化验证方法可有效保证代码生成器的可靠性.本文介绍了用于构建形式化验证代码生成器的方法,针对Lustre到C的代码生成器给出Lustre和C的抽象语法,并定义Lust...     

磷石膏料浆专用阀设计

介绍了磷石膏料浆的生产过程及其工况特性,给出了磷石膏料浆专用阀的技术参数、工作原理、结构特点和设计方法。