Analysis of the extreme diversity of salivary alpha-amylase isoforms generated by physiological proteolysis using liquid chromatography-tandem mass spectrometry

Saliva is a crucial biofluid for oral health and is also of increasing importance as a non-invasive source of disease biomarkers. Salivary alpha-amylase is an abundant protein in saliva, and changes in amylase expression have been previously associated with a variety of diseases and conditions. Salivary alpha-amylase is subject to a high diversity of post-translational modifications, including physiological proteolysis in the oral cavity. Here we developed methodology for rapid sample preparation and non-targeted LC-ESI-MS/MS analysis of saliva from healthy subjects and observed an extreme diversity of alpha-amylase proteolytic isoforms. Our results emphasize the importance of consideration of post-translational events such as proteolysis in proteomic studies, biomarker discovery and validation, particularly in saliva. (C) 2012 Elsevier B.V. All rights reserved.

Reading and writing PE-files with PERWAPI

PERWAPI is a component for reading and writing .NET PE-files. The name is a compound acronym for Program Executable – Reader/Writer – Application Programming Interface. The code was written by one of us (Diane Corney) with some contributions from some of the early users of the tool. PERWAPI is a managed component, written entirely in safe C#. The design of the writer part of the component is loosely based on Diane Corney’s previous PEAPI component. It is open source software, and is released under a “FreeBSD-like” license. The source may be downloaded from “http://plas.fit.qut.edu.au/perwapi/” As of the date of this document the code has facilities for reading and writing PEfiles compatible with the latest (beta-2) release of the ”Whidbey” version of .NET, that is, the Visual Studio 2005 framework. An invocation option allows earlier versions of the framework to be targeted.

Vitronectin modulates human mesenchymal stem cell response to insulin-like growth factor-I and transforming growth factor beta 1 in a serum-free environment

The use of animal sera for the culture of therapeutically important cells impedes the clinical use of the cells. We sought to characterize the functional response of human mesenchymal stem cells (hMSCs) to specific proteins known to exist in bone tissue with a view to eliminating the requirement of animal sera. Insulin-like growth factor-I (IGF-I), via IGF binding protein-3 or -5 (IGFBP-3 or -5) and transforming growth factor-beta 1 (TGF-beta(1)) are known to associate with the extracellular matrix (ECM) protein vitronectin (VN) and elicit functional responses in a range of cell types in vitro. We found that specific combinations of VN, IGFBP-3 or -5, and IGF-I or TGF-beta(1) could stimulate initial functional responses in hMSCs and that IGF-I or TGF-beta(1) induced hMSC aggregation, but VN concentration modulated this effect. We speculated that the aggregation effect may be due to endogenous protease activity, although we found that neither IGF-I nor TGF-beta(1) affected the functional expression of matrix metalloprotease-2 or -9, two common proteases expressed by hMSCs. In summary, combinations of the ECM and growth factors described herein may form the basis of defined cell culture media supplements, although the effect of endogenous protease expression on the function of such proteins requires investigation.

An adolescent injury intervention : selecting targeted behaviours with implications for program design and evaluation

Objectives: This paper sought to identify the behaviour change targets for an injury prevention program; Skills for Preventing Injury in Youth, SPIY. The aim was to explore how such behaviours could subsequently be implemented and evaluated in the program. Methods and Design: The quantitative procedure involved a survey with 267 Year 8 and 9 students (mean age 13.23 years) regarding their engagement in risk-taking behaviours that may lead to injury. The qualitative study involved 30 students aged 14 to 17 years reporting their experiences of injury and risk-taking. Results: Injury risk behaviours co-occurred among three-quarters of those who reported engaging in any alcohol use or transport or violence related injury risk behaviour. Students described in detail some of these experiences. Conclusions: The selection process of identifying target behaviours for change for an injury prevention program is described. Adolescents’ description of such risk behaviours can inform the process of operationalising and contextualising program content and deciding on evaluation methodology. The design of an effective injury prevention program involves considerable preparatory work and this paper was able to describe the process of identifying the behavioural targets for change that can be operationalised and evaluated in the injury prevention program, SPIY.

A functional screen identifies lateral transfer of beta-glucuronidase (gus) from bacteria to fungi

Lateral gene transfer (LGT) from prokaryotes to microbial eukaryotes is usually detected by chance through genome-sequencing projects. Here, we explore a different, hypothesis-driven approach. We show that the fitness advantage associated with the transferred gene, typically invoked only in retrospect, can be used to design a functional screen capable of identifying postulated LGT cases. We hypothesized that beta-glucuronidase (gus) genes may be prone to LGT from bacteria to fungi (thought to lack gus) because this would enable fungi to utilize glucuronides in vertebrate urine as a carbon source. Using an enrichment procedure based on a glucose-releasing glucuronide analog (cellobiouronic acid), we isolated two gus(+) ascomycete fungi from soils (Penicillium canescens and Scopulariopsis sp.). A phylogenetic analysis suggested that their gus genes, as well as the gus genes identified in genomic sequences of the ascomycetes Aspergillus nidulans and Gibberella zeae, had been introgressed laterally from high-GC gram(+) bacteria. Two such bacteria (Arthrobacter spp.), isolated together with the gus(+) fungi, appeared to be the descendants of a bacterial donor organism from which gus had been transferred to fungi. This scenario was independently supported by similar substrate affinities of the encoded beta-glucuronidases, the absence of introns from fungal gus genes, and the similarity between the signal peptide-encoding 5' extensions of some fungal gus genes and the Arthrobacter sequences upstream of gus. Differences in the sequences of the fungal 5' extensions suggested at least two separate introgression events after the divergence of the two main Euascomycete classes. We suggest that deposition of glucuronides on soils as a result of the colonization of land by vertebrates may have favored LGT of gus from bacteria to fungi in soils.