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Background: Rapid reperfusion with percutaneous coronary intervention (PCI) is vital for patients with ST segment elevation myocardial infarction (STEMI). However, the guideline-recommended time targets are regularly exceeded. The goal of this study was to gain insight into how Dutch PCI centres try to achieve these time targets by comparing their care processes with one another and with the European guideline-recommended process. In addition, accelerating factors perceived by care providers were identified., Methods: In this multiple case study, interviews with STEMI care providers were conducted, transcribed and used to create process descriptions per centre. Analyses consisted of within-case and between-case analyses of the processes. Accelerating factors were identified by means of open and axial coding., Results: In total, 28 interviews were conducted in six PCI centres. The centres differed from the guideline-recommended process on, for example, additional, unavoidable patient routings and monitoring delays, and from one another on the communication of diagnostic information (eg, transmitting all, only ambiguous or no ECGs) and catheterisation room preparation. These differences indicated diverging choices to maintain a balance between speed and diagnostic accuracy. Factors perceived by care providers as accelerating the process included trust in the tentative diagnosis, and avoiding unnecessary intercaregiver consultations. The combination of processes and accelerating factors were summarised in a model., Conclusions: Numerous differences in processes between PCI centres were identified. Several time-saving strategies were applied by PCI centres, however, in different configurations. To further improve the care for patients with STEMI, best practices can be shared between centres and countries., Competing Interests: Competing interests: None declared.

Background: A short delay between diagnosis and treatment for patients diagnosed with ST-elevation myocardial infarction (STEMI) is vital to prevent cardiac damage and mortality. The objective of this study was to explore the treatment delay and associated factors in the management of patients diagnosed with STEMI going for percutaneous coronary intervention (PCI)., Methods: In a cross-sectional multicenter study, the treatment delay (time between first electrocardiogram and start of PCI procedure) of STEMI patients in seven PCI centers in the Netherlands was measured. Data were analyzed by means of multivariable generalized linear models, accounting for a non-normally distributed outcome and clustering of patients within centers., Results: In total, 1017 patient charts were included. The majority of the patients (78.7%) were treated within the guideline recommended time target of 90 min. Overall, the median treatment delay was 64 min (interquartile range 47-82). A significantly prolonged delay was found among patients of whom their first electrocardiogram was performed at a general practitioner's practice (+23.9 min; 95% confidence interval 9.9-40.8) or in-hospital (+9.5 min; 95% confidence interval 2.5-17.3), patients requiring interhospital transfer (+14.6 min; 95% confidence interval 7.6-22.4) or presenting with acute heart failure on admission (+17.6 min; 95% confidence interval 7.9-28.7)., Conclusions: Despite a short median delay between first electrocardiogram and PCI, the time targets are occasionally exceeded for patients diagnosed with STEMI. To further improve the process of care, PCI centers should focus on improving regional STEMI care networks, involving general practitioners, emergency departments and referring hospitals.

An underexposed ethical issue raised by tissue engineering is the use of laboratory animals in tissue engineering research. Even though this research results in suffering and loss of life in animals, tissue engineering also has great potential for the development of alternatives to animal experiments. With the objective of promoting a joint effort of tissue engineers and alternative experts to fully realise this potential, this study provides the first comprehensive overview of the possibilities of using tissue-engineered constructs as a replacement of laboratory animals. Through searches in two large biomedical databases (PubMed, Embase) and several specialised 3R databases, 244 relevant primary scientific articles, published between 1991 and 2011, were identified. By far most articles reviewed related to the use of tissue-engineered skin/epidermis for toxicological applications such as testing for skin irritation. This review article demonstrates, however, that the potential for the development of alternatives also extends to other tissues such as other epithelia and the liver, as well as to other fields of application such as drug screening and basic physiology. This review discusses which impediments need to be overcome to maximise the contributions that the field of tissue engineering can make, through the development of alternative methods, to the reduction of the use and suffering of laboratory animals., (Copyright © 2013 John Wiley & Sons, Ltd.)

Cardiovascular disease is the leading cause of death in the western world. Heart failure is a heterogeneous and complex syndrome, arising from various etiologies, which result in cellular phenotypes that vary from patient to patient. The ability to utilize genetic manipulation and biochemical experimentation in animal models has made them indispensable in the study of this chronic condition. Similarly, proteomics has been helpful for elucidating complicated cellular and molecular phenotypes and has the potential to identify circulating biomarkers and drug targets for therapeutic intervention. In this review, the use of human samples and animal model systems (pig, dog, rat, mouse, zebrafish, and fruit fly) in cardiac research is discussed. Additionally, the protein sequence homology between these species and the extent of conservation at the level of the phospho-proteome in major kinase signaling cascades involved in heart failure are investigated., (© The Authors - PROTEOMICS Clinical Applications Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

Serum- and plasma-based biomarker discovery requires technologies with specific capabilities: sufficient proteome coverage and depth, technical reproducibly, and the scalability to enable analysis on a large number of samples at reasonable cost. We have shown that plasma samples processed using IgY LC10 Proteome Partitioning kits to remove the most highly abundant proteins selectively, followed by intact protein separation by two-dimensional liquid chromatography (2DLC, chromatofocusing, and reversed phase) can uniquely enrich for middle to lower-abundant proteins. Equally, 1DLC (reversed phase) separation of intact proteins is complementary to 2DLC. The serial use of a single piece of equipment can be prohibitively time consuming and thus, this chapter also describes the harmonization of multiple LC instruments in order to minimize technical variation and ensure reproducibility. These technical improvements allow large numbers of individual clinical samples to be analyzed with multiple instruments in a timely manner, while retaining optimal reproducibility and allowing precise differential analysis at the proteome scale.

We have implemented an orthogonal 3-D intact protein analysis system (IPAS) to quantitatively profile protein differences between human serum and plasma. Reference specimens consisting of pooled Caucasian-American serum, citrate-anticoagulated plasma, and EDTA-anticoagulated plasma were each depleted of six highly abundant proteins, concentrated, and labeled with a different Cy dye (Cy5, Cy3, or Cy2). A mixture consisting of each of the labeled samples was subjected to three dimensions of separation based on charge, hydrophobicity, and molecular mass. Differences in the abundance of proteins between each of the three samples were determined. More than 5000 bands were found to have greater than two-fold difference in intensity between any pair of labeled specimens by quantitative imaging. As expected, some of the differences in band intensities between serum and plasma were attributable to proteins related to coagulation. Interestingly, many proteins were identified in multiple fractions, each exhibiting different pI, hydrophobicity, or molecular mass. This is likely reflective of the expression of different protein isoforms or specific protein cleavage products, as illustrated by complement component 3 precursor and clusterin. IPAS provides a high resolution, high sensitivity, and quantitative approach for the analysis of serum and plasma proteins, and allows assessment of PTMs as a potential source of biomarkers.

The substantial complexity and vast dynamic range of protein abundance in biological fluids, notably serum and plasma, present a formidable challenge for comprehensive protein analysis. Integration of multiple technologies is required to achieve high-resolution and high-sensitivity proteomics analysis of biological fluids. We have implemented an orthogonal three-dimensional intact-protein analysis system (IPAS), coupled with protein tagging and immunodepletion of abundant proteins, to quantitatively profile the human plasma proteome. Following immunodepletion, plasma proteins in each of paired samples are concentrated and labeled with a different Cy dye, before mixing. Proteins are subsequently separated in three dimensions according to their charge, hydrophobicity, and molecular mass. Differences in the abundance of resolved proteins are determined based on Cy dye ratios. We have applied this strategy to profile the plasma proteome for changes that occur with acute graft-versus-host disease (GVHD), following allogeneic bone marrow transplantation (BMT). Using capillary HPLC ESI Q-TOF MS, we identified 75 proteins in the micromolar to femtomolar range that exhibited quantitative differences between the pre- and post-GVHD samples. These proteins included serum amyloid A, apolipoproteins A-I/A-IV, and complement C3 that are well-known acute-phase reactants likely reflecting the post-BMT inflammatory state. In addition, we identified some potentially interesting immunologically relevant molecules including vitamin D-binding protein, fetuin, vitronectin, proline-rich protein 3 and 4, integrin-alpha, and leukocyte antigen CD97. IPAS provides a combination of comprehensive profiling and quantitative analysis, with a substantial dynamic range, for disease-related applications.

There is currently limited data available pertaining to the global characterization of the cell surface proteome. We have implemented a strategy for the comprehensive profiling and identification of surface membrane proteins. This strategy has been applied to cancer cells, including the SH-SY5Y neuroblastoma, the A549 lung adenocarcinoma, the LoVo colon adenocarcinoma, and the Sup-B15 acute lymphoblastic leukemia (B cell) cell lines and ovarian tumor cells. Surface membrane proteins of viable, intact cells were subjected to biotinylation then affinity-captured and purified on monomeric avidin columns. The biotinylated proteins were eluted from the monomeric avidin columns as intact proteins and were subsequently separated by two-dimensional PAGE, transferred to polyvinylidene difluoride membranes, and visualized by hybridization with streptavidin-horseradish peroxidase. Highly reproducible, but distinct, two-dimensional patterns consisting of several hundred biotinylated proteins were obtained for the different cell populations analyzed. Identification of a subset of biotinylated proteins among the different cell populations analyzed using matrix-assisted laser desorption ionization and tandem mass spectrometry uncovered proteins with a restricted expression pattern in some cell line(s), such as CD87 and the activin receptor type IIB. We also identified more widely expressed proteins, such as CD98, and a sushi repeat-containing protein, a member of the selectin family. Remarkably, a set of proteins identified as chaperone proteins were found to be highly abundant on the cell surface, including GRP78, GRP75, HSP70, HSP60, HSP54, HSP27, and protein disulfide isomerase. Comprehensive profiling of the cell surface proteome provides an effective approach for the identification of commonly occurring proteins as well as proteins with restricted expression patterns in this compartment.

Hypermethylation of CpG islands, resulting in the inactivation of tumor suppressor genes, is an early event in the development of some malignancies. Recent studies suggest that this abnormal methylation may be a function of aging. The number of CpG islands that methylate with age is unknown. We used restriction landmark genome scanning (RLGS) to approximate the extent to which CpG islands change methylation status during aging. Comparison of more than 2000 loci in T lymphocytes isolated from newborn, middle age, and elderly people revealed that 29 loci ( approximately 1%) changed methylation status during aging, with 23 increasing methylation, and six decreasing. The same subset also changed methylation status with age in the esophagus, lung, and pancreas, but in variable directions. Virtual genome scanning identified one of these loci as a member of the forkhead family, recently implicated in aging, and another as an EST fragment. The methylation status of both correlated with level of expression. Confirming studies in multiple tissues from normal and DNMT1(+/-) mice demonstrated only one age dependent change in the methylation of more than 2000 loci, occurring in liver and kidney. These results indicate that the methylation status of the majority of CpG islands in both mice and humans is tightly controlled during aging, and that changes are infrequent and in humans confined to a specific subset of genes.

We have developed a comprehensive approach to identifying molecular changes in lung cancer that includes both genomic and proteomic analyses. The related effort has produced a large amount of data pertaining to gene expression at the RNA and protein levels. As a result, we have constructed a database that contains protein expression data on lung cancer as well as other relevant data including DNA microarray derived data. A large number of proteins that are expressed in different types of lung cancer have been identified and have been correlated with the expression measures for their corresponding genes at the RNA level. The database is intended to facilitate our effort at developing novel classification schemes for lung cancer and the identification of novel markers for early diagnosis.