Organismal differences in post-translational modifications in histones H3 and H4

Post-translational modifications (PTMs) of histones play an important role in many cellular processes, notably gene regulation. Using a combination of mass spectrometric and immunobiochemical approaches, we show that the PTM profile of histone H3 differs significantly among the various model organisms examined. Unicellular eukaryotes, such as Saccharomyces cerevisiae (yeast) and Tetrahymena thermophila (Tet), for example, contain more activation than silencing marks as compared with mammalian cells (mouse and human), which are generally enriched in PTMs more often associated with gene silencing. Close examination reveals that many of the better-known modified lysines (Lys) can be either methylated or acetylated and that the overall modification patterns become more complex from unicellular eukaryotes to mammals. Additionally, novel species-specific H3 PTMs from wild-type asynchronously grown cells are also detected by mass spectrometry. Our results suggest that some PTMs are more conserved than previously thought, including H3K9me1 and H4K20me2 in yeast and H3K27me1, -me2, and -me3 in Tet. On histone H4, methylation at Lys-20 showed a similar pattern as H3 methylation at Lys-9, with mammals containing more methylation than the unicellular organisms. Additionally, modification profiles of H4 acetylation were very similar among the organisms examined.     

Replication and fine mapping of asthma-associated loci in individuals of African ancestry

Asthma originates from genetic and environmental factors with about half the risk of disease attributable to heritable causes. Genome-wide association studies, mostly in populations of European ancestry, have identified numerous asthma-associated single nucleotide polymorphisms (SNPs). Studies in populations with diverse ancestries allow both for identification of robust associations that replicate across ethnic groups and for improved resolution of associated loci due to different patterns of linkage disequilibrium between ethnic groups. Here we report on an analysis of 745 African-American subjects with asthma and 3,238 African-American control subjects from the Candidate Gene Association Resource (CARe) Consortium, including analysis of SNPs imputed using 1,000 Genomes reference panels and adjustment for local ancestry. We show strong evidence that variation near RAD50/IL13, implicated in studies of European ancestry individuals, replicates in individuals largely of African ancestry. Fine mapping in African ancestry populations also refined the variants of interest for this association. We also provide strong or nominal evidence of replication at loci near ORMDL3/GSDMB, IL1RL1/IL18R1, and 10p14, all previously associated with asthma in European or Japanese populations, but not at the PYHIN1 locus previously reported in studies of African-American samples. These results improve the understanding of asthma genetics and further demonstrate the utility of genetic studies in populations other than those of largely European ancestry.     

Phenylalanine promotes interaction of transmembrane domains via GxxxG motifs

Interactions of transmembrane helices play a crucial role in the folding and oligomerisation of integral membrane proteins. In order to uncover novel sequence motifs mediating these interactions, we randomised one face of a transmembrane helix with a set of non-polar or moderately polar amino acids. Those sequences capable of self-interaction upon integration into bacterial inner membranes were selected by means of the ToxR/POSSYCCAT system. A comparison between low/medium-affinity and high-affinity sequences reveals that high-affinity sequences are strongly enriched in phenylalanine residues that are frequently observed at the − 3 position of GxxxG motifs, thus yielding FxxGxxxG motifs. Mutation of Phe or GxxxG in selected sequences significantly reduces self-interaction of the transmembrane domains without affecting their efficiency of membrane integration. Conversely, grafting FxxGxxxG onto unrelated transmembrane domains strongly enhances their interaction. Further, we find that FxxGxxxG is significantly over-represented in transmembrane domains of bitopic membrane proteins. The same motif contributes to self-interaction of the vesicular stomatitis virus G protein transmembrane domain. We conclude that Phe stabilises membrane-spanning GxxxG motifs. This is one example of how the role of certain side-chains in helix-helix interfaces is modulated by sequence context.     

Quantitative proteomics analysis of detergent-resistant membranes from chemical synapses: evidence for cholesterol as spatial organizer of synaptic vesicle cycling

Synaptic vesicles (SVs) in the central nervous system upon stimulation undergo rapid calcium-triggered exoendocytic cycling within the nerve terminal that at least in part depends on components of the clathrin- and dynamin-dependent endocytosis machinery. How exocytic SV fusion and endocytic retrieval are temporally and spatially coordinated is still an open question. One possibility is that specialized membrane microdomains characterized by their high content in membrane cholesterol may assist in the spatial coordination of synaptic membrane protein recycling. Quantitative proteomics analysis of detergent-resistant membranes (DRMs) isolated from rat brain synapses or cholesterol-depleted control samples by liquid chromatography-tandem mass spectrometry identified a total of 159 proteins. Among these 122 proteins were classified as cholesterol-dependent DRM or DRM-associated proteins, many of which with proven or hypothesized functions in exoendocytic vesicle cycling including clathrin, the clathrin adaptor complex AP-2, and a variety of SV proteins. In agreement with this, SV membrane and endocytic proteins displayed a partial resistance to extraction with cold Triton X-100 in cultured rat hippocampal neurons where they co-localized with labeled cholera toxin B, a marker for cholesterol-enriched DRMs. Moreover SV proteins formed cholesterol-dependent complexes in CHAPS-extracted synaptic membrane lysates. Our combined data suggest that lipid microdomains may act as spatial coordinators for exoendocytic vesicle cycling at synapses.     

用简并寡核苷酸探针筛选对虾白斑综合征病毒DNA多聚酶基因片段

根据一些病毒的DNA多聚酶氨基酸序列中特有的保守序列VYGDTD设计的简并寡核苷酸,经地高辛标记后与对虾白斑综合征病毒基因库克隆杂交,筛选出一段长度为707 bp的EcoR I基因片段,该片段在一个开放阅读框内.并含DNA多聚酶B家族特有的保守序列YGDTDS.经与基因库比较,其氨基酸序列与藻类DNA病毒科(Phycodnaviridae)的几株藻类病毒的DNA多聚酶片段有部分相似,因此推测该核苷酸片段为对虾白斑综合征病毒DNA多聚酶基因的部分序列.     

A 10-miRNA risk score-based prediction model for pathological complete response to neoadjuvant chemotherapy in hormone receptor-positive breast cancer

Patients with hormone receptor(HR)-positive tumors breast cancer usually experience a relatively low pathological complete response(p CR) to neoadjuvant chemotherapy(NAC). Here, we derived a 10-micro RNA risk score(10-mi RNA RS)-based model with better performance in the prediction of p CR and validated its relation with the disease-free survival(DFS) in 755 HRpositive breast cancer patients(273, 265, and 217 in the training, internal, and external validation sets, respectively). This model,pres...     

A lateral-displacement flume for fish behavior and stranding studies during simulated pulsed flows

In regulated rivers, fluctuating water depths associated with pulsed discharges may strand small fish in side channels and pools. Quantitative assessments of stranded fish are difficult in field studies (e.g., due to unknown effects of avian and terrestrial vertebrate predators). To assess such lateral displacement and stranding on juvenile stream fishes, we designed, constructed, and tested (with three species) a 2 × 1-m, lateral-displacement flume. The flume featured a main channel that never drained and a raised, wide “floodplain” channel that alternately flooded, with a simulated pulse, and became dewatered. The floodplain contained four pools, with different shapes and draining capacities, in which fish could become stranded as the water level subsided. Fish-stranding rates (8%) in this relatively compact laboratory flume, after exposure to simulated pulsed stream flows, were comparable to those observed in past investigations using larger, artificial streams.     

Development, validation and implementation of immobilized metal affinity for phosphochemicals (IMAP)-based high-throughput screening assays for low-molecular-weight compound libraries

This protocol describes assay development, validation and implementation of automated immobilized metal affinity for phosphochemicals (IMAP)-based fluorescence polarization (FP) and time-resolved fluorescence resonance energy transfer (TR-FRET) high-throughput screening (HTS) assays for identification of low-molecular-weight kinase inhibitors. Both procedures are performed in miniaturized kinase reaction volumes and involve the stepwise addition of test or control compounds, enzyme and substrate/ATP. Kinase reactions are stopped by subsequent addition of IMAP-binding buffer. Assay attributes of the IMAP FP and TR-FRET methodologies are described. HTS assays developed using these procedures should result in Z-factors and low assay variability necessary for robust HTS assays. Providing that the required reagents and equipment are available, one scientist should be able to develop a 384-well, miniaturized HTS assay in approximately 6-8 weeks. Specific automated HTS assay conditions will determine the number of assay plates processed in a screening session, but two scientists should expect to process between 100 and 150 assay plates in one 8-h screening day.