Antioxidants attenuate hyperglycaemia-mediated brain endothelial cell dysfunction and blood–brain barrier hyperpermeability

Aims: Hyperglycaemia (HG), in stroke patients, is associated with worse neurological outcome by compromising endothelial cell function and the blood–brain barrier (BBB) integrity. We have studied the contribution of HG-mediated generation of oxidative stress to these pathologies and examined whether antioxidants as well as normalization of glucose levels following hyperglycaemic insult reverse these phenomena. Methods: Human brain microvascular endothelial cell (HBMEC) and human astrocyte co-cultures were used to simulate the human BBB. The integrity of the BBB was measured by transendothelial electrical resistance using STX electrodes and an EVOM resistance meter, while enzyme activities were measured by specific spectrophotometric assays. Results: After 5 days of hyperglycaemic insult, there was a significant increase in BBB permeability that was reversed by glucose normalization. Co-treatment of cells with HG and a number of antioxidants including vitamin C, free radical scavengers and antioxidant enzymes including catalase and superoxide dismutase mimetics attenuated the detrimental effects of HG. Inhibition of p38 mitogen-activated protein kinase (p38MAPK) and protein kinase C but not phosphoinositide 3 kinase (PI3 kinase) also reversed HG-induced BBB hyperpermeability. In HBMEC, HG enhanced pro-oxidant (NAD(P)H oxidase) enzyme activity and expression that were normalized by reverting to normoglycaemia. Conclusions: HG impairs brain microvascular endothelial function through involvements of oxidative stress and several signal transduction pathways.

Towards local cultures of sustainability : facilitating community created environment education centres through design

Ecological sustainability has been proposed to address the problem of human impacts increasingly degrading planetary resources and ecosystems, threatening biodiversity, eco-services and human survival. Ecological sustainability is an imperative, with Australia having one of the highest eco-footprints per person worldwide. While significant progress has been made via implementation of ecologically sustainable design in urban communities, relatively little has been undertaken in small, disparate regional communities in Australia. Regional communities are disadvantaged by rural economic decline associated with structural change and inequities of resource transfer. The ecologically sustainable solution is holistic, so all settlements need to be globally wise, richly biodiverse yet locally specific. As a regional solution to this global problem, this research offers the practical means by which a small regional community can contribute. It focuses on the design and implementation of a community centre and the fostering of transformative community learning through an integrated ‘learning community’ awareness of ecologically sustainable best practice. Lessons learned are documented by the participant researcher who as a designer, facilitator, local resident and social narrator has been deeply connected with the Tweed-Caldera region over a period since 1980. The collective action of the local community of Chillingham has been diligently recorded over a decade of design and development. Over this period, several positive elements emerged in terms of improvements to the natural and built environment, greater social cohesion and co-operative learning along with a shift towards a greener local economy. Behavioural changes in the community were noted as residents strived to embrace ecological ideals and reduce fossil fuel dependency. They found attractive local solutions to sourcing of food and using local employment opportunities to up skill their residents via transformative learning as a community in transition. Finally, the catalytic impact of external partnering has also been documented. How well the region as a whole has achieved its ecologically sustainable objectives is measured in terms of the delivered success of private and public partnering with the community, the creation of a community centre cum environment education centre, the restoration of local heritage buildings, the repair of riparian forests and improved water conditions in local river systems, better roads and road safety, local skills and knowledge transfer, support of local food and local/regional growers markets to attract tourists via the integrated trails network. In aggregate, each and every element contributes to a measure of eco-positive development for the built environment, its social organisation and its economy that has guided the local community to find its own pathway to sustainability. Within the Tweed-Caldera bioregion in northern New South Wales, there has been a lack of strategic planning, ecologically sustainable knowledge and facilities in isolated communities that could support the development of a local sustained green economy, provide a hub for socio-cultural activities and ecology based education. The first challenge in this research was to model a whole systems approach to eco-positive development in Chillingham, NSW, a small community where Nature and humanity know no specific boundary. The net result was the creation of a community environment education centre featuring best-affordable ecological practice and regionally distinctive, educational building form from a disused heritage building (cow bale). This development, implemented over a decade, resonated with the later regional wide programs that were linked in the Caldera region by the common purpose of extending the reach of local and state government assistance to regional NSW in economic transition coupled with sustainability. The lessons learned from these linked projects reveal that subsequent programs have been significantly easier to initiate, manage, develop and deliver results. In particular, pursuing collaborative networks with all levels of government and external private partners has been economically effective. Each community’s uniqueness has been celebrated and through drawing out these distinctions, has highlighted local vision, strategic planning, sense of belonging and connection of people with place. This step has significantly reduced the level of friction between communities that comes from natural competition for the finite pool of funds. Following the pilot Tweed-Caldera study, several other NSW regional communities are now undertaking a Community Economic Transition Program based on the processes, trials and positive experiences witnessed in the Tweed-Caldera region where it has been demonstrated that regional community transition programs can provide an opportunity to plan and implement effective long term strategies for sustainability, empowering communities to participate in eco-governance. This thesis includes the design and development of a framework for community created environment education centres to provide an equal access place for community to participate to meet their essential needs locally. An environment centre that facilitates community transition based on easily accessible environmental education, skills and infrastructure is necessary to develop local cultures of sustainability. This research draws upon the literatures of ecologically sustainable development, environmental education and community development in the context of regional community transition towards ‘strong sustainability’. The research approach adapted is best described as a four stage collaborative action research cycle where the participant researcher (me) has a significant involvement in the process to foster local cultures of sustainability by empowering its citizens to act locally and in doing so, become more self reliant and socially resilient. This research also draws upon the many fine working exemplars, such as the resilience of the Cuban people, the transition town initiative in Totnes, U.K. and the models of Australian Community Gardens, such as CERES (Melbourne) and Northey Street (Brisbane). The objectives of this study are to research and evaluate exemplars of ecologically sustainable environment education centres, to facilitate the design and development of an environment education centre created by a small regional community as an ecologically sustainable learning environment; to facilitate a framework for community transition based on environmental education, skills and infrastructure necessary to develop local cultures of sustainability. The research was undertaken as action research in the Tweed Caldera in Northern NSW. This involved the author as participant researcher, designer and volunteer in two interconnected initiatives: the Chillingham Community Centre development and the Caldera Economic Transition Program (CETP). Both initiatives involved a series of design-led participatory community workshops that were externally facilitated with the support of government agency partnerships, steering committees and local volunteers. Together the Caldera research programs involved communities participating in developing their own strategic planning process and outcomes. The Chillingham Community Centre was developed as a sustainable community centre/hub using a participatory design process. The Caldera Economic Transition Program (CETP) prioritised Caldera region projects: the Caldera farmer’s market; community gardens and community kitchens; community renewable energy systems and an integrated trails network. The significant findings were: the CETP projects were capable of moving towards an eco-positive design benchmark through transformative learning. Community transition to sustainability programs need to be underpinned by sustainability and environmental education based frameworks and practical on ground experience in local needs based projects through transformative learning. The actioned projects were successfully undertaken through community participation and teamwork. Ecological footprint surveys were undertaken to guide and assess the ongoing community transition process, however the paucity of responses needs to be revisited. The concept of ecologically sustainable development has been adopted internationally, however existing design and planning strategies do not assure future generations continued access to healthy natural life support systems. Sustainable design research has usually been urban focussed, with little attention paid to regional communities. This study seeks to redress this paucity through the design of ecologically sustainable (deep green) learning environments for small regional communities. Through a design-led process of environmental education, this study investigates how regional communities can be facilitated to model the principles of eco-positive development to support transition to local cultures of sustainability. This research shows how community transition processes and projects can incorporate sustainable community development as transformative learning through design. Regional community transition programs can provide an opportunity to plan long term strategies for sustainability, empowering people to participate in eco-governance. A framework is developed for a community created environment education centre to provide an equal access place for the local community to participate in implementing ways to meet their essential needs locally. A community environment education centre that facilitates community transition based on holistic environmental education, skills and infrastructure is necessary to develop local cultures of sustainability.

Engraftment outcomes after HPC co-culture with mesenchymal stromal cells and osteoblasts

Haematopoietic stem cell (HSC) transplantation is an established cell-based therapy for a number of haematological diseases. To enhance this therapy, there is considerable interest in expanding HSCs in artificial niches prior to transplantation. This study compared murine HSC expansion supported through co-culture on monolayers of either undifferentiated mesenchymal stromal cells (MSCs) or osteoblasts. Sorted Lineage− Sca-1+ c-kit+ (LSK) haematopoietic stem/progenitor cells (HPC) demonstrated proliferative capacity on both stromal monolayers with the greatest expansion of LSK shown in cultures supported by osteoblast monolayers. After transplantation, both types of bulk-expanded cultures were capable of engrafting and repopulating lethally irradiated primary and secondary murine recipients. LSKs co-cultured on MSCs showed comparable, but not superior, reconstitution ability to that of freshly isolated LSKs. Surprisingly, however, osteoblast co-cultured LSKs showed significantly poorer haematopoietic reconstitution compared to LSKs co-cultured on MSCs, likely due to a delay in short-term reconstitution. We demonstrated that stromal monolayers can be used to maintain, but not expand, functional HSCs without a need for additional haematopoietic growth factors. We also demonstrated that despite apparently superior in vitro performance, co-injection of bulk cultures of osteoblasts and LSKs in vivo was detrimental to recipient survival and should be avoided in translation to clinical practice.

Complete genomic sequences for hepatitis C virus subtypes 6e and 6g isolated from Chinese patients with injection drug use and HIV-1 co-infection

In one of our recent studies, two HCV genotype 6 variants were identified in patients from Hong Kong and Guangxi in southern China, with injection drug use and HIV-1 co-infection. We report the complete genomic sequences for these two variants: GX004 and

Plasmid DNA analysis of Staphylococcus epidermidis isolated from blood and colonization cultures in very low birth weight neonates

We prospectively studied the course of colonization and sepsis with Staphylococcus epidermidis among 29 very low birth weight neonates undergoing prolonged umbilical catheterization. S. epidermidis bacteremia occurred in 7 patients. In 6 bacteremia was preceded by positive colonization cultures. Isolates obtained from nares, base of umbilicus, umbilical catheter entry sites, catheter tips and blood were examined for plasmid DNA profiles. In 4 patients the plasmid profiles of the catheter entry site isolates were identical with those of the blood isolates. In the other 3 bacteremic patients plasmid profiles of the catheter entry site and blood isolates were different. No correlation was observed in the plasmid DNA patterns of isolates obtained from catheter tip cultures as compared to the corresponding blood cultures. The blood isolates from bacteremic patients had different plasmid profiles.

Co-60 gamma irradiation prevents Bothrops jararacussu venom neurotoxicity and myotoxicity in isolated mouse neuromuscular junction

The ability of gamma radiation from Co-60 (2000 Gy) to attenuate the toxic effects of Bothrops jararacussu venom was investigated on mouse neuromuscular preparations in vitro. A comparative study between the effects of native and irradiated venoms was performed on both phrenic-diaphragm (PD) and extensor digitorum longus (EDL) preparations by means of myographic, biochemical and morphological techniques. Native venom (10 and 20 mug/ml) induced a concentration-dependent paralysis of both directly and indirectly evoked contractions on PD preparations. At 20 mug/ml, it also caused a pronounced myotoxic effect on the EDL muscle preparation that was characterized by an increase of creatine kinase release and by several morphological changes of this preparation. By contrast, irradiated venom, even at concentrations as high as 40 mug/ml, induced neither paralyzing nor myotoxic effects. It was concluded that Co-60 gamma radiation is able to abolish both the paralyzing and the myotoxic effects of B. jararacussu venom on the mouse neuromuscular junction. These findings support the hypothesis that gamma radiation could be an important toot to improve antisera production by reducing toxicity while preserving immunogenicity. (C) 2002 Elsevier B.V. Ltd. All rights reserved.

Coagulase-negative staphylococci strains resistant to oxacillin isolated from neonatal blood cultures

Coagulase-negative staphylococci (CoNS) are the microorganisms most frequently isolated from clinical samples and are commonly found in neonatal blood cultures. Oxacillin is an alternative treatment of choice for CoNS infections; however, resistance to oxacillin can have a substantial impact on healthcare by adversely affecting morbidity and mortality. The objective of this study was to detect and characterise oxacillin-resistant CoNS strains in blood cultures of newborns hospitalised at the neonatal ward of the University Hospital of the Faculty of Medicine of Botucatu. One hundred CoNS strains were isolated and the mecA gene was detected in 69 of the CoNS strains, including 73.2% of Staphylococcus epidermidis strains, 85.7% of Staphylococcus haemolyticus strains, 28.6% of Staphylococcus hominis strains and 50% of Staphylococcus lugdunensis strains. Among these oxacillin-resistant CoNS strains, staphylococcal cassette chromosome mec (SCCmec) type I was identified in 24.6%, type II in 4.3%, type III in 56.5% and type IV in 14.5% of the strains. The data revealed an increase in the percentage of CoNS strains isolated from blood cultures from 1991-2009. Furthermore, a predominant SCCmec profile of the oxacillin-resistant CoNS strains isolated from neonatal intensive care units was identified with a prevalence of SCCmec types found in hospital-acquired strains.

Reduced susceptibility to vancomycin and biofilm formation in methicillin-resistant Staphylococcus epidermidis isolated from blood cultures

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Probiotic properties of lactic acid bacteria isolated from water-buffalo mozzarella cheese

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Produção de progesterona pelas células luteínicas esteroidogênicas bovina cultivadas e co-cultivadas in vitro

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

The effects of elevated CO2 on the growth and toxicity of field populations and cultures of the saxitoxin-producing dinoflagellate, Alexandrium fundyense

The effects of coastal acidification on the growth and toxicity of the saxitoxin-producing dinoflagellate Alexandrium fundyense were examined in culture and ecosystem studies. In culture experiments, Alexandrium strains isolated from Northport Bay, New York, and the Bay of Fundy, Canada, grew significantly faster (16-190%; p < 0.05) when exposed to elevated levels of PCO2 ( 90-190 Pa=900-1900 µatm) compared to lower levels ( 40 Pa=400 µatm). Exposure to higher levels of PCO2 also resulted in significant increases (71-81%) in total cellular toxicity (fg saxitoxin equivalents/cell) in the Northport Bay strain, while no changes in toxicity were detected in the Bay of Fundy strain. The positive relationship between PCO2 enrichment and elevated growth was reproducible in natural populations from New York waters. Alexandrium densities were significantly and consistently enhanced when natural populations were incubated at 150 Pa PCO2 compared to 39 Pa. During natural Alexandrium blooms in Northport Bay, PCO2 concentrations increased over the course of a bloom to more than 170 Pa and were highest in regions with the greatest Alexandrium abundances, suggesting Alexandrium may further exacerbate acidification and/or be especially adapted to these acidi-fied conditions. The co-occurrence of Alexandrium blooms and elevated PCO2 represents a previously unrecognized, compounding environmental threat to coastal ecosystems. The ability of elevated PCO2 to enhance the growth and toxicity of Alexandrium indicates that acidification promoted by eutrophication or climate change can intensify these, and perhaps other, harmful algal blooms.

Co-Permeability of 3H-Labeled Water and 14C-Labeled Organic Acids across Isolated Plant Cuticles1 : Investigating Cuticular Paths of Diffusion and Predicting Cuticular Transpiration

Penetration of 3H-labeled water (3H2O) and the 14C-labeled organic acids benzoic acid ([14C]BA), salicylic acid ([14C]SA), and 2,4-dichlorophenoxyacetic acid ([14C]2,4-D) were measured simultaneously in isolated cuticular membranes of Prunus laurocerasus L., Ginkgo biloba L., and Juglans regia L. For each of the three pairs of compounds (3H2O/[14C]BA, 3H2O/[14C]SA, and 3H2O/[14C]2,4-D) rates of cuticular water penetration were highly correlated with the rates of penetration of the organic acids. Therefore, water and organic acids penetrated the cuticles by the same routes. With the combination 3H2O/[14C]BA, co-permeability was measured with isolated cuticles of nine other plant species. Permeances of 3H2O of all 12 investigated species were highly correlated with the permeances of [14C]BA (r2 = 0.95). Thus, cuticular transpiration can be predicted from BA permeance. The application of this experimental method, together with the established prediction equation, offers the opportunity to answer several important questions about cuticular transport physiology in future investigations.

Effect of commercial starter cultures and native yeasts on ochratoxin a production by aspergillus westerdijkiae and penicillium nordicum in meat products

Processed meat products are of worldwide importance and, because of their intrinsic factors as well as the processing methods, they are highly prone to fungal and mycotoxin contamination. Ochratoxin A (OTA) is the most significant mycotoxin in processed meat products. Penicillium nordicum is considered to be responsible for OTA contamination of meat products, as it is highly adapted to salt and protein-rich matrices and is moderately psycrotrophic. However, another OTA-producing fungus, Aspergillus westerdijkiae, adapted to carbon-rich matrices such as cereals and coffee beans, has been recently associated with high levels of OTA in meat products. Several Lactic Acid Bacteria (LAB) and yeasts have been tested as biocontrol agents against P. nordicum growth and OTA production in meat products, with promising results, but none of the studies have considered A. westerdijkiae. The aim of this work was to evaluate in vitro the effect of a commercial starter culture used in sausage fermentation and four yeasts isolated from dry-cured sausage on these two OTA-producing fungi, both in terms of fungal growth and of OTA production, using different meat-based culture media as model systems. The mechanisms underlying the observed effect were also studied. For this purpose, C. krusei, C. zeylanoides, R. mucilaginosa, R. glutinis, a mix of these yeasts and the starter culture were co-inoculated with P. nordicum and A. westerdijkiae in industrial sausage, traditional sausage, and ham-based media, under conditions of water activity, salt concentration and temperature that mimic real conditions at beginning and end of sausage curing process. Fungal growth was determined by measuring colony diameter, and OTA production was quantified by HPLC-FLD after extraction with methanol. Yeasts where found to inhibit significantly the growth of both fungi. P. nordicum was unable to produce detectable OTA in both sausage-based media under any condition. In ham, yeasts reduced OTA production, while the starter culture significantly increased it. Unexpectedly, OTA production by A. westerdijkiae was significantly stimulated in all media tested by all microorganisms. Matrix has a significant effect on OTA production by P. nordicum, but not by A. westerdijkiae, for which only temperature showed to have effect. By testing the mechanisms of action by which starter culture and C. zeylanoides influenced fungal responses, we were able to determine that direct contact and simultaneous growth of test organisms were the mechanisms more significantly involved in the responses. In conclusion, ochratoxigenic fungi do not all respond to antagonistic microorganisms in the same way. The use of biocontrol agents with the intent of reducing fungal growth and mycotoxin production by one fungus can have unexpected effects on others, thus leading to unforeseen safety problems. Further experiments are recommended to properly understand the reasons behind the different effects of microorganisms, to ensure their safe as biocontrol agents.