Probing cellular protein complexes using single-molecule pull-down.

Proteins perform most cellular functions in macromolecular complexes. The same protein often participates in different complexes to exhibit diverse functionality. Current ensemble approaches of identifying cellular protein interactions cannot reveal physiological permutations of these interactions. Here we describe a single-molecule pull-down (SiMPull) assay that combines the principles of a conventional pull-down assay with single-molecule fluorescence microscopy and enables direct visualization of individual cellular protein complexes. SiMPull can reveal how many proteins and of which kinds are present in the in vivo complex, as we show using protein kinase A. We then demonstrate a wide applicability to various signalling proteins found in the cytosol, membrane and cellular organelles, and to endogenous protein complexes from animal tissue extracts. The pulled-down proteins are functional and are used, without further processing, for single-molecule biochemical studies. SiMPull should provide a rapid, sensitive and robust platform for analysing protein assemblies in biological pathways

Post-translational modifications regulate ??2-Adrenoceptor signaling in cardiac myocytes

??2AR (??2 adrenoceptor) is a prototypical G-protein coupled receptor (GPCR) that plays an important role in cardiovascular and pulmonary physiology through activation of the classic Gs-adenylate cyclase-cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) signaling pathway. In the mammalian heart, increasing cAMP-PKA activity leads to phosphorylation of an array of proteins involved in increasing heart contractility and rate. ??2AR also has a cardiac protective role through utilizing multiple mechanisms to reduce receptor signaling. This includes ??2AR desensitization, ??2AR coupling to G??i, and ??2AR degradation, all of which are tightly regulated by post-translational modifications of the C-terminal region of ??2AR. Over past decades, these modifications have been extensively characterized biochemically in fibroblasts, such as phosphorylation by PKA (serines 261, 262, 345 and 346) and G protein coupled receptor kinases (GRKs) (serines 355, 356 and 364), ubiquitination (lysines 348, 372 and 375) and palmitoylation (cysteine 341). However, the physiological role of these modifications on ??2AR signaling regulation in the heart remains unclear. This study provides new insight into the role of three post-translational modifications on ??2AR signaling regulation in cardiac myocytes. We find that palmitoylation, the fatty acid modification of ??2AR at cysteine 341 is not required for receptor targeting to the plasma membrane caveolae. Instead, both palmitoylation and GRK phosphorylation are required to mediate the association of ??2AR with ??-arrestin 2/ phosphodiesterase 4D complexes to regulate cAMP signaling. In addition, we provide a new mechanism explaining ??2AR coupling from Gas to Gai, which is agonist dose dependent and controlled by both PKA and GRK phosphorylation of the receptor. Moreover, we demonstrate that mutation of either PKA or GRK phosphorylation sites on ??2AR leads to rapid receptor degradation than that of wild type ??2AR. Interestingly, our data also suggest that degradation of ??2AR is coordinated by both lysosomes and proteasomes: the extracellular domains are degraded by lysosomes and the intracellular domains are degraded by proteasomes. Together, all three post-translational modifications coordinate to regulate ??2AR signaling in cardiac tissue under physiological conditions

Comparing genotyping algorithms for Illumina's Infinium whole-genome SNP BeadChips.

BACKGROUND: Illumina's Infinium SNP BeadChips are extensively used in both small and large-scale genetic studies. A fundamental step in any analysis is the processing of raw allele A and allele B intensities from each SNP into genotype calls (AA, AB, BB). Various algorithms which make use of different statistical models are available for this task. We compare four methods (GenCall, Illuminus, GenoSNP and CRLMM) on data where the true genotypes are known in advance and data from a recently published genome-wide association study. RESULTS: In general, differences in accuracy are relatively small between the methods evaluated, although CRLMM and GenoSNP were found to consistently outperform GenCall. The performance of Illuminus is heavily dependent on sample size, with lower no call rates and improved accuracy as the number of samples available increases. For X chromosome SNPs, methods with sex-dependent models (Illuminus, CRLMM) perform better than methods which ignore gender information (GenCall, GenoSNP). We observe that CRLMM and GenoSNP are more accurate at calling SNPs with low minor allele frequency than GenCall or Illuminus. The sample quality metrics from each of the four methods were found to have a high level of agreement at flagging samples with unusual signal characteristics. CONCLUSIONS: CRLMM, GenoSNP and GenCall can be applied with confidence in studies of any size, as their performance was shown to be invariant to the number of samples available. Illuminus on the other hand requires a larger number of samples to achieve comparable levels of accuracy and its use in smaller studies (50 or fewer individuals) is not recommended.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

In-Orbit Instrument Performance Study and Calibration for POLAR Polarization Measurements

POLAR is a compact space-borne detector designed to perform reliable measurements of the polarization for transient sources like Gamma-Ray Bursts in the energy range 50-500keV. The instrument works based on the Compton Scattering principle with the plastic scintillators as the main detection material along with the multi-anode photomultiplier tube. POLAR has been launched successfully onboard the Chinese space laboratory TG-2 on 15th September, 2016. In order to reliably reconstruct the polarization information a highly detailed understanding of the instrument is required for both data analysis and Monte Carlo studies. For this purpose a full study of the in-orbit performance was performed in order to obtain the instrument calibration parameters such as noise, pedestal, gain nonlinearity of the electronics, threshold, crosstalk and gain, as well as the effect of temperature on the above parameters. Furthermore the relationship between gain and high voltage of the multi-anode photomultiplier tube has been studied and the errors on all measurement values are presented. Finally the typical systematic error on polarization measurements of Gamma-Ray Bursts due to the measurement error of the calibration parameters are estimated using Monte Carlo simulations.Comment: 43 pages, 30 figures, 1 table; Preprint accepted by NIM

Antibiotics in the offshore waters of the Bohai Sea and the Yellow Sea in China: Occurrence, distribution and ecological risks

The ocean is an important sink of land-based pollutants. Previous studies showed that serious antibiotic pollution occurred in the coastal waters, but limited studies focused on their presence in offshore waters. In this study, eleven antibiotics in three different categories were investigated in offshore waters of the Bohai Sea and the Yellow Sea in China. The results indicated that three antibiotics dehydration erythromycin, sulfamethoxazole and trimethoprim occurred throughout the offshore waters at concentrations of 0.10-16.6 ng L-1 and they decreased exponentially from the rivers to the coastal and offshore waters. The other antibiotics all presented very low detection rates (<10%) and concentrations (<0.51 ng L-1). Although the concentrations were very low, risk assessment based on the calculated risk quotients (RQs) showed that sulfamethoxazole, dehydration erythromycin and clarithromycin at most of sampling sites posed medium or low ecological risks (0.01 < RQ < 1) to some sensitive aquatic organisms, including Synechococcus leopoliensis and Pseudokirchneriella subcapitata. (C) 2012 Elsevier Ltd. All rights reserved.The ocean is an important sink of land-based pollutants. Previous studies showed that serious antibiotic pollution occurred in the coastal waters, but limited studies focused on their presence in offshore waters. In this study, eleven antibiotics in three different categories were investigated in offshore waters of the Bohai Sea and the Yellow Sea in China. The results indicated that three antibiotics dehydration erythromycin, sulfamethoxazole and trimethoprim occurred throughout the offshore waters at concentrations of 0.10-16.6 ng L-1 and they decreased exponentially from the rivers to the coastal and offshore waters. The other antibiotics all presented very low detection rates (<10%) and concentrations (<0.51 ng L-1). Although the concentrations were very low, risk assessment based on the calculated risk quotients (RQs) showed that sulfamethoxazole, dehydration erythromycin and clarithromycin at most of sampling sites posed medium or low ecological risks (0.01 < RQ < 1) to some sensitive aquatic organisms, including Synechococcus leopoliensis and Pseudokirchneriella subcapitata. (C) 2012 Elsevier Ltd. All rights reserved

Effect of (0001) Strain on the Electronic and Magnetic Properties of the Half-Metallic Ferromagnet Fe 2

The electronic and magnetic properties of the half-metallic ferromagnet Fe2Si under (0001) strain have been evaluated by the first-principles density functional theory method. The spin-up band structure shows that bulk Fe2Si has metallic character, whereas the spin-down band structure shows that bulk Fe2Si is an S-L indirect band gap of 0.518 eV in the vicinity of Fermi surface. Indirect-to-direct band gaps and an unstable-to-stable transition are observed in bulk Fe2Si as strain is applied. In the range −11% to 11% (excluding zero strain), bulk Fe2Si has stable half-metallic ferromagnetism, the spin polarization at the Fermi surface is 100%, and the magnetic moment of the Fe2Si unit cell is 4.0 μB. The density distribution shows that the spin states of bulk Fe2Si mainly come from the Fe1-3d and Fe3-3d states, indicating that bulk Fe2Si has spin-polarized ferromagnetism. The half-metallic ferromagnetism of bulk Fe2Si is mainly caused by d–d exchange and p–d hybridization, which are not sensitive to strain. It is very important to investigate the effect of changes in the lattice constant on the half-metallic ferromagnetic properties of bulk Fe2Si