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Food borne pathogens, namely the Gram-positive bacterium Bacillus subtilis and the Gram- negative bacterium Escherichia coli, were grown under the inhibition with four different disinfectants based on chloride and bromide salts of pyridinium oxime. Bacterial samples were subjected to the sequential extraction of proteins and the in-solution tryptic digestion of obtained extracts was performed prior to the identification of proteins with LC-ESI-MS/MS. Proteomic analysis identified up- and down- regulated proteins in these bacteria after treatment with each compound. The tables with differently expressed proteins are presented with this article.

Expanding the sequencing depth of the peptides with a statistically significant quantitative change derived from a biological stimulation is critical. Here we demonstrate that optimization of LC gradient and analytical column construction can reveal over 30,000 unique peptides and 23,000 phosphopeptides at high confidence. The quantitative reproducibility of different analytical workflows was evaluated by comparing the phosphoproteome of CD3/4 stimulated and unstimulated T-cells as a model system. A fritless, 50 cm-long column packed with 1.9 μm particles operated with a standard pressure HPLC significantly improved the sequencing depth 51% and decreased the selected ion chromatogram peak spreading. Most importantly, under the optimal workflow we observed an improvement of over 300% in detection of significantly changed phosphopeptides in the stimulated cells compared with the other workflows. The discovery power of the optimized column configuration was illustrated by identification of significantly altered phosphopeptides harboring novel sites from proteins previously established as important in T cell signaling including A-Raf, B-Raf, c-Myc, CARMA1, Fyn, ITK, LAT, NFAT1/2/3, PKCα, PLCγ1/2, RAF1, and SOS1. Taken together, our results reveal the analytical power of optimized chromatography using sub 2 μm particles for the analysis of the T cell phosphoproteome to reveal a vast landscape of significantly altered phosphorylation changes in response to T cell receptor stimulation.

This study investigated relationships between contact experiences and perceived threat on attitudes toward same-sex marriage and its beneficiaries. 367 self-identified heterosexual college students completed a series of measures addressing contact experiences (both positive and negative), realistic and symbolic threat from same-sex marriage, and eight outcome measures including attitudes toward same-sex marriage, stereotypes, feelings, and emotional reactions. Broadly, findings support Intergroup Contact Theory’s proposition that favorable contact experiences relate to less bias and Intergroup Threat Theory’s proposition that feelings of threat relate to prejudice. Threats demonstrated an indirect effect on the contact-prejudice relationship with more consistent effect for realistic threat.

Proteomic analysis of foodborne pathogen Listeria monocytogenes after treatment with three disinfectants based on ammonium salts of pyridoxal oxime (POD) reveal perturbation of cellular processes. These inhibitors caused disturbance in the synthesis of plasma membrane proteins and cell wall proteoglycans. Some of key proteins and proteoglycans from these two groups that are important for bacterial growth are down-regulated. Additionally, we demonstrated that the main bacterial toxin Listeriolysin O (LLO) is significantly down- regulated after treatment with each of three investigated inhibitors. These investigations confirm already postulated mechanism of action of POD-based inhibitors that results in disturbance of key cell surface proteins and proteoglycans in Gram-positive bacteria. Additionally, the use of some proteins such as LLO, as potential biomarker candidates of food poisoning with this bacterium is discussed.

A team of renowned scholars examines how sacred art and artefacts responded to the demands of a world stage in the age of reform, demonstrating the significance of religious systems for a global art history.

A comprehensive proteomic analysis of food borne pathogens after treatment with disinfectants based on ammonium salts of pyridinium oxime was performed. Changes in proteomes of the Gram-positive bacterium Bacillus subtilis and the Gram-negative one, Escherichia coli, were evaluated. Up and down- regulated proteins in these bacteria after growth under the inhibition with four different disinfectants based on chloride and bromide salts of pyridinium oxime were identified and their cellular localizations and functions were determined by gene ontology searching. Proteome changes presented here demonstrate different mechanisms of action of these disinfectants. In the Gram-positive food pathogen Bacillus subtilis, the inhibitory substances seem to act mainly at the cell surface and cause significant alterations of membrane and cell surface proteins. On the other hand, intracellular proteins were more affected in the Gram-negative pathogen Escherichia coli. This research is a contribution to the investigation of the virulence and pathogenicity of food borne bacteria and their survival under stress conditions, and can also lead the way for further development of new inhibitors of microbial growth and studies of mechanism of their actions.

The antimicrobial activity of quaternary ammonium salts of pyridinium oxime against four food pathogenic bacteria, the Gram positive Bacillus subtilis and Listeria monocytogenes, as well as the Gram negative Escherichia coli and Yersinia enterocolitica, were evaluated. Changes of proteome in these bacteria grown under stress conditions were identified. By the application of a new method for sample preparation, followed by both in-gel and in-solution digestion and LC–MS/MS, both characterization and comparison of proteomes of these food pathogens were achieved. It was shown in all investigated bacteria that some of the proteins of key importance for protein turnover and bacterial metabolism are down regulated. Some stress proteins involved in protein folding and degradation were up regulated. Most of both up- and down-regulated proteins belong to the group of proteins with high abundance. Flagellin is the only protein of lower abundance that was found to be down regulated in two strains, B. subtilis and E. coli. The presented results give the better view into the proteome of food pathogens, and pave the way for further investigation of their virulence, pathogenicity and detection of biomarkers for tracing the ways and sources of food contamination.

Strong indications have been presented that dietary poisoning with aristolochic acids (AA) is responsible for Endemic Nephropathy (EN) and AA associated cancer of the upper urinary tract (UUTC). Our recent investigation showed drastic urinary proteome changes in AA treated mice. This study was designed to identify proteome changes associated with AA nephrotoxicity in experimental animal model. The DBA and C57BL mice, which differ in AA sensitivity, were exposed to AA for 4 days. The strategy for urinary, plasma and kidney tissue proteome study of AA exposed and control mice integrated gel-based and in-solution tryptic digestion combined with LC-ESI-MS/MS. To maximize proteome coverage, plasma fractionation scheme was developed and MS compatible sequential tissue extraction procedure was established. Proteomic analyses of urinary, plasma and kidney tissue tryptic digests resulted in identification of several cytoskeletal proteins, as well as proteins involved in kidney development and inflammatory response, that are differentially expressed in both AA exposed and control mice. These proteins are consistent with renal pathogenesis of endotoxicity and cancer. This proteomic strategy could be effectively translated for unbiased discovery of potential biomarkers for EN and associated UUTC in humans. At the same time, these results highlight the significance of AA exposure with EN. This article is part of a Special Issue entitled: Integrated omics.

Sample displacement chromatography (SDC) in reversed-phase and ion-exchange modes was introduced approximately 20 years ago. This method was first used for the preparative purification of peptides and proteins. Recently, SDC in ion-exchange mode was also successfully used for enrichment of low-abundance proteins from human plasma. In this paper, the use of SDC for the separation of plasma proteins in hydrophobic interaction mode is demonstrated. By use of two or more columns coupled in series during sample application, and subsequent elution of detached columns in parallel, additional separation of bound proteins was achieved. Further low-abundance, physiologically active proteins could be highly enriched and detected by ESI-MS/MS.

Sample displacement chromatography (SDC) in reversed-phase and ion-exchange modes was introduced approximately twenty years ago. This method takes advantage of relative binding affinities of components in a sample mixture. During loading, there is a competition among different sample components for the sorption on the surface of the stationary phase. SDC was first used for the preparative purification of proteins. Later, it was demonstrated that this kind of chromatography can also be performed in ion- exchange, affinity and hydrophobic-interaction mode. It has also been shown that SDC can be performed on monoliths and membrane-based supports in both analytical and preparative scale. Recently, SDC in ion-exchange and hydrophobic interaction mode was also employed successfully for the removal of trace proteins from monoclonal antibody preparations and for the enrichment of low abundance proteins from human plasma. In this review, the principals of SDC are introduced, and the potential for separation of proteins and peptides in micro-analytical, analytical and preparative scale is discussed.

Human serum albumin (HSA) and immunoglobulin G (IgG) represent over 75% of all proteins present in human plasma. These two proteins frequently interfere with detection, determination and purification of low abundance proteins that can be potential biomarkers and biomarker candidates for various diseases. Some low abundance plasma proteins such as clotting factors and inhibitors are also important therapeutic agents. In this paper, the characterization of ion-exchange monolithic supports under overloading conditions was performed by use of sample displacement chromatography (SDC). If these supports were used for separation of human plasma, the composition of bound and eluted proteins in both anion and cation-exchange mode is dependent on column loading. Under overloading conditions, the weakly bound proteins such as HSA in anion exchange and IgG in cation-exchange mode seem to be are displaced by stronger binding proteins, and this phenomenon was not dependent on column size. Consequently, small monolithic columns with a column volume of 100 and 200 μL are ideal supports for high throughput screening in order to develop new methods for separation of complex mixtures, and for sample preparation in proteomic technology.

Proteomic methods were used to identify the levels of impurities in three commercial plasma-derived clotting factor VIII-von Willebrand factor (FVIII/VWF) concentrates. In all three concentrates, significant amounts of other plasma proteins were found. In Octanate and Haemoctin, two concentrates developed in the 1990s, the major impurities identified were inter-alpha inhibitor proteins, fibrinogen and fibronectin. These two concentrates were also found to contain additional components such as clotting factor II (prothrombin) that are known activators of FVIII. In Wilate, a recently developed FVIII/VWF concentrate, the amount of these impurities was significantly reduced. Batch-to-batch variations and differences between three investigated products were detected using iTRAQ, an isotope labeling technique for comparative mass spectrometry, demonstrating the potential value of this technique for quality control analysis. The importance of thorough proteomic investigations of therapeutic F VIII/VWF preparations from human plasma is also discussed.

Aristolochic acids (AAs) present in Aristolochia plants are substances responsible for Chinese herbs nephropathy. Recently, strong indications have also been presented, which dietary poisoning with AA is responsible for endemic (Balkan) nephropathy, an enigmatic renal disease that affects rural population living in some countries in Southeastern Europe. A mouse model was applied to follow the effects of two forms of AA, AAI and AAII. SDS-PAGE and SELDI-TOF mass spectrometry with normal phase chips were used to evaluate changes in the urine of treated animals. These two methods are demonstrated to be comparable. The use of SELDI-TOF MS for rapid analysis of a large number of samples and the combination of this method with nano-LC-ESI MS/MS for protein identification were demonstrated. Biomarker discovery after analysis of large cohort of endemic nephropathy patients will be the final aim of these investigations

La Saincte Bible published by Martin Lempereur in Antwerp in 1530 has been called the last Bible to be published without a confessional label. But its title-page carries an allegorical scene that derives from Lucas Cranach compositions, usually called Law and Grace, that are specifically Lutheran. Lempereur reused the image on his second edition of La Saincte Bible and a Latin Bible, both published in 1534; only these later editions were proscribed in the Indexes of Leuven and Antwerp, apparently because of their paratextual material rather than the title-page image. As inoffensive as Lempereur's 1530 Bible may have been to the authorities, its title-page allegory may have stimulated illicit discussion among laypersons in conventicles.

Seed coat fragments (SCF) in lint cause problems in textile mills during spinning and dyeing, so it is important to prevent or remove them during ginning. The objective of this experiment was to quantify SCF as they were formed and removed during ginning so that future studies can focus on preventing SCF formation. Samples of cotton, seed, and trash were collected throughout a gin to analyze SCF, motes, seed meats, and seed. The lint cleaner did not change the number of SCF in lint, but it reduced the weight of SCF by 56% as it removed larger SCF or broke them into smaller SCF. Seed meats in the ginned seed and small seed missing after the gin stand were estimated to be 0.8% and 2.3%, respectively, of all seed ginned, accounting for 32% and 15% of SCF formed in the gin stand, so a significant portion of SCF was attributed to seed damage not revealed by simply observing damaged seed in the seed roll. Damaged seed observed in the seed roll was 7% higher than in seed cotton, and these were thought to be a major source of SCF found in lint. The SCF mass entering the gin stand was 24% of the SCF mass exiting the gin stand, so a small but significant portion of SCF were formed before the gin stand. Seed meats found in the seed cotton cleaner waste were estimated to account for 22% of SCF found entering the gin stand. Seed damage occurred in the field, in seed cotton cleaners, and in the gin stand, so there are opportunities at each of these steps to prevent SCF in lint.

Plasma membrane proteins serve essential functions for cells, interacting with both cellular and extracellular components, structures and signaling molecules. Additionally, plasma membrane proteins comprise more than two-thirds of the known protein targets for existing drugs. Consequently, defining membrane proteomes is crucial to understanding the role of plasma membranes in fundamental biological processes and for finding new targets for action in drug development. MS-based identification methods combined with chromatographic and traditional cell-biology techniques are powerful tools for proteomic mapping of proteins from organelles. However, the separation and identification of plasma membrane proteins remains a challenge for proteomic technology because of their hydrophobicity and microheterogeneity. Creative approaches to solve these problems and potential pitfalls will be discussed. Finally, a representative overview of the impressive achievements in this field will also be given.

This chapter addresses the principles of transparency and openness and focusses on the prime application of these principles in the EEA which is access to documents. It seeks to provide the practitioner with an initial ready-reference guide to the regimes in place, including Regulation 1049/2001, but with emphasis on ESA’s Rules on Access to Documents 2012 (RAD 2012). The chapter also considers the transparency of the Court of Justice of the European Union and the EFTA Court, focussing on their archives, and also gives ideas on potential future developments in this fast-developing area of law.

Inter-alpha inhibitor proteins (IaIp) are a family of structurally related serine protease inhibitors found in relatively high concentrations in human plasma. Recent studies have implicated a role for IaIp in sepsis, and have demonstrated their potential as biomarkers in sepsis and cancer. For characterization of isolated IaI proteins and contaminating proteins during the last steps of the purification process, SELDI-TOF MS and HPLC– ESI-MS/MS were used. After separation by SDSPAGE or 2-DE, polypeptide bands of 80, 125 and 250 kDa were excised from gels and digested by trypsin. The tryptic peptides were analyzed by bothMS methods. The main contamination during the purification process, a band of 80 kDa, contains mainly IaIp heavy chain (HC) H3.HCH1 and H2 were also found in this band. In addition, some vitamin K-dependent clotting factors and inhibitors and other plasma proteins were identified. The 125-kDa band, representing the pre-alpha inhibitor, was found to contain both bikunin and HC H3. The presence of other HC H1, H2 and the recently described HC H4 was also detected by SELDI-TOF MS. The presence of HC H1, H2, and H3 in the 125-kDa band was confirmed by ESI-MS/MS, but not the presence of the H4. Three polypeptides, H1 and H2 together with bikunin, were identified in the 250-kDa band, representing the ITI, by bothMS techniques. Once again, the presence ofH4 was detected in this band only by SELDI-TOF MS, but the number of corresponding peptides was still not sufficient for final identification of this polypeptide. The importance of the application of proteomic methods for the proper evaluation of therapeutic drugs based on human plasma is discussed.

A model system for selective solubilization and fast separation of proteins from the rat liver membrane fraction and purified rat liver plasma membranes for their further proteomic analysis is presented. For selective solubilization, high-pH solutions and a concentrated urea solution, combined with different detergents, are used. After extraction, proteins are separated by anion-exchange chromatography or a combination of anion- and cation-exchange chromatography with convective interaction monolithic supports. This separation method enables fast and effective prefractionation of membrane proteins based on their hydrophobicity and charge prior to one-dimensional (1-D) and 2-D electrophoresis and mass spectrometry. By use of this sample preparation method, the less-abundant proteins can be detected and identified.

Defines on‐book and off‐book fraud: the former occurs when theft takes place after the recording of money on the victim company’s books, the latter when it occurs after recording, and types of such fraud can involve billing, payroll, expenses, cheque tampering, and register disbursement. Describes methods of investigating off‐book fraud: financial statement analysis, undercover surveillance, invigilation, and admission‐seeking interviews. Gives a case study concerning the Northern Exposure club, where so much skimming occurred that only 10% profits were being made after tax instead of the 30% expected; details how the fraud investigators proceeded by generating fraud theories based on the controls, or lack of, for monitoring cash flows, then collecting and evaluating evidence, estimating the losses incurred, and so on.

A distinct feature of human prostate cancer (PCa) is the development of osteoblastic (bone- forming) bone metastases. Metastatic growth in the bone is supported by factors secreted by PCa cells that activate signaling networks in the tumor microenvironment that augment tumor growth. To better understand these signaling networks and identify potential targets for therapy of bone metastases, we characterized the secretome of a patient-derived xenograft, MDA-PCa-118b (PCa-118b), generated from osteoblastic bone lesion. PCa-118b induces osteoblastic tumors when implanted either in mouse femurs or subcutaneously. To study signaling molecules critical to these unique tumor/microenvironment-mediated events, we performed mass spectrometry on conditioned media of isolated PCa-118b tumor cells, and identified 26 secretory proteins, such as TGF- β2, GDF15, FGF3, FGF19, CXCL1, galectins, and β2-microglobulin, which represent both novel and previously published secreted proteins. RT- PCR using human versus mouse-specific primers showed that TGFβ2, GDF15, FGF3, FGF19, and CXCL1 were secreted from PCa-118b cells. TGFβ2, GDF15, FGF3, and FGF19 function as both autocrine and paracrine factors on tumor cells and stromal cells, that is, endothelial cells and osteoblasts. In contrast, CXCL1 functions as a paracrine factor through the CXCR2 receptor expressed on endothelial cells and osteoblasts. Thus, our study reveals a complex PCa bone metastasis secretome with paracrine and autocrine signaling functions that mediate cross-talk among multiple cell types within the tumor microenvironment.