Phage library screening for the rapid identification and in vivo testing of candidate genes for a DNA vaccine against Mycoplasma mycoides subsp. mycoides small colony biotype

A new strategy for rapidly selecting and testing genetic vaccines has been developed, in which a whole genome library is cloned into a bacteriophage λ ZAP Express vector which contains both prokaryotic (Plac) and eukaryotic (PCMV) promoters upstream of the insertion site. The phage library is plated on Escherichia coli cells, immunoblotted, and probed with hyperimmune and/or convalescent-phase antiserum to rapidly identify vaccine candidates. These are then plaque purified and grown as liquid lysates, and whole bacteriophage particles are then used directly to immunize the host, following which PCMV-driven expression of the candidate vaccine gene occurs. In the example given here, a semirandom genome library of the bovine pathogen Mycoplasma mycoides subsp. mycoides small colony (SC) biotype was cloned into λ ZAP Express, and two strongly immunodominant clones, λ-A8 and λ-B1, were identified and subsequently tested for vaccine potential against M. mycoides subsp. mycoides SC biotype-induced mycoplasmemia. Sequencing and immunoblotting indicated that clone λ-A8 expressed an isopropyl-β-d-thiogalactopyranoside (IPTG)-inducible M. mycoides subsp. mycoides SC biotype protein with a 28-kDa apparent molecular mass, identified as a previously uncharacterized putative lipoprotein (MSC_0397). Clone λ-B1 contained several full-length genes from the M. mycoides subsp. mycoides SC biotype pyruvate dehydrogenase region, and two IPTG-independent polypeptides, of 29 kDa and 57 kDa, were identified on immunoblots. Following vaccination, significant anti-M. mycoides subsp. mycoides SC biotype responses were observed in mice vaccinated with clones λ-A8 and λ-B1. A significant stimulation index was observed following incubation of splenocytes from mice vaccinated with clone λ-A8 with whole live M. mycoides subsp. mycoides SC biotype cells, indicating cellular proliferation. After challenge, mice vaccinated with clone λ-A8 also exhibited a reduced level of mycoplasmemia compared to controls, suggesting that the MSC_0397 lipoprotein has a protective effect in the mouse model when delivered as a bacteriophage DNA vaccine. Bacteriophage-mediated immunoscreening using an appropriate vector system offers a rapid and simple technique for the identification and immediate testing of putative candidate vaccines from a variety of pathogens.

Enhancement of bacterial mutagenicity of bifunctional alkylating agents by expression of mammalian glutathione s-transferase

Recently, we inserted the plasmid vector pKK233-2 containing rat GSH S-transferase (GST) 5-5 cDNA into Salmonella typhimurium TA1535 and found that these bacteria [GST 5-5(+)] expressed the protein and produced mutations when ethylene or methylene dihalides were added [Thier, R., Taylor, J. B., Pemble, S. E., Ketterer, B., Persmark, M., Humphreys, W. G., and Guengerich, F. P. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 8576-8580]. After exposure to the known GST 5-5 substrate 1,2-epoxy-3-(4′-nitrophenoxy)propane, the GST 5-5(+) strain showed fewer mutants than the bacteria transfected with the cDNA clone in a reverse orientation [GST 5-5(-)], suggesting a protective role of GST 5-5. However, mutations were considerably enhanced in the GST 5-5(+) strain [as compared to GST 5-5(-)] when 1,2,3,4-diepoxybutane (butadiene diepoxide) or 1,2-epoxy-4-bromobutane was added. The GST 5-5(+) and GST 5-5(-) bacterial stains showed similar responses to 1,2-epoxypropane, 3,4-epoxy-1-butene, and 1,4-dibromobutane. The results suggest that some bifunctional activated butanes are transformed to mutagenic products through GSH conjugation. We also found that the GST 5-5(+) strain showed enhanced mutagenicity with 1,4-dibromo-2,3-epoxybutane, 1,2-epoxy-3-bromopropane (epibromohydrin), and (±)-1,4-dibromo-2,3-dihydroxybutane. The possibility was considered that a 5-membered thialonium ion may be involved in the mutagenicity. Model thialonium compounds were rather stable to hydrolysis in aqueous solution at pH 7.4 and slowly alkylated 4-(4-nitrobenzyl)pyridine. The presence of a hydroxyl group β to the sulfur did not enhance reactivity. Mechanisms involving episulfonium ions are considered more likely. Potential oxidation products of the toxic pesticide 1,2-dibromo-3-chloropropane (DBCP) were also considered in this system. DBCP itself gave rather similar results in the two strains. Others have reported that oxidation of DBCP is required for mutagenicity, along with GST-catalyzed GSH conjugation [Simula, T. P., Glancey, M. J., Söderlund, E. J., Dybing, E., and Wolf, C. R. (1993) Carcinogenesis 14, 2303-2307]. The putative oxidation product 1,2-dibromopropional did not show a difference between the two strains. However, 1,3-dichloroacetone, a model for the putative oxidation product 1-bromo-3-chloroacetone, was considerably more mutagenic in the GST 5-5(+) strain.

Boosting the effects of a curriculum based injury prevention program through a school connectedness intervention : final report to NRMA-ACT Road Safety Trust

Injury is the leading cause of death among young people (AIHW, 2008). A primary contributing factor to injury among adolescents is risk taking behaviour, including road related risks such as risky bicycle and motorcycle use and riding with dangerous or drink-drivers. Injury rates increase dramatically throughout adolescence, at the same time as adolescents are becoming more involved in risk taking behaviour. Also throughout this period, adolescents‟ connectedness to school is decreasing (Monahan, Oesterle & Hawkins, 2010; Whitlock, 2004). School connectedness refers to „the extent to which students feel personally accepted, respected, included, and supported by others in the school‟ (Goodenow, 1993, p. 80), and has been repeatedly shown to be a critical protective factor in adolescent development. For example, school connectedness has been shown to be associated with decreased risk taking behaviour, including violence and alcohol and other drug use (e.g., Resnick et al., 1997), as well as with decreased transport risk taking and vehicle related injuries (Chapman et al., accepted April 2011). This project involved the pilot evaluation of a school connectedness intervention (a professional development program for teachers) to reduce adolescent risk taking behaviour and injury. This intervention has been developed for use as a component of the Skills for Preventing Injury in Youth (SPIY) curriculum based injury prevention program for early adolescents. The objectives of this research were to: 1. Implement a trial School Connectedness intervention (professional development program for teachers) in ACT high schools, and evaluate using comparison high schools. 2. Determine whether the School Connectedness program impacts on adolescent risk taking behaviour and associated injuries (particularly transport risks and injuries). 3. Evaluate the process effectiveness of the School Connectedness program.

From bench to bedside : immunotherapy for prostate cancer

The mainstay therapeutic strategy for metastatic castrate-resistant prostate cancer (CRPC) continues to be androgen deprivation therapy usually in combination with chemotherapy or androgen receptor targeting therapy in either sequence, or recently approved novel agents such as Radium 223. However, immunotherapy has also emerged as an option for the treatment of this disease following the approval of sipuleucel-T by the FDA in 2010. Immunotherapy is a rational approach for prostate cancer based on a body of evidence suggesting these cancers are inherently immunogenic and, most importantly, that immunological interventions can induce protective antitumour responses. Various forms of immunotherapy are currently being explored clinically, with the most common being cancer vaccines (dendritic-cell, viral, and whole tumour cell-based) and immune checkpoint inhibition. This review will discuss recent clinical developments of immune-based therapies for prostate cancer that have reached the phase III clinical trial stage. A perspective of how immunotherapy could be best employed within current treatment regimes to achieve most clinical benefits is also provided.

Changes in outdoor workers' sun-related attitudes, beliefs, and behaviors : a pre-post workplace intervention

OBJECTIVE To evaluate changes in outdoor workers' sun-related attitudes, beliefs, and behaviors in response to a health promotion intervention using a participatory action research process. METHODS Fourteen workplaces across four outdoor industry types worked collaboratively with the project team to develop tailored sun protection action plans. Workers were assessed before and after the 18-month intervention. RESULTS Outdoor workers reported increases in workplace support for sun protection (P < 0.01) and personal use of sun protection (P < 0.01). More workers reported seeking natural shade (+20%) and wearing more personal protective equipment, including broad-brimmed hats (+25%), long-sleeved collared shirts (+19%), and long trousers (+16%). The proportion of workers reporting sunburn over the past 12 months was lower at postintervention (-14%) (P = 0.03); however, the intensity of reported sunburn increased. CONCLUSIONS This intervention was successful in increasing workers' sun protective attitudes, beliefs, and behaviors.

Co-branding strategies for luxury fashion brands : Missoni for Target

Missoni is a luxury Italian knitwear brand that partnered with Target in September 2011 releasing a large, one off, mass-market collection that ranged from apparel to home wares. The collaboration received extensive media coverage and was consequently extremely sought after. The online sales site crashed within hours of opening while shelves were cleared in stores minutes after trading began. Within hours more than 40000 items from the collection were posted for sale online at greatly inflated prices. Evaluation of the case study revealed that sales of the Missoni collection increased following the collaboration and the value of the publicity generated at estimated US$100 million. The lack of available stock, despite the enormous hype created, reinforced Missoni’s luxury image. Missoni was able to gain massive awareness of the brand despite not employing any of its own communication channels in the promotion of the collaboration. However the co-branded collaboration was distinctively Missoni, potentially inciting comparison and confusion with the signature line. Nevertheless, this study shows that co-branding strategies can offer a viable opportunity for luxury brands to increase their market share, while they maintain their market position.

Beyond Employment: The Legal Regulation of Work Relationships

Australian labour law, at least from the mid-twentieth century, was dominated by the employment paradigm: the assumption that labour law’s scope was the regulation of employment relationships –full-time and part-time, and continuing, fixed term or casual – with a single (usually corporate) entity employer. But no sooner had the employment paradigm established and consolidated its shape, it began to fall apart. Since the 1980s there has been a significant growth of patterns of work that fall outside this paradigm, driven by organisational restructuring and management techniques such as labour hire, sub-contracting and franchising. Beyond Employment analyses the way in which Australian labour law is being reframed in this shift away from the pre-eminence of the employment paradigm. Its principal concern is with the legal construction and regulation of various forms of contracting, including labour hire arrangements, complex contractual chains and modern forms like franchising, and of casual employment. It outlines the current array of work relationships in Australia, and describes and analyses the way in which those outside continuous and fixed term employment are regulated. The book seeks to answer the central question: How does law (legal rules and principles) construct these work relationships, and how does it regulate these relationships? The book identifies the way in which current law draws the lines between the various work relationships through the use of contract and property ownership, and describes, analyses and synthesises the legal rules that govern these different forms of work relationships. The legal rules that govern work relationships are explored through the traditional lens of labour law’s protective function, principally in four themes: control of property, and the distribution of risks and rewards; maintenance of income security; access to collective voice mechanisms, focusing on collective bargaining; and health, safety and welfare. The book critically evaluates the gaps in the coverage and content of these rules and principles, and the implications of these gaps for workers. It also reflects upon the power relationships that underpin the work arrangements that are the focus of the book and that are enhanced through the laws of contract and property. Finally, it frames an agenda to address the gaps and identified weaknesses insofar as they affect the economic wellbeing, democratic voice, and health and safety of workers.

Late reactivation of herpes zoster keratitis results in band keratopathy

Purpose To report an unusual case of a late-stage reactivation of immune stromal keratitis associated with herpes zoster ophthalmicus (HZO), occurring without any apparent predisposing factors, more than 4 years after an acute zoster dermatomal rash. Significant corneal hypoesthesia and a central band keratopathy developed within 6 months of the late-stage reactivation. The clinical case management, issues associated with management, and management options are discussed, including the use of standardized, regulatory approved, antibacterial medical honey. Case Report An 83-year-old woman presented for routine review with a reactivation of right anterior stromal keratitis and mild anterior uveitis, occurring more than 4 years after an acute HZO dermatomal rash and an associated initial episode of anterior stromal keratitis. Corneal sensation became markedly impaired, and over the subsequent 6 months, a right central band keratopathy developed despite oral antiviral and topical steroid therapy. Visual acuity with pinhole was reduced to 20/100 in the affected eye and moderate irritation and epiphora were experienced. The patient declined the surgical intervention options of chelation, lamellar keratectomy, and phototherapeutic keratectomy to treat the band keratopathy. Longer-term management has involved preservative-free artificial tears, eyelid hygiene, standardized antibacterial medical honey, topical nonpreserved steroid, and UV-protective wraparound sunglasses. The clinical condition has improved over 14 months with this ocular surface management regimen, and visual acuity of 20/30 is currently achieved in a comfortable eye. Conclusions The chronic and recurrent nature of HZO can be associated with significant corneal morbidity, even many years after the initial zoster episode. Long-term review and management of patients with a history of herpes zoster stromal keratitis are indicated following the initial corneal involvement. Standardized antibacterial medical honey can be considered in the management of the chronic ocular surface disease associated with HZO and warrants further evaluation in clinical trials.

Caveating a mortgagee sale - The need for a uniform approach

The ability of a mortgagor to lodge a caveat, where an allegation is raised of serious impropriety by a mortgagee in exercising power of sale, may be a critical protective tool for a mortgagor. The mortgagor’s capacity, as caveator, to lodge a caveat over the mortgagor’s own title and the nature of the interest necessary to support the caveat are contentious issues. This article examines the different judicial approaches that have been adopted to date and states a case for a uniform approach to this issue across Australia in the future.

Predictors of chronic bronchitis in South African adults

SETTING National household survey of adults in South Africa, a middle income country. OBJECTIVE To determine the prevalence and predictors of chronic bronchitis. DESIGN A stratified national probability sample of households was selected. All adults in the selected households were interviewed. Chronic bronchitis was defined as chronic productive cough. Socio-demographic predictors were wealth, education, race, age and urban residence. Personal and exposure variables included history of tuberculosis, domestic exposure to smoky fuels, occupational exposures, smoking and body mass index. RESULTS The overall prevalence of chronic bronchitis was 2.3% in men and 2.8% in women. The strongest predictor of chronic bronchitis was a history of tuberculosis (men, odds ratio [OR] 4.9; 95% confidence interval [CI] 2.6-9.2; women, OR 6.6; 95% CI 3.7-11.9). Other risk factors were smoking, occupational exposure (in men), domestic exposure to smoky fuel (in women) and (in univariate analysis only) being underweight. Wealth and particularly education were protective. CONCLUSION The pattern of chronic bronchitis in South Africa suggests a combination of risk factors that includes not only smoking but also tuberculosis, occupational exposures in men and domestic fuel exposure in women. Control of these risk factors requires public health action across a broad front. The protective role of education requires elucidation.

Pathogenesis of Fallopian tube damage caused by Chlamydia trachomatis infections

Chlamydia trachomatis is the leading cause of bacterial sexually transmitted disease worldwide resulting in 4–5 million new cases of Chlamydia annually and an estimated 100 million cases per annum. Infections of the lower female genital tract (FGT) frequently are asymptomatic so they often remain undiagnosed or untreated. If infections are either not resolved, or are left untreated, chlamydia can ascend to the upper FGT and infect the fallopian tubes (FTs) causing salpingitis that may lead to functional damage of the FTs and tubal factor infertility (TFI). Clinical observations and experimental data have indicated a role for antibodies against C. trachomatis proteins such as the 60 kDa heat-shock protein 60 (cHSP60) in the immunopathogenesis of TFI. When released from infected cells cHSP60 can induce pro-inflammatory immune responses that may functionally impair the FTs leading to fibrosis and luminal occlusion. Chlamydial pathogenesis of irreversible and permanent tubal damage is a consequence of innate and adaptive host immune responses to ongoing or repeated infections. The extracellular matrix (ECM) that is regulated by metalloproteinases (MMPs) may also be modified by chlamydial infections of the FGT. This review will highlight protective and pathogenic immune responses to ongoing and repeated chlamydial infections of the FGT. It will also present two recent hypotheses to explain mechanisms that may contribute to FT damage during a C. trachomatis infection. If Chlamydia immunopathology can be controlled it might yield a method of inducing fibrosis and thus provide a means of non-surgical permanent contraception for women.

Mississippi Delta subsidence primarily caused by compaction of Holocene strata

Coastal subsidence causes sea-level rise, shoreline erosion and wetland loss, which poses a threat to coastal populations. This is especially evident in the Mississippi Delta in the southern United States, which was devastated by Hurricane Katrina in 2005. The loss of protective wetlands is considered a critical factor in the extensive flood damage. The causes of subsidence in coastal Louisiana, attributed to factors as diverse as shallow compaction and deep crustal processes, remain controversial. Current estimates of subsidence rates vary by several orders of magnitude. Here, we use a series of radiocarbon-dated sediment cores from the Mississippi Delta to analyse late Holocene deposits and assess compaction rates. We find that millennial-scale compaction rates primarily associated with peat can reach 5mm per year, values that exceed recent model predictions. Locally and on timescales of decades to centuries, rates are likely to be 10 mm or more per year. We conclude that compaction of Holocene strata contributes significantly to the exceptionally high rates of relative sea-level rise and coastal wetland loss in the Mississippi Delta, and is likely to cause subsidence in other organic-rich and often densely populated coastal plains.

How do Australian print media representations of child abuse and neglect inform the public and system reform?

The print media play a vital role in informing the public about child abuse and neglect. This information helps build broad support for laws and system developments that enable the state to intervene into private family lives and ensure that children are protected from maltreatment. Print media coverage usually sets the daily media agenda. It therefore influences public understandings of child abuse and neglect and what people believe should be done about it. Media impact on policy agendas should not be underestimated. This article outlines the results of a study of all major Australian newspaper stories covering abuse and neglect matters during an 18-month period in 2008–2009. A range of issues are identified concerning how well these stories inform the public about the nature of the problem and the current national reform agenda for protective systems that promotes early intervention and prevention...

Process engineering aspects of plasmid-based biopharmaceuticals production : tackling the threatening vaccine shortages to prevent global pandemics

Infectious diseases such as SARS, influenza and bird flu have the potential to cause global pandemics; a key intervention will be vaccination. Hence, it is imperative to have in place the capacity to create vaccines against new diseases in the shortest time possible. In 2004, The Institute of Medicine asserted that the world is tottering on the verge of a colossal influenza outbreak. The institute stated that, inadequate production system for influenza vaccines is a major obstruction in the preparation towards influenza outbreaks. Because of production issues, the vaccine industry is facing financial and technological bottlenecks: In October 2004, the FDA was caught off guard by the shortage of flu vaccine, caused by a contamination in a US-based plant (Chiron Corporation), one of the only two suppliers of US flu vaccine. Due to difficulties in production and long processing times, the bulk of the world's vaccine production comes from very small number of companies compared to the number of companies producing drugs. Conventional vaccines are made of attenuated or modified forms of viruses. Relatively high and continuous doses are administered when a non-viable vaccine is used and the overall protective immunity obtained is ephemeral. The safety concerns of viral vaccines have propelled interest in creating a viable replacement that would be more effective and safer to use.

The timing of solid introduction in an ‘obesogenic’ environment : a narrative review of the evidence and methodological issues.

Objective To evaluate the evidence for association between obesity risk outcomes >12 months of age and timing of solid introduction in healthy term infants in developed countries, the large majority of whom are not exclusively breastfed to 6 months of age. Methods Studies included were published 1990-March 2013. Results Twenty-six papers with weight status or obesity prevalence outcomes were identified. Studies were predominantly cohort design, most with important methodological limitations. Ten studies reported a positive association. Of these only two were large good quality studies and both examined the outcome of early (<4 months) solid introduction. None of the four good quality studies that directly evaluated current guidelines provided evidence of any clinically relevant protective effect of solid introduction from 4-5 versus ≥ 6 months of age. Conclusion Overall the introduction of solids prior to 4 months may result in increased risk of childhood obesity but there is little evidence of adverse weight status outcomes associated with introducing solids at 4-6 rather than at 6 months. Implications More and better quality evidence is required to inform guidelines on the ‘when, what and how’ of complementary feeding.