Chironomus riparius (Diptera) genome sequencing reveals the impact of minisatellite transposable elements on population divergence

Active transposable elements (TEs) may result in divergent genomic insertion and abundance patterns among conspecific populations. Upon secondary contact, such divergent genetic backgrounds can theoretically give rise to classical Dobzhansky–Muller incompatibilities (DMI), thus contributing to the evolution of endogenous genetic barriers and eventually causing population divergence. We investigated differential TE abundance among conspecific populations of the nonbiting midge Chironomus riparius and evaluated their potential role in causing endogenous genetic incompatibilities between these populations. We focussed on a Chironomus‐specific TE, the minisatellite‐like Cla‐element, whose activity is associated with speciation in the genus. Using a newly generated and annotated draft genome for a genomic study with five natural C. riparius populations, we found highly population‐specific TE insertion patterns with many private insertions. A significant correlation of the pairwise F_ST estimated from genomewide single‐nucleotide polymorphisms (SNPs) and the F_ST estimated from TEs is consistent with drift as the major force driving TE population differentiation. However, the significantly higher Cla‐element F_ST level due to a high proportion of differentially fixed Cla‐element insertions also indicates selection against segregating (i.e. heterozygous) insertions. With reciprocal crossing experiments and fluorescent in situ hybridization of Cla‐elements to polytene chromosomes, we documented phenotypic effects on female fertility and chromosomal mispairings. We propose that the inferred negative selection on heterozygous Cla‐element insertions may cause endogenous genetic barriers and therefore acts as DMI among C. riparius populations. The intrinsic genomic turnover exerted by TEs may thus have a direct impact on population divergence that is operationally different from drift and local adaptation.     

Toward a Choice-Based Judgment Bias Task for Horses

Judgment bias tasks for nonhuman animals are promising tools to assess emotional valence as a measure of animal welfare. In view of establishing a valid judgment bias task for horses, the present study aimed to evaluate 2 versions (go/no-go and active choice) of an auditory judgment bias task for horses in terms of acquisition learning and discrimination of ambiguous cues. Five mares and 5 stallions were randomly assigned to the 2 designs and trained for 10 trials per day to acquire different operant responses to a low-frequency tone and a high-frequency tone, respectively. Following acquisition learning, horses were tested on 4 days with 3 ambiguous-tone trials interspersed between the 10 high-tone and low-tone trials. All 5 go/no-go horses but only one active-choice horse successfully learned their task, indicating that it is more difficult to train horses on an active choice task than on a go/no-go task. During testing, however, go/no-go horses did not differentiate between the 3 different ambiguous cues, thereby making the validity of the test results questionable in terms of emotional valence.     

Exclusive and complete introduction of amino groups and their N-sulfo and N-carboxymethyl groups into the 6-position of cellulose without the use of protecting groups

A new regioselective synthesis of 6-amino-6-deoxycellulose with a DS 1.0 (degree of substitution) at C-6, and its 6-N-sulfonated and its 6-N-carboxymethylated derivatives, without using protecting groups is described in this paper. The reaction conditions were optimized for preparing cellulose tosylate with full tosylation at C-6 and partial tosylation at C-2 and C-3. The nucleophilic substitution (S(N)) reaction of the tosyl group by NaN(3) at low temperature of 50 degrees C in Me(2)SO was achieved completely at C-6, whereas the tosyl groups at C-2 and C-3 were not displaced. In contrast to this, at 100 degrees C the tosyl groups at C-6, and also those at C-2 and C-3, were replaced by azido groups. This regioselective reaction that depends on temperature makes it possible to reach a selective and quantitative S(N) reaction at C-6 at low temperatures. In the subsequent reduction step with LiAlH(4), the azido group at C-6 was reduced to the amino group, and the tosyl groups at C-2 and C-3 were simultaneously completely removed. Also reported is a temperature-dependent, regioselective and complete iodination by nucleophilic substitution of the tosyl group at C-6 at 60 degrees C. At higher temperatures from 75 to 130 degrees C, substitution is also observed to occur at C-2. The selective iodination at 60 degrees C was employed to confirm the complete tosylation at C-6 of cellulose. The reaction products were identified by four different independent quantitative methods, namely 13C NMR, elemental analysis, ESCA, and fluorescence spectroscopy. 6-N-Sulfonated and 6-N-carboxymethylated cellulose derivatives were also synthesized. The new derivatives are potent candidates for structure-function studies, e.g., studies in relation to regioselectively 2-N-sulfonated and 2-N-carboxymethylated chitosan derivatives.     

Interstitial cystitis-epidemiology, diagnostic criteria, clinical markers

Knowledge of the epidemiology of interstitial cystitis (IC), the burden of the disease in the population, and the identification of possible risk factors remains largely fragmentary. The most reliable information comes from the few population-based studies that have appeared in the literature over the past 25 years, but two major issues urgently need to be addressed by appropriate epidemiologic studies: although studies find that only 10% of IC occurs in men, the hallmark symptoms of chronic prostatitis-pelvic pain, voiding dysfunction, and pain associated with sexual activity-overlap with those in men who carry the IC diagnosis. In addition, it is not known whether children suffer from the condition. The definition of IC is grounded in the symptomatology of pelvic pain and urinary frequency of a chronic nature and unexplained by any known urologic or other system pathology, but undue reliance on cystoscopic criteria has undoubtedly led to significant underdiagnosis. Efforts to identify clinical markers for diagnosis of IC are continuing and may lead the way to ascertaining the etiology and pathophysiology of IC.     

Stable isotope labeling by amino acids in cell culture,SILAC, as a simple and accurate approach to expression proteomics

Quantitative proteomics has traditionally been performed by two-dimensional gel electrophoresis, but recently, mass spectrometric methods based on stable isotope quantitation have shown great promise for the simultaneous and automated identification and quantitation of complex protein mixtures. Here we describe a method, termed SILAC, for stable isotope labeling by amino acids in cell culture, for the in vivo incorporation of specific amino acids into all mammalian proteins. Mammalian cell lines are grown in media lacking a standard essential amino acid but supplemented with a non-radioactive, isotopically labeled form of that amino acid, in this case deuterated leucine (Leu-d3). We find that growth of cells maintained in these media is no different from growth in normal media as evidenced by cell morphology, doubling time, and ability to differentiate. Complete incorporation of Leu-d3 occurred after five doublings in the cell lines and proteins studied. Protein populations from experimental and control samples are mixed directly after harvesting, and mass spectrometric identification is straightforward as every leucine-containing peptide incorporates either all normal leucine or all Leu-d3. We have applied this technique to the relative quantitation of changes in protein expression during the process of muscle cell differentiation. Proteins that were found to be up-regulated during this process include glyceraldehyde-3-phosphate dehydrogenase, fibronectin, and pyruvate kinase M2. SILAC is a simple, inexpensive, and accurate procedure that can be used as a quantitative proteomic approach in any cell culture system.     

Progression of arteriovenous bypass restenosis in mice exposed to a 50 Hz magnetic field

The controversy over whether magnetic fields (MF) produced by electrical wiring and appliances contribute to diseases such as cancer has been debated in the literature for more than 2 decades. These extremely low frequency fields at 50 or 60 Hz are omnipresent in the industrialized world and have been linked to various forms of cancer by epidemiological studies. Little has been published investigating any possible role of MF and cardiovascular disease, and this is the first study looking specifically at the effect of exposure to high-intensity MF on the development and progression of restenosis. A mouse arteriovenous bypass model was used, and mice were exposed to MF for periods of 1, 2, or 3 weeks. Neointima formation, infiltration of mononuclear cells, and heat shock protein 60 expression were all studied at the conclusion of the exposure regimen. Animals exposed to the MF for 1 week showed significantly smaller neointima formation compared with control mice exposed to a null field, although this difference was not observed in mice exposed for 2 or 3 weeks. No difference was found between mice exposed to MF and controls in any of the other parameters investigated.     

Phosphotyrosine mapping in Bcr/Abl oncoprotein using phosphotyrosine-specific immonium ion scanning

Bcr/Abl is a fusion oncoprotein that is of paramount importance in chronic myelogenous leukemia and acute lymphocytic leukemia. The tyrosine-phosphorylated fraction of the p185 form of Bcr/Abl was isolated by immunoprecipitation with an anti-phosphotyrosine antibody and SDS-PAGE. The tryptic digest of the gel-separated protein was prefractionated on POROS R2/OLIGO R3 microcolumns and subjected to phosphotyrosine mapping by precursor ion scanning in positive ion mode utilizing the immonium ion of phosphotyrosine, also called phosphotyrosine-specific immonium ion scanning, on a quadrupole time-of-flight tandem mass spectrometer. In total, nine different phosphorylated tyrosine residues were unambiguously localized in 12 different precursor ions. These phosphorylation sites correspond to three previously described phosphotyrosine residues and six novel tyrosine phosphorylation sites, and most of them were not predicted by the phosphorylation motif prediction programs ProSite, NetPhos, or ScanSite. This study shows the power of phosphotyrosine-specific immonium ion scanning for sensitive phosphotyrosine mapping when limited amounts of samples are available.     

Production of selenomethionine-labelled proteins using simplified culture conditions and generally applicable host/vector systems

The amino acid analogue selenomethionine (SeMet) is shown to be efficiently incorporated into recombinant proteins expressed in Escherichia coli grown in a simple minimal medium without the addition of synthetic amino acids. Furthermore, satisfactory SeMet incorporation is obtained with a methionine-prototrophic strain transformed with commonly used vector systems. As examples, purified tryparedoxin 1 from Crithidia fasciculata, alkylhydroperoxide reductase (AhpC) from Mycobacterium marinum and the 16-kDa antigen from M. tuberculosis are shown to be efficiently labelled with SeMet, using the culture conditions and the host/vector systems described here. Enzymatic analysis reveals no differences between native and SeMet-labelled tryparedoxin 1 enzyme. Both proteins yield crystals under similar conditions. The culture conditions and host vector systems described greatly facilitate selenium-labelling of proteins for 3-D structure determination.     

Experimental evidence of impingement induced strains at the interface and the periphery of an embedded acetabular cup implant

After total hip arthroplasty, impingement of implant components may occur during every-day patient activities causing increased shear stresses at the acetabular implant-bone interface. In the literature, impingement related lever-out moments were noted for a number of acetabular components. But there is little information about pelvic load transfer. The aim of the current study was to measure the three-dimensional strain distribution at the macrostructured hemispherical interface and in the periphery of a standard acetabular press-fit cup in an experimental implant-bone substitute model. An experimental setup was developed to simulate impingement loading via a lever arm representing the femoral component and the lower limb. In one experimental setup 12 strain gauges were embedded at predefined positions in the periphery of the acetabular cup implant inside a tray, using polyurethane composite resin as a bone substitute material. By incremental rotation of the implant tray in steps of 10 and 30 deg, respectively, the strains were measured at evenly distributed positions. With the described method 288 genuine strain values were measured in the periphery of an embedded acetabular cup implant in one experimental setup. In two additional setups the strains were evaluated at different distances from the implant interface. Both in radial and meridional interface directions strain magnitudes reach their peak near the rim of the cup below the impingement site. Values of equatorial strains vary near zero and reach their peaks near the rim of the cup on either side and in some distance from the impingement site. Interestingly, the maximum of averaged radial strains does not occur, as expected, close to the interface but at an interface offset of 5.6 mm. With the described experimental setup it is now possible to measure and display the three-dimensional strain distribution in the interface and the periphery of an embedded acetabular cup implant. The current study provides the first experimental proof of the high local stresses gradients in the direct vicinity of the impingement site. The results of the current study help for a better understanding of the impingement mechanism and its impact on acetabular cup stability.     

Recommendations for mass spectrometry data quality metrics for open access data (corollary to the Amsterdam principles)

Policies supporting the rapid and open sharing of proteomic data are being implemented by the leading journals in the field. The proteomics community is taking steps to ensure that data are made publicly accessible and are of high quality, a challenging task that requires the development and deployment of methods for measuring and documenting data quality metrics. On September 18, 2010, the U.S. National Cancer Institute (NCI) convened the "International Workshop on Proteomic Data Quality Metrics" in Sydney, Australia, to identify and address issues facing the development and use of such methods for open access proteomics data. The stakeholders at the workshop enumerated the key principles underlying a framework for data quality assessment in mass spectrometry data that will meet the needs of the research community, journals, funding agencies, and data repositories. Attendees discussed and agreed upon two primary needs for the wide use of quality metrics: (i) an evolving list of comprehensive quality metrics and (ii) standards accompanied by software analytics. Attendees stressed the importance of increased education and training programs to promote reliable protocols in proteomics. This workshop report explores the historic precedents, key discussions, and necessary next steps to enhance the quality of open access data. By agreement, this article is published simultaneously in Proteomics, Proteomics Clinical Applications, Journal of Proteome Research, and Molecular and Cellular Proteomics, as a public service to the research community. The peer review process was a coordinated effort conducted by a panel of referees selected by the journals.