Comparative analysis of FimB and FimE recombinase activity

FimB and FimE are site-specific recombinases, part of the λ integrase family, and invert a 314 bp DNA switch that controls the expression of type 1 fimbriae in Escherichia coli. FimB and FimE differ in their activity towards the fim switch, with FimB catalysing inversion in both directions in comparison to the higher-frequency but unidirectional on-to-off recombination catalysed by FimE. Previous work has demonstrated that FimB, but not FimE, recombination is completely inhibited in vitro and in vivo by a regulator, PapB, expressed from a distinct fimbrial locus. The aim of this work was to investigate differences between FimB and FimE activity by exploiting the differential inhibition demonstrated by PapB. The research focused on genetic changes to the fim switch that alter recombinase binding and its structural context. FimB and FimE still recombined a switch in which the majority of fimS DNA was replaced with a larger region of non-fim DNA. This demonstrated a minimal requirement for FimB and FimE recombination of the Fim binding sites and associated inverted repeats. With the original leucine-responsive regulatory protein (Lrp) and integration host factor (IHF)-dependent structure removed, PapB was now able to inhibit both recombinases. The relative affinities of FimB and FimE were determined for the four ‘half sites’. This analysis, along with the effect of extensive swaps and duplications of the half sites on recombination frequency, demonstrated that FimB recruitment and therefore subsequent activity was dependent on a single half site and its context, whereas FimE recombination was less stringent, being able to interact initially with two half sites with equally high affinity. While increasing FimB recombination frequencies failed to overcome PapB repression, mutations made in recombinase binding sites resulted in inhibition of FimE recombination by PapB. Overall, the data support a model in which the recombinases differ in loading order and co-operative interactions. PapB exploits this difference and FimE becomes susceptible when its normal loading is restricted or changed.

Chromosomal genotoxicity of nitrobenzene and benzonitrile

In order to investigate the chromosomal genotoxicity of nitrobenzene and benzonitrile, we studied the induction of micronuclei (MN) by these test compounds in V79 cells, as well as effects on the formation and stability of microtubules and on motor protein functions. No cytotoxicity was seen in V79 cell cultures in terms of Neutral red uptake after 18 h treatment with up to 1 mM nitrobenzene or 1 mM benzonitrile. Subsequently, a concentration range up to 100 μM was used in the experiments on induction of MN. Both test compounds exhibit a weak, but definitely positive test result compared to the solvent (DMSO) control. Minimal effect concentrations of nitrobenzene and benzonitrile appeared as low as 0.01 μM, and no-effect-concentrations were between 0.001 and 0.005 μM. Clearly enhanced MN rates were found at 0.1 μM and higher. Both, nitrobenzene and benzonitrile, induced mostly kinetochor (CREST)-positive micronuclei, thus characterising the chromosomal effects as aneugenic. In cell-free assays, a slight effect on tubulin assembly was observed at 1 mM nitrobenzene without addition of DMSO. Higher concentrations (5 mM) led to secondary effects. In presence of 1% DMSO, nitrobenzene exerted no detectable effect on tubulin assembly up to the solubility limit in water of about 15 mM. For benzonitrile in presence of DMSO, a clear dose-response of inhibition of tubulin assembly at 37°C was seen above the no-effect-concentration of 2 mM, with an IC50 of 13 mM and protein denaturation starting above a level of about 20 mM. The nature of the effects of nitrobenzene and benzonitrile on the association of tubulin to form microtubules was confirmed by electron microscopy. Treatment by either 5 mM nitrobenzene or 13 mM benzonitrile plus 1% DMSO left the microtubular structure intact whereas 5 mM nitrobenzene, in absence of DMSO, led to irregular cluster formations. The experiments demonstrate that both nitrobenzene and benzonitrile, in millimolar concentration ranges, may lead to interference with tubulin assembly in a cell-free system. The functionality of the tubulin-kinesin motor protein system was assessed using the microtubule gliding assay. Nitrobenzene affected the gliding velocity in a concentration-dependent manner, starting at about 7.5 μM and reaching complete inhibition of motility at 30 μM, whereas benzonitrile up to 200 μM did not affect the kinesin-driven gliding velocity. The micronucleus assay data demonstrate a chromosomal endpoint of genotoxicity of nitrobenzene and benzonitrile. Aneugenic effects of both compounds occur at remarkably low concentrations, with lowest-effect-concentrations being 0.1 μM. This points to the relevance of interactions with the cellular spindle apparatus.

Development of a polymer theranostic for prostate cancer

Theranostics offers an improved treatment strategy for prostate cancer by facilitating simultaneous targeting of tumour cells with subsequent drug delivery and imaging. In this report we describe the synthesis of hyperbranched polymers that are biocompatible, can specifically target and be internalised by prostate cancer cells (through targeting of prostate-specific membrane antigen – PSMA) and ultimately facilitate controlled delivery of a model drug. The theranostic also incorporates a far-red fluorescent dye that allows tracking of the polymer via optical imaging. Controlled synthesis of the polymer is achieved via reversible addition fragmentation chain transfer polymerisation of polyethylene glycol monomethyl methacrylate, with ethylene glycol dimethacrylate as the branching agent. Incorporation of 20 mol% of an hydrazide-methacrylate monomer allows post-ligation of a model drug, fluorene-2-carboxaldehyde, through a hydrolytically-degradable hydrazone linkage. The rate of degradation of this particular linker was enhanced at endosomal pH (pH = 5.5) where [similar]95% of the model drug was released in 4 hours compared to less than 5% released over the same period at physiological pH. The theranostic showed high uptake into prostate cancer cells expressing prostate-specific membrane antigen, while minimal uptake was observed in PC3 cells negative for PSMA, highlighting the enhanced efficacy of the targeting ligand.

Intravenous injection of leconotide, an omega conotoxin : synergistic antihyperalgesic effects with morphine in a rat model of bone cancer pain

Objective.  Leconotide (CVID, AM336, CNSB004) is an omega conopeptide similar to ziconotide, which blocks voltage sensitive calcium channels. However, unlike ziconotide, which must be administered intrathecally, leconotide can be given intravenously because it is less toxic. This study investigated the antihyperalgesic potency of leconotide given intravenously alone and in combinations with morphine-administered intraperitoneally, in a rat model of bone cancer pain. Design.  Syngeneic rat prostate cancer cells AT3B-1 were injected into one tibia of male Wistar rats. The tumor expanded within the bone causing hyperalgesia to heat applied to the ipsilateral hind paw. Measurements were made of the maximum dose (MD) of morphine and leconotide given alone and in combinations that caused no effect in an open-field activity monitor, rotarod, and blood pressure and heart rate measurements. Paw withdrawal thresholds from noxious heat were measured. Dose response curves for morphine (0.312–5.0 mg/kg intraperitoneal) and leconotide (0.002–200 µg/kg intravenous) given alone were plotted and responses compared with those caused by morphine and leconotide in combinations. Results.  Leconotide caused minimal antihyperalgesic effects when administered alone. Morphine given alone intraperitoneally caused dose-related antihyperalgesic effects (ED50 = 2.40 ± 1.24 mg/kg), which were increased by coadministration of leconotide 20 µg/kg (morphine ED50 = 0.16 ± 1.30 mg/kg); 0.2 µg/kg (morphine ED50 = 0.39 ± 1.27 mg/kg); and 0.02 µg/kg (morphine ED50 = 1.24 ± 1.30 mg/kg). Conclusions.  Leconotide caused a significant increase in reversal by morphine of the bone cancer-induced hyperalgesia without increasing the side effect profile of either drug. Clinical Implication.  Translation into clinical practice of the method of analgesia described here will improve the quantity and quality of analgesia in patients with bone metastases. The use of an ordinary parenteral route for administration of the calcium channel blocker (leconotide) at low dose opens up the technique to large numbers of patients who could not have an intrathecal catheter for drug administration. Furthermore, the potentiating synergistic effect with morphine on hyperalgesia without increased side effects will lead to greater analgesia with improved quality of life.

Pharmacokinetics and tumor disposition of PEGylated, methotrexate conjugated poly-l-lysine dendrimers

Dendrimers have potential for delivering chemotherapeutic drugs to solid tumours via the enhanced permeation and retention (EPR) effect. The impact of conjugation of hydrophobic anticancer drugs to hydrophilic PEGylated dendrimer surfaces, however, has not been fully investigated. The current study has therefore characterised the effect on dendrimer disposition of conjugating α-carboxyl protected methotrexate (MTX) to a series of PEGylated 3H-labelled poly-L-lysine dendrimers ranging in size from generation 3 (G3) to 5 (G5) in rats. Dendrimers contained 50% surface PEG and 50% surface MTX. Conjugation of MTX generally increased plasma clearance when compared to conjugation with PEG alone. Conversely, increasing generation reduced clearance, increased metabolic stability and reduced renal elimination of the administered radiolabel. For constructs with molecular weights >20 kDa increasing the molecular weight of conjugated PEG also reduced clearance and enhanced metabolic stability but had only a minimal effect on renal elimination. Tissue distribution studies revealed retention of MTX conjugated smaller (G3-G4) PEG570 dendrimers (or their metabolic products) in the kidneys. In contrast, the larger G5 dendrimer was concentrated more in the liver and spleen. The G5 PEG1100 dendrimer was also shown to accumulate in solid Walker 256 and HT1080 tumours and comparative disposition data in both rats (1 to 2% dose/g in tumour) and mice (11% dose/g in tumour) are presented. The results of this study further illustrate the potential utility of biodegradable PEGylated poly-L-lysine dendrimers as long circulating vectors for the delivery and tumour-targeting of hydrophobic drugs.

Connecting the person with dementia and family : a feasibility study of a telepresence robot

Background Maintenance of communication is important for people with dementia living in long-term care. The purpose of this study was to assess the feasibility of using “Giraff”, a telepresence robot to enhance engagement between family and a person with dementia living in long-term care. Methods A mixed-methods approach involving semi-structured interviews, call records and video observational data was used. Five people with dementia and their family member participated in a discussion via the Giraff robot for a minimum of six times over a six-week period. A feasibility framework was used to assess feasibility and included video analysis of emotional response and engagement. Results Twenty-six calls with an average duration of 23 mins took place. Residents showed a general state of positive emotions across the calls with a high level of engagement and a minimal level of negative emotions. Participants enjoyed the experience and families reported that the Giraff robot offered the opportunity to reduce social isolation. A number of software and hardware challenges were encountered. Conclusions Participants perceived this novel approach to engage families and people with dementia as a feasible option. Participants were observed and also reported to enjoy the experience. The technical challenges identified have been improved in a newer version of the robot. Future research should include a feasibility trial of longer duration, with a larger sample and a cost analysis.

Diagnostic potential of saliva : current state and future applications

BACKGROUND: Over the past 10 years, the use of saliva as a diagnostic fluid has gained attention and has become a translational research success story. Some of the current nanotechnologies have been demonstrated to have the analytical sensitivity required for the use of saliva as a diagnostic medium to detect and predict disease progression. However, these technologies have not yet been integrated into current clinical practice and work flow. CONTENT: As a diagnostic fluid, saliva offers advantages over serum because it can be collected noninvasively by individuals with modest training, and it offers a cost-effective approach for the screening of large populations. Gland-specific saliva can also be used for diagnosis of pathology specific to one of the major salivary glands. There is minimal risk of contracting infections during saliva collection, and saliva can be used in clinically challenging situations, such as obtaining samples from children or handicapped or anxious patients, in whom blood sampling could be a difficult act to perform. In this review we highlight the production of and secretion of saliva, the salivary proteome, transportation of biomolecules from blood capillaries to salivary glands, and the diagnostic potential of saliva for use in detection of cardiovascular disease and oral and breast cancers. We also highlight the barriers to application of saliva testing and its advancement in clinical settings. SUMMARY: Saliva has the potential to become a first-line diagnostic sample of choice owing to the advancements in detection technologies coupled with combinations of biomolecules with clinical relevance. (C) 2011 American Association for Clinical Chemistry

Diagnostic potential of saliva : state of the art and future applications [Potenziale diagnostico della saliva: stato dell’arte e applicazioni future]

Over the past 10 years, the use of saliva as a diagnostic fluid has gained attention and has become a translational research success story. Some of the current nanotechnologies have been demonstrated to have the analytical sensitivity required for the use of saliva as a diagnostic medium to detect and predict disease progression. However, these technologies have not yet been integrated into current clinical practice and work flow. As a diagnostic fluid, saliva offers advantages over serum because it can be collected noninvasively by individuals with modest training, and it offers a cost-effective approach for the screening of large populations. Gland-specific saliva can also be used for diagnosis of pathology specific to one of the major salivary glands. There is minimal risk of contracting infections during saliva collection, and saliva can be used in clinically challenging situations, such as obtaining samples from children or handicapped or anxious patients, in whom blood sampling could be a difficult act to perform. In this review we highlight the production of and secretion of saliva, the salivary proteome, transportation of biomolecules from blood capillaries to salivary glands, and the diagnostic potential of saliva for use in detection of cardiovascular disease and oral and breast cancers. We also highlight the barriers to application of saliva testing and its advancement in clinical settings. Saliva has the potential to become a first-line diagnostic sample of choice owing to the advancements in detection technologies coupled with combinations of biomolecules with clinical relevance.

Treatment for prevention of HIV transmission in a localised epidemic : the case for South Australia

Background: Discussion is currently taking place among international HIV/AIDS groups around increasing HIV testing and initiating earlier use of antiretroviral therapy (ART) among people diagnosed with HIV as a method to reduce the spread of HIV. In this study, we explore the expected epidemiological impact of this strategy in a small population in which HIV transmission is predominantly confined to men who have sex with men (MSM). Methods: A deterministic mathematical transmission model was constructed to investigate the impacts of strategies that increase testing and treatment rates, and their likely potential to mitigate HIV epidemics among MSM. Our novel model distinguishes men in the population who are more easily accessible to prevention campaigns through engagement with the gay community from men who are not. This model is applied to the population of MSM in South Australia. Results: Our model-based findings suggest that increasing testing rates alone will have minimal impact on reducing the expected number of infections compared to current conditions. However, in combination with increases in treatment coverage, this strategy could lead to a 59–68% reduction in the number of HIV infections over the next 5 years. Targeting men who are socially engaged with the gay community would result in the majority of potential reductions in incidence, with only minor improvements possible by reaching all other MSM. Conclusions: Investing in strategies that will achieve higher coverage and earlier initiation of treatment to reduce infectiousness of HIV-infected individuals could be an effective strategy for reducing incidence in a population of MSM.

Triumphs and tribulations in the psychotherapy of schizophrenia : reflections on a pilot study of metacognitive narrative psychotherapy

Schizophrenia results in a profound disruption of one’s capacity to make sense of mental states, coherently narrate self-experiences, and meaningfully relate to others. While current treatment options for people with schizophrenia tend to be symptom-focused, experience in designing and implementing a study focusing on enhancing sense of self demonstrates the feasibility of developing and implementing models of treatment that prioritize the subjective distress and self-experience of people with schizophrenia. There is emerging research evidence, based upon dialogical theory of self, that posits the potential of people with deficits of self to engage in meaningful therapeutic relationships and work toward greater integrity of self and degrees of recovery. The challenge is to translate these ideas into a research methodology that can be successfully applied within therapeutic contexts with people who meet the diagnostic criteria for schizophrenia. Based upon dialogical theory, we developed a principle-based manual for metacognitive narrative psychotherapy: a psychological approach to the treatment of people with schizophrenia, which aims to enhance metacognitive capacity and ability to narrate self-experiences. Five phases of treatment were identified: (1) developing a therapeutic relationship, (2) eliciting narratives, (3) enhancing metacognitive capacity, (4) enriching narratives, and (5) living enriched stories. Proscribed practices were also identified. We then implemented the manual within a university clinic context. Six therapists were trained to implement the model and, in turn, provided therapy to 11 patients who completed 12 to 24 months of treatment. Participants were assessed on metacognitive capacity, narrative coherence, narrative richness, self-reported recovery, and symptomatology at three points in time over the course of therapy. Contrary to expectations, participants were highly engaged in the therapeutic process, with minimal dropout. Overall, over 75% of participants evidenced improvement in their level of recovery over the course of therapy. The manualization and outcome findings demonstrate the feasibility of applying such interventions to a broader clinical population.

Saliva as an emerging biofluid for clinical diagnosis and applications of MEMS/NEMS in salivary diagnostics

Saliva as a biological fluid is gaining wider acceptance for diagnosing diseases. The growing interest in saliva as a biological fluid is due to its noninvasiveness, ease of use, cost-effectiveness, and multiple sample collection possibilities as well as minimal risk to health care professionals of contracting infectious organisms such as HIV and Hep B. However, the clinical translation of saliva is hampered by our lack of understanding of the biomolecular transportation from blood into saliva, the diurnal variations of biomolecules present in saliva, and relatively low levels of analytes (100th to a 1000th fold less than in blood). We provide information on the current status of salivary research, salivary diagnostics empowered by nanotechnology, and future prospects in this emerging field of saliva diagnostics.

Theorizing the use of multiple conceptual models in combination

Conceptual modelling continues to be an important means for graphically capturing the requirements of an information system. Observations of modelling practice suggest that modellers often use multiple conceptual models in combination, because they articulate different aspects of real-world domains. Yet, the available empirical as well as theoretical research in this area has largely studied the use of single models, or single modelling grammars. We develop a Theory of Combined Ontological Coverage by extending an existing theory of ontological expressiveness of conceptual modelling grammars. Our new theory posits that multiple conceptual models are used to increase the maximum coverage of the real-world domain being modelled, whilst trying to minimize the ontological overlap between the models. We illustrate how the theory can be applied to analyse sets of conceptual models. We develop three propositions of the theory about evaluations of model combinations in terms of users’ selection, understandability and usefulness of conceptual models.

Subprime agriculture, and Australia?

Today, Australian agriculture is not where we hoped it would be. Despite being highly productive and the nation's only 'strongly competitive industry', it is struggling across the country. There are successes, as there always will be, but the bulk of our food and fibre production is from enterprises with minimal profitability and unstable or unsound finances. A debt-deflation spiral and subprime mortgage crisis are now being fuelled by property fire sales while leading bankers proclaim no problem and governments dance at the edges. However, it is not just the bush that has problems. National economic conditions are deteriorating with per capita incomes falling and real interest rates still high. Well-informed policy strategies and effective responses are needed quickly if Australians are to avoid needless losses of capacity and wealth destruction in the cities and the bush.

Computation of Mathematical Models for Complex Industrial Processes

Designed for undergraduate and postgraduate students, academic researchers and industrial practitioners, this book provides comprehensive case studies on numerical computing of industrial processes and step-by-step procedures for conducting industrial computing. It assumes minimal knowledge in numerical computing and computer programming, making it easy to read, understand and follow. Topics discussed include fundamentals of industrial computing, finite difference methods, the Wavelet-Collocation Method, the Wavelet-Galerkin Method, High Resolution Methods, and comparative studies of various methods. These are discussed using examples of carefully selected models from real processes of industrial significance. The step-by-step procedures in all these case studies can be easily applied to other industrial processes without a need for major changes and thus provide readers with useful frameworks for the applications of engineering computing in fundamental research problems and practical development scenarios.

Women’s perceptions of their lifestyle and quality of life several years after a diagnosis of endometrial cancer

Background Few studies have examined the long-term changes experienced by women treated for endometrial cancer. Objective The objectives of this study were to describe what women perceived important to their lifestyle and quality of life in the years following their diagnosis and to provide new insights that might inform healthcare practice. Methods This was a thematic analysis of 237 open-ended comments from Australian women diagnosed with endometrial cancer 3 to 5 years previously. Results We identified 3 main themes: (1) personal change, in which women spoke about cancer as permanently altering their lives in mostly negative but sometimes positive ways; (2) continuity of former life, which described both the minimal impact of cancer on women's lives and identities and the difficulties negotiating this within the dominant "cancer survivorship" culture; (3) social support, where women wrote about how the quality of their relationships shaped their cancer trajectory. Conclusions While typical "survivorship" issues exist for many women with endometrial cancer (eg, physical, emotional, sexual health changes), a proportion of women will not be focused on their cancer and can be encouraged to form lives and identities that are not situated within the "cancer survivorship" culture. Implications for Practice A network of support, sensitive to women's responses to having cancer, may benefit women's long-term adjustment. Regular standardized assessment of women's needs may facilitate appropriate support for those with concerns, whereas those without concerns could be reassured by health professionals that their experience is normal and shared by other people with cancer. This may encourage women to form lives that are personally meaningful.