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1.  Collapsing Aged Culture of the Cyanobacterium Synechococcus elongatus Produces Compound(s) Toxic to Photosynthetic Organisms 
PLoS ONE  2014;9(6):e100747.
Phytoplankton mortality allows effective nutrient cycling, and thus plays a pivotal role in driving biogeochemical cycles. A growing body of literature demonstrates the involvement of regulated death programs in the abrupt collapse of phytoplankton populations, and particularly implicates processes that exhibit characteristics of metazoan programmed cell death. Here, we report that the cell-free, extracellular fluid (conditioned medium) of a collapsing aged culture of the cyanobacterium Synechococcus elongatus is toxic to exponentially growing cells of this cyanobacterium, as well as to a large variety of photosynthetic organisms, but not to eubacteria. The toxic effect, which is light-dependent, involves oxidative stress, as suggested by damage alleviation by antioxidants, and the very high sensitivity of a catalase-mutant to the conditioned medium. At relatively high cell densities, S. elongatus cells survived the deleterious effect of conditioned medium in a process that required de novo protein synthesis. Application of conditioned medium from a collapsing culture caused severe pigment bleaching not only in S. elongatus cells, but also resulted in bleaching of pigments in a cell free extract. The latter observation indicates that the elicited damage is a direct effect that does not require an intact cell, and therefore, is mechanistically different from the metazoan-like programmed cell death described for phytoplankton. We suggest that S. elongatus in aged cultures are triggered to produce a toxic compound, and thus, this process may be envisaged as a novel regulated death program.
PMCID: PMC4069110  PMID: 24959874
2.  Economic Analysis of Greenhouse Lighting: Light Emitting Diodes vs. High Intensity Discharge Fixtures 
PLoS ONE  2014;9(6):e99010.
Lighting technologies for plant growth are improving rapidly, providing numerous options for supplemental lighting in greenhouses. Here we report the photosynthetic (400–700 nm) photon efficiency and photon distribution pattern of two double-ended HPS fixtures, five mogul-base HPS fixtures, ten LED fixtures, three ceramic metal halide fixtures, and two fluorescent fixtures. The two most efficient LED and the two most efficient double-ended HPS fixtures had nearly identical efficiencies at 1.66 to 1.70 micromoles per joule. These four fixtures represent a dramatic improvement over the 1.02 micromoles per joule efficiency of the mogul-base HPS fixtures that are in common use. The best ceramic metal halide and fluorescent fixtures had efficiencies of 1.46 and 0.95 micromoles per joule, respectively. We also calculated the initial capital cost of fixtures per photon delivered and determined that LED fixtures cost five to ten times more than HPS fixtures. The five-year electric plus fixture cost per mole of photons is thus 2.3 times higher for LED fixtures, due to high capital costs. Compared to electric costs, our analysis indicates that the long-term maintenance costs are small for both technologies. If widely spaced benches are a necessary part of a production system, the unique ability of LED fixtures to efficiently focus photons on specific areas can be used to improve the photon capture by plant canopies. Our analysis demonstrates, however, that the cost per photon delivered is higher in these systems, regardless of fixture category. The lowest lighting system costs are realized when an efficient fixture is coupled with effective canopy photon capture.
PMCID: PMC4048233  PMID: 24905835
3.  Local caspase activation interacts with Slit-Robo signaling to restrict axonal arborization 
The Journal of Cell Biology  2013;203(4):657-672.
Local caspase activation at axonal branch points restricts arbor growth and synaptogenesis by interacting with Slit1a-Robo2 signaling in the central nervous system.
In addition to being critical for apoptosis, components of the apoptotic pathway, such as caspases, are involved in other physiological processes in many types of cells, including neurons. However, very little is known about their role in dynamic, nonphysically destructive processes, such as axonal arborization and synaptogenesis. We show that caspases were locally active in vivo at the branch points of young, dynamic retinal ganglion cell axonal arbors but not in the cell body or in stable mature arbors. Caspase activation, dependent on Caspase-3, Caspase-9, and p38 mitogen-activated protein kinase (MAPK), rapidly increased at branch points corresponding with branch tip addition. Time-lapse imaging revealed that knockdown of Caspase-3 and Caspase-9 led to more stable arbors and presynaptic sites. Genetic analysis showed that Caspase-3, Caspase-9, and p38 MAPK interacted with Slit1a-Robo2 signaling, suggesting that localized activation of caspases lie downstream of a ligand receptor system, acting as key promoters of axonal branch tip and synaptic dynamics to restrict arbor growth in vivo in the central nervous system.
PMCID: PMC3840933  PMID: 24385488
4.  Oxidative Stress Is a Mediator for Increased Lipid Accumulation in a Newly Isolated Dunaliella salina Strain 
PLoS ONE  2014;9(3):e91957.
Green algae offer sustainable, clean and eco-friendly energy resource. However, production efficiency needs to be improved. Increasing cellular lipid levels by nitrogen depletion is one of the most studied strategies. Despite this, the underlying physiological and biochemical mechanisms of this response have not been well defined. Algae species adapted to hypersaline conditions can be cultivated in salty waters which are not useful for agriculture or consumption. Due to their inherent extreme cultivation conditions, use of hypersaline algae species is better suited for avoiding culture contamination issues. In this study, we identified a new halophilic Dunaliella salina strain by using 18S ribosomal RNA gene sequencing. We found that growth and biomass productivities of this strain were directly related to nitrogen levels, as the highest biomass concentration under 0.05 mM or 5 mM nitrogen regimes were 495 mg/l and 1409 mg/l, respectively. We also confirmed that nitrogen limitation increased cellular lipid content up to 35% under 0.05 mM nitrogen concentration. In order to gain insight into the mechanisms of this phenomenon, we applied fluorometric, flow cytometric and spectrophotometric methods to measure oxidative stress and enzymatic defence mechanisms. Under nitrogen depleted cultivation conditions, we observed increased lipid peroxidation by measuring an important oxidative stress marker, malondialdehyde and enhanced activation of catalase, ascorbate peroxidase and superoxide dismutase antioxidant enzymes. These observations indicated that oxidative stress is accompanied by increased lipid content in the green alga. In addition, we also showed that at optimum cultivation conditions, inducing oxidative stress by application of exogenous H2O2 leads to increased cellular lipid content up to 44% when compared with non-treated control groups. Our results support that oxidative stress and lipid overproduction are linked. Importantly, these results also suggest that oxidative stress mediates lipid accumulation. Understanding such relationships may provide guidance for efficient production of algal biodiesels.
PMCID: PMC3961284  PMID: 24651514
5.  First Report of Pseudobodo sp, a New Pathogen for a Potential Energy-Producing Algae: Chlorella vulgaris Cultures 
PLoS ONE  2014;9(3):e89571.
Chlorella vulgaris, is a kind of single-celled green algae, which could serve as a potential source of food and energy because of its photosynthetic efficiency. In our study, a pathogenic organism targeting C. vulgaris was discovered. The algae-lytic activity relates to a fraction from lysates of infected C. vulgaris that was blocked upon filtration through a 3 µm filter. 18S rRNA gene sequence analysis revealed that it shared 99.0% homology with the protist Pseudobodo tremulans. Scanning electron microscope analysis showed that Pseudobodo sp. KD51 cells were approximately 4–5 µm long, biflagellate with an anterior collar around the anterior part of the cell in unstressed feeding cells. Besides the initial host, Pseudobodo sp. KD51 could also kill other algae, indicating its relatively wide predatory spectrum. Heat stability, pH and salinity tolerance experiments were conducted to understand their effects on its predatory activities, and the results showed that Pseudobodo sp. KD51 was heat-sensitive, and pH and salinity tolerant.
PMCID: PMC3943784  PMID: 24599263
6.  Nutrient Limitation in Northern Gulf of Mexico (NGOM): Phytoplankton Communities and Photosynthesis Respond to Nutrient Pulse 
PLoS ONE  2014;9(2):e88732.
Although the Mississippi-Atchafalaya River system exports large amounts of nutrients to the Northern Gulf of Mexico annually, nutrient limitation of primary productivity still occurs offshore, acting as one of the major factors controlling local phytoplankton biomass and community structure. Bioassays were conducted for 48 hrs at two stations adjacent to the river plumes in April and August 2012. High Performance of Liquid Chromatography (HPLC) combined with ChemTax and a Fluorescence Induction and Relaxation (FIRe) system were combined to observe changes in the phytoplankton community structure and photosynthetic activity. Major fluorescence parameters (Fo, Fv/Fm) performed well to reveal the stimulating effect of the treatments with nitrogen (N-nitrate) and with nitrogen plus phosphate (+NPi). HPLC/ChemTax results showed that phytoplankton community structure shifted with nitrate addition: we observed an increase in the proportion of diatoms and prasinophytes and a decrease in cyanobacteria and prymnesiophytes. These findings are consistent with predictions from trait-based analysis which predict that phytoplankton groups with high maximum growth rates (μmax) and high nutrient uptake rates (Vmax) readily take advantage of the addition of limiting nutrients. Changes in phytoplankton community structure, if persistent, could trigger changes of particular organic matter fluxes and alter the micro-food web cycles and bottom oxygen consumption.
PMCID: PMC3925166  PMID: 24551144
7.  Up-Regulated Expression of AOS-LOXa and Increased Eicosanoid Synthesis in Response to Coral Wounding 
PLoS ONE  2014;9(2):e89215.
In octocorals, a catalase–like allene oxide synthase (AOS) and an 8R-lipoxygenase (LOX) gene are fused together encoding for a single AOS-LOX fusion protein. Although the AOS-LOX pathway is central to the arachidonate metabolism in corals, its biological function in coral homeostasis is unclear. Using an acute incision wound model in the soft coral Capnella imbricata, we here test whether LOX pathway, similar to its role in plants, can contribute to the coral damage response and regeneration. Analysis of metabolites formed from exogenous arachidonate before and after fixed time intervals following wounding indicated a significant increase in AOS-LOX activity in response to mechanical injury. Two AOS-LOX isoforms, AOS-LOXa and AOS-LOXb, were cloned and expressed in bacterial expression system as active fusion proteins. Transcription levels of corresponding genes were measured in normal and stressed coral by qPCR. After wounding, AOS-LOXa was markedly up-regulated in both, the tissue adjacent to the incision and distal parts of a coral colony (with the maximum reached at 1 h and 6 h post wounding, respectively), while AOS-LOXb was stable. According to mRNA expression analysis, combined with detection of eicosanoid product formation for the first time, the AOS-LOX was identified as an early stress response gene which is induced by mechanical injury in coral.
PMCID: PMC3925239  PMID: 24551239
8.  Distal Ulna Hook Plate: Angular Stable Implant for Fixation of Distal Ulna 
Journal of Wrist Surgery  2013;2(1):87-92.
Distal ulna fractures, especially styloid injuries, classically have not been repaired, and only recently have these injuries been considered important. Certain fracture patterns of the distal ulna contribute to distal radioulnar joint (DRUJ) incongruity and potential instability. Appropriate fixation of the distal ulna is frequently difficult for several reasons: (1) high incidence of osteoporois in the affected patient population, (2) proximity of the injury to articular surfaces, and (3) lack of a proper implant to treat these injuries. The 2.0-mm locking compression distal ulna plate (LC-DUP) is an anatomically contoured implant with a low profile and fixed angle that provides proper stability to treat injuries of the distal ulna. The plate was designed for the treatment of distal ulna fractures, but its success has led to an extension of its indications to be used in treating symptomatic basistyloid ulnar nonunions and in ulnar shortening osteotomy for ulnocarpal abutment syndrome. The authors' description of the techniques used for each indication as well as their perspectives in the treatment of distal ulna injuries are described in detail in this report.
PMCID: PMC3656578  PMID: 24436795
distal ulna fracture; DUP; LC-DUP; distal ulna hook plate; ulnar styloid; ulnocarpal abutment syndrome; ulnar impaction syndrome; ulna nonunion; ulna fracture; ulnar shortening osteotomy
9.  Effects of Marine and Freshwater Macroalgae on In Vitro Total Gas and Methane Production 
PLoS ONE  2014;9(1):e85289.
This study aimed to evaluate the effects of twenty species of tropical macroalgae on in vitro fermentation parameters, total gas production (TGP) and methane (CH4) production when incubated in rumen fluid from cattle fed a low quality roughage diet. Primary biochemical parameters of macroalgae were characterized and included proximate, elemental, and fatty acid (FAME) analysis. Macroalgae and the control, decorticated cottonseed meal (DCS), were incubated in vitro for 72 h, where gas production was continuously monitored. Post-fermentation parameters, including CH4 production, pH, ammonia, apparent organic matter degradability (OMd), and volatile fatty acid (VFA) concentrations were measured. All species of macroalgae had lower TGP and CH4 production than DCS. Dictyota and Asparagopsis had the strongest effects, inhibiting TGP by 53.2% and 61.8%, and CH4 production by 92.2% and 98.9% after 72 h, respectively. Both species also resulted in the lowest total VFA concentration, and the highest molar concentration of propionate among all species analysed, indicating that anaerobic fermentation was affected. Overall, there were no strong relationships between TGP or CH4 production and the >70 biochemical parameters analysed. However, zinc concentrations >0.10−1 may potentially interact with other biochemical components to influence TGP and CH4 production. The lack of relationship between the primary biochemistry of species and gas parameters suggests that significant decreases in TGP and CH4 production are associated with secondary metabolites produced by effective macroalgae. The most effective species, Asparagopsis, offers the most promising alternative for mitigation of enteric CH4 emissions.
PMCID: PMC3898960  PMID: 24465524
10.  Metabolic Flexibility as a Major Predictor of Spatial Distribution in Microbial Communities 
PLoS ONE  2014;9(1):e85105.
A better understand the ecology of microbes and their role in the global ecosystem could be achieved if traditional ecological theories can be applied to microbes. In ecology organisms are defined as specialists or generalists according to the breadth of their niche. Spatial distribution is often used as a proxy measure of niche breadth; generalists have broad niches and a wide spatial distribution and specialists a narrow niche and spatial distribution. Previous studies suggest that microbial distribution patterns are contrary to this idea; a microbial generalist genus (Desulfobulbus) has a limited spatial distribution while a specialist genus (Methanosaeta) has a cosmopolitan distribution. Therefore, we hypothesise that this counter-intuitive distribution within generalist and specialist microbial genera is a common microbial characteristic. Using molecular fingerprinting the distribution of four microbial genera, two generalists, Desulfobulbus and the methanogenic archaea Methanosarcina, and two specialists, Methanosaeta and the sulfate-reducing bacteria Desulfobacter were analysed in sediment samples from along a UK estuary. Detected genotypes of both generalist genera showed a distinct spatial distribution, significantly correlated with geographic distance between sites. Genotypes of both specialist genera showed no significant differential spatial distribution. These data support the hypothesis that the spatial distribution of specialist and generalist microbes does not match that seen with specialist and generalist large organisms. It may be that generalist microbes, while having a wider potential niche, are constrained, possibly by intrageneric competition, to exploit only a small part of that potential niche while specialists, with far fewer constraints to their niche, are more capable of filling their potential niche more effectively, perhaps by avoiding intrageneric competition. We suggest that these counter-intuitive distribution patterns may be a common feature of microbes in general and represent a distinct microbial principle in ecology, which is a real challenge if we are to develop a truly inclusive ecology.
PMCID: PMC3897421  PMID: 24465487
11.  Response of the Unicellular Diazotrophic Cyanobacterium Crocosphaera watsonii to Iron Limitation 
PLoS ONE  2014;9(1):e86749.
Iron (Fe) is widely suspected as a key controlling factor of N2 fixation due to the high Fe content of nitrogenase and photosynthetic enzymes complex, and to its low concentrations in oceanic surface seawaters. The influence of Fe limitation on the recently discovered unicellular diazotrophic cyanobacteria (UCYN) is poorly understood despite their biogeochemical importance in the carbon and nitrogen cycles. To address this knowledge gap, we conducted culture experiments on Crocosphaera watsonii WH8501 growing under a range of dissolved Fe concentrations (from 3.3 to 403 nM). Overall, severe Fe limitation led to significant decreases in growth rate (2.6-fold), C, N and chlorophyll a contents per cell (up to 4.1-fold), N2 and CO2 fixation rates per cell (17- and 7-fold) as well as biovolume (2.2-fold). We highlighted a two phased response depending on the degree of limitation: (i) under a moderate Fe limitation, the biovolume of C. watsonii was strongly reduced, allowing the cells to keep sufficient energy to maintain an optimal growth, volume-normalized contents and N2 and CO2 fixation rates; (ii) with increasing Fe deprivation, biovolume remained unchanged but the entire cell metabolism was affected, as shown by a strong decrease in the growth rate, volume-normalized contents and N2 and CO2 fixation rates. The half-saturation constant for growth of C. watsonii with respect to Fe is twice as low as that of the filamentous Trichodesmium indicating a better adaptation of C. watsonii to poor Fe environments than filamentous diazotrophs. The physiological response of C. watsonii to Fe limitation was different from that previously shown on the UCYN Cyanothece sp, suggesting potential differences in Fe requirements and/or Fe acquisition within the UCYN community. These results contribute to a better understanding of how Fe bioavailability can control the activity of UCYN and explain the biogeography of diverse N2 fixers in ocean.
PMCID: PMC3897776  PMID: 24466221
12.  The Dimethylsulfide Cycle in the Eutrophied Southern North Sea: A Model Study Integrating Phytoplankton and Bacterial Processes 
PLoS ONE  2014;9(1):e85862.
We developed a module describing the dimethylsulfoniopropionate (DMSP) and dimethylsulfide (DMS) dynamics, including biological transformations by phytoplankton and bacteria, and physico-chemical processes (including DMS air-sea exchange). This module was integrated in the MIRO ecological model and applied in a 0D frame in the Southern North Sea (SNS). The DMS(P) module is built on parameterizations derived from available knowledge on DMS(P) sources, transformations and sinks, and provides an explicit representation of bacterial activity in contrast to most of existing models that only include phytoplankton process (and abiotic transformations). The model is tested in a highly productive coastal ecosystem (the Belgian coastal zone, BCZ) dominated by diatoms and the Haptophyceae Phaeocystis, respectively low and high DMSP producers. On an annual basis, the particulate DMSP (DMSPp) production simulated in 1989 is mainly related to Phaeocystis colonies (78%) rather than diatoms (13%) and nanoflagellates (9%). Accordingly, sensitivity analysis shows that the model responds more to changes in the sulfur:carbon (S:C) quota and lyase yield of Phaeocystis. DMS originates equally from phytoplankton and bacterial DMSP-lyase activity and only 3% of the DMS is emitted to the atmosphere. Model analysis demonstrates the sensitivity of DMS emission towards the atmosphere to the description and parameterization of biological processes emphasizing the need of adequately representing in models both phytoplankton and bacterial processes affecting DMS(P) dynamics. This is particularly important in eutrophied coastal environments such as the SNS dominated by high non-diatom blooms and where empirical models developed from data-sets biased towards open ocean conditions do not satisfactorily predict the timing and amplitude of the DMS seasonal cycle. In order to predict future feedbacks of DMS emissions on climate, it is needed to account for hotspots of DMS emissions from coastal environments that, if eutrophied, are dominated not only by diatoms.
PMCID: PMC3895025  PMID: 24465753
13.  Membrane Labeling of Coral Gastrodermal Cells by Biotinylation: The Proteomic Identification of Surface Proteins Involving Cnidaria-Dinoflagellate Endosymbiosis 
PLoS ONE  2014;9(1):e85119.
The cellular and molecular-scale processes underlying the stability of coral-Symbiodinium endosymbioses remain unclear despite decades of investigation. As the coral gastroderm is the only tissue layer characterized by this unique symbiotic association, the membranes of these symbiotic gastrodermal cells (SGCs) may play important roles in the initiation and maintenance of the endosymbiosis. In order to elucidate the interactions between the endosymbiotic dinoflagellates and their coral hosts, a thorough characterization of SGC membranes is therefore required. Cell surface proteins of isolated SGCs were biotinylated herein by a cell impermeant agent, biotin-XX sulfosuccinimidyl ester. The in situ distribution of these biotinylated proteins was uncovered by both fluorescence and transmission electron microscopic imaging of proteins bound to Alexa Fluor® 488-conjugated streptavidin. The identity of these proteins was then determined by two-dimensional gel electrophoresis followed by liquid chromatography-tandem mass spectrometry. Nineteen (19) proteins were identified, and they are known to be involved in the molecular chaperone/stress response, cytoskeletal remodeling, and energy metabolism. These results not only reveal the molecular characters of the host SGC membrane, but also provide critical insight into understanding the possible role of host membranes in this ecologically important endosymbiotic association.
PMCID: PMC3883709  PMID: 24409319
14.  Carbon Dynamics within Cyclonic Eddies: Insights from a Biomarker Study 
PLoS ONE  2013;8(12):e82447.
It is generally assumed that episodic nutrient pulses by cyclonic eddies into surface waters support a significant fraction of the primary production in subtropical low-nutrient environments in the northern hemisphere. However, contradictory results related to the influence of eddies on particulate organic carbon (POC) export have been reported. As a step toward understanding the complex mechanisms that control export of material within eddies, we present here results from a sediment trap mooring deployed within the path of cyclonic eddies generated near the Canary Islands over a 1.5-year period. We find that, during summer and autumn (when surface stratification is stronger, eddies are more intense, and a relative enrichment in CaCO3 forming organisms occurs), POC export to the deep ocean was 2–4 times higher than observed for the rest of the year. On the contrary, during winter and spring (when mixing is strongest and the seasonal phytoplankton bloom occurs), no significant enhancement of POC export associated with eddies was observed. Our biomarker results suggest that a large fraction of the material exported from surface waters during the late-winter bloom is either recycled in the mesopelagic zone or bypassed by migrant zooplankton to the deep scattering layer, where it would disaggregate to smaller particles or be excreted as dissolved organic carbon. Cyclonic eddies, however, would enhance carbon export below 1000 m depth during the summer stratification period, when eddies are more intense and frequent, highlighting the important role of eddies and their different biological communities on the regional carbon cycle.
PMCID: PMC3875410  PMID: 24386098
15.  Enhanced Production of Green Tide Algal Biomass through Additional Carbon Supply 
PLoS ONE  2013;8(12):e81164.
Intensive algal cultivation usually requires a high flux of dissolved inorganic carbon (Ci) to support productivity, particularly for high density algal cultures. Carbon dioxide (CO2) enrichment can be used to overcome Ci limitation and enhance productivity of algae in intensive culture, however, it is unclear whether algal species with the ability to utilise bicarbonate (HCO3−) as a carbon source for photosynthesis will benefit from CO2 enrichment. This study quantified the HCO3− affinity of three green tide algal species, Cladophora coelothrix, Cladophora patentiramea and Chaetomorpha linum, targeted for biomass and bioenergy production. Subsequently, we quantified productivity and carbon, nitrogen and ash content in response to CO2 enrichment. All three species had similar high pH compensation points (9.7–9.9), and grew at similar rates up to pH 9, demonstrating HCO3− utilization. Algal cultures enriched with CO2 as a carbon source had 30% more total Ci available, supplying twenty five times more CO2 than the control. This higher Ci significantly enhanced the productivity of Cladophora coelothrix (26%), Chaetomorpha linum (24%) and to a lesser extent for Cladophora patentiramea (11%), compared to controls. We demonstrated that supplying carbon as CO2 can enhance the productivity of targeted green tide algal species under intensive culture, despite their clear ability to utilise HCO3−.
PMCID: PMC3852247  PMID: 24324672
16.  Physiological Responses of Zostera marina and Cymodocea nodosa to Light-Limitation Stress 
PLoS ONE  2013;8(11):e81058.
The effects of light-limitation stress were investigated in natural stands of the seagrasses Zostera marina and Cymodocea nodosa in Ria Formosa coastal lagoon, southern Portugal. Three levels of light attenuation were imposed for 3 weeks in two adjacent meadows (2–3 m depth), each dominated by one species. The response of photosynthesis to light was determined with oxygen electrodes. Chlorophylls and carotenoids were determined by high-pressure liquid chromatography (HPLC). Soluble protein, carbohydrates, malondialdehyde and phenol contents were also analysed. Both species showed evident signs of photoacclimation. Their maximum photosynthetic rates were significantly reduced with shading. Ratios between specific light harvesting carotenoids and the epoxidation state of xanthophyll cycle carotenoids revealed significantly higher light harvesting efficiency of C. nodosa, a competitive advantage in a low light environment. The contents of both soluble sugars and starch were considerably lower in Z. marina plants, particularly in the rhizomes, decreasing even further with shading. The different carbohydrate energy storage strategies found between the two species clearly favour C. nodosa's resilience to light deprivation, a condition enhanced by its intrinsic arrangement of the pigment pool. On the other hand, Z. marina revealed a lower tolerance to light reduction, mostly due to a less plastic arrangement of the pigment pool and lower carbohydrate storage. Our findings indicate that Z. marina is close to a light-mediated ecophysiological threshold in Ria Formosa.
PMCID: PMC3842938  PMID: 24312260
17.  Best of Both Worlds: Simultaneous High-Light and Shade-Tolerance Adaptations within Individual Leaves of the Living Stone Lithops aucampiae 
PLoS ONE  2013;8(10):e75671.
“Living stones” (Lithops spp.) display some of the most extreme morphological and physiological adaptations in the plant kingdom to tolerate the xeric environments in which they grow. The physiological mechanisms that optimise the photosynthetic processes of Lithops spp. while minimising transpirational water loss in both above- and below-ground tissues remain unclear. Our experiments have shown unique simultaneous high-light and shade-tolerant adaptations within individual leaves of Lithops aucampiae. Leaf windows on the upper surfaces of the plant allow sunlight to penetrate to photosynthetic tissues within while sunlight-blocking flavonoid accumulation limits incoming solar radiation and aids screening of harmful UV radiation. Increased concentration of chlorophyll a and greater chlorophyll a∶b in above-ground regions of leaves enable maximum photosynthetic use of incoming light, while inverted conical epidermal cells, increased chlorophyll b, and reduced chlorophyll a∶b ensure maximum absorption and use of low light levels within the below-ground region of the leaf. High NPQ capacity affords physiological flexibility under variable natural light conditions. Our findings demonstrate unprecedented physiological flexibility in a xerophyte and further our understanding of plant responses and adaptations to extreme environments.
PMCID: PMC3806800  PMID: 24194825
18.  Behavioral and Physiological Changes during Benthic-Pelagic Transition in the Harmful Alga, Heterosigma akashiwo: Potential for Rapid Bloom Formation 
PLoS ONE  2013;8(10):e76663.
Many species of harmful algae transition between a motile, vegetative stage in the water column and a non-motile, resting stage in the sediments. Physiological and behavioral traits expressed during benthic-pelagic transition potentially regulate the timing, location and persistence of blooms. The roles of key physiological and behavioral traits involved in resting cell emergence and bloom formation were examined in two geographically distinct strains of the harmful alga, Heterosigma akashiwo. Physiological measures of cell viability, division and population growth, and cell fatty acid content were made using flow cytometry and gas chromatography – mass spectrometry techniques as cells transitioned between the benthic resting stage and the vegetative pelagic stage. Video-based tracking was used to quantify cell-level swimming behaviors. Data show increased temperature and light triggered rapid emergence from the resting stage and initiated cell swimming. Algal strains varied in important physiological and behavioral traits, including survivorship during life-stage transitions, population growth rates and swimming velocities. Collectively, these traits function as “population growth strategies” that can influence bloom formation. Many resting cells regained the up-swimming capacity necessary to cross an environmentally relevant halocline and the ability to aggregate in near-surface waters within hours after vegetative growth supporting conditions were restored. Using a heuristic model, we illustrate how strain-specific population growth strategies can govern the timescales over which H. akashiwo blooms form. Our findings highlight the need for identification and quantification of strain-specific physiological and behavioral traits to improve mechanistic understanding of bloom formation and successful bloom prediction.
PMCID: PMC3790758  PMID: 24124586
19.  Aureochrome 1a Is Involved in the Photoacclimation of the Diatom Phaeodactylum tricornutum 
PLoS ONE  2013;8(9):e74451.
Aureochromes constitute a family of blue light (BL) receptors which are found exclusively in heterokont algae such as diatoms (Bacillariophyceae) and yellow-green algae (Xanthophyceae). Previous studies on the diatom Phaeodactylum tricornutum indicate that the formation of a high light acclimated phenotype is mediated by the absorption of BL and that aureochromes might play an important role in this process. P. tricornutum possesses four genes encoding aureochromes. In this study we confirm the nuclear localisation of the PtAUREO1a, 1b and 2 proteins. Furthermore we studied the physiology of light quality acclimation in genetically transformed P. tricornutum cell lines with reduced expression of the aureochrome 1a gene. The results demonstrate that the AUREO1a protein has a distinct function in light acclimation. However, rather unexpectedly AUREO1a seems to repress high light acclimation which resulted in a state of ‘hyper’ high light acclimation in aureo1a silenced strains. This was indicated by characteristic changes of several photosynthetic parameters, including increased maximum photosynthesis rates, decreased chlorophyll a contents per cell and increased values of non-photochemical quenching in AUREO1a silenced strains compared to wild type cultures. Strikingly, AUREO1a silenced strains exhibited phenotypic differences compared to wild type cells during cultivation under BL as well as under red light (RL) conditions. Therefore, AUREO1a might influence the RL signalling process, suggesting an interaction of AUREO1a with RL perception pathways.
PMCID: PMC3779222  PMID: 24073211
20.  Desiccation Induces Accumulations of Antheraxanthin and Zeaxanthin in Intertidal Macro-Alga Ulva pertusa (Chlorophyta) 
PLoS ONE  2013;8(9):e72929.
For plants and algae, exposure to high light levels is deleterious to their photosynthetic machineries. It also can accelerate water evaporation and thus potentially lead to drought stress. Most photosynthetic organisms protect themselves against high light caused photodamages by xanthophyll cycle-dependent thermal energy dissipation. It is generally accepted that high light activates xanthophyll cycle. However, the relationship between xanthophyll cycle and drought stress remains ambiguous. Herein, Ulva pertusa (Chlorophyta), a representative perennial intertidal macro-algae species with high drought-tolerant capabilities and simple structures, was used to investigate the operation of xanthophyll cycle during desiccation in air. The results indicate that desiccation under dim light induced accumulation of antheraxanthin (Ax) and zeaxanthin (Zx) at the expense of violaxanthin (Vx). This accumulation could be arrested by dithiothreitol completely and by uncoupler (carbonyl cyanide p-trifluoromethoxyphenylhydrazone) partially, implying the participation of Vx de-epoxidase in conversion of Vx to Ax and Zx. Treatment with inhibitors of electron transport along thylakoid membrane, e.g. DCMU, PG and DBMIB, did not significantly arrest desiccation-induced accumulation of Ax and Zx. We propose that for U. pertusa, besides excess light, desiccation itself could also induce accumulation of Ax and Zx. This accumulation could proceed without electron transport along thylakoid membrane, and is possibly resulting from the reduction of thylakoid lumen volume during desiccation. Considering the pleiotropic effects of Ax and Zx, accumulated Ax and Zx may function in protecting thylakoid membrane and enhancing thermal quenching during emersion in air.
PMCID: PMC3764160  PMID: 24039824
21.  Variable Angle Locking Intercarpal Fusion System for Four-Corner Arthrodesis: Indications and Surgical Technique 
Journal of Wrist Surgery  2012;1(1):73-78.
Four-corner fusion (4CF) is an accepted and regularly performed procedure when managing posttraumatic degenerative disorders in the wrist. This procedure consists of excision of the entire scaphoid in association with midcarpal fusion of the remaining four ulnar carpal bones (hamate, capitate, lunate, and triquetrum). In the majority of cases, the long-term outcome is a functional painless wrist. However, the exact procedure to best achieve a rapid solid bone union of the fusion mass without hardware complications remains controversial. The authors have developed a precise system to ensure precise positioning, firm fixation, and fusion at the midcarpal joint together with an early postoperative recovery, avoiding some of the issues reported with other implants used for 4CF. The described implant is a circular plate accommodating variable angle locking screws as well as compression screws that can firmly fix the plate to the carpal bones. The locking technology produces a very solid construct. A special reaming-distraction-compression guide has also been developed to both countersink the plate on the underlying carpal bone mass and allow distraction of the midcarpal joint for debridement and cancellous bone graft interposition. The features of the implant, its surgical technique, and a relevant case are described.
PMCID: PMC3658673  PMID: 23904983
four-corner fusion; SLAC wrist; SNAC wrist; wrist
22.  Global Transcriptional Responses of the Toxic Cyanobacterium, Microcystis aeruginosa, to Nitrogen Stress, Phosphorus Stress, and Growth on Organic Matter 
PLoS ONE  2013;8(7):e69834.
Whole transcriptome shotgun sequencing (RNA-seq) was used to assess the transcriptomic response of the toxic cyanobacterium Microcystis aeruginosa during growth with low levels of dissolved inorganic nitrogen (low N), low levels of dissolved inorganic phosphorus (low P), and in the presence of high levels of high molecular weight dissolved organic matter (HMWDOM). Under low N, one third of the genome was differentially expressed, with significant increases in transcripts observed among genes within the nir operon, urea transport genes (urtBCDE), and amino acid transporters while significant decreases in transcripts were observed in genes related to photosynthesis. There was also a significant decrease in the transcription of the microcystin synthetase gene set under low N and a significant decrease in microcystin content per Microcystis cell demonstrating that N supply influences cellular toxicity. Under low P, 27% of the genome was differentially expressed. The Pho regulon was induced leading to large increases in transcript levels of the alkaline phosphatase phoX, the Pst transport system (pstABC), and the sphX gene, and transcripts of multiple sulfate transporter were also significantly more abundant. While the transcriptional response to growth on HMWDOM was smaller (5–22% of genes differentially expressed), transcripts of multiple genes specifically associated with the transport and degradation of organic compounds were significantly more abundant within HMWDOM treatments and thus may be recruited by Microcystis to utilize these substrates. Collectively, these findings provide a comprehensive understanding of the nutritional physiology of this toxic, bloom-forming cyanobacterium and the role of N in controlling microcystin synthesis.
PMCID: PMC3720943  PMID: 23894552
23.  Axonal Protein Synthesis and Degradation Are Necessary for Efficient Growth Cone Regeneration 
Axonal regeneration can occur within hours of injury, the first step being the formation of a new growth cone. For sensory and retinal axons, regenerative ability in vivo correlates with the potential to form a new growth cone after axotomy in vitro. We show that this ability to regenerate a new growth cone depends on local protein synthesis and degradation within the axon. Axotomy in vitro leads to a fourfold to sixfold increase in 3H-leucine incorporation in both neurones and axons, starting within 10 min and peaking 1 h after axotomy. Application of protein synthesis inhibitors (cycloheximide and anisomycin) to cut axons, including axons whose cell bodies were removed, or proteasome inhibitors (lactacystin and N-acetyl-Nor-Leu-Leu-Al) all result in a reduction in the proportion of transected axons able to reform growth cones. Similar inhibition of growth cone formation was observed on addition of target of rapamycin (TOR), p38 MAPK (mitogen-activated protein kinase), and caspase-3 inhibitors. Comparing retinal and sensory axons of different developmental stages, levels of ribosomal protein P0 and phosphorylated translation initiation factor are high in sensory axons, lower in embryonic axons, and absent in adult retinal axons. Conditioning lesions, which increase the regenerative ability of sensory axons, lead to increases in intra-axonal protein synthetic and degradative machinery both in vitro and in vivo. Collectively, these findings suggest that local protein synthesis and degradation, controlled by various TOR-, p38 MAPK-, and caspase-dependent pathways, underlie growth cone initiation after axotomy.
PMCID: PMC3687202  PMID: 15647476
growth cone formation; axotomy; local protein synthesis; proteasome-mediated degradation; conditioning lesion; axon regeneration
24.  Photophysiological and Photosynthetic Complex Changes during Iron Starvation in Synechocystis sp. PCC 6803 and Synechococcus elongatus PCC 7942 
PLoS ONE  2013;8(3):e59861.
Iron is an essential component in many protein complexes involved in photosynthesis, but environmental iron availability is often low as oxidized forms of iron are insoluble in water. To adjust to low environmental iron levels, cyanobacteria undergo numerous changes to balance their iron budget and mitigate the physiological effects of iron depletion. We investigated changes in key protein abundances and photophysiological parameters in the model cyanobacteria Synechococcus PCC 7942 and Synechocystis PCC 6803 over a 120 hour time course of iron deprivation. The iron stress induced protein (IsiA) accumulated to high levels within 48 h of the onset of iron deprivation, reaching a molar ratio of ∼42 IsiA : Photosystem I in Synechococcus PCC 7942 and ∼12 IsiA : Photosystem I in Synechocystis PCC 6803. Concomitantly the iron-rich complexes Cytochrome b6f and Photosystem I declined in abundance, leading to a decrease in the Photosystem I : Photosystem II ratio. Chlorophyll fluorescence analyses showed a drop in electron transport per Photosystem II in Synechococcus, but not in Synechocystis after iron depletion. We found no evidence that the accumulated IsiA contributes to light capture by Photosystem II complexes.
PMCID: PMC3602374  PMID: 23527279
25.  Rising CO2 Interacts with Growth Light and Growth Rate to Alter Photosystem II Photoinactivation of the Coastal Diatom Thalassiosira pseudonana 
PLoS ONE  2013;8(1):e55562.
We studied the interactive effects of pCO2 and growth light on the coastal marine diatom Thalassiosira pseudonana CCMP 1335 growing under ambient and expected end-of-the-century pCO2 (750 ppmv), and a range of growth light from 30 to 380 µmol photons·m−2·s−1. Elevated pCO2 significantly stimulated the growth of T. pseudonana under sub-saturating growth light, but not under saturating to super-saturating growth light. Under ambient pCO2 susceptibility to photoinactivation of photosystem II (σi) increased with increasing growth rate, but cells growing under elevated pCO2 showed no dependence between growth rate and σi, so under high growth light cells under elevated pCO2 were less susceptible to photoinactivation of photosystem II, and thus incurred a lower running cost to maintain photosystem II function. Growth light altered the contents of RbcL (RUBISCO) and PsaC (PSI) protein subunits, and the ratios among the subunits, but there were only limited effects on these and other protein pools between cells grown under ambient and elevated pCO2.
PMCID: PMC3561317  PMID: 23383226

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