Expression of mammalian glutathione S-transferase 5-5 in Salmonella typhimurium TA1535 leads to base-pair mutations upon exposure to dihalomethanes

Dihalomethanes can produce liver tumors in mice but not in rats, and concern exists about the risk of these compounds to humans. Glutathione (GSH) conjugation of dihalomethanes has been considered to be a critical event in the bioactivation process, and risk assessment is based upon this premise; however, there is little experimental support for this view or information about the basis of genotoxicity. A plasmid vector containing rat GSH S-transferase 5-5 was transfected into the Salmonella typhimurium tester strain TA1535, which then produced active enzyme. The transfected bacteria produced base-pair revertants in the presence of ethylene dihalides or dihalomethanes, in the order CH2Br2 > CH2BrCl > CH2Cl2. However, revertants were not seen when cells were exposed to GSH, CH2Br2, and an amount of purified GSH S-transferase 5-5 (20-fold excess in amount of that expressed within the cells). HCHO, which is an end product of the reaction of GSH with dihalomethanes, also did not produce mutations. S-(1-Acetoxymethyl)GSH was prepared as an analog of the putative S-(1-halomethyl)GSH reactive intermediates. This analog did not produce revertants, consistent with the view that activation of dihalomethanes must occur within the bacteria to cause genetic damage, presenting a model to be considered in studies with mammalian cells. S-(1-Acetoxymethyl)GSH reacted with 2′-deoxyguanosine to yield a major adduct, identified as S-[1-(N2-deoxyguanosinyl)methyl]GSH. Demonstration of the activation of dihalomethanes by this mammalian GSH S-transferase theta class enzyme should be of use in evaluating the risk of these chemicals, particularly in light of reports of the polymorphic expression of a similar activity in humans.

Ecionines A and B, two new cytotoxic pyridoacridine alkaloids from the Australian marine sponge, Ecionemia geodides

Chemical investigations of the Australian marine sponge Ecionemia geodides resulted in the isolation of two new pyridoacridine alkaloids, ecionines A (1) and B (2), along with the previously isolated marine natural products, biemnadin (3) and meridine (4). Compounds 1 and 2 both contain an imine moiety, which is rare for the pyridoacridine structure class. The chemical structures of 1 and 2 were determined by extensive 1D and 2D NMR and MS data analyses. All compounds were tested against a panel of human bladder cancer cell lines, the increasingly metastatic TSU-Pr1 series (TSU-Pr1, TSU-Pr1-B1 and TSU-Pr1- B2) and the superficial bladder cancer cell line 5637. Ecionine A (1) displayed cytotoxicity against all cell lines, with IC50 values ranging from 3 to 7 mM. This is the first report of chemistry from the sponge genus Ecionemia.

B-lactamase-mediated resistance to antimicrobials : the relationship between genotype and phenotype

This thesis examined the ability to predict the emergence of bacteria resistant to antibiotics using genetic markers in the bacteria. Bacteria containing the genetic markers were able to become resistant to antibiotics, whereas bacteria that did not have the genetic markers remained susceptible. Existing techniques can identify the presence of resistance by looking at the characteristics of the bacteria during growth. However, having the ability to predict antibiotic resistance before it emerges could improve the preservation of currently available antibiotics and minimise treatment failure.

Identification of eusynstyelamide B as a potent cell cycle inhibitor following the generation and screening of an ascidian-derived extract library using a real time cell analyzer

Ascidians are marine invertebrates that have been a source of numerous cytotoxic compounds. Of the first six marine-derived drugs that made anticancer clinical trials, three originated from ascidian specimens. In order to identify new anti-neoplastic compounds, an ascidian extract library (143 samples) was generated and screened in MDA-MB-231 breast cancer cells using a real-time cell analyzer (RTCA). This resulted in 143 time-dependent cell response profiles (TCRP), which are read-outs of changes to the growth rate, morphology, and adhesive characteristics of the cell culture. Twenty-one extracts affected the TCRP of MDA-MB-231 cells and were further investigated regarding toxicity and specificity, as well as their effects on cell morphology and cell cycle. The results of these studies were used to prioritize extracts for bioassay-guided fractionation, which led to the isolation of the previously identified marine natural product, eusynstyelamide B (1). This bis-indole alkaloid was shown to display an IC50 of 5 μM in MDA-MB-231 cells. Moreover, 1 caused a strong cell cycle arrest in G2/M and induced apoptosis after 72 h treatment, making this molecule an attractive candidate for further mechanism of action studies.

Circulating fragments of N-Terminal Pro-B-Type Natriuretic Peptides in plasma of heart failure patients

BACKGROUND: The use of nonstandardized N-terminal pro-B-type natriuretic peptide (NT-proBNP) assays can contribute to the misdiagnosis of heart failure (HF). Moreover, there is yet to be established a common consensus regarding the circulating forms of NT-proBNP being used in current assays. We aimed to characterize and quantify the various forms of NT-proBNP in the circulation of HF patients. METHODS: Plasma samples were collected from HF patients (n = 20) at rest and stored at -80 degrees C. NT-proBNP was enriched from HF patient plasma by use of immunoprecipitation followed by mass spectrometric analysis. Customized homogeneous sandwich AlphaLISA (R) immunoassays were developed and validated to quantify 6 fragments of NT-proBNP. RESULTS: Mass spectrometry identified the presence of several N- and C-terminally processed forms of circulating NT-proBNP, with physiological proteolysis between Pro2-Leu3, Leu3-Gly4, Pro6-Gly7, and Pro75-Arg76. Consistent with this result, AlphaLISA immunoassays demonstrated that antibodies targeting the extreme N or C termini measured a low apparent concentration of circulating NT-proBNP. The apparent circulating NT-proBNP concentration was increased with antibodies targeting nonglycosylated and nonterminal epitopes (P < 0.05). CONCLUSIONS: In plasma collected from HF patients, immunoreactive NT-proBNP was present as multiple N- and C-terminally truncated fragments of the full length NT-proBNP molecule. Immunodetection of NT-proBNP was significantly improved with the use of antibodies that did not target these terminal regions. These findings support the development of a next generation NT-proBNP assay targeting nonterminal epitopes as well as avoiding the central glycosylated region of this molecule. (c) 2013 American Association for Clinical Chemistry

In vitro investigations on the effect of dermal fibroblasts on keratinocyte responses to ultraviolet B radiation

Exposure to ultraviolet radiation is closely linked to the development of skin cancers in humans. The ultraviolet B (UVB) radiation wavelength (280-320 nm), in particular, causes DNA damage in epidermal keratinocytes, which are linked to the generation of signature premalignant mutations. Interactions between dermal fibroblasts and keratinocytes play a role in epidermal repair and regeneration after UVB-induced damage. To investigate these processes, established two and three-dimensional culture models were utilized to study the impact of fibroblast-keratinocyte crosstalk during the acute UVB response. Using a coculture system it was observed that fibroblasts enhanced keratinocyte survival and the repair of cyclobutane pyrimidine dimers (CPDs) after UVB radiation exposure. These findings were also mirrored in irradiated human skin coculture models employed in this study. Fibroblast coculture was shown to play a role in the expression and activation of members of the apoptotic cascade, including caspase-3 and Bad. Interestingly, the expression and phosphorylation of p53, a key player in the regulation of keratinocyte cell fate postirradiation, was also shown to be influenced by fibroblast-produced factors. This study highlights the importance of synergistic interactions between fibroblasts and keratinocytes in maintaining a functional epidermis while promoting repair and regeneration following UVB radiation-induced damage.

Principles of goal-directed spatial robot navigation in biomimetic models

Mobile robots and animals alike must effectively navigate their environments in order to achieve their goals. For animals goal-directed navigation facilitates finding food, seeking shelter or migration; similarly robots perform goal-directed navigation to find a charging station, get out of the rain or guide a person to a destination. This similarity in tasks extends to the environment as well; increasingly, mobile robots are operating in the same underwater, ground and aerial environments that animals do. Yet despite these similarities, goal-directed navigation research in robotics and biology has proceeded largely in parallel, linked only by a small amount of interdisciplinary research spanning both areas. Most state-of-the-art robotic navigation systems employ a range of sensors, world representations and navigation algorithms that seem far removed from what we know of how animals navigate; their navigation systems are shaped by key principles of navigation in ‘real-world’ environments including dealing with uncertainty in sensing, landmark observation and world modelling. By contrast, biomimetic animal navigation models produce plausible animal navigation behaviour in a range of laboratory experimental navigation paradigms, typically without addressing many of these robotic navigation principles. In this paper, we attempt to link robotics and biology by reviewing the current state of the art in conventional and biomimetic goal-directed navigation models, focusing on the key principles of goal-oriented robotic navigation and the extent to which these principles have been adapted by biomimetic navigation models and why.

Methylproamine protects against ionizing radiation by preventing DNA double-strand breaks

Purpose The majority of cancer patients will receive radiotherapy (RT), therefore, investigations into advances of this modality are important. Conventional RT dose intensities are limited by adverse responses in normal tissues and a primary goal is to ameliorate adverse normal tissue effects. The aim of these experiments is to further our understanding regarding the mechanism of radioprotection by the DNA minor groove binder, methylproamine, in a cellular context at the DNA level. Materials and methods We used immunocytochemical methods to measure the accumulation of phosphorylated H2AX (γH2AX) foci following ionizing radiation (IR) in patient-derived lymphoblastoid cells exposed to methylproamine. Furthermore, we performed pulsed field gel electrophoresis DNA damage and repair assays to directly interrogate the action of methylproamine on DNA in irradiated cells. Results We found that methylproamine-treated cells had fewer γH2AX foci after IR compared to untreated cells. Also, the presence of methylproamine decreased the amount of lower molecular weight DNA entering the gel as shown by the pulsed field gel electrophoresis assay. Conclusions These results suggest that methylproamine acts by preventing the formation of DNA double-strand breaks (dsbs) and support the hypothesis that radioprotection by methylproamine is mediated, at least in part, by decreasing initial DNA damage.

What is plan B? Using Foucault's archaeology to enhance policy analysis

Many governments in western democracies conduct the work of leading their societies forward through policy generation and implementation. Despite government attempts at extensive negotiation, collaboration and debate, the general populace in these same countries frequently express feelings of disempowerment and undue pressure to be compliant, often leading to disengagement. Here we outline Plan B: a process for examining how policies that emerge from good intentions are frequently interpreted as burdensome or irrelevant by those on whom they have an impact. Using a case study of professional standards for teachers in Australia, we describe how we distilled Foucault’s notions of archaeology into a research approach centring on the creation of ‘polyhedrons of intelligibility’ as an alternative approach by which both policy makers and those affected by their policies may understand how their respective causes are supported and adversely affected.

Factors influencing robustness and effectiveness of conditional random fields in active learning frameworks

Active learning approaches reduce the annotation cost required by traditional supervised approaches to reach the same effectiveness by actively selecting informative instances during the learning phase. However, effectiveness and robustness of the learnt models are influenced by a number of factors. In this paper we investigate the factors that affect the effectiveness, more specifically in terms of stability and robustness, of active learning models built using conditional random fields (CRFs) for information extraction applications. Stability, defined as a small variation of performance when small variation of the training data or a small variation of the parameters occur, is a major issue for machine learning models, but even more so in the active learning framework which aims to minimise the amount of training data required. The factors we investigate are a) the choice of incremental vs. standard active learning, b) the feature set used as a representation of the text (i.e., morphological features, syntactic features, or semantic features) and c) Gaussian prior variance as one of the important CRFs parameters. Our empirical findings show that incremental learning and the Gaussian prior variance lead to more stable and robust models across iterations. Our study also demonstrates that orthographical, morphological and contextual features as a group of basic features play an important role in learning effective models across all iterations.

Autologous human kidney proximal tubule epithelial cells (PTEC) modulate dendritic cell (DC), T cell and B cell responses

This is a comprehensive study of human kidney proximal tubular epithelial cells (PTEC) which are known to respond to and mediate the pathological process of a range of kidney diseases. It identifies various molecules expressed by PTEC and how these molecules participate in down-regulating the inflammatory process, thereby highlighting the clinical potential of these molecules to treat various kidney diseases. In the disease state, PTEC gain the ability to regulate the immune cell responses present within the interstitium. This down-regulation is a complex interaction of contact dependent/independent mechanisms involving various immuno-regulatory molecules including PD-L1, sHLA-G and IDO. The overall outcome of this down-regulation is suppressed DC maturation, decreased number of antibody producing B cells and low T cell responses. These manifestations within a clinical setting are expected to dampen the ongoing inflammation, preventing the damage caused to the kidney tissue.

Different correlation of Sphingosine-1-Phosphate receptor 1 with receptor activator of nuclear factor kappa B ligand and regulatory T cells in rat periapical lesions

Introduction Sphingosine-1-phosphate receptor 1 (S1P1) is crucial for regulation of immunity and bone metabolism. This study aimed to investigate the expression of S1P1 in rat periapical lesions and its relationship with receptor activator of nuclear factor kappa B ligand (RANKL) and regulatory T (Treg) cells. Methods Periapical lesions were induced by pulp exposure in the first lower molars of 55 Wistar rats. Thirty rats were killed on days 0, 7, 14, 21, 28, and 35, and their mandibles were harvested for x-ray imaging, micro–computed tomography scanning, histologic observation, immunohistochemistry, enzyme histochemistry, and double immunofluorescence analysis. The remaining 25 rats were killed on days 0, 14, 21, 28, and 35, and mandibles were harvested for flow cytometry. Results The volume and area of the periapical lesions increased from day 0 to day 21 and then remained comparably stable after day 28. S1P1-positive cells were observed in the inflammatory periapical regions; the number of S1P1-positive cells peaked at day 14 and then decreased from day 21 to day 35. The distribution of S1P1-positive cells was positively correlated with the dynamics of RANKL-positive cells but was negatively correlated with that of Treg cells. Conclusions S1P1 expression was differentially correlated with RANKL and Treg cell infiltration in the periapical lesions and is therefore a contributing factor to the pathogenesis of such lesions.

Translating biomechanics research into clinical practice

There is a large amount of research conducted each year examining every aspect of the mechanics of the human body and its interaction with medical devices and the environment; from the cellular level through to the whole body. While, as researchers, we obtain great pleasure from conducting studies and creating new knowledge we need to keep in mind that while this is a good thing it is even better if this new knowledge can lead to improvement in the quality of life for individuals suffering from biomechanical disorders. Such that while commercialisation is a good aim, not all research leads to marketable outcomes. However, it can lead to improvements in surgical techniques and clinical practice. It is important for us to identify and promote how the outcomes of research lead to improvements in quality of care, as this is perhaps the most important outcome for individual patients.

B-adrenoceptor determinants of contractility in the human heart: The role of phosphodiesterase enzymes

This thesis investigated how enzymes called phosphodiesterases control changes in contractility mediated by noradrenaline and adrenaline through activation of β1- and β2-adrenoceptors in live human heart tissue from patients with advanced heart failure undergoing transplantation. The study compared patients who had been administered β-blocker medicines metoprolol or carvedilol or no β-blocker treatment. This work helped to further elucidate the complex roles of target receptors and enzymes that are integral to the progression of heart failure, to compare the mechanisms of action of β-blockers currently used to manage heart failure and to identify new drug targets for heart failure treatment.