Is simulation training effective in increasing podiatrists' confidence in foot ulcer management?

Background: Foot ulcers are a frequent reason for diabetes-related hospitalisation. Clinical training is known to have a beneficial impact on foot ulcer outcomes. Clinical training using simulation techniques has rarely been used in the management of diabetes-related foot complications or chronic wounds. Simulation can be defined as a device or environment that attempts to replicate the real world. The few non-web-based foot-related simulation courses have focused solely on training for a single skill or “part task” (for example, practicing ingrown toenail procedures on models). This pilot study aimed to primarily investigate the effect of a training program using multiple methods of simulation on participants’ clinical confidence in the management of foot ulcers. Methods: Sixteen podiatrists participated in a two-day Foot Ulcer Simulation Training (FUST) course. The course included pre-requisite web-based learning modules, practicing individual foot ulcer management part tasks (for example, debriding a model foot ulcer), and participating in replicated clinical consultation scenarios (for example, treating a standardised patient (actor) with a model foot ulcer). The primary outcome measure of the course was participants’ pre- and post completion of confidence surveys, using a five-point Likert scale (1 = Unacceptable-5 = Proficient). Participants’ knowledge, satisfaction and their perception of the relevance and fidelity (realism) of a range of course elements were also investigated. Parametric statistics were used to analyse the data. Pearson’s r was used for correlation, ANOVA for testing the differences between groups, and a paired-sample t-test to determine the significance between pre- and post-workshop scores. A minimum significance level of p < 0.05 was used. Results: An overall 42% improvement in clinical confidence was observed following completion of FUST (mean scores 3.10 compared to 4.40, p < 0.05). The lack of an overall significant change in knowledge scores reflected the participant populations’ high baseline knowledge and pre-requisite completion of web-based modules. Satisfaction, relevance and fidelity of all course elements were rated highly. Conclusions: This pilot study suggests simulation training programs can improve participants’ clinical confidence in the management of foot ulcers. The approach has the potential to enhance clinical training in diabetes-related foot complications and chronic wounds in general.

Do people gamble with their lives : The relationship between risky driving behaviour and other risky behaviours

General risky behaviour is explored for correlation with risky driving behaviour in light of two theories, self-control and cross-situational consistency. Identification of general risky behaviours associated with risky driving behaviour, and the theory that best predicts the behaviours, will enable better targeting of intervention and education strategies to reduce driving related fatalities and injuries. A correlational study using participants (N=152) drawn from first year university undergraduates and the public surveyed their lifestyle and behaviours. Relationships were found between risky driving behaviours and other risky behaviours such as alcohol consumption, cannabis use and performing unlawful activities. No significant differences were found between genders, with the exception that males were more likely to believe that they were at risk of injury from their employment, χ2 (1, N = 152) = 4.49, p = .03, were more likely to have performed an unlawful offence, χ2 (1, N = 152) = 11.77, p = .001 and were more likely to drink drive, t (55.41) = -3.87, p < .001, mean difference = -0.63, CI 95% (-0.9, -0.37). People engaged in risky driving behaviours were more likely to engage in other risky behaviours. The theories that were explored were unable to accurately predict an association between general risky behaviour and driving without a license or when disqualified. Cross-situational consistency explained 20% (R2adj = .16) of the variance in which people engaged in risky driving with low self-control theory explaining an additional 0.3% variance (R2change = .003), F (8,143) = 6.92, p < .001. Driving while under the influence of alcohol could be predicted by risky behaviours in lifestyle, health, smoking, cannabis use and alcohol consumption, F (8,143) = 6.92, p < .001. The addition of self-control was not significant.

GABAergic modification of myopic and hyperopic ocular tissue effects on scleral fibroblast growth

Purpose: Myopia is a common eye disorder affecting up to 90% of children in South East Asia and 30% of the population worldwide. Myopia of high severity is a leading cause of blindness around the world (4th to 5th most common). Changes and remodelling of the sclera i.e. increase cellular proliferation & increase protein synthesis within scleral cells (↑ scleral DNA) and thinning and lose of extracellular matrix of sclera (↓ scleral GAG synthesis) have been linked to myopic eye growth in animal models. Signals acting on the sclera are thought to originate in the retina, and are modulated by the retinal pigment epithelium (RPE) with limited evidence suggesting that the RPE can modify scleral cell growth in culture. However, the mechanism of retinal signal transmission and the role of posterior eye cup tissue, including the RPE, in mediating changes in scleral fibroblast growth during myopia development are unclear. Retinal transmitter systems are critically involved in pathways regulating eye growth, which ultimately lead to alterations in the sclera if eye size is to change. A dopaminergic agonist and muscarinic antagonists decrease the proliferation of scleral chondrocytes when co-cultured with chick’s retinal pigment epithelium (RPE). GABA receptors have recently been localised to chick sclera. We therefore hypothesised that posterior eye cup tissue from myopic eyes would stimulate and from hyperopic eyes would inhibit growth of scleral fibroblasts in vitro and that GABAergic agents could directly interact with scleral cells or indirectly modify the effects of myopic and hyperopic posterior eye cup tissue on scleral fibroblast growth. Method: Fibroblastic cells obtained from 8-day-old chick sclera were used to establish cell banks. Two major experiments were performed. Experiment 1: To determine if posterior eye cup tissues from myopic eye stimulates and hyperopic eye inhibits scleral cell proliferation, when co-cultured with scleral cells in vitro. This study comprised two linked experiments, i) monocular visual treatments of FDM (form-deprivation myopia), LIM (lens-induced myopia) and LIH (lens-induced hyperopia) with assessment of the effect of full punch eye cup tissue on DNA and GAG synthesis by cultured chick scleral fibroblasts, and ii) binocular visual treatments comprising LIM and LIH with assessment of the effect of individual layers of eye cup tissues (neural retina, RPE and choroid) on cultured chick scleral fibroblasts. Visual treatment was applied for 3 days. Experiment 2: To determine the direct interaction of GABA agents on scleral cell growth and to establish whether GABA agents modify the stimulatory/inhibitory effect of myopic and hyperopic posterior eye cup tissues on cultured scleral cell growth in vitro. Two linked experiments were performed. i) GABA agonists (muscimol and baclofen) and GABA antagonists (bicuculine (-), CGP46381 and TPMPA) were added to scleral cell culture medium to determine their direct effect on scleral cells. ii) GABAergic agents (agonists and antagonists) were administered to scleral fibroblasts co-cultured with posterior eye cup tissue (retina, RPE, retina/RPE, RPE/choroid). Ocular tissues were obtained from chick eyes wearing +15D (LIH) or -15D lenses (LIM) for 3 days. In both experiments, tissues were added to hanging cell culture insert (pore size 1.0ìm) placed over each well of 24 well plates while scleral cells were cultured in DMEM/F12, Glutamax (Gibco) plus 10% FBS and penicillin/streptomycin (50U/ml)) and fungizone (1.25ug/ml) (Gibco), at seeding density of 30,000 cells/well at the bottom of the well and allowed to grow for 3 days. Scleral cells proliferation rate throughout the study was evaluated by determining GAG and DNA content of scleral cells using Dimethylmethylene blue (DMMB) dye and Quant-iTTm Pico Green® dsDNA reagent respectively. Results and analysis: Based on DNA and GAG content, there was no significant difference in tissue effect of LIM and LIH eyes on scleral fibroblast growth (DNA: 8.4 ± 1.1μg versus 9.3 ± 2.3 μg, p=0.23; GAG: 10.13 ± 1.4 μg versus 12.67 ± 1.2 μg, F2,23=6.16, p=0.0005) when tissues were obtained from monocularly treated chick eyes (FDM or +15D lens or -15D lens over right eyes with left eyes untreated) and co-cultured as full punch. When chick eyes were treated binocularly with -15D lens (LIM) right eye and +15D lens (LIH) left eyes and tissue layers were separated, the retina from LIM eyes did not stimulate scleral cell proliferation compared to LIH eyes (DNA: 27.2 ± 6.7 μg versus 23.2 ± 1.5 μg, p=0.23; GAG: 28.1 ±3.7 μg versus 28.7 ± 4.2 μg, p=0.21). Similarly, the LIH and LIM choroid did not produce a differential effect based on DNA (LIM 46.9 ± 6.4 μg versus LIH 53.5 ± 4.7 μg, p=0.18), however the choroid from LIH eyes induced higher scleral GAG content than from LIM eyes (32.5 ± 6.7 μg versus 18.9 ± 1.2 μg, p=0.023). In contrast, the RPE from LIM eyes caused a significant increase in fibroblast proliferation whereas the RPE from LIH eyes was relatively inhibitory (72.4 ± 6.3 μg versus 27.9 ± 2.3 μg, F1, 6=69.99, p=0.0005). GAG data were opposite to DNA data e.g. the RPE from LIH eyes increased (33.7 ± 7.9 μg) while the RPE from LIM eyes decreased (28.2 ± 3.0 μg) scleral cell growth (F1, 6=13.99, p=0.010). Based on DNA content, GABA agents had a small direct effect on scleral cell growth; GABA agonists increased (21.4 ± 1.0% and 18.3 ± 1.0% with muscimol and baclofen, p=0.0021), whereas GABA antagonists decreased fibroblast proliferation (-23.7 ± 0.9% with bicuculine & CGP46381 and -28.1 ± 0.5% with TPMPA, p=0.0004). GABA agents also modified the effect of LIM and LIH tissues (p=0.0005).The increase in proliferation rate of scleral fibroblasts co-cultured with tissues (RPE, retina, RPE/retina and RPE/choroid) from LIM treated eyes was enhanced by GABA agonists (muscimol: 27.4 ± 1.2%, 35.8 ± 1.6%, 8.4 ± 0.3% and 11.9 ± 0.6%; baclofen: 27.0 ± 1.0%, 15.8 ± 1.5%, 16.8 ± 1.2% and 15.4 ± 0.4%, p=0.014) whereas GABA antagonists further reduced scleral fibroblasts growth (bicuculine: -52.5 ± 2.5%, -36.9 ± 1.4%, -37.5 ± 0.6% and -53.7 ± 0.9%; TPMPA: 57.3 ± 1.3%, -15.7 ± 1.2%, -33.5 ± 0.4% and -45.9 ± 1.5%; CGP46381: -51.9 ± 1.6%, -28.5 ± 1.5%, -25.4 ± 2.0% and -45.5 ± 1.9% respectively, p=0.0034). GAG data were opposite to DNA data throughout the experiment e.g. GABA agonists further inhibited while antagonists relatively enhanced scleral fibroblasts growth for both LIM and LIH tissue co-culture. The effect of GABA agents was relatively lower (p=0.0004) for tissue from LIH versus LIM eyes but was in a similar direction. There was a significant drug effect on all four tissue types e.g. RPE, retina, RPE/retina and RPE/choroid for both LIM and LIH tissue co-culture (F20,92=3.928, p=0.0005). However, the effect of GABA agents was greatest in co-culture with RPE tissue (F18,36=4.865, p=0.0005). Summary and Conclusion: 1) Retinal defocus signals are transferred to RPE and choroid which then exert their modifying effect on scleral GAG and DNA synthesis either through growth stimulating factors or directly interacting with scleral cells in process of scleral remodeling during LIM and LIH visual conditions. 2) GABAergic agents affect the proliferation of scleral fibroblasts both directly and when co-cultured with ocular tissues in vitro.

Book Review : Bellanta, Melissa (2012) Larrikins : A History. Brisbane: University of Queensland Press.

This book has been painstakingly researched by a scholar whose intellectual competencies span several disciplines: history, sociology, criminology, culture, drama and film studies. It is theoretically sophisticated and yet not dense as it reads like a novel with an abundance of interesting complex characters.

The development of a supportive care needs assessment tool for Indigenous people with cancer

Background: Little is known about the supportive care needs of Indigenous people with cancer and to date, existing needs assessment tools have not considered cultural issues for this population. We aimed to adapt an existing supportive care needs assessment tool for use with Indigenous Australians with cancer. Methods: Face-to-face interviews with Indigenous cancer patients (n = 29) and five focus groups with Indigenous key-informants (n = 23) were conducted to assess the face and content validity, cultural acceptability, utility and relevance of the Supportive Care Needs Survey - Short Form 34 (SCNS-SF34) for use with Indigenous patients with cancer. Results: All items from the SCNS-SF34 were shortened and changed to use more appropriate language (e.g. the word 'anxiety' was substituted with 'worry'). Seven questions were omitted (e.g. items on death and future considerations) as they were deemed culturally inappropriate or irrelevant and 12 items were added (e.g. accessible transport). Optional instructions were added before the sexual items. The design and response format of the SCNS-SF34 was modified to make it easier to use for Indigenous cancer patients. Given the extensive modifications to the SCNS-SF34 and the liklihood of a different factor structure we consider this tool to be a new tool rather than a modification. The Supportive care needs assessment tool for Indigenous people (SCNAT-IP) shows promising face and content validity and will be useful in informing services where they need to direct their attention for these patients. Conclusions: Indigenous people with cancer have language, customs and specific needs that are not accommodated within the standard SCNS-SF34. Our SCNAT-IP improves acceptability, relevance and face validity for Indigenous-specific concerns. Our SCNAT-IP will allow screening for supportive care needs that are specific to Indigenous cancer patients' and greatly inform targeted policy development and practice.

Update on exercise and weight control

Recent analyses of population data reveal that obesity rates continue to rise, and are projected to reach unprecedented levels over the next decade 1. Despite concerted efforts to impede obesity progression, as of today, weight loss and weight maintenance strategies remain at best partially successful endeavours. Regardless of the observation that weight loss strategies can produce significant weight loss 2 and substantial improvements of the determinants of the metabolic risk profile 3, 4, it is clear that actual weight loss tends to be lower than the anticipated weight loss, and most individuals who achieve weight loss will likely regain some weight 5 and even overshoot 6 their pre-intervention body weight. As such, an improved understanding of the factors that contribute to lower than expected weight loss, and poor weight maintenance would improve the effectiveness of weight loss interventions.

The interactions between insulin and androgens in progression to castrate resistant prostate cancer

An association between the metabolic syndrome and reduced testosterone levels has been identified, and a specific inverse relationship between insulin and testosterone levels suggests that an important metabolic crosstalk exists between these two hormonal axes; however, the mechanisms by which insulin and androgens may be reciprocally regulated are not well described. Androgen-dependant gene pathways regulate the growth and maintenance of both normal and malignant prostate tissue, and androgen-deprivation therapy (ADT) in patients exploits this dependence when used to treat recurrent and metastatic prostate cancer resulting in tumour regression. A major systemic side effect of ADT includes induction of key features of the metabolic syndrome and the consistent feature of hyperinsulinaemia. Recent studies have specifically identified a correlation between elevated insulin and high-grade PCa and more rapid progression to castrate resistant disease. This paper examines the relationship between insulin and androgens in the context of prostate cancer progression. Prostate cancer patients present a promising cohort for the exploration of insulin stabilising agents as adjunct treatments for hormone deprivation or enhancers of chemosensitivity for treatment of advanced prostate cancer.

S-allylmercaptocysteine reduces carbon tetrachloride-induced hepatic oxidative stress and necroinflammation via nuclear factor kappa B-dependent pathways in mice.

Purpose To study the protective effects and underlying molecular mechanisms of SAMC on carbon tetrachloride (CCl4)-induced acute hepatotoxicity in the mouse model. Methods Mice were intraperitoneally injected with CCl4 (50 μl/kg; single dose) to induce acute hepatotoxicity with or without a 2-h pre-treatment of SAMC intraperitoneal injection (200 mg/kg; single dose). After 8 h, the blood serum and liver samples of mice were collected and subjected to measurements of histological and molecular parameters of hepatotoxicity. Results SAMC reduced CCl4-triggered cellular necrosis and inflammation in the liver under histological analysis. Since co-treatment of SAMC and CCl4 enhanced the expressions of antioxidant enzymes, reduced the nitric oxide (NO)-dependent oxidative stress, and inhibited lipid peroxidation induced by CCl4. SAMC played an essential antioxidative role during CCl4-induced hepatotoxicity. Administration of SAMC also ameliorated hepatic inflammation induced by CCl4 via inhibiting the activity of NF-κB subunits p50 and p65, thus reducing the expressions of pro-inflammatory cytokines, mediators, and chemokines, as well as promoting pro-regenerative factors at both transcriptional and translational levels. Conclusions Our results indicate that SAMC mitigates cellular damage, oxidative stress, and inflammation in CCl4-induced acute hepatotoxicity mouse model through regulation of NF-κB. Garlic or garlic derivatives may therefore be a potential food supplement in the prevention of liver damage.

Can slide positivity rates predict malaria transmission?

Background: Malaria is a significant threat to population health in the border areas of Yunnan Province, China. How to accurately measure malaria transmission is an important issue. This study aimed to examine the role of slide positivity rates (SPR) in malaria transmission in Mengla County, Yunnan Province, China. Methods: Data on annual malaria cases, SPR and socio-economic factors for the period of 1993 to 2008 were obtained from the Center for Disease Control and Prevention (CDC) and the Bureau of Statistics, Mengla, China. Multiple linear regression models were conducted to evaluate the relationship between socio-ecologic factors and malaria incidence. Results: The results show that SPR was significantly positively associated with the malaria incidence rates. The SPR (beta = 1.244, p = 0.000) alone and combination (SPR, beta = 1.326, p < 0.001) with other predictors can explain about 85% and 95% of variation in malaria transmission, respectively. Every 1% increase in SPR corresponded to an increase of 1.76/100,000 in malaria incidence rates. Conclusion: SPR is a strong predictor of malaria transmission, and can be used to improve the planning and implementation of malaria elimination programmes in Mengla and other similar locations. SPR might also be a useful indicator of malaria early warning systems in China.

Quality of work life among primary health care nurses in the Jazan region, Saudi Arabia : a crosssectional study

Background: Quality of work life (QWL) is defined as the extent to which employee is satisfied with personal and working needs through participating in the workplace while achieving the organisation’s goals. QWL has been found to influence the commitment and productivity of employees in healthcare organisations, as well as in other industries. However, reliable information on the QWL of PHC nurses is limited. The purpose of this study was to assess the QWL among PHC nurses in the Jazan region, Saudi Arabia. Methods: A descriptive research design, namely, a cross-sectional survey was used in this study. Data were collected using Brooks’ survey of quality of nursing work life (QNWL) and demographic questions. A convenience sample was recruited from 143 PHC centres in Jazan, Saudi Arabia. The Jazan region is located in the southern part of Saudi Arabia. A response rate of 91% (N = 532/585) was achieved (effective RR = 87%, n = 508). Data analysis consisted of descriptive statistics, t-test and one way-analysis of variance. Total scores and sub-scores for QWL Items and item summary statistics were computed and reported, using SPSS version 17 for Windows. Results: Findings suggested that the respondents were dissatisfied with their work life. The major influencing factors were unsuitable working hours/shifts, lack of facilities for nurses, inability to balance work with family needs, inadequacy of family-leave time, poor staffing, management and supervision practices, lack of professional development opportunities, and inappropriate working environment in terms of the level of security, patient care supplies and equipment, and recreation facilities (Break-area). Other essential factors include the community’s view of nursing and inadequate salary. More positively, the majority of nurses were satisfied with their co-workers, satisfied to be nurses and had a sense of belonging in their workplaces. Significant differences were found according to gender, age, marital status, dependent children, dependent adults, nationality, ethnicity, nursing tenure, organisational tenure, positional tenure, and payment per month. No significant differences were found according to education level and location of PHC. Conclusions: These findings can be used by PHC managers and policy makers for developing and appropriately implementing successful plans to improve the QWL. This will help to enhance the home and work environments, improve individual and organisation performance and increase nurses’ commitment.

The relationship between quality of work life and turnover intention of primary health care nurses in Saudi Arabia

Background Quality of work life (QWL) has been found to influence the commitment of health professionals including nurses. However, reliable information on the QWL and turnover intention of primary health care (PHC) nurses is limited. The aim of this study was to examine the relationship between QWL and turnover intention of PHC nurses in Saudi Arabia. Methods A cross-sectional survey was used in this study. Data were collected using Brooks’ survey of Quality of Nursing Work life (QNWL), the Anticipated Turnover Scale and demographic data questions. A total of 508 PHC nurses in the Jazan region, Saudi Arabia completed the questionnaire (RR = 87%). Descriptive statistics, t-test, ANOVA, General Linear Model (GLM) univariate analysis, standard multiple regression (SMR), and hierarchical multiple regression (HMR) were applied for analysis using SPSS v17 for Windows. Results Findings suggested that the respondents were dissatisfied with their work life, with almost 40% indicating a turnover intention from their current PHC centres. Turnover intention was significantly related to QWL. Using SMR, 26% of the variance in turnover intention was explained by the QWL, p < 0.001, with R² = .263. Further analysis using HMR found that the total variance explained by the model as a whole (demographics and QWL) was 32.1%, p < 0.001. QWL explained an additional 19% of the variance in turnover intention, after controlling for demographic variables. Conclusions Creating and maintaining a healthy work life for PHC nurses is very important to improve their work satisfaction, reduce turnover, enhance productivity and improve nursing care outcomes.

Next-generation sequencing of cervical DNA detects human papillomavirus types not detected by commercial kits

Background Human papillomavirus (HPV) is the aetiological agent for cervical cancer and genital warts. Concurrent HPV and HIV infection in the South African population is high. HIV positive (+) women are often infected with multiple, rare and undetermined HPV types. Data on HPV incidence and genotype distribution are based on commercial HPV detection kits, but these kits may not detect all HPV types in HIV + women. The objectives of this study were to (i) identify the HPV types not detected by commercial genotyping kits present in a cervical specimen from an HIV positive South African woman using next generation sequencing, and (ii) determine if these types were prevalent in a cohort of HIV-infected South African women. Methods Total DNA was isolated from 109 cervical specimens from South African HIV + women. A specimen within this cohort representing a complex multiple HPV infection, with 12 HPV genotypes detected by the Roche Linear Array HPV genotyping (LA) kit, was selected for next generation sequencing analysis. All HPV types present in this cervical specimen were identified by Illumina sequencing of the extracted DNA following rolling circle amplification. The prevalence of the HPV types identified by sequencing, but not included in the Roche LA, was then determined in the 109 HIV positive South African women by type-specific PCR. Results Illumina sequencing identified a total of 16 HPV genotypes in the selected specimen, with four genotypes (HPV-30, 74, 86 and 90) not included in the commercial kit. The prevalence's of HPV-30, 74, 86 and 90 in 109 HIV positive South African women were found to be 14.6 %, 12.8 %, 4.6 % and 8.3 % respectively. Conclusions Our results indicate that there are HPV types, with substantial prevalence, in HIV positive women not being detected in molecular epidemiology studies using commercial kits. The significance of these types in relation to cervical disease remains to be investigated.

Stability studies of HIV-1 Pr55gag virus-like particles made in insect cells after storage in various formulation media

Background: HIV-1 Pr55gag virus-like particles (VLPs) expressed by baculovirus in insect cells are considered to be a very promising HIV-1 vaccine candidate, as they have been shown to elicit broad cellular immune responses when tested in animals, particularly when used as a boost to DNA or BCG vaccines. However, it is important for the VLPs to retain their structure for them to be fully functional and effective. The medium in which the VLPs are formulated and the temperature at which they are stored are two important factors affecting their stability. FINDINGS We describe the screening of 3 different readily available formulation media (sorbitol, sucrose and trehalose) for their ability to stabilise HIV-1 Pr55gag VLPs during prolonged storage. Transmission electron microscopy (TEM) was done on VLPs stored at two different concentrations of the media at three different temperatures (4[degree sign]C, --20[degree sign]C and -70[degree sign]C) over different time periods, and the appearance of the VLPs was compared. VLPs stored in 15% trehalose at -70[degree sign]C retained their original appearance the most effectively over a period of 12 months. VLPs stored in 5% trehalose, sorbitol or sucrose were not all intact even after 1 month storage at the temperatures tested. In addition, we showed that VLPs stored under these conditions were able to be frozen and re-thawed twice before showing changes in their appearance. Conclusions Although the inclusion of other analytical tools are essential to validate these preliminary findings, storage in 15% trehalose at -70[degree sign]C for 12 months is most effective in retaining VLP stability.

Setting up a platform for plant-based influenza virus vaccine production in South Africa

Background During a global influenza pandemic, the vaccine requirements of developing countries can surpass their supply capabilities, if these exist at all, compelling them to rely on developed countries for stocks that may not be available in time. There is thus a need for developing countries in general to produce their own pandemic and possibly seasonal influenza vaccines. Here we describe the development of a plant-based platform for producing influenza vaccines locally, in South Africa. Plant-produced influenza vaccine candidates are quicker to develop and potentially cheaper than egg-produced influenza vaccines, and their production can be rapidly upscaled. In this study, we investigated the feasibility of producing a vaccine to the highly pathogenic avian influenza A subtype H5N1 virus, the most generally virulent influenza virus identified to date. Two variants of the haemagglutinin (HA) surface glycoprotein gene were synthesised for optimum expression in plants: these were the full-length HA gene (H5) and a truncated form lacking the transmembrane domain (H5tr). The genes were cloned into a panel of Agrobacterium tumefaciens binary plant expression vectors in order to test HA accumulation in different cell compartments. The constructs were transiently expressed in tobacco by means of agroinfiltration. Stable transgenic tobacco plants were also generated to provide seed for stable storage of the material as a pre-pandemic strategy. Results For both transient and transgenic expression systems the highest accumulation of full-length H5 protein occurred in the apoplastic spaces, while the highest accumulation of H5tr was in the endoplasmic reticulum. The H5 proteins were produced at relatively high concentrations in both systems. Following partial purification, haemagglutination and haemagglutination inhibition tests indicated that the conformation of the plant-produced HA variants was correct and the proteins were functional. The immunisation of chickens and mice with the candidate vaccines elicited HA-specific antibody responses. Conclusions We managed, after synthesis of two versions of a single gene, to produce by transient and transgenic expression in plants, two variants of a highly pathogenic avian influenza virus HA protein which could have vaccine potential. This is a proof of principle of the potential of plant-produced influenza vaccines as a feasible pandemic response strategy for South Africa and other developing countries.

Recombination hotspots and host susceptibility modulate the adaptive value of recombination during maize streak virus evolution

Background Maize streak virus -strain A (MSV-A; Genus Mastrevirus, Family Geminiviridae), the maize-adapted strain of MSV that causes maize streak disease throughout sub-Saharan Africa, probably arose between 100 and 200 years ago via homologous recombination between two MSV strains adapted to wild grasses. MSV recombination experiments and analyses of natural MSV recombination patterns have revealed that this recombination event entailed the exchange of the movement protein - coat protein gene cassette, bounded by the two genomic regions most prone to recombination in mastrevirus genomes; the first surrounding the virion-strand origin of replication, and the second around the interface between the coat protein gene and the short intergenic region. Therefore, aside from the likely adaptive advantages presented by a modular exchange of this cassette, these specific breakpoints may have been largely predetermined by the underlying mechanisms of mastrevirus recombination. To investigate this hypothesis, we constructed artificial, low-fitness, reciprocal chimaeric MSV genomes using alternating genomic segments from two MSV strains; a grass-adapted MSV-B, and a maize-adapted MSV-A. Between them, each pair of reciprocal chimaeric genomes represented all of the genetic material required to reconstruct - via recombination - the highly maize-adapted MSV-A genotype, MSV-MatA. We then co-infected a selection of differentially MSV-resistant maize genotypes with pairs of reciprocal chimaeras to determine the efficiency with which recombination would give rise to high-fitness progeny genomes resembling MSV-MatA. Results Recombinants resembling MSV-MatA invariably arose in all of our experiments. However, the accuracy and efficiency with which the MSV-MatA genotype was recovered across all replicates of each experiment depended on the MSV susceptibility of the maize genotypes used and the precise positions - in relation to known recombination hotspots - of the breakpoints required to re-create MSV-MatA. Although the MSV-sensitive maize genotype gave rise to the greatest variety of recombinants, the measured fitness of each of these recombinants correlated with their similarity to MSV-MatA. Conclusions The mechanistic predispositions of different MSV genomic regions to recombination can strongly influence the accessibility of high-fitness MSV recombinants. The frequency with which the fittest recombinant MSV genomes arise also correlates directly with the escalating selection pressures imposed by increasingly MSV-resistant maize hosts.