Identification of senescence and death in Emiliania huxleyi and Thalassiosira pseudonana: Cell staining chlorophyll alterations and dimethylsulfoniopropionate (DMSP) metabolism

We measured membrane permeability, hydrolytic enzyme, and caspase-like activities using fluorescent cell stains to document changes caused by nutrient exhaustion in the coccolithophore Emiliania huxleyi and the diatom Thalassiosira pseudonana, during batch-culture nutrient limitation. We related these changes to cell death, pigment alteration, and concentrations of dimethylsulfide (DMS) and dimethylsulfoniopropionate (DMSP) to assess the transformation of these compounds as cell physiological condition changes. E. huxleyi persisted for 1 month in stationary phase; in contrast, T. pseudonana cells rapidly declined within 10 d of nutrient depletion. T. pseudonana progressively lost membrane integrity and the ability to metabolize 5-chloromethylfluorescein diacetate (CMFDA; hydrolytic activity), whereas E. huxleyi developed two distinct CMFDA populations and retained membrane integrity (SYTOX Green). Caspase-like activity appeared higher in E. huxleyi than in T. pseudonana during the post-growth phase, despite a lack of apparent mortality and cell lysis. Photosynthetic pigment degradation and transformation occurred in both species after growth; chlorophyll a (Chl a) degradation was characterized by an increase in the ratio of methoxy Chl a : Chl a in T. pseudonana but not in E. huxleyi, and the increase in this ratio preceded loss of membrane integrity. Total DMSP declined in T. pseudonana during cell death and DMS increased. In contrast, and in the absence of cell death, total DMSP and DMS increased in E. huxleyi. Our data show a novel chlorophyll alteration product associated with T. pseudonana death, suggesting a promising approach to discriminate nonviable cells in nature.

Protection of cells from salinity stress by extracellular polymeric substances in diatom biofilms

Diatom biofilms are abundant in the marine environment. It is assumed (but untested) that extracellular polymeric substances(EPS), produced by diatoms, enable cells to cope with fluctuating salinity. To determine the protective role of EPS, Cylindrotheca closterium was grown in xanthan gum at salinities of 35, 50, 70 and 90 ppt. A xanthan matrix significantly increased cell viability (determined by SYTOX-Green), growth rate and population density by up to 300, 2, 300 and 200%, respectively. Diatoms grown in 0.75% w/v xanthan, subjected to acute salinity shock treatments (at salinities 17.5, 50, 70 and 90 ppt) maintained photosynthetic capacity, F_q′/F_m′, within 4% of pre-shock values, whereas F_q′/F_m′ in cells grown without xanthan declined by up to 64% with hypersaline shock. Biofilms that developed in xanthan at standard salinity helped cells to maintain function during salinity shock. These results provide evidence of the benefits of living in an EPS matrix for biofilm diatoms.

Quantification and viability analyses of Pseudokirchneriella subcapitata algal cells using image-based cytometry

This work aims to evaluate the feasibility of using image-based cytometry (IBC) in the analysis of algal cell quantification and viability, using Pseudokirchneriella subcapitata as a cell model. Cell concentration was determined by IBC to be in a linear range between 1 × 105 and 8 × 106 cells mL−1. Algal viability was defined on the basis that the intact membrane of viable cells excludes the SYTOX Green (SG) probe. The disruption of membrane integrity represents irreversible damage and consequently results in cell death. Using IBC, we were able to successfully discriminate between live (SG-negative cells) and dead algal cells (heat-treated at 65 °C for 60 min; SG-positive cells). The observed viability of algal populations containing different proportions of killed cells was well correlated (R 2 = 0.994) with the theoretical viability. The validation of the use of this technology was carried out by exposing algal cells of P. subcapitata to a copper stress test for 96 h. IBC allowed us to follow the evolution of cell concentration and the viability of copper-exposed algal populations. This technology overcomes several main drawbacks usually associated with microscopy counting, such as labour-intensive experiments, tedious work and lack of the representativeness of the cell counting. In conclusion, IBC allowed a fast and automated determination of the total number of algal cells and allowed us to analyse viability. This technology can provide a useful tool for a wide variety of fields that utilise microalgae, such as the aquatic toxicology and biotechnology fields.

Use of a fluorescence-based approach to assess short-term responses of the alga Pseudokirchneriella subcapitata to metal stress

This work explores the use of fluorescent probes to evaluate the responses of the green alga Pseudokirchneriella subcapitata to the action of three nominal concentrations of Cd(II), Cr(VI), Cu(II) and Zn(II) for a short time (6 h). The toxic effect of the metals on algal cells was monitored using the fluorochromes SYTOX Green (SG, membrane integrity), fluorescein diacetate (FDA, esterase activity) and rhodamine 123 (Rh123, mitochondrial membrane potential). The impact of metals on chlorophyll a (Chl a) autofluorescence was also evaluated. Esterase activity was the most sensitive parameter. At the concentrations studied, all metals induced the loss of esterase activity. SG could be used to effectively detect the loss of membrane integrity in algal cells exposed to 0.32 or 1.3 μmol L−1 Cu(II). Rh123 revealed a decrease in the mitochondrial membrane potential of algal cells exposed to 0.32 and 1.3 μmol L−1 Cu(II), indicating that mitochondrial activity was compromised. Chl a autofluorescence was also affected by the presence of Cr(VI) and Cu(II), suggesting perturbation of photosynthesis. In conclusion, the fluorescence-based approach was useful for detecting the disturbance of specific cellular characteristics. Fluorescent probes are a useful diagnostic tool for the assessment of the impact of toxicants on specific targets of P. subcapitata algal cells.

Characterisation, immunolocalisation and antifungal activity of a lipid transfer protein from chili pepper (Capsicum annuum) seeds with novel alpha-amylase inhibitory properties

Lipid transfer proteins (LTPs) were thus named because they facilitate the transfer of lipids between membranes in vitro. This study was triggered by the characterization of a 9-kDa LTP from Capsicum annuum seeds that we call Ca-LTP(1). Ca-LTP(1) was repurified, and in the last chromatographic purification step, propanol was used as the solvent in place of acetonitrile to maintain the protein`s biological activity. Bidimensional electrophoresis of the 9-kDa band, which corresponds to the purified Ca-LTP(1), showed the presence of three isoforms with isoelectric points (pIs) of 6.0, 8.5 and 9.5. Circular dichroism (CD) analysis suggested a predominance of alpha-helices, as expected for the structure of an LTP family member. LTPs immunorelated to Ca-LTP(1) from C. annuum were also detected by western blotting in exudates released from C. annuum seeds and also in other Capsicum species. The tissue and subcellular localization of Ca-LTP(1) indicated that it was mainly localized within dense vesicles. In addition, isolated Ca-LTP(1) exhibited antifungal activity against Colletotrichum lindemunthianum, and especially against Candida tropicalis, causing several morphological changes to the cells including the formation of pseudohyphae. Ca-LTP(1) also caused the yeast plasma membrane to be permeable to the dye SYTOX green, as verified by fluorescence microscopy. We also found that Ca-LTP(1) is able to inhibit mammalian alpha-amylase activity in vitro.

Leishmanicidal activity of an alkenylphenol from Piper malacophyllum is related to plasma membrane disruption

Leishmaniasis and Chagas disease are parasitic protozoan infections that affect the poorest population in the world, causing high mortality and morbidity. As a result of highly toxic and long-duration treatments, novel, safe and more efficacious drugs are essential. In this work, the methanol (MeOH) extract from the leaves of Piper malacophyllum (Piperaceae) was fractioned to afford one alkenylphenol, which was characterized as 4-[(3'E)-decenyl]phenol (gibbilimbol B) by spectroscopic methods. Anti-protozoan in vitro assays demonstrated for the first time that Leishmania (L.) infantum chagasi was susceptible to gibbilimbol B. with an in vitro EC50 of 23 mu g/mL against axenic promastigotes and an EC50 of 22 mu g/mL against intracellular amastigotes. Gibbilimbol B was also tested for anti-trypanosomal activity (Trypanosoma cruzi) and showed an EC50 value of 17 mu g/mL against trypomastigotes. To evaluate the cytotoxic parameters, this alkenylphenol was tested in vitro against NCTC cells, showing a CC50 of 59 mu g/mL and absent hemolytic activity at the highest concentration of 75 mu g/mL. Using the fluorescent probe SYTOX Green suggested that the alkenylphenol disrupted the Leishmania plasma membrane upon initial incubation. Further drug design studies aiming at derivatives could be a promising tool for the development of new therapeutic agents for leishmaniasis and Chagas disease. (C) 2012 Elsevier Inc. All rights reserved.

Antimicrobial activity of peach and grapevine defensins

Antimicrobial peptides (AMPs) are an important component of the innate immune system of the plants. Plant defensins are a large family of antimicrobial peptides with several interesting features, such as small dimension, high stability and broad spectrum of action. The discovery of new molecules and the study of their mechanism of action allow to consider them attractive for biotechnological applications. In this PhD thesis a defensin from Prunus persica (PpDFN1) and four novel DEFensin Like (DEFL) peptides from Vitis vinifera have been studied. In order to characterize the antimicrobial activity of these molecules, the recombinant mature peptides have been expressed in Escherichia coli and purified to homogeneity by chromatography techniques. PpDFN1 is able to inhibit the growth of B. cinerea, P. expansum and M. laxa with different intensity. The recombinant peptide is capable of membrane permeabilization as demonstrated by SYTOX green fluorescence uptake in treated mycelia. Its interaction with membranes containing sphingolipid species has been shown by artificial lipid monolayers. Furthermore, PpDFN1 displays stronger interaction with monolayers composed by lipids extracted from sensitive fungi with the highest interaction against P. expansum, the most sensitive fungi to PpDFN1 action. DEFL 13, a defensin from grapevine, resulted the strongest antibotrytis peptides. It is electrostatically attracted to the fungal membranes as shown by the antagonist effect of the cations and is able to membrane permeabilization in B. cinerea hyphae. DEFL 13 is internalized in fungal cells and leads to fungal death by activation of some signaling pathways as demonstrated by screening of a mutant collection of B. cinerea

Cell death and degeneration in the symbiotic dinoflagellates of the coral Stylophora pistillata during bleaching

Rising sea temperatures are increasing the incidences of mass coral bleaching (the dissociation of the coral-algal symbiosis) and coral mortality. In this study, the effects of bleaching (induced by elevated light and temperature) on the condition of symbiotic dinoflagellates (Symbiodinium sp.) within the tissue of the hard coral Stylophora pistillata (Esper) were assessed using a suite of techniques. Bleaching of S. pistillata was accompanied by declines in the maximum potential quantum yield of photosynthesis (F-v/F-m, measured using pulse amplitude modulated [PAM] fluorometry), an increase in the number of Sytox-green-stained algae (indicating compromised algal membrane integrity and cell death), an increase in 2',7'-dichlorodihydrofluroscein diacetate (H(2)DCFDA)stained algae (indicating increased oxidative stress), as well as ultrastructural changes (vacuolisation, losses of chlorophyll, and an increase in accumulation bodies). Algae expelled from S. pistillata exhibited a complete disorganisation of cellular contents; expelled cells contained only amorphous material. In situ samples taken during a natural mass coral bleaching event on the Great Barrier Reef in February 2002 also revealed a high number of Sytox-labelled algae cells in symbio. Dinoflagellate degeneration during bleaching seems to be similar to the changes resulting from senescence-phase cell death in cultured algae. These data support a role for oxidative stress in the mechanism of coral bleaching and highlight the importance of algal degeneration during the bleaching of a reef coral.

Capability green

Tract consultants are a landscape architecture practice, founded in 1973 as an offshoot to the highly innovative, interdisciplinary design and build company Merchant Builders, and was perhaps the first truly corporate practice of this type in Australia. Founding directors Rodney Wulff and Steve Calhoun were both instrumental in establishing the undergraduate landscape architecture course at RMIT University, and bringing our Jim Sinatra, who had taught Calhoun at the University of Iowa. Wulff remained for many years the holder of the only doctorate in landscape architecture in the country. This combination of an academic, design and professional agenda was a rich one for Tract in their early days. This founding generosity and interest in the intellectual aspects of landscape architecture continues in relation to the university in a number of ways, including information ones, such as the regular employment of applicants who fail to get into the course at RMIT. In preparing them for re-applying, he has given a number of individuals a way into the profession that the university could not allow.

Village green

For a communal garden in Copenhagen, Stig L. Andersson uses grasses of varying texture and height, creating a new view or spatial experience from every angle. The idea of vegetation texture being an important constituent of planting design is pervasive. Gardening books tell aspiring designers that "colour, texture and form" are the central aspects of planting arrangements. While these elements contribute to this language, they have tended to limit the language of planting to a singular, two dimensional paradigm, where planting is designed in static elevation. This has developed from a perennial-border approach demonstrated by the early 20th century garden designer Gertrude Jekyll, where the viewer is parallel to the bed, and the planting is layered to address this view. If one were to characterise the difference between a garden design and a landscape architectural approach, the latter would seem self-conscious in its use of space, movement and vision.

Living walls - a way to green the built environment

Actions Towards Sustainable Outcomes Environmental Issues/Principal Impacts The increasing urbanisation of cities brings with it several detrimental consequences, such as: • Significant energy use for heating and cooling many more buildings has led to urban heat islands and increased greenhouse gas emissions. • Increased amount of hard surfaces, which not only contributes to higher temperatures in cities, but also to increased stormwater runoff. • Degraded air quality and noise. • Health and general well-being of people is frequently compromised, by inadequate indoor air quality. • Reduced urban biodiversity. Basic Strategies In many design situations, boundaries and constraints limit the application of cutting EDGe actions. In these circumstances, designers should at least consider the following: • Living walls are an emerging technology, and many Australian examples function more as internal feature walls. However,as understanding of the benefits and construction of living walls develops this technology could be part of an exterior facade that enhances a building’s thermal performance. • Living walls should be designed to function with an irrigation system using non-potable water. Cutting EDGe Strategies • Living walls can be part of a design strategy that effectively improves the thermal performance of a building, thereby contributing to lower energy use and greenhouse gas emissions. • Including living walls in the initial stages of design would provide greater flexibility to the design, especially of the facade, structural supports, mechanical ventilation and watering systems, thus lowering costs. • Designing a building with an early understanding of living walls can greatly reduce maintenance costs. • Including plant species and planting media that would be able to remove air impurities could contribute to improved indoor air quality, workplace productivity and well-being. Synergies and References • Living walls are a key research topic at the Centre for Subtropical Design, Queensland University of Technology: http://www.subtropicaldesign.bee.qut.edu.au • BEDP Environment Design Guide: DES 53: Roof and Facade Gardens • BEDP Environment Design Guide: GEN 4: Positive Development – Designing for Net Positive Impacts (see green scaffolding and green space frame walls). • Green Roofs Australia: www.greenroofs.wordpress.com • Green Roofs for Healthy Cities USA: www.greenroofs.org

Green a city grow a wall

As Brisbane grows, it is rapidly becoming akin to any other city in the world with its typical stark grey concrete buildings rather than being characterized by its subtropical element of abundant green vegetation. Living Walls can play a vital role in restoring the loss of this distinct local element of a subtropical city. This paper will start by giving an overview of the traditional methods of greening subtropical cities with the use of urban parks and street trees. Then, by examining a recent heat imaging map of Brisbane, the effect of green cover with the built environment will be shown. With this information from a macro level, this paper will proceed to examine a typical urban block within the Central Business District (CBD) to demonstrate urban densification in relation to greenery in the city. Then, this paper will introduce the new technology where Living Walls have the untapped potential of effectively greening a city where land is scarce and given over to high density development. Living Walls incorporated into building design does not only enhance the subtropical lifestyle that is being lost in modern cities but is also an effective means for addressing climate change. This paper will serve as a preliminary investigation into the effects of incorporating Living Walls into cities. By growing a Living Wall onto buildings, we can be part of an effective design solution for countering global warming and at the same time, Living Walls can return local character to subtropical cities, thereby greening the city as well.

Public awareness of ‘green’ residential property – an empirical survey based on data from New Zealand and Germany

Over the last few years more stringent environmental laws (e.g. the German “Energie¬ein-sparverordnung ENEV” - Energy Performance of Buildings Directive) and soaring energy prices has increased the need for the real estate industry to react and participate in overall energy reduction through efficient house construction and design, as well as upgrading the existing housing stock to be more energy efficient. Therefore the Property Economics Group at Queensland University of Technology in Australia and Nuertingen-Geislingen University in Germany are carrying out research in relation to sustainable housing construction and public awareness of “green” residential property. Part of this research is to gain an understanding of the level of knowledge and importance of these issues to the house buyer and to determine the importance of sustainable housing to the general public. The paper compares data from two different empirical studies; one of studies analyzes the situation in New Zealand, the other is focused on Germany.