Proteomics Approaches in the Identification of Molecular Signatures of Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) are undifferentiated, multi-potent stem cells with the ability to renew. They can differentiate into many types of terminal cells, such as osteoblasts, chondrocytes, adipocytes, myocytes, and neurons. These cells have been applied in tissue engineering as the main cell type to regenerate new tissues. However, a number of issues remain concerning the use of MSCs, such as cell surface markers, the determining factors responsible for their differentiation to terminal cells, and the mechanisms whereby growth factors stimulate MSCs. In this chapter, we will discuss how proteomic techniques have contributed to our current knowledge and how they can be used to address issues currently facing MSC research. The application of proteomics has led to the identification of a special pattern of cell surface protein expression of MSCs. The technique has also contributed to the study of a regulatory network of MSC differentiation to terminal differentiated cells, including osteocytes, chondrocytes, adipocytes, neurons, cardiomyocytes, hepatocytes, and pancreatic islet cells. It has also helped elucidate mechanisms for growth factor–stimulated differentiation of MSCs. Proteomics can, however, not reveal the accurate role of a special pathway and must therefore be combined with other approaches for this purpose. A new generation of proteomic techniques have recently been developed, which will enable a more comprehensive study of MSCs. Keywords

(Ultra)fast catalyst-free macromolecular conjugation in aqueous environment at ambient temperature

Tailor-made water-soluble macromolecules, including a glycopolymer, obtained by living/controlled RAFT-mediated polymerization are demonstrated to react in water with diene-functionalized poly(ethylene glycol)s without pre- or post-functionalization steps or the need for a catalyst at ambient temperature. As previously observed in organic solvents, hetero-Diels-Alder (HDA) conjugations reached quantitative conversion within minutes when cyclopentadienyl moieties were involved. However, while catalysts and elevated temperatures were previously necessary for open-chain diene conjugation, additive-free HDA cycloadditions occur in water within a few hours at ambient temperature. Experimental evidence for efficient conjugations is provided via unambiguous ESI-MS, UV/vis, NMR, and SEC data.

Molecular phylogeny supports the paraphyletic nature of the genus Trogoderma (Coleoptera: Dermestidae) collected in the Australasian ecozone

To date, a molecular phylogenetic approach has not been used to investigate the evolutionary structure of Trogoderma and closely related genera. Using two mitochondrial genes, Cytochrome Oxidase I and Cytochrome B, and the nuclear gene, 18S, the reported polyphyletic positioning of Trogoderma was examined. Paraphyly in Trogoderma was observed, with one Australian Trogoderma species reconciled as sister to all Dermestidae and the Anthrenocerus genus deeply nested within the Australian Trogoderma clade. In addition, time to most recent common ancestor for a number of Dermestidae was calculated. Based on these estimations, the Dermestidae origin exceeded 175 million years, placing the origins of this family in Pangaea.

Efficient safety information retrieval on construction sites: a preliminary methodology

IT-supported field data management benefits on-site construction management by improving accessibility to the information and promoting efficient communication between project team members. However, most of on-site safety inspections still heavily rely on subjective judgment and manual reporting processes and thus observers’ experiences often determine the quality of risk identification and control. This study aims to develop a methodology to efficiently retrieve safety-related information so that the safety inspectors can easily access to the relevant site safety information for safer decision making. The proposed methodology consists of three stages: (1) development of a comprehensive safety database which contains information of risk factors, accident types, impact of accidents and safety regulations; (2) identification of relationships among different risk factors based on statistical analysis methods; and (3) user-specified information retrieval using data mining techniques for safety management. This paper presents an overall methodology and preliminary results of the first stage research conducted with 101 accident investigation reports.

Molecular investigation of the mechanical properties of single actin filaments based on vibration analyses

The mechanical vibration properties of single actin filaments from 50 to 288 nm are investigated by the molecular dynamics simulation in this study. The natural frequencies obtained from the molecular simulations agree with those obtained from the analytical solution of the equivalent Euler–Bernoulli beam model. Through the convergence study of the mechanical properties with respect to the filament length, it was found that the Euler–Bernoulli beam model can only be reliably used when the single actin filament is of the order of hundreds of nanometre scale. This molecular investigation not only provides the evidence for the use of the continuum beam model in characterising the mechanical properties of single actin filaments, but also clarifies the criteria for the effective use of the Euler–Bernoulli beam model.

Purine Nucleoside Phosphorylase mediated molecular chemotherapy and conventional chemotherapy: A tangible union against chemoresistant cancer

Background Late stage Ovarian Cancer is essentially incurable primarily due to late diagnosis and its inherent heterogeneity. Single agent treatments are inadequate and generally lead to severe side effects at therapeutic doses. It is crucial to develop clinically relevant novel combination regimens involving synergistic modalities that target a wider repertoire of cells and lead to lowered individual doses. Stemming from this premise, this is the first report of two- and three-way synergies between Adenovirus-mediated Purine Nucleoside Phosphorylase based gene directed enzyme prodrug therapy (PNP-GDEPT), docetaxel and/or carboplatin in multidrug-resistant ovarian cancer cells. Methods The effects of PNP-GDEPT on different cellular processes were determined using Shotgun Proteomics analyses. The in vitro cell growth inhibition in differentially treated drug resistant human ovarian cancer cell lines was established using a cell-viability assay. The extent of synergy, additivity, or antagonism between treatments was evaluated using CalcuSyn statistical analyses. The involvement of apoptosis and implicated proteins in effects of different treatments was established using flow cytometry based detection of M30 (an early marker of apoptosis), cell cycle analyses and finally western blot based analyses. Results Efficacy of the trimodal treatment was significantly greater than that achieved with bimodal- or individual treatments with potential for 10-50 fold dose reduction compared to that required for individual treatments. Of note was the marked enhancement in apoptosis that specifically accompanied the combinations that included PNP-GDEPT and accordingly correlated with a shift in the expression of anti- and pro-apoptotic proteins. PNP-GDEPT mediated enhancement of apoptosis was reinforced by cell cycle analyses. Proteomic analyses of PNP-GDEPT treated cells indicated a dowregulation of proteins involved in oncogenesis or cancer drug resistance in treated cells with accompanying upregulation of apoptotic- and tumour- suppressor proteins. Conclusion Inclusion of PNP-GDEPT in regular chemotherapy regimens can lead to significant enhancement of the cancer cell susceptibility to the combined treatment. Overall, these data will underpin the development of regimens that can benefit patients with late stage ovarian cancer leading to significantly improved efficacy and increased quality of life.

Molecular structure of the mineral svanbergite SrAl3(PO 4,SO4)2(OH)6 - A vibrational spectroscopic study

The mineral svanbergite SrAl 3(PO 4,SO 4) 2(OH) 6 is a hydroxy phosphate-sulphate mineral belonging to the beudantite subgroup of alunites and has been characterised by vibrational spectroscopy. Bands at various wavenumbers were assigned to the different vibrational modes of svanbergite, which were then associated with the structure of the mineral. Bands were primarily assigned to phosphate and sulphate stretching and bending modes. Two symmetric stretching modes for both phosphate and sulphate supported the concept of non-equivalent phosphate and sulphate units in the mineral structure. Bands in the OH stretching region enabled hydrogen bond distances to be calculated. Comparison of the hydrogen bond distances and the calculated hydrogen bond distances from the structure models indicates that hydrogen bonding in svanbergite occurs between the two OH units rather than OH to SO42- units.

Novel molecular markers of Chlamydia pecorum genetic diversity in the koala (Phascolarctos cinereus)

Background Chlamydia pecorum is an obligate intracellular bacterium and the causative agent of reproductive and ocular disease in several animal hosts including koalas, sheep, cattle and goats. C. pecorum strains detected in koalas are genetically diverse, raising interesting questions about the origin and transmission of this species within koala hosts. While the ompA gene remains the most widely-used target in C. pecorum typing studies, it is generally recognised that surface protein encoding genes are not suited for phylogenetic analysis and it is becoming increasingly apparent that the ompA gene locus is not congruent with the phylogeny of the C. pecorum genome. Using the recently sequenced C. pecorum genome sequence (E58), we analysed 10 genes, including ompA, to evaluate the use of ompA as a molecular marker in the study of koala C. pecorum genetic diversity. Results Three genes (incA, ORF663, tarP) were found to contain sufficient nucleotide diversity and discriminatory power for detailed analysis and were used, with ompA, to genotype 24 C. pecorum PCR-positive koala samples from four populations. The most robust representation of the phylogeny of these samples was achieved through concatenation of all four gene sequences, enabling the recreation of a "true" phylogenetic signal. OmpA and incA were of limited value as fine-detailed genetic markers as they were unable to confer accurate phylogenetic distinctions between samples. On the other hand, the tarP and ORF663 genes were identified as useful "neutral" and "contingency" markers respectively, to represent the broad evolutionary history and intra-species genetic diversity of koala C. pecorum. Furthermore, the concatenation of ompA, incA and ORF663 sequences highlighted the monophyletic nature of koala C. pecorum infections by demonstrating a single evolutionary trajectory for koala hosts that is distinct from that seen in non-koala hosts. Conclusions While the continued use of ompA as a fine-detailed molecular marker for epidemiological analysis appears justified, the tarP and ORF663 genes also appear to be valuable markers of phylogenetic or biogeographic divisions at the C. pecorum intra-species level. This research has significant implications for future typing studies to understand the phylogeny, genetic diversity, and epidemiology of C. pecorum infections in the koala and other animal species.