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Cooled antiproton beams of unprecedented intensities in the momentum range of 1.5–15 GeV/c will be used for the P ¯ ANDA experiment at FAIR to perform high precision experiments in the charmed quark sector. The proposed P ¯ ANDA detector is a 4 π internal target spectrometer at the HESR allowing the detection and identification of neutral and charged particles generated within the total energy range of the antiproton annihilation products. The detector is divided in a forward spectrometer and a target spectrometer. The charged particle identification in the latter is performed by ring imaging Cherenkov counters employing the DIRC principle.

The PANDA experiment at the FAIR upgrade to GSI, Darmstadt, aims to study modern hadronic physics with unprecedented precision. Excellent particle identification of both charged and neutral particles is necessary to achieve the physics aims of PANDA. Two DIRC detectors are foreseen for charged particle identification, one in a barrel configuration for the central region, and the second in a disc configuration for the forward endcap. The design of the forward disc DIRC centres around the novel application of passive chromatic dispersion correction elements, allied with focussing optics, to achieve excellent resolution of Chernekov angle. A system of comprehensive prototype development will show the feasibility of the system. Key to the success of the prototype is fully understanding the production, transport and detection of the Cherenkov photons within the detector. Photon yield and its dependence on the polar angle of the detector have been measured. Observed photon yield is also the basis for subsequent studies into the benefit of chromatic dispersion correction elements, focussing and overall detector performance.

18 pags, 11 figs, 2 tabs. -- Additional Supporting Information may be found in the online version of this article, Characterization of all members of a gene family established by gene divergence is essential to delineate distinct or overlapping expression profiles and functionalities. Their activity as potent modulators of diverse physiological processes directs interest to galectins (endogenous lectins with β-sandwich fold binding β-galactosides and peptide motifs), warranting their study with the long-term aim of a comprehensive analysis. The comparatively low level of complexity of the galectin network in chicken with five members explains the choice of this organism as model. Previously, the three proto-type chicken galectins CG-1A, CG-1B, and CG-2 as well as the tandem-repeat-type CG-8 had been analyzed. Our study fills the remaining gap to determine gene structure, protein characteristics and expression profile of the fifth protein, that is, chimera-type chicken galectin-3 (CG-3). Its gene has a unique potential to generate variants: mRNA production stems from two promoters, alternative splicing of the form from the second transcription start point (tsp) can generate three mRNAs. The protein with functional phosphorylation sites in the N-terminus generated by transcription from the first tsp (tsp1CG-3) is the predominant CG-3 type present in adult tissues. Binding assays with neoglycoproteins and cultured cells disclose marked similarity to properties of human galectin-3. The expression and localization profiles as well as proximal promoter regions have characteristic features distinct from the other four CGs. This information on CG-3 completes the description of the panel of CGs, hereby setting the stage for detailed comparative analysis of the entire CG family, e.g., in embryogenesis. © 2011 Wiley-Liss, Inc., Spanish Ministry of Science and Innovation Grant number: BFU2009-10052; Grant sponsor: CIBER of Respiratory Diseases (CIBERES) (ISCII)

Reversible protein phosphorylation is a key mediator for intracellular signal transduction. Here we report an innovative method for accurate, site-specific protein phosphorylation degree determination by nanoLC-ESI-MS/MS. A stable isotope-labeled pair of peptide/phosphopeptide standards with volumetrically defined molar ratio is used as reference, providing an internal standard for both the analyte peptide and the phosphopeptide. For the preparation of one-source peptide/phosphopeptide standards, an aliquot of the labeled phosphopeptide standard is quantitatively dephosphorylated, yielding an equimolar solution of the peptide standard. Subsequently, the two solutions are mixed at a 1:1 or other volumetric ratio, which equals the molar ratio. This procedure assures a defined concentration ratio of both components that is independent from their absolute concentration. We demonstrate the applicability of the one-source peptide/phosphopeptide standard method by determining the phosphorylation degree of the signalling proteins STAT5A/B and STAT6.

During pregnancy, trophoblasts grow to adapt the feto-maternal unit to fetal requirements. Aldosterone and cortisol levels increase, the latter being inactivated by a healthy placenta. By contrast, preeclamptic placental growth is reduced while aldosterone levels are low and placental cortisol tissue levels are high due to improper deactivation. Aldosterone acts as a growth factor in many tissues, whereas cortisol inhibits growth. We hypothesized that in preeclampsia low aldosterone and enhanced cortisol availability might mutually affect placental growth and function. Proliferation of cultured human trophoblasts was time- and dose-dependently increased with aldosterone (P < 0.04 to P < 0.0001) and inhibited by spironolactone and glucocorticoids (P < 0.01). Mineralo- and glucocorticoid receptor expression and activation upon agonist stimulation was verified by visualization of nuclear translocation of the receptors. Functional aldosterone deficiency simulated in pregnant mice by spironolactone treatment (15 μg/g body weight/day) led to a reduced fetal umbilical blood flow (P < 0.05). In rat (P < 0.05; R2 = 0.2055) and human (X2 = 3.85; P = 0.0249) pregnancy, placental size was positively related to plasma aldosterone. Autocrine production of these steroid hormones was excluded functionally and via the absence of specific enzymatic transcripts for CYP11B2 and CYP11B1. In conclusion, activation of mineralocorticoid receptors by maternal aldosterone appears to be required for trophoblast growth and a normal feto-placental function. Thus, low aldosterone levels and enhanced cortisol availability may be one explanation for the reduced placental size in preeclampsia and related disorders.

Background: Currently, there are targeted therapies for estrogen receptor (ER) positive and HER2-amplified breast cancers. However, treatment options for patients with triple negative breast cancers (TNBCs), tumors that are hormone receptor negative (ER and progesterone, PR) and lack HER2 amplification, has been limited by the heterogeneity and lack of moleclular understanding of the subtypes of this disease. Use of genomic information (gene expression, copy number analysis, methylation arrays, and next-generation sequencing) will unravel the complexity of the disease and identify novel targeted therapies. Methods and Findings: In order to identify biologically distinct TNBC subgroups and associated molecular drivers, we compiled an extensive TNBC transcriptome dataset from 21 independent breast cancer studies. We performed reiterative k-means clustering analysis on a 386 TNBC gene expression profile training set and identified six stable clusters that display unique gene expression patterns and gene ontologies. An independent set of 201 TNBC gene expression profiles was used to validate the six TNBC subgroups. We identified two basal-like subgroups (BL1 and BL2) characterized by cell cycle and DNA damage response genes, two mesenchymal-like subgroups (M and MSL) enriched in cell differentiation, epithelial-mesenchymal transition and growth factor pathways, an immunomodulatory subgroup (IM) defined by immune cell surface antigens, receptors and signal transduction genes, and a luminal subgroup (LAR) driven by androgen receptor signaling. Using tumor gene expression signatures, we identified representative cell lines to model each of the TNBC subgroups and also pharmacologically targeted prominent signaling pathways. Conclusions: Through transciptome analysis we subclassified TNBC into six biological subgroups and identified potential therapeutic targets for future exploration. The data generated in this study have significant value for target selection in drug discovery, clinical trial design and selection of biomarkers that will enable the alignment of TNBC patients to the appropriate targeted therapy. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr PD01-07.

It is hypothesized that metal ion-mediated adsorption of phosphorylated peptides on stationary phases of LC-columns is the major cause for their frequently observed poor detection efficiency in LC-MS. To study this phenomenon in more detail, sample solutions spiked with metal ion-mobilizing additives were analyzed by reversed phase μLC-ICP-MS or nanoLC-ESI-MS. Using μLC-ICP-MS, metal ions were analyzed directly as atomic ions. Using electrospray ionization, either metal ion chelates or phosphopeptide standard mixtures injected in subpicomole amounts were analyzed. Deferoxamine, imidazole, ascorbate, citrate, EDTA, and the tetrapeptide pSpSpSpS were tested as sample additives for the interlinked purposes of metal ion-mobilization and improvement of phosphopeptide recovery. Iron probably represents the major metal ion contamination of reversed phase columns. Based on the certified iron level in LC-grade solvents, a daily metal ion load of10 pmol was estimated for typical nanoLC flow rates. In addition, phosphopeptide fractions from IMAC columns were identified as source for metal ion contamination of the LC column, as demonstrated for Ga(3+)-IMAC. The three metal ion-chelating additives, EDTA, citrate and pSpSpSpS, were found to perform best for improving the LC recovery of multiply phosphorylated peptides injected at subpicomole amounts. The benefits of metal ion-mobilizing LC (mimLC) characterized by metal ion complexing sample additives is demonstrated for three different instrumental setups comprising (a) a nanoUPLC-system with direct injection on the analytical column, (b) a nanoLC system with inclusion of a trapping column, and (c) the use of a HPLC-Chip system with integrated trapping and analytical column.

Iron-platinum nanoparticles embedded in a poly(methacrylic acid) (PMA) polymer shell and fluorescently labeled with the dye ATTO 590 (FePt-PMA-ATTO-2%) are investigated in terms of their intracellular localization in lung cells and potential to induce a proinflammatory response dependent on concentration and incubation time. A gold core coated with the same polymer shell (Au-PMA-ATTO-2%) is also included. Using laser scanning and electron microscopy techniques, it is shown that the FePt-PMA-ATTO-2% particles penetrate all three types of cell investigated but to a higher extent in macrophages and dendritic cells than epithelial cells. In both cell types of the defense system but not in epithelial cells, a particle-dose-dependent increase of the cytokine tumor necrosis factor alpha (TNFalpha) is found. By comparing the different nanoparticles and the mere polymer shell, it is shown that the cores combined with the shells are responsible for the induction of proinflammatory effects and not the shells alone. It is concluded that the uptake behavior and the proinflammatory response upon particle exposure are dependent on the time, cell type, and cell culture.

The ANDA experiment at FAIR will use DIRC detectors for the separation of hadrons. The compactness of the ANDA detector requires the image planes of these detectors to be placed inside the magnetic field of the solenoid. Due to this and other boundary conditions MCP-PMTs were identified as the only suitable photon sensors. Until recently the major obstacle for an application of MCP-PMTs in high rate experiments like ANDA were serious aging problems which led to damage at the photo-cathode and a fast declining quantum efficiency as the integrated anode charge (IAC) increased. With new countermeasures against the aging, in particular due to the application of an atomic layer deposition (ALD) technique to coat the MCP pores, the lifetime of MCP-PMTs has meanwhile increased by a factor >50 which is fully sufficient for ANDA. The recent results of our long-term lifetime measurements are discussed. New 2-inch MCP-PMT prototypes from Hamamatsu show an encouraging behavior. However, the currently best performing MCP-PMT is a 2-inch PHOTONIS tube with two ALD-layers which reaches an IAC of >16 C/cm2 without any visible sign of aging. In the second part of these proceedings a new data acquisition system of the PADIWA/TRB type is presented which allows a quasi-parallel measurement of many MCP-PMT performance parameters. Especially unwanted effects like dark-count rate, crosstalk, ion after-pulsing, and recoil electrons can be studied in more detail than ever before. Exemplary results for these parameters are shown. The discussed DAQ system will be used for the comprehensive data quality checks of the MCP-PMTs being built into the DIRCs.

A novel, widely applicable method for the production of absolutely quantified proteins is described, which can be used as internal standards for quantitative proteomic studies based on mass spectrometry. These standards are recombinant proteins containing an isotope label and selenomethionine. For recombinant protein expression, assembly of expression vectors fitted to cell-free protein synthesis was conducted using the gateway technology which offers fast access to a variety of genes via open reading frame libraries and an easy shuttling of genes between vectors. The proteins are generated by cell-free expression in a medium in which methionine is exchanged against selenomethionine and at least one amino acid is exchanged by a highly stable isotope labeled analogue. After protein synthesis and purification, selenium is used for absolute quantification by element mass spectrometry, while the heavy amino acids in the protein serve as reference in subsequent analyses by LC-ESI-MS or MALDI-MS. Accordingly, these standards are denominated RISQ (for recombinant isotope labeled and selenium quantified) proteins. In this study, a protein was generated containing Lys+6 ([(13)C(6)]-lysine) and Arg+10 ([(13)C(6),(15)N(4)]-arginine) so that each standard tryptic peptide contains a labeled amino acid. Apolipoprotein A1 was synthesized as RISQ protein, and its use as internal standard led to quantification of a reference material within the specified value. Owing to their cell-free expression, RISQ proteins do not contain posttranslational modifications. Thus, correct quantitative data by ESI- or MALDI-MS are restricted to quantifications based on peptides derived from unmodified regions of the analyte protein. Therefore, besides serving as internal standards, RISQ proteins stand out as new tools for quantitative analysis of covalent protein modifications.

Upon stimulation of cells with transforming growth factor beta (TGF-beta), Smad proteins form trimeric complexes and activate a broad spectrum of target genes. It remains unresolved which of the possible Smad complexes are formed in cellular contexts and how these contribute to gene expression. By combining quantitative mass spectrometry with a computational selection strategy, we predict and provide experimental evidence for the three most relevant Smad complexes in the mouse hepatoma cell line Hepa1-6. Utilizing dynamic pathway modeling, we specify the contribution of each Smad complex to the expression of representative Smad target genes, and show that these contributions are conserved in human hepatoma cell lines and primary hepatocytes. We predict, based on gene expression data of patient samples, increased amounts of Smad2/3/4 proteins and Smad2 phosphorylation as hallmarks of hepatocellular carcinoma and experimentally verify this prediction. Our findings demonstrate that modeling approaches can disentangle the complexity of transcription factor complex formation and its impact on gene expression.

A novel type of isobaric internal peptide standard for quantitative proteomics is described. The standard is a synthetic peptide derived from the target peptide by positional permutation of two amino acids. This type of internal standard is denominated minimally permutated peptide analog (MIPA). MIPA can be differentiated from their target analytes by LC-MS due to individual retention times and/or by MS/MS due to specific fragment ions. Both quantification methods are demonstrated using peptide mixtures of low and high complexity.

Advanced ceramic and metallic coatings for wear and corrosion protection of engineered structures applied by thermal spraying techniques have become a mainstay of surface engineering. Estimated global sales of thermally sprayed coatings are in the US$5 billion range per year, 50% of which are achieved by atmospheric plasma spraying. The development of suitable deposition equipment, sophisticated on-line process control, ease of application to complex surfaces, availability of a broad range of tailored feedstock materials, and sensitive characterization and quality assurance methods make novel surface coatings and their improved deposition techniques increasingly attractive. This review will cover patented progress in the field of wear- and corrosion-resistant coatings achieved during the past few years.

Purpose: Alpine ski performance relates closely to both anaerobic and aerobic capacities. During their competitive season, skiers greatly reduce endurance and weight training, and on-snow training becomes predominant. To typify this shift, we compared exhaustive ramp cycling and squat (SJ) and countermovement jumping (CMJ) performance in elite males before and after their competitive season. Results: In postseason compared with preseason: 1) maximal oxygen uptake (V[spacing dot above]O2max) normalized to bodyweight was higher (55.2 +/- 5.2 vs 52.7 +/- 3.6 mL[middle dot]kg-1[middle dot]min-1, P < 0.01), but corresponding work rate (W) was unchanged; 2) at ventilatory thresholds (VT), absolute and relative work rates were similar but heart rates were lower; 3) V[spacing dot above]O2/W slope was greater (9.59 +/- 0.6 vs 9.19 +/- 0.4 mL O2[middle dot]min-1[middle dot]W-1, P = 0.02), with similar flattening (P < 0.01) above VT1 at both time points; and 4) jump height was greater in SJ (47.4 +/- 4.4 vs 44.7 +/- 4.3 cm, P < 0.01) and CMJ (52.7 +/- 4.6 vs 50.4 +/- 5.0 cm, P < 0.01). Discussion: We believe that aerobic capacity and leg power were constrained in preseason and that improvements primarily reflected an in-season recovery from a fatigued state, which was caused by incongruous preseason training. Residual adaptations to high-altitude exposure in preseason could have also affected the results. Nonetheless, modern alpine skiing seemingly provides an ample cardiovascular training stimulus for skiers to maintain their aerobic capacities during the racing season. Conclusions: We conclude that aerobic fitness and leg explosiveness can be maintained in-season but may be compromised by heavy or excessive preseason training. In addition, ramp test V[spacing dot above]O2/W slope analysis could be useful for monitoring both positive and negative responses to training.

Plasmodium falciparum glideosome-associated protein 45 (PfGAP45) was in vitro phosphorylated by P. falciparum calcium-dependent protein kinase (PfCDPK1) and digested using the four proteases trypsin, chymotrypsin, AspN, and elastase. Subsequently, phosphopeptide enrichment using Ga(III) immobilized metal affinity chromatography (IMAC) was performed. The resulting fractions were analyzed using ultra performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UPLC-ESI-MS/MS), resulting in the identification of a total of nine phosphorylation sites: Ser31, Ser89, Ser103, Ser109, Ser121, Ser149, Ser156, Thr158, and Ser173. During in-depth analyses of the detected phosphopeptides, it was observed that phosphorylation alters the properties of PfGAP45 as kinase and protease substrate. The closely adjacent phosphorylation sites Ser156 (major site) and Thr158 (minor site) were analyzed in detail because at first glance the specific proteases gave highly variable results with respect to the relative abundance of these sites. It was observed that (i) formation of pSer156 and pThr158 was mutually exclusive and (ii) phosphorylation at Ser156 or Thr158 interfered specifically with proteolysis by chymotrypsin or trypsin, respectively. The latter effect was studied in detail using synthetic phosphopeptides carrying either pSer156 or pThr158 as substrate for chymotrypsin or trypsin, respectively.

Several cutaneotropic human papillomavirus (HPV) types seem to be involved in the early onset of cutaneous squamous-cell carcinoma. To test the hypothesis that cutaneotropic HPV infections are facilitated because of close and frequent skin contact (for example, between child and mother), we examined HPV prevalence in hair follicle cells from 134 volunteers (1-89 years of age, median 42 years) from 13 families. We used a high-throughput HPV-typing approach with a sensitive beta-/gamma-cutaneous PCR method, followed by reverse line blotting, to detect 30 cutaneotropic HPV types. HPV prevalence in all individuals was 42% and increased with age from 5% ator =20 years to 27% at 21-40 years, 53% at 41-60 years, and 76% at60 years. The effect of life age was significant, independent of couples and family members shown by regression analyses (Por =10(-8)). A higher similarity of HPV infection patterns was observed in couples versus two randomly chosen individuals (P/=0.05). However, the same specific HPV type was rarely found within couples or between children and their parents. Cutaneotropic HPV types are occasionally exchanged between family members during the entire lifetime, but other donors should also be considered in viral transmission.

Phospholipase C (PLC) is one signalling effector enzyme whose activity is directly modulated by opioids. Several physiological studies have implicated PLC-linked pathways in in-vivo pain regulation and opioid tolerance. Co-administration of PLC-β 2/3 activity blocker M119 with morphine resulted in a dramatic increase in morphine-induced amnesic effect in mice, proving a role for β subunit of PLC enzyme in these processes. Administration of morphine to mice at amnesic dose increased PLC-β3 activity, with respect to basal value, in the membrane-soluble material from anterior cortex and hippocampal formation in brain areas. PLC-β3 appears to be simultaneously implicated in both analgesic and amnesic effects induced by administration of morphine to mice suggesting a commonality in the molecular mechanisms of morphine-induced analgesia and memory impairment.

The powerful particle identification detection system of the P ¯ ANDA (antiProton ANihilations at DArmstadt) experiment contains three Cherenkov detectors. In the forward direction a conventional RICH detector is planned. In the high solenoid field of the target spectrometer thin Cherenkov detectors using the DIRC principle are mandatory and are envisaged in the form of a barrel and a vertical disc. The combination of the time of arrival of the photons with their spatial image determines not only the particle velocity, but also the wavelength of the photons. Therefore dispersion corrections on the lower and upper detection threshold is possible. Investigations concerning the development of a 3D-Barrel-DIRC are discussed as well as two different studies realizing a new Disc-DIRC for the forward angles. One is based on the Time of Propagation (ToP) method the other on the readout with focusing light guides.

Incomplete recovery from the LC column is identified as a major cause for poor detection efficiency of phosphopeptides by LC-MS/MS. It is proposed that metal ions adsorbed on the stationary phase interact with the phosphate group of phosphopeptides via an ion-pairing mechanism related to IMAC (IMAC: immobilized metal ion affinity chromatography). This may result in their partial or even complete retention. Addition of phosphate, EDTA or citrate to the phosphopeptide sample was tested to overcome the detrimental phosphopeptide suppression during gradient LC-MS/MS analysis, while the standard solvent composition (water, acetonitrile, formic acid) of the LC system was left unchanged. With the use of UPLC, a citrate additive was found to be highly effective in increasing the phosphopeptide detection sensitivity. Addition of EDTA was found to be comparable with respect to sensitivity enhancement, but led to fast clogging and destruction of the spray needle and analytical columns due to precipitation. In contrast, a citrate additive is compatible with prolonged and stable routine operation. A 50 mM citrate additive was tested successfully for UPLC-MS analysis of a commercial four-component phosphopeptide mixture, a tryptic beta-casein digest, and several digests of the 140 kDa protein SETDB1. In this protein, 27 phosphorylation sites could be identified by UPLC-MS/MS using addition of citrate, including the detection of several phosphopeptides carrying 3-5 pSer/pThr residues, compared to identification of only 10 sites without citrate addition. A 50 mM citrate additive particularly increases the recovery of multiply phosphorylated peptides, thus, extending the scope of phosphopeptide analysis by LC-MS/MS.

Males of advanced age represent a rapidly growing population at risk for prostate cancer. In the contemporary setting of earlier detection, a majority of prostate carcinomas are still clinically localized and often treated using radiation therapy. Our recent studies have shown that premature cellular senescence, rather than apoptosis, accounts for most of the clonogenic death induced by clinically relevant doses of irradiation in prostate cancer cells. We show here that this treatment-induced senescence was associated with a significantly increased release of exosome-like microvesicles. In premature senescence, this novel secretory phenotype was dependent on the activation of p53. In addition, the release of exosome-like microvesicles also increased during proliferative senescence in normal human diploid fibroblasts. These data support the hypothesis that senescence, initiated either by telomere attrition (e.g., aging) or DNA damage (e.g., radiotherapy), may induce a p53-dependent increase in the biogenesis of exosome-like vesicles. Ultrastructural analysis and RNA interference–mediated knockdown of Tsg101 provided significant evidence that the additional exosomes released by prematurely senescent prostate cancer cells were principally derived from multivesicular endosomes. Moreover, these exosomes were enriched in B7-H3 protein, a recently identified diagnostic marker for prostate cancer, and an abundance of what has recently been termed “exosomal shuttle RNA.” Our findings are consistent with the proposal that exosomes can transfer cargos, with both immunoregulatory potential and genetic information, between cells through a novel mechanism that may be recruited to increase exosome release during accelerated and replicative cellular senescence. [Cancer Res 2008;68(19):7864–71]

The P¯ANDA experiment at the planned FAIR facility at GSI, Darmstadt aims at measuring hadronic final states with unprecedented precision and luminosity. Superior particle identification of charged and neutral particles is mandatory to fulfil P¯ANDA's physics aims. DIRC (detection of internally reflected Cherenkov light) counters are foreseen for charged particle identification. A barrel DIRC will cover the central region while a disc DIRC will provide particle identification in the forward region. Three DIRC concepts differing in the radiator geometry and method for dispersion correction are studied. Results are presented on the common issues of radiator material and quality. Photon detection will be discussed as well.

Cooled antiproton beams of unprecedented intensities in the energy range of 1–15 GeV/ c will be used at the P ¯ ANDA experiment at FAIR to perform high precision experiments in the charmed quark sector. The charged particle identification in the barrel region needs a thin detector operating in a strong magnetic field. Both requirements can be met by a Cherenkov detector using the DIRC principle. Combining the time of arrival of the photons with their spatial image determines not only the particles velocity, but also the wavelength of the photons. Therefore, dispersion correction at the lower and upper detection thresholds is possible. Special care has to be taken to couple the photon detector to the barrel radiator bars.

Most of the proposed electron accelerator projects for future FELs, ERLs or 4th generation light sources require electron beams with an unprecedented combination of high brightness, low emittance, and high average current. In all projects photoguns will be applied: DC-photoguns, normal conducting RF-photoguns (NC-guns), and superconducting RF photoguns (SRF-guns). While the concepts of DC- and NC-guns are well proofed, the SRF-gun development still possesses a high risk. Challenges are the design of the superconducting cavity, the choice of the photocathode type, its life time, a possible cavity contamination, the difficulty of coupling high average power into the gun, and, finally, the risk of beam excitation of higher-order cavity modes. In combination with SRF linacs, the SRF-guns seem to be the best solution for high average currents. Several R&D projects of SRF-gun have been launched. In this paper, we will give an overview of the progress of the SRF photoinjector development. In detail, the technical concept, the performance and the status of the Dresden Rossendorf SRF-gun project, a collaboration of BESSY, DESY, MBI and FZD, will be presented. The main design parameters of this SRF-gun are the final electron energy of 9.5 MeV, 1 mA average current, and transverse normalized emittances (rms) of 1 mm mrad at 77 pC and 2.5 mm mrad at 1 nC bunch charge. The 1.3 GHz cavity consists of three TESLA-shaped cells, a specially designed half-cell where the photocathode is placed and a choke filter in order to prevent RF losses at the cathode side. The normal-conducting photocathode with a Cs2Te photoemission layer is cooled by liquid nitrogen. The SRF-gun cryostat consists of a stainless steel vacuum vessel, a warm magnetic shield, a liquid nitrogen-cooled thermal shield and a titanium He tank with a two-phase supply tube. The 10 kW fundamental power coupler is adopted from the ELBE cryomodule. In a first commissioning and test period the gun will be operated in parallel to the accelerator. A diagnostic beamline will allow beam parameter measurement and further optimization of the SRF-gun. In a final step, the gun will be connected to the ELBE superconducting linear accelerator to deliver an improved electron beam to the user labs.

beta-Papillomaviruses (PV) seem to be involved in the pathogenesis of cutaneous squamous cell carcinoma and its early stage actinic keratosis. In this study, typing was extended of a previously described consensus primer-mediated beta- and gamma-cutaneous HPV PCR method followed by reverse-line-blotting (BGC-PCR/RLB) to detect all 25 known beta-PV and to examine their prevalence in actinic keratosis. The typing format of the BGC-PCR assay was extended by adding hybridization probes of six beta-PV (HPV 75, 76, 80, 92, 93, and 96) to the RLB system. Subsequently, tumor and normal skin tissues were collected from 75 patients with actinic keratosis, allowing typing for a total of 25 beta- and 5 gamma-types. The analytical sensitivity was between 10 copies (HPV 75, 80, 92, 93, and 96) and 100 copies (HPV 76). Except for that of HPV 76, none of the added probes showed any cross-hybridization with other beta-HPV. HPV DNA was detected in 45% of actinic keratosis and in 33% of normal skin by BGC-PCR, and at least one of the six added beta-types was present in 19% of actinic keratoses and in 13% of normal skin. Six beta-HPV types were added successfully to the typing format of the BGC-PCR/RLB system. The potential role of these types in the development of non-melanoma skin cancer awaits further studies.

A dramatic stage-migration in diagnosis of prostate cancer has led to earlier detection of clinically localized carcinoma and an increased use of radiation therapy. The p53 protein responds to irradiation-induced DNA damage by removing critically damaged cells from the proliferative pool. This review will focus on the dominant role that p53-dependent cellular senescence, rather than cell death, plays in determining the radiosensitivity of human prostate cancer cells in vitro. The finding that senescence is a primary mechanism of tumor regression indicates that p53 activators or downstream effectors may prove effective in radiosensitizing some carcinoma of the prostate.

Introduction: AIDA v4 is an interactive educational diabetes simulator that has been made available, for over a decade, without charge via the Internet. The software is currently freely accessible at http://www.2aida.org . This report sets out a collaborative development plan to enhance the program with a new model of subcutaneous insulin absorption, which permits the simulation of rapidly acting and very long-acting insulin analogues, as well as insulin injection doses larger than 40 units. Methods: A novel, generic, physiological subcutaneous insulin absorption model is overviewed and a methodology is proposed by which this can be substituted in place of the previously adopted insulin absorption model utilized within AIDA v4.3a. Apart from this substitution it is proposed to retain the existing model of the glucoregulatory system currently used in AIDA v4.3a. Results: Initial simulation results based on bench testing of this approach using MATLAB are presented for the exogenous insulin flow profile ( Iex) following subcutaneous injections of a rapidly acting insulin analogue, a short-acting (regular) insulin preparation, intermediate-acting insulins (both Semilente and neutral protamine Hagedorn types), and a very long-acting insulin analogue. Discussion It is proposed to implement this collaborative development plan—first by bench testing the approach in MATLAB and then by integrating the generic subcutaneous insulin absorption Iex model into the AIDA simulator in Pascal. The aim is to provide enhanced functionality and educational simulations of regimens utilizing novel insulin analogues, as well as injections larger than 40 units of insulin.

Summary Background Ultraviolet radiation induces DNA damage and is the major risk factor for the development of non-melanoma skin cancer (NMSC). Different mutation rates of p53, p16INK4a and Ha-ras in cutaneous squamous cell carcinoma (SCC) and the earlier stage actinic keratosis (AK) have been reported. Objectives To assess the presence of missense mutations in hotspot exons of p53, p16INK4a and Ha-ras in low-graded AK. Patients/methods Cryo-biopsies of 75 sun-exposed AK lesions and 75 sun-shielded areas of normal skin from 75 AK patients were analysed to identify mutations in p53 (exons 7 and 8), p16INK4a (exon 2) and Ha-ras (exon 1) using polymerase chain reaction (PCR) followed by direct sequencing. As a representative subset of the specimens, ten mutation-negative AK were also micro-dissected in order to exclude the possibility that additional mutations were undetected. Results Eight missense and one nonsense point mutations were found in the 75 AK lesions examined (12%), of which seven (9%) were tumour-specific (i.e. present in AK lesions only) and two (3%) were p16INK4a mutations (i.e. also detected in normal skin). Three of the tumour-specific mutations (42%) were cytosine (C) to thymine (T) transitions at pyrimidine-rich sequences. Tumour-specific mutations were identified in 1% of p16INK4a (exon 2), 1% of Ha-ras (exon 1) and at a higher rate of 7% in p53 (exons 7 and 8), including one nonsense mutation. Conclusions The evaluation of a large number of AK specimens in this study have found a low gene mutation rate in low-graded AK lesions. p53 mutations rather than p16INK4a and/or Ha-ras mutations may be an early event in the development of AK to cutaneous SCC.

To evaluate the efficacy of AcrySof SN60WF aspheric intraocular lens (IOL) in decreasing spherical aberration and total higher order aberrations (HOAs) after cataract surgery compared to the spherical SN60AT lens.Wavefront analysis was conducted on 28 eyes of 28 patients that underwent un-complicated phacoemulsification with implantation of either SN60WF (15 eyes) or SN60AT lenses (13 eyes). Eyes with a history of uveitis, retinal diseases, and previous surgery were excluded.SN60WF eyes had less mean absolute spherical aberration than SN60AT eyes both at 4 mm (0.04+/-0.03 vs 0.11+/-0.03 RMS, p0.0001) and 6 mm pupils (0.09+/-0.04 vs 0.43+/-0.12 RMS, p0.0001). Mean total HOAs was lower in the SN60WF group at 6 mm pupils (0.44+/-0.14 vs 0.56+/-0.13 RMS, p=0.0274), while no difference was seen at 4 mm pupils (0.20+/-0.10 vs 0.25+/-0.08 RMS, p=0.160). There were no clinically significant differences between the SN60WF and SN60AT IOLs both at 4 and 6 mm pupils in terms of coma (0.16+/-0.07 vs 0.18+/-0.09 RMS, p=0.514 and 0.25+/-0.12 vs 0.23+/-0.12 RMS, p=0.664) and trefoil (0.14+/-0.09 vs 0.10+/-0.05 RMS, p=0.167 and 0.28+/-0.12 vs 0.23+/-0.07 RMS, p=0.199). There were no differences be-tween groups in mean age, axial length, postoperative spherical equivalent, IOL power, or corneal curvature.An aspheric posterior optic IOL design with thinner center effectively reduces the positive ocular spherical aberration observed in the pseudophakic and elderly eyes, especially at larger pupillary diameters (6 mm), with no notable increase in coma. However, reduction in total ocular HOAs was only significant at 6 mm pupils.

International audience; The SH2 domain-containing leukocyte protein of 76 kD (SLP-76) is a pivotal element of the signaling machinery controlling T cell receptor (TCR)-mediated activation. Here, we identify 14-3-3epsilon and zeta proteins as SLP-76 binding partners. This interaction was induced by TCR ligation and required phosphorylation of SLP-76 at serine 376. Ribonucleic acid interference and in vitro phosphorylation experiments showed that serine 376 is the target of the hematopoietic progenitor kinase 1 (HPK-1). Interestingly, either S376A mutation or HPK-1 knockdown resulted in increased TCR-induced tyrosine phosphorylation of SLP-76 and phospholipase C-gamma1. Moreover, an SLP-76-S376A mutant induced higher interleukin 2 gene transcription than wild-type SLP-76. These data reveal a novel negative feedback loop involving HPK-1-dependent serine phosphorylation of SLP-76 and 14-3-3 protein recruitment, which tunes T cell activation.

Many essential cellular functions such as growth rate, motility, and metabolic activity are linked to reversible protein phosphorylation, since they are controlled by signaling cascades based mainly on phosphorylation/dephosphorylation events. Quantification of global or site-specific protein phosphorylation is not straightforward with standard proteomic techniques. The coupling of capillary liquid chromatography (μLC) with ICP-MS (inductively coupled plasma-mass spectrometry) is a method which allows a quantitative screening of protein extracts for their phosphorus and sulfur content, and thus provides access to the protein phosphorylation degree. In extension of a recent pilot study, we analyzed protein extracts from the model organisms Arabidopsis thaliana and Chlamydomonas reinhardtii as representatives for multicellular and unicellular green photosynthetically active organisms. The results indicate that the average protein phosphorylation level of the algae C. reinhardtii is higher than that of A. thaliana. Both the average phosphorylation levels were found to be between the extreme values determined so far for prokaryotes (C. glutamicum, lowest levels) and eukaryotes (Mus musculus, highest levels). Tissue samples of A. thaliana representing different stages of plant development showed varying levels of protein phosphorylation indicating a different adjustment of the kinase/phosphatase system. We also utilized the μLC–ICP-MS technology to estimate the efficiency of a novel phosphoprotein enrichment method based on aluminum hydroxide, since the enrichment of phosphorylated species is often an essential step for their molecular characterization.

Bovine fetuin-A is a member of a glycoprotein family with a wide spectrum of functions. Until now the bovine protein has been thought to be a single-chain protein. Recently we have shown that native bovine plasma fetuin-A partially exists as a disulfide-bridged two-chain protein with a heavy N-terminal and a lighter C-terminal chain similar to the structure of human fetuin-A homologue (α2HS glycoprotein), and also is partially phosphorylated at residues Ser120, Ser302, Ser305 and Ser306 (Wind et al., Anal. Biochem. 317 (2003) 26–33). Both fetuin-A modifications, the phosphorylation at the four sites as well as the proteolysis which causes longer or shorter light chains (termed lc-1 and lc-2, respectively), are probably brought about by targeted enzymatic activities which still need to be defined. In this study we show that authentic bovine fetuin-A disulfide-bridged two-chain forms, which include the original C-terminus, were liberated from the single-chain precursor by metalloproteinases MMP-3 (stromelysin-1) and MMP-7 (matrilysin), but not by elastase, cathepsin E and cathepsin G. Peptide sequencing suggested cleavage sites chiefly at the Pro277–Ser278 or Arg294–His295 peptide bonds. Fetuin-A radioactive phosphorylation in vitro by protein kinase CK2 caused 32 P incorporation into the fetuin-A light chain lc-1 but not lc-2 or the fetuin-A heavy chain, as revealed by MMP assisted proteolysis. Analysis by nanoESI-MS pinpointed phosphorylation at the native phospho-residues Ser302, Ser305 and Ser306 by increased relative abundance following in vitro phosphorylation. Moreover, CK2 phosphorylation of synthetic C-terminal fetuin-A peptides, used as effective controls to the native protein, strongly implies that CK2 is involved in the in vivo phosphorylation of fetuin-A. The phosphorylation of N-terminally truncated peptide homologs seemed highly dependent on the sequence context N-terminal of the phosphorylation sites, thus providing a likely explanation for the non-phosphorylation of the light chain lc-2 in native fetuin-A.

Because p53 inactivation may limit the effectiveness of radiation therapy for localized prostate cancer, it is important to understand how this gene regulates clonogenic survival after an exposure to ionizing radiation. Here, we show that premature cellular senescence is the principal mode of cell death accounting for the radiosensitivity of human prostate cancer cell lines retaining p53 function. Alternative stress response pathways controlled by this tumor suppressor, including cell cycle arrest, DNA damage repair, mitotic catastrophe and apoptosis, contributed significantly less to radiation-induced clonogenic death. Using a dominant negative C-terminal fragment of p53, we present the first evidence that a complete loss of endogenous p53 function is sufficient to limit the irradiation-induced senescence and clonogenic death of prostate cancer cells. Conversely, inheritance of wild-type p53 by prostate cancer cells lacking a functional allele of this gene (i.e., DU145) significantly increases clonogenic death through p53-dependent cellular senescence and apoptotic pathways. Our data provide evidence that mutations of even one p53 allele may be sufficient to alter their clonogenic fate. In addition, they support the idea that the p53 pathway can be used as a specific target for enhancing the radiosensitivity of prostate cancer cells. Activation of p53 by the drug nutlin-3 is shown to be an effective radiosensitizer of prostate cancer cells retaining functional alleles of p53 and this effect was entirely attributable to an increased induction of p53-dependent cellular senescence.

This column continues the report of a detailed, questionnaire-based, post-release feedback survey of 200 users of the AIDA version 4 educational diabetes simulator. AIDA is a software program—avail...

The lifetime risk of developing diabetes for students born in the new millennium in the United States is estimated to be 27% to 52%. Many students need to learn about diabetes for their personal care, or desire to learn about diabetes to develop a career in healthcare. Most teenagers are adept at learning through Web-based computer tools.Twenty-one students entering 8(th) and 9(th) grades (aged 12 to 14 years old) enrolled in a Biotechnology Summer Camp focused on diabetes. Lectures on pathophysiology and clinical aspects of diabetes were followed by simulated cases using the AIDA online diabetes software simulator accessed via the internet at www.2aida.net. Two cases demonstrated glycemic effects and pharmacokinetics of insulin administration, diet, and exercise in insulin-dependent (Type 1) diabetes and non-insulin-dependent (Type 2) diabetes. Students filled out standardized evaluations at the end of the session to assess receptiveness to this type of learning; opinions on the utility, information, and ease of use; and perceived risks of using the online simulator to understand diabetes.All students were receptive to this educational tool. The majority found AIDA online useful (17/21 [81%]), educational (21/21 [100%]), worthy of wider distribution (20/21 [95%]), and would recommend the program to others with diabetes or wanting to learn about diabetes (18/21 [86%]). A minority (2/21 [9.5%]) found the program risky regarding the information given to the students. Positive comments included the ability to visualize concepts being taught in earlier lectures, and recognized the rigors required to manage diabetes. Fewer negative comments reflected frustration with the web-based user interface, the course materials, or difficulty in achieving good simulated glycemic control.Teaching pathophysiology of diabetes and pharmacology of insulin to middle school students is enhanced with the AIDA online diabetes simulator. Future versions of this program, and development of similar programs, could be useful in teaching adolescents who have diabetes, and might help stimulate interested students to learn more about the care of people with diabetes.

Proteins, such as the raf kinase inhibitory protein (RKIP), serve as modulators of signalling pathways by either promoting or inhibiting the formation of productive signalling complexes through protein–protein interactions. In the present study, the plasmodial RKIP ortholog, PfPE-PB1, was cloned, recombinantly expressed and purified to homogeneity. The purified protein was used to investigate the effect of plasmodial RKIP on the autophosphorylation and substrate phosphorylation activity of Plasmodium falciparum calcium-dependent protein kinase 1, PfCDPK1. Phosphorylation of RKIP by PfCDPK1 in in vitro kinase assays suggests that RKIP may be an in vivo substrate of this kinase, although the specific activity of PfCDPK1 is approximately seven-fold lower when RKIP, instead of casein, an exogenous substrate of this enzyme, is used as a substrate. In addition to the observed phosphorylation of RKIP itself, its presence in the assays greatly enhanced the autophosphorylation capacity of PfCDPK1 by approximately 5.5-fold. This substantial increase in autophosphorylation activity was associated with a diminished substrate phosphorylation activity of PfCDPK1 when casein was used. At the same time, RKIP phosphorylation slightly increased when casein was included into the assays. Thus, RKIP is recognized as a substrate under in vitro conditions and appears to act as a regulator of PfCDPK1 kinase activity, which possibly is one of its actual functions in the parasite.

Strong, sequence-specific gas-phase bindings between proline-rich peptides and alkaline earth metal ions in nanoESI-MS experiments were reported by Lehmann et al. (Rapid Commun. Mass Spectrom. 2006, 20, 2404-2410), however its relevance for physiological-like aqueous phase is uncertain. Therefore, the complexes should also be studied in aqueous solution and the relevance of the MS method for binding studies be evaluated. A mobility shift ACE method was used for determining the binding between the small peptide GAPAGPLIVPY and various metal ions in aqueous solution. The findings were compared to the MS results and further explained using computational methods. While the MS data showed a strong alkaline earth ion binding, the ACE results showed nonsignificant binding. The proposed vacuum state complex also decomposed during a molecular dynamic simulation in aqueous solution. This study shows that the formed stable peptide-metal ion adducts in the gas phase by ESI-MS does not imply the existence of analogous adducts in the aqueous phase. Comparing peptide-metal ion interaction under the gaseous MS and aqueous ACE conditions showed huge difference in binding behavior.

This review retraces the role of stable isotopes and mass spectrometry in the life sciences. The timeline is divided into four segments covering the years 1920-1950, 1950-1980, 1980-2000, and 2000 until today. For each period methodic progress and typical applications are discussed. Application of stable isotopes is driven by improvements of mass spectrometry, chromatography, and related fields in sensitivity, mass accuracy, structural specificity, complex sample handling ability, data output, and data evaluation. We currently experience the vision of omics-type analyses, that is, the comprehensive identification and quantification of a complete compound class within one or a few analytical runs. This development is driven by stable isotopes without competition by radioisotopes. In metabolic studies as classic field of isotopic tracer experiments, stable isotopes and radioisotopes were competing solutions, with stable isotopes as the long-term junior partner. Since the 1990s the number of metabolic studies with radioisotopes decreases, whereas stable isotope studies retain their slow but stable upward tendency. Unique fields of stable isotopes are metabolic tests in newborns, metabolic experiments in healthy controls, newborn screening for inborn errors, quantification of drugs and drug metabolites in doping control, natural isotope fractionation in geology, ecology, food authentication, or doping control, and more recently the field of quantitative omics-type analyses. There, cells or whole organisms are systematically labeled with stable isotopes to study proteomic differences or specific responses to stimuli or genetic manipulation. The duo of stable isotopes and mass spectrometry will probably continue to grow in the life sciences, since it delivers reference-quality quantitative data with molecular specificity, often combined with informative isotope effects. © 2016 Wiley Periodicals, Inc. Mass Spec Rev 36:58-85, 2017.

Triple negative breast cancer (TNBC) is a molecularly heterogeneous disease lacking recurrent targetable alterations and thus therapeutic advances have been challenging. The absence of ER, PR and HER2 amplifications, leaves combination chemotherapy as the standard of care treatment option in the adjuvant, neoadjuvant and metastatic settings. Recently, multiple studies have shed some light on the heterogeneity of TNBC and identified distinct transcriptional subtypes with unique biologies. Herein we review the molecular heterogeneity and the impact on previous and future clinical trials.

The rapid progress in high-throughput sequencing has significantly enriched our capacity for studying the mitochondrial DNA (mtDNA). In addition to performing specific mitochondrial targeted sequencing, an increasingly popular alternative approach is using the off-target reads from exome sequencing to infer mtDNA variants, including single nucleotide polymorphisms (SNPs) and heteroplasmy. However, the effectiveness and practicality of this approach have not been tested. Recently, RNAseq data have also been suggested as a good source for alternative data mining, but whether mitochondrial variants can be detected from RNAseq data has not been validated. We designed a study to evaluate the practicability of mtDNA variant detection using exome and RNA sequencing data. Five breast cancer cell lines were sequenced through mitochondrial targeted, exome, and RNA sequencing. Mitochondrial targeted sequencing was used as the gold standard to compute the validation and false discovery rates of SNP and heteroplasmy detection in exome and RNAseq data. We found that exome and RNA sequencing can accurately detect mitochondrial SNPs. However, the lower false discovery rate makes exome sequencing a better choice for heteroplasmy detection than RNAseq. Furthermore, we examined three alignment strategies and found that aligning reads directly to the mitochondrial reference genome or aligning reads to the nuclear and mitochondrial references genomes simultaneously produced the best results, and that aligning to the nuclear genome first and afterwards to the mitochondrial genome performed poorly. In conclusion, our study provides important guidelines for future studies that intend to use either exome sequencing or RNAseq data to infer mitochondrial SNPs and heteroplasmy.

The role of AnxA6 in breast cancer and in particular, the mechanisms underlying its contribution to tumor cell growth and/or motility remain poorly understood. In this study, we established the tumor suppressor function of AnxA6 in triple negative breast cancer (TNBC) cells by showing that loss of AnxA6 is associated with early onset and rapid growth of xenograft TNBC tumors in mice. We also identified the Ca2+ activated RasGRF2 as an effector of AnxA6 mediated TNBC cell growth and motility. Activation of Ca2+ mobilizing oncogenic receptors such as epidermal growth factor receptor (EGFR) in TNBC cells or pharmacological stimulation of Ca2+ influx led to activation, subsequent degradation and altered effector functions of RasGRF2. Inhibition of Ca2+ influx or overexpression of AnxA6 blocked the activation/degradation of RasGRF2. We also show that AnxA6 acts as a scaffold for RasGRF2 and Ras proteins and that its interaction with RasGRF2 is modulated by GTP and/or activation of Ras proteins. Meanwhile, down-regulation of RasGRF2 and treatment of cells with the EGFR-targeted tyrosine kinase inhibitor (TKI) lapatinib strongly attenuated the growth of otherwise EGFR-TKI resistant AnxA6 high TNBC cells. These data not only suggest that AnxA6 modulated Ca2+ influx and effector functions of RasGRF2 underlie at least in part, the AnxA6 mediated TNBC cell growth and/or motility, but also provide a rationale to target Ras-driven TNBC with EGFR targeted therapies in combination with inhibition of RasGRF2.