Engineering plant cysteine protease inhibitors for the transgenic control of banana weevil, Cosmopolites sordidus (Germar) (Coleoptera : Curculionidae) and other coleopteran insects in transgenic plants

Cysteine protease inhibitors (cystatins) are expressed in plants in response to wounding and insect herbivory and they form part of the native host-plant defence system. Cysteine proteases are enzymes important in the break down of dietary proteins mainly in the mid gut of coleopteran insects such as the banana weevil. The inhibition of these proteases has a direct effect on the digestive activity of the insect resulting in protein deficiency. This significantly affects insect development and survival. Based on these observations, strategies have been designed involving expression of cysteine protease inhibitors for the transgenic control of insect pests of several crop plants. For this study, it was hypothesized that the major proteases in banana weevil are cysteine proteases and can be effectively targeted by plant cystatins. It was further hypothesised that since plant cystatins are defense related, certain amino acid residues may have undergone positive selection. This provides an opportunity to increase their inhibitory potential to the weevil gut proteases via protein engineering. To prove the hypotheses, both in-vitro and in-vivo assays were set up thus allowing us to demonstrate the presence of cysteine type proteases banana weevil as well as the effect of cystatins on the weevil proteases and early development. Initial in-vitro experiments were able to characterize the proteolytic activity of the banana weevil gut proteases, which are mostly of the cysteine type, and in particular cathepsin B and L like. Two recombinant phytocystatins were further successfully produced using a 6xHis-tagged affinity chromatogephy system in Escherichia coli bacteria. The recombinant phytocystatins were used in a newly developed vacuum infiltration assay system using banana stems. Young weevil larvae were allowed to develop on phytocystatin-treated stems for up to 10 days. They had a 60% reduction in body weight and rate of growth compared to those that grew in untreated stems. By carrying out sitedirected mutagenesis to improve the inhibition efficiency of a model papaya cystatin, more than 8 amino acid residues were found to be subjected to positive selection. Mutation of amino acids yielded improved the inhibition potential of papaya cystatin against the model cysteine protease papain. Increased inhibition was greatest when amino acids were changed in the highly variable regions of the amino acid sequence very closely to the conserved regions. This study has been able to show for the first time that banana weevils use cysteine protease as major protein hydrolysis enzymes and that these can be effectively targeted by plant cystatins. It has also created novel phytocystatins using engineering of single amino acid sites following an evolutionary approach to modulate them for improved activity and targeting specific proteases.Thesis (PhD)--University of Pretoria, 2009.Plant Scienceunrestricte

Building a bi-directional promoter binary vector from the intergenic region of Arabidopsis thaliana cab1 and cab2 divergent genes useful for plant transformation

The ability to express genes in a controlled and limited domain is essential to succeed in targeted genetic modification. Having tools by which to rapidly and conveniently generate constructs which can be assayed in a diverse array of plant species expedites research and end-product development. Targeting specifically green plant tissues offers an opportunity to effect changes to diverse processes such as water use efficiency, photosynthesis, predation and nutrition. To facilitate the generation of transgenes to be expressed in this domain, we created a series of plasmids called p2CABA based on the Arabidopsis thaliana chlorophyll a/b gene promoter, a single natural bidirectional promoter that can drive and express two different genes at the same time. Studies we carried out showed reporter gene, GUS expressed in leaves and stems but not in the roots, as expected since this endogenous promoter controls the expression of two photosynthetic genes in A. thaliana. We, therefore, utilized the intergenic region between the A. thaliana cab1 and cab2 divergent genes to design and construct a bidirectional promoter vector containing two multiple cloning sites and a gateway recombination cassette. This in turn will help minimize gene silencing and achieve desirable expression pattern of transgenes, a critical issue in plant genetic engineering and in this report we show their use in Medicago and tomato.Key words: Arabidopsis cab genes, bidirectional promoters, gene expression, plant genetic engineering, reporter genes, photosynthetic genes

Enhancing banana weevil (Cosmopolites sordidus) resistance by plant genetic modification: A perspective

Banana weevil is a serious pest of bananas and plantains in Africa. The development of resistant cultivars is seen as the long term and more sustainable control strategy. The difficulty in conventional breeding of bananas and plantains has prompted efforts towards the use of genetic transformation for banana and plantain improvement. In this review, the current status of banana weevil resistance, sources of resistance and resistance mechanisms is assessed. Further, current efforts and future prospects for identifying resistance genes outside the genus Musa with potential to control banana weevil in a transgenic approach are outlined and discussed. Key words: Banana weevil, host plant resistance, pest resistance genes, transgenic plants African Journal of Biotechnology Vol. 2 (12), pp. 563-569, December 200

Identity of Fusarium species associated with collar rot and wilt in passion fruit (Passiflora edulis)

Background: Despite the immense contribution of passion fruits to people’s livelihood on a global scale, the crop’s productivity remains low owing to fungal diseases causing up to 100% loss. Fungi are highly variable and the identity of species or variates responsible for recently devastating passion fruit wilt and collar rot diseases had not been characterized. This study was aimed at identifying pathogens causing wilt and collar rot symptoms in passion fruits. Methodology: Fungi were isolated from diseased samples collected from three locations in Central Uganda to identify Fusarium spp associated with collar rot and wilting of passion fruit. This was established by differentiating mycelium pigmentation on Potato Dextrose Agar (PDA), examining slides at X40 magnification under a light microscope for specific macro and microconidia, and amplification with specific Transcription Elongation Factor-1α, TEF 1α primers for identification of Fusarium spp. Results: It was revealed that wilting was associated with a single species, out of 6 selected isolates from the suspected wilted plant, 3 were Fusarium spp associated with the disease in the field but only one of these isolates was proved to be a pathogenic type Fusarium oxysporium. Collar rot was associated with one pathogenic Fusarium spp out of the 6 selected isolates. Conclusion: The results indicate that collar rot and Fusarium wilt are each caused by specific strains of Fusarium pathogens. Recommendation: The identification of pathogenic Fusarium in farmers’ orchards is a starting point for designing effective disease management measures against the predominant fungal pathogenic variants in passion fruits. 

Agrobacterium mediated transformation of banana (Musa sp.) cv. Sukali Ndiizi (ABB) with a modified Carica papaya cystatin (CpCYS) gene

Conventional banana breeding for pest and disease resistance is a very difficult and slow process due to the limited sources of resistance, sterility of cultivated banana varieties, high polyploidy levels, long cropping cycle and the lack of rapid screening methods. Molecular breeding using the transgenic approach with candidate genes such as cystatins offers an alternative method to banana improvement. Cystatin proteins inhibit the activity of cysteine proteases responsible for the breakdown of dietary proteins in the gut of many pests including nematodes resulting in protein deficiency. In this study, the papaya cystatin gene was introduced into the banana genome. Embryogenic cell suspension (ECS) cultures of the banana cultivar Sukali Ndiizi (ABB) were used as explants material for the successful transformation of banana. The Carica papaya cystatin gene (CpCYS-Mut89) previously modified to improve its inhibitory potential against banana pests was introduced into this cultivar using Agrobacterium tumefaciens, strain LBA4404 and the gus reporter gene was used to observe successful transformation process. We report the successful protocol for routine transformation of this cultivar, which was completed in six months with plant regeneration observed at a frequency of 23%. An additional four months was required to multiply the regenerant lines in order to have at least 20 plants per line for downstream challenging studies. Putatively transgenic plants were analyzed by PCR using hpt and CpCYS-Mut89 specific primers to confirm the presence of transgenes. Out of 28 selected lines, 27 were positive for both hpt and CpCYS-Mut89 transgenes giving 96.4% transformation efficiency. Five lines were then selected on the basis of putative PCR positives and a Southern blot analysis gave hybridization signals with 1 to 4 copy number integration patterns characteristic of Agrobacterium mediated transformation. These results confirm stable gene integration in East African banana cultivar cv. Sukali Ndiizi (genome group ABB) through an efficient Agrobacterium-mediated transformation protocol described for routine use in future improvement of this crop with genes of economic importance.Bioversity International through the National Banana Research Programmme of the National Agricultural Research Organization, Uganda.http://www.academicjournals.org/AJBam201

The Prevalence of Asymptomatic Bacteriuria and Associated Factors among Women Attending Antenatal Clinics in Lower Mulago Hospital, Uganda

Asymptomatic bacteriuria (ASB) complicates 2-14% of pregnancies. If not treated in pregnancy it may progress to symptomatic urinary tract infection in 25 % of the cases. Some of the complications of untreated ASB in pregnancy include maternal anaemia, pregnancy and premature rupture of membranes.  In the fetus it may cause abortion, and premature labour. Our objective was to determine the prevalence of asymptomatic bacteriuria in pregnancy and associated factors.  This cross sectional study was carried out in lower Mulago hospital antenatal clinic. We consecutively recruited 385 women with no symptoms of urinary tract infection. The outcome of interest was asymptomatic bacteriuria. A questionnaire was used to record clients’ data. Urine specimens were taken for culture and sensitivity. The prevalence of asymptomatic bacteria was determined. Bivariate analysis was done to find the association between asymptomatic bacteriuria, with maternal risk factors. Four hundred and eight (408) pregnant women were enrolled in the study. The prevalence of ASB+ was found to be 12.2%.The factors associated with asymptomatic bacteriuria were maternal age ≥35 years, OR 2.84, 95 % CI ( 1.2-6.4), Gravidity≥5, OR 2.2, 95%CI  (1.1-4.4), history of UTI, OR 2.6, 95 % CI (1.3-5.1). The prevalence of asymptomatic bacteriuria among women attending antenatal clinic in lower Mulago hospital is high.  Screening for asymptomatic bacteriuria should be done for all women attending lower Mulago hospital antenatal clinic with particular emphasis in all women of the age of  ³ 35 years, multiparous women and those with history of urinary tract infection.

Outcome of a workshop

International audience; In tackling agricultural challenges, policy-makers in sub-Saharan Africa (SSA) have increasingly considered genetically modified (GM) crops as a potential tool to increase productivity and to improve product quality. Yet, as elsewhere in the world, the adoption of GM crops in SSA has been marked by controversy, encompassing not only the potential risks to animal and human health, and to the environment, but also other concerns such as ethical issues, public participation in decision-making, socio-economic factors and intellectual property rights. With these non-scientific factors complicating an already controversial situation, disseminating credible information to the public as well as facilitating stakeholder input into decision-making is essential. In SSA, there are various and innovative risk communication approaches and strategies being developed, yet a comprehensive analysis of such data is missing. This gap is addressed by giving an overview of current strategies, identifying similarities and differences between various country and institutional approaches and promoting a way forward, building on a recent workshop with risk communicators working in SSA

CD4+ T cell recovery during suppression of HIV replication: an international comparison of the immunological efficacy of antiretroviral therapy in North America, Asia and Africa

Background: Even among HIV-infected patients who fully suppress plasma HIV RNA replication on antiretroviral therapy, genetic (e.g. CCL3L1 copy number), viral (e.g. tropism) and environmental (e.g. chronic exposure to microbial antigens) factors influence CD4 recovery. These factors differ markedly around the world and therefore the expected CD4 recovery during HIV RNA suppression may differ globally. Methods: We evaluated HIV-infected adults from North America, West Africa, East Africa, Southern Africa and Asia starting non-nucleoside reverse transcriptase inhibitor-based regimens containing efavirenz or nevirapine, who achieved at least one HIV RNA level <500/µl in the first year of therapy and observed CD4 changes during HIV RNA suppression. We used a piecewise linear regression to estimate the influence of region of residence on CD4 recovery, adjusting for socio-demographic and clinical characteristics. We observed 28 217 patients from 105 cohorts over 37 825 person-years. Results: After adjustment, patients from East Africa showed diminished CD4 recovery as compared with other regions. Three years after antiretroviral therapy initiation, the mean CD4 count for a prototypical patient with a pre-therapy CD4 count of 150/µl was 529/µl [95% confidence interval (CI): 517-541] in North America, 494/µl (95% CI: 429-559) in West Africa, 515/µl (95% CI: 508-522) in Southern Africa, 503/µl (95% CI: 478-528) in Asia and 437/µl (95% CI: 425-449) in East Africa. Conclusions: CD4 recovery during HIV RNA suppression is diminished in East Africa as compared with other regions of the world, and observed differences are large enough to potentially influence clinical outcomes. Epidemiological analyses on a global scale can identify macroscopic effects unobservable at the clinical, national or individual regional leve

Cost-effectiveness of voluntary medical male circumcision for HIV prevention across sub-Saharan Africa : results from five independent models

BACKGROUND: Voluntary medical male circumcision (VMMC) has been a recommended HIV prevention strategy in sub-Saharan Africa since 2007, particularly in countries with high HIV prevalence. However, given the scale-up of antiretroviral therapy programmes, it is not clear whether VMMC still represents a cost-effective use of scarce HIV programme resources. METHODS: Using five existing well described HIV mathematical models, we compared continuation of VMMC for 5 years in men aged 15 years and older to no further VMMC in South Africa, Malawi, and Zimbabwe and across a range of setting scenarios in sub-Saharan Africa. Outputs were based on a 50-year time horizon, VMMC cost was assumed to be US$90, and a cost-effectiveness threshold of US$500 was used. FINDINGS: In South Africa and Malawi, the continuation of VMMC for 5 years resulted in cost savings and health benefits (infections and disability-adjusted life-years averted) according to all models. Of the two models modelling Zimbabwe, the continuation of VMMC for 5 years resulted in cost savings and health benefits by one model but was not as cost-effective according to the other model. Continuation of VMMC was cost-effective in 68% of setting scenarios across sub-Saharan Africa. VMMC was more likely to be cost-effective in modelled settings with higher HIV incidence; VMMC was cost-effective in 62% of settings with HIV incidence of less than 0·1 per 100 person-years in men aged 15-49 years, increasing to 95% with HIV incidence greater than 1·0 per 100 person-years. INTERPRETATION: VMMC remains a cost-effective, often cost-saving, prevention intervention in sub-Saharan Africa for at least the next 5 years. FUNDING: Bill & Melinda Gates Foundation for the HIV Modelling Consortium

Ten golden rules for optimal antibiotic use in hospital settings: the WARNING call to action

Antibiotics are recognized widely for their benefits when used appropriately. However, they are often used inappropriately despite the importance of responsible use within good clinical practice. Effective antibiotic treatment is an essential component of universal healthcare, and it is a global responsibility to ensure appropriate use. Currently, pharmaceutical companies have little incentive to develop new antibiotics due to scientific, regulatory, and financial barriers, further emphasizing the importance of appropriate antibiotic use. To address this issue, the Global Alliance for Infections in Surgery established an international multidisciplinary task force of 295 experts from 115 countries with different backgrounds. The task force developed a position statement called WARNING (Worldwide Antimicrobial Resistance National/International Network Group) aimed at raising awareness of antimicrobial resistance and improving antibiotic prescribing practices worldwide. The statement outlined is 10 axioms, or “golden rules,” for the appropriate use of antibiotics that all healthcare workers should consistently adhere in clinical practice