Analysis of Human Proteome Organization Plasma Proteome Project (HUPO PPP) reference specimens using surface enhanced laser desorption/ionization-time of flight (SELDI-TOF) mass spectrometry: multi-institution correlation of spectra and identification of biomarkers

We report on a multicenter analysis of HUPO reference specimens using SELDI-TOF MS. Eight sites submitted data obtained from serum and plasma reference specimen analysis. Spectra from five sites passed preliminary quality assurance tests and were subjected to further analysis. Intralaboratory CVs varied from 15 to 43%. A correlation coefficient matrix generated using data from these five sites demonstrated high level of correlation, with values >0.7 on 37 of 42 spectra. More than 50 peaks were differentially present among the various sample types, as observed on three chip surfaces. Additionally, peaks at approximately 9200 and approximately 15,950 m/z were present only in select reference specimens. Chromatographic fractionation using anion-exchange, membrane cutoff, and reverse phase chromatography, was employed for protein purification of the approximately 9200 m/z peak. It was identified as the haptoglobin alpha subunit after peptide mass fingerprinting and high-resolution MS/MS analysis. The differential expression of this protein was confirmed by Western blot analysis. These pilot studies demonstrate the potential of the SELDI platform for reproducible and consistent analysis of serum/plasma across multiple sites and also for targeted biomarker discovery and protein identification. This approach could be exploited for population-based studies in all phases of the HUPO PPP.     

Endothelin-1 promotes myofibroblast induction through the ETA receptor via a rac/phosphoinositide 3-kinase/Akt-dependent pathway and is essential for the enhanced contractile phenotype of fibrotic fibroblasts

The endothelins are a family of endothelium-derived peptides that possess a variety of functions, including vasoconstriction. Endothelin-1 (ET-1) is up-regulated during tissue repair and promotes myofibroblast contraction and migration, hence contributing to matrix remodeling during tissue repair. Here, we show that addition of ET-1 to normal lung fibroblasts induces expression of proteins that contribute to a contractile phenotype, including alpha-smooth muscle actin (alpha-SMA), ezrin, moesin, and paxillin. We confirm that ET-1 enhances the ability of lung fibroblasts to contract extracellular matrix, a function essential for tissue repair, through induction of de novo protein synthesis. Blockade of the Akt/phosphoinositide 3-kinase (PI3-kinase) pathway with LY294002 and wortmannin prevents the ability of ET-1 to induce alpha-SMA, ezrin, paxillin, and moesin and to promote matrix contraction. Dominant negative rac and Akt blocked the ability of ET-1 to promote formation of alpha-SMA stress fibers. Using specific ET-1 receptor inhibitors, we show that ET-1 induces collagen matrix contraction through the ETA, but not the ETB, receptor. Relative to normal pulmonary fibroblasts, fibroblasts cultured from scars of patients with the fibrotic disease systemic sclerosis (scleroderma) show enhanced ET-1 expression and binding. Systemic sclerosis lung fibroblasts show increased ability to contract a collagen matrix and elevated expression of the procontractile proteins alpha-SMA, ezrin, paxillin, and moesin, which are greatly reduced by antagonizing endogenous ET-1 signaling. Thus, blocking ET-1 or the PI3-kinase/Akt cascades might be beneficial in reducing scar formation in pulmonary fibrosis.     

Cell proliferation rates in an artificial tissue-engineered environment

Worldwide, and particularly in Europe, Japan and the USA, cardiovascular disease is a major killer. It can be treated using tissue or organ transplant surgery, but donor organs may be scarce. Tissue engineering is the integration of engineering principles and biology to produce satisfactory synthetic replacement body parts, using viable cells in a suitable matrix, for regenerative medicine. The aim of this study was to measure and compare cell proliferation kinetics after different time intervals of myofibroblasts in a synthetic matrix, thus to be able to deduce the period that a transplanted-cell population can be expected to survive in a tissue-engineered environment. Porcine aortic wall cells were grown in a porous sponge scaffold, that later could be fashioned into aortic or heart valve substitutes. Freshly acquired cells were seeded on identical sponges and were grown under normal culture conditions for a period of 4 weeks. Seeding concentration was a million cells per sponge. Cells progressively populated the sponges, both covering the surface and infiltrating the depth of the matrix, via sponge pores. Samples were taken at 1 week and at 4 weeks, and the rate of cell proliferation was determined by the metaphase arrest technique. Specimens were also taken for light and electron microscopy to determine whether these transplanted cells were capable of synthesizing their own extracellular matrix.     

Impact of Cre1, Cre8 and Cre3 genes on cereal cyst nematode resistance in wheat

The cereal cyst nematode (CCN; Heterodera avenae), a root disease of cereal crops, is a major economic constraint in many wheat (Triticum aestivum)-growing areas of the world. The objective of this study was to assess the impact of the Cre1, Cre8 and Cre3 genes on CCN resistance. A population of 92 doubled-haploid (DH) lines derived from a cross between wheat cvs. Frame and Silverstar as well as 1,851 wheat breeding lines were screened for CCN resistance at the Primary Industries Research Victoria (PIRVic). A second population of 9,470 wheat breeding lines was screened at the South Australian Research and Development Institute (SARDI). Cre3 had the largest impact on reducing the number of female cysts, followed by Cre1 and Cre8. There was no significant difference in number of cysts between DH lines with or without the Cre8 marker, suggesting that the marker is not perfectly linked to Cre8. The estimated heritabilities were 0.32 in the DH population, 0.48 in the PIRVic data set and 0.32 in the SARDI data set, which confirm that this is a trait of low heritability. The repeatability of CCN resistance improved with an increase in the number of plants assessed per line—up to ten. However, 85–88% of the improvement was achieved with the assessments of the first five plants.     

Planning for psychiatric emergencies.

Hospital emergency services have been used increasingly in recent years. This has resulted in questions as to the true nature of patients'' complaints and the appropriateness of this type of care. Since the increase in the number of psychiatric emergency patients has paralleled that for patients at other types of emergency clinics a study was conducted at the Clarke Institute of Psychiatry, Toronto, to examine the situation prior to the inception in 1977 of a crisis intervention unit. It was found that most patients had both psychiatric and social difficulties, and it was considered that planning should concentrate on strategies for efficient management of the clinical problems.