Francisca Fernández-piñas
Universidad Autónoma de Madrid, Biology, Faculty Member
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The toxic action of graphene oxide (GO) and the micropollutants contained in a biologically-treated wastewater were studied alone and in combination.
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Microorganisms have been very useful in environmental monitoring due to their constant sensing of the surrounding environment, their easy maintenance and low cost. Some freeze-dried toxicity kits based on naturally bioluminescent bacteria... more
Microorganisms have been very useful in environmental monitoring due to their constant sensing of the surrounding environment, their easy maintenance and low cost. Some freeze-dried toxicity kits based on naturally bioluminescent bacteria are commercially available and commonly used to assess the toxicity of environmental samples such as Microtox (Aliivibrio fischeri) or ToxScreen (Photobacterium leiognathi), however, due to the marine origin of these bacteria, they could not be the most appropriate for fresh-waters monitoring. Cyanobacteria are one of the most representative microorganisms of aquatic environments, and are well suited for detecting contaminants in aqueous samples. This study presents the development and application of the first freeze-dried cyanobacterial bioassay for fresh-water contaminants detection. The effects of different cell growth phases, cryoprotectant solutions, freezing protocols, rehydration solutions and incubation conditions methods were evaluated and the best combination of these parameters for freeze-drying was selected. The study includes detailed characterization of sensitivity towards reference pollutants, as well as, comparison with the standard assays. Moreover, long-term viability and sensitivity were evaluated after 3 years of storage. Freeze-dried cyanobacteria showed, in general, higher sensitivity than the standard assays and viability of the cells remained after 3 years of storage. Finally, the validation of the bioassay using a wastewater sample was also evaluated. Freeze-drying of cyanobacteria in 96-well plates presents a simple, fast and multi-assay method for environmental monitoring.
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The present study was aimed at investigating the role of intracellular free calcium, [Ca(2+)]c, in the early cellular response of the green alga Chlamydomonas reinhardtii to the emergent pollutant Triclosan (13.8μM; 24h of exposure).... more
The present study was aimed at investigating the role of intracellular free calcium, [Ca(2+)]c, in the early cellular response of the green alga Chlamydomonas reinhardtii to the emergent pollutant Triclosan (13.8μM; 24h of exposure). There is a growing concern about the persistence and toxicity of this antimicrobial in aquatic environments, where non-target organisms such as C. reinhardtii, a primary producer of ecological relevance, might be severely impacted. A mechanistic study was undertaken which combined flow cytometry protocols, physiological as well as gene expression analysis. As an early response, Triclosan strongly altered [Ca(2+)]c homeostasis which could be prevented by prechelation with the intracellular calcium chelator BAPTA-AM. Triclosan induced ROS overproduction which ultimately leads to oxidative stress with loss of membrane integrity, membrane depolarization, photosynthesis inhibition and mitochondrial membrane depolarization; within this context, Triclosan also induced an increase in caspase 3/7 activity and altered the expression of metacaspase genes which are indicative of apoptosis. All these adverse outcomes were dependent on [Ca(2+)]c. Interestingly, an interconnection between [Ca(2+)]c alterations and increased ROS formation by Triclosan was found. Taken altogether these results shed light on the mechanisms behind Triclosan toxicity in the green alga Chlamydomonas reinhardtii and demonstrate the role of [Ca(2+)]c in mediating the observed toxicity.
Research Interests: Photosynthesis, Flow Cytometry, Aquatic Toxicology, Calcium, Oxidative Stress, and 15 moreApoptosis, Biological Sciences, Caspases, Environmental Sciences, Calcium Signaling, Lipid peroxidation, TRICLOSAN, CHEMICAL SCIENCES, Hydrogen Peroxide, Chlamydomonas Reinhardtii, Intracellular Space, Superoxides, Gene Expression Regulation, Cell Membrane, and fluorescent dyes
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The ecological impacts of emerging pollutants such as pharmaceuticals are not well understood. The lack of experimental approaches for the identification of pollutant effects in realistic settings (that is, low doses, complex mixtures,... more
The ecological impacts of emerging pollutants such as pharmaceuticals are not well understood. The lack of experimental approaches for the identification of pollutant effects in realistic settings (that is, low doses, complex mixtures, and variable environmental conditions) supports the widespread perception that these effects are often unpredictable. To address this, we developed a novel screening method (GSA-QHTS) that couples the computational power of global sensitivity analysis (GSA) with the experimental efficiency of quantitative high-throughput screening (QHTS). We present a case study where GSA-QHTS allowed for the identification of the main pharmaceutical pollutants (and their interactions), driving biological effects of low-dose complex mixtures at the microbial population level. The QHTS experiments involved the integrated analysis of nearly 2700 observations from an array of 180 unique low-dose mixtures, representing the most complex and data-rich experimental mixture e...
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Cerium oxide nanoparticles (nanoceria; CNPs) have been found to have both pro-oxidant and anti-oxidant effects on different cell systems or organisms. In order to untangle the mechanisms which underlie the biological activity of... more
Cerium oxide nanoparticles (nanoceria; CNPs) have been found to have both pro-oxidant and anti-oxidant effects on different cell systems or organisms. In order to untangle the mechanisms which underlie the biological activity of nanoceria, we have studied the effect of five different CNPs on a model relevant aquatic microorganism. Neither shape, concentration, synthesis method, surface charge (ζ-potential), nor nominal size had any influence in the observed biological activity. The main driver of toxicity was found to be the percentage of surface content of Ce(3+) sites: CNP1 (58%) and CNP5 (40%) were found to be toxic whereas CNP2 (28%), CNP3 (36%) and CNP4 (26%) were found to be non-toxic. The colloidal stability and redox chemistry of the most and least toxic CNPs, CNP1 and CNP2, respectively, were modified by incubation with iron and phosphate buffers. Blocking surface Ce(3+) sites of the most toxic CNP, CNP1, with phosphate treatment reverted toxicity and stimulated growth. Col...
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Global temperature is expected to rise throughout this century, and blooms of cyanobacteria in lakes and estuaries are predicted to increase with the current level of global warming. The potential environmental, economic and sanitation... more
Global temperature is expected to rise throughout this century, and blooms of cyanobacteria in lakes and estuaries are predicted to increase with the current level of global warming. The potential environmental, economic and sanitation repercussions of these blooms have attracted considerable attention among the world's scientific communities, water management agencies and general public. Of particular concern is the worldwide occurrence of hepatotoxic cyanobacteria posing a serious threat to global public health. Here, we highlight plausible effects of global warming on physiological and molecular changes in these cyanobacteria and resulting effects on hepatotoxin production. We also emphasize the importance of understanding the natural biological function(s) of hepatotoxins, various mechanisms governing their synthesis, and climate-driven changes in food-web interactions, if we are to predict consequences of the current and projected levels of global warming for production and accumulation of hepatotoxins in aquatic ecosystems.
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The individual and combined toxicities of amoxicillin, erythromycin, levofloxacin, norfloxacin and tetracycline have been examined in two organisms representative of the aquatic environment, the cyanobacterium Anabaena CPB4337 as a target... more
The individual and combined toxicities of amoxicillin, erythromycin, levofloxacin, norfloxacin and tetracycline have been examined in two organisms representative of the aquatic environment, the cyanobacterium Anabaena CPB4337 as a target organism and the green alga Pseudokirchneriella subcapitata as a non-target organism. The cyanobacterium was more sensitive than the green alga to the toxic effect of antibiotics. Erythromycin was highly toxic for both organisms; tetracycline was more toxic to the green algae whereas the quinolones levofloxacin and norfloxacin were more toxic to the cyanobacterium than to the green alga. Amoxicillin also displayed toxicity to the cyanobacterium but showed no toxicity to the green alga. The toxicological interactions of antibiotics in the whole range of effect levels either in binary or multicomponent mixtures were analyzed using the Combination Index (CI) method. In most cases, synergism clearly predominated both for the green alga and the cyanobacterium. The CI method was compared with the classical models of additivity Concentration Addition (CA) and Independent Action (IA) finding that CI could accurately predict deviations from additivity. Risk assessment was performed by calculating the ratio between Measured Environmental Concentration (MEC) and the Predicted No Effect Concentration (PNEC). A MEC/PNEC ratio higher than 1 was found for the binary erythromycin and tetracycline mixture in wastewater effluents, a combination which showed a strong synergism at low effect levels in both organisms. From the tested antibiotic mixtures, it can be concluded that certain specific combinations may pose a potential ecological risk for aquatic ecosystems with the present environmentally measured concentrations.
Research Interests: Photosynthesis, Risk assessment, Phytoplankton, Multidisciplinary, Spain, and 15 moreAquatic organisms, Amoxicillin, Fresh water, Risk Assessment, Erythromycin, Cyanobacterium, Anabaena, Antibiotic Toxicity, Combination Index, Green Alga, Risk Quotient, Chlorophyta, Drug Stability, Fluoroquinolones, and Drug synergism
Cyanobacteria are the main primary producers and are responsible for the blooms and eutrophication processes caused by excess nutrients in surface waters. The aim of this paper is to use cyanobacteria to monitor the presence and... more
Cyanobacteria are the main primary producers and are responsible for the blooms and eutrophication processes caused by excess nutrients in surface waters. The aim of this paper is to use cyanobacteria to monitor the presence and bioavailability of different chemical species of nitrogen in freshwater. Cyanobacteria have mechanisms which can detect the presence of nutrients in their environment and can activate or repress specific genes, or operons, depending on nutrient bioavailability. Therefore, monitoring the expression of these genes can facilitate measurement of the availability of nutrients. To achieve this we have constructed self-bioluminescent reporter strains of the filamentous nitrogen-fixing cyanobacterium Nostoc sp. PCC 7120 expressing promoters of genes responsive to nitrogen fused to the luxCDABE operon. Three promoters were selected to direct the expression of luxCDABE: The first, the glnA promoter, is activated in the absence of combined nitrogen. We found that it responded linearly to the addition of known amounts of combined N in the range 50-500 μM NH₄(+) or NO₃(-). The second, the nirA operon promoter, turns on in the presence of nitrate being inhibited by ammonium. The bioreporter responded linearly in the range of 10-100 μM NO₃. The third, the gifA promoter, is activated in the presence of ammonium, responding linearly in the range 100-600 μM NH4(+). We also used a previously described strain of Synechococcus elongatus PCC 7942 expressing glnN (glutamine synthetase type III) fused to luxAB. We found that the glnN promoter responded linearly to the addition of known amounts of N in the range 50-500 μM NH₄(+) or NO₃(-). These cyanobacterial bioreporters were tested in real environmental samples (i.e. river waters) which confirmed their validity and showed a broad spectrum response. They are therefore useful in the detection of both total N-bioavailability and specific nitrogen species.
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... César Poza-Carrión a , Eduardo Fernández-Valiente a , Francisca Fernández Piñas a and Francisco Leganés Corresponding Author Contact Information ... RJ Ritchie, Membrane potential and pH control in the cyanobacterium Synechococcus R-2... more
... César Poza-Carrión a , Eduardo Fernández-Valiente a , Francisca Fernández Piñas a and Francisco Leganés Corresponding Author Contact Information ... RJ Ritchie, Membrane potential and pH control in the cyanobacterium Synechococcus R-2 PCC7242, J Plant Physiol 137 ...
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Anabaena sp. PCC7120 contains a gene, mrpA (all1838), which forms part of a seven gene-cluster (all1843-all1837) with significant sequence similarity to bacterial operons that putatively code for a multicomponent cation/proton antiporter... more
Anabaena sp. PCC7120 contains a gene, mrpA (all1838), which forms part of a seven gene-cluster (all1843-all1837) with significant sequence similarity to bacterial operons that putatively code for a multicomponent cation/proton antiporter involved in alkaline pH adaptation and salt resistance. We previously showed that growth and photosynthesis were inhibited in a strain mutated in mrpA, denoted as PHB11, particularly at alkaline pH. Here, we show that respiration was also impaired in the mutant independently of the external pH. In addition, at high pH, less ATP and vegetative cell ferredoxin were present in PHB11, which also showed lower levels of ferredoxin-NADP(+) oxidoreductase (FNR). Ferredoxin and FNR are involved in the generation of reductant NADPH in cyanobacteria. These results suggest an energetic role of mrpA (and perhaps of the whole mrp-gene cluster) in Anabaena sp. PCC 7120 that is further supported by the significant similarity of putative Anabaena Mrp proteins to membrane subunits of complex I.
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Research Interests: Ecotoxicology, Daphnia, Nitrogen Cycle, Biological Sciences, Environmental Sciences, and 15 moreAnimals, Luminescence, Lipid metabolism, CHEMICAL SCIENCES, Particulate Matter, Daphnia Magna, Nutrient Availability, Anabaena, Acute Toxicity, Biological Assay, Blood Lipids, Pollution Science, Toxicity Testing, Aliivibrio fischeri, and Risk estimation
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The cyanobacterial phycobilisome (PBS) is a giant pigment-protein complex which harvests light energy for photosynthesis and comprises two structures: a core and peripheral rods. Most studies on PBS structure and function are based on... more
The cyanobacterial phycobilisome (PBS) is a giant pigment-protein complex which harvests light energy for photosynthesis and comprises two structures: a core and peripheral rods. Most studies on PBS structure and function are based on mutants of unicellular strains. In this report, we describe the phenotypic and genetic characterization of a transposon mutant of the filamentous Anabaena sp. strain PCC 7120, denoted LC1, which cannot synthesize the phycobiliprotein phycocyanin (PC), the main component of the rods; in this mutant, the transposon had inserted into the cpcB gene (orf alr0528) which putatively encodes PC-β chain. Mutant LC1 was able to synthesize phycoerythrocyanin (PEC), a phycobiliprotein (PBP) located at the terminal region of the rods; but in the absence of PC, PEC did not attach to the PBSs that only retained the allophycocyanin (APC) core; ferredoxin: NADP+-oxidoreductase (FNR) that is associated with the PBS in the wild type, was not found in isolated PBSs from LC1. The performance of the mutant exposed to different environmental conditions was evaluated. The mutant phenotype was successfully complemented by cloning and transfer of the wild type complete cpc operon to mutant LC1. Interestingly, LC1 compensated its mutation by significantly increasing the number of its core-PBS and the effective quantum yield of photosystem II (PSII) photochemistry; this feature suggests a more efficient energy conversion in the mutant which may be useful for biotechnological applications.
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A genomic analysis of putative penicillin-binding proteins (PBPs) that are involved in the synthesis of the peptidoglycan layer of the cell wall and are encoded in 12 cyanobacterial genomes was performed in order to help elucidate the... more
A genomic analysis of putative penicillin-binding proteins (PBPs) that are involved in the synthesis of the peptidoglycan layer of the cell wall and are encoded in 12 cyanobacterial genomes was performed in order to help elucidate the role(s) of these proteins in peptidoglycan synthesis, especially during cyanobacterial cellular differentiation. The analysis suggested that the minimum set of PBPs needed to assemble the peptidoglycan layer in cyanobacteria probably does not exceed one bifunctional transpeptidase-transglycosylase Class A high-molecular-weight PBP; two Class B high-molecular-weight PBPs, one of them probably involved in cellular elongation and the other in septum formation; and one low-molecular-weight PBP. The low-molecular-weight PBPs of all of the cyanobacteria analyzed are putative endopeptidases and are encoded by fewer genes than in Escherichia coli. We show that in Anabaena sp. strain PCC 7120, predicted proteins All2981 and Alr4579, like Alr5101, are Class A hi...
Research Interests: Microbiology, Medical Microbiology, Archives, Cyanobacteria, Phylogeny, and 12 moreCell Differentiation, Escherichia coli, Cellular differentiation, Genome Analysis, Software Requirement Specification, Penicillin-Binding Proteins, Cell Shape, Multienzyme complexes, Amino Acid Sequence, Cell Wall, Anabaena, and Low molecular weight
We investigated the possibility of Ca(2+) signaling in cyanobacteria (blue-green algae) by measuring intracellular free Ca(2+) levels ([Ca(2+)](i)) in a recombinant strain of the nitrogen fixing cyanobacterium Anabaena strain sp. PCC7120,... more
We investigated the possibility of Ca(2+) signaling in cyanobacteria (blue-green algae) by measuring intracellular free Ca(2+) levels ([Ca(2+)](i)) in a recombinant strain of the nitrogen fixing cyanobacterium Anabaena strain sp. PCC7120, which constitutively expresses the Ca(2+)-binding photoprotein apoaequorin. The homeostasis of intracellular Ca(2+) in response to increasing external Ca(2+) has been studied in this strain. The resting level of free Ca(2+) in Anabaena was found to be between 100 and 200 nM. Additions of increasing concentrations of external Ca(2+) gave a transient burst of [Ca(2+)](i) followed by a very quick decline, reaching a plateau within seconds that brought the level of [Ca(2+)](i) back to the resting value. These results indicate that Anabaena strain sp. PCC7120 is able to regulate its internal Ca(2+) levels. We also monitored Ca(2+) transients in our recombinant strain in response to heat and cold shock. The cell's response to both stresses was depend...