Publications by authors named "Cecilia Giacomini"

13 Publications

  • Page 1 of 1

Structural insights in galectin-1-glycan recognition: Relevance of the glycosidic linkage and the N-acetylation pattern of sugar moieties.

Bioorg Med Chem 2021 Aug 14;44:116309. Epub 2021 Jul 14.

Laboratorio de Bioquímica, Departamento de Biociencias, Facultad de Química, UdelaR, Gral. Flores, 2124, 11800 Montevideo, Uruguay. Electronic address:

Galectins, soluble lectins widely expressed intra- and extracellularly in different cell types, play major roles in deciphering the cellular glycocode. Galectin-1 (Gal-1), a prototype member of this family, presents a carbohydrate recognition domain (CRD) with specific affinity for β-galactosides such as N-acetyllactosamine (β-d-Galp-(1 → 4)-d-GlcpNAc), and mediate numerous physiological and pathological processes. In this work, Gal-1 binding affinity for β-(1 → 6) galactosides, including β-d-Galp-(1 → 6)-β-d-GlcpNAc-(1 → 4)-d-GlcpNAc was evaluated, and their performance was compared to that of β-(1 → 4) and β-(1 → 3) galactosides. To this end, the trisaccharide β-d-Galp-(1 → 6)-β-d-GlcpNAc-(1 → 4)-d-GlcpNAc was enzymatically synthesized, purified and structurally characterized. To evaluate the affinity of Gal-1 for the galactosides, competitive solid phase assays (SPA) and isothermal titration calorimetry (ITC) studies were carried out. The experimental dissociation constants and binding energies obtained were compared to those calculated by molecular docking. These analyses evidenced the critical role of the glycosidic linkage between the terminal galactopyranoside residue and the adjacent monosaccharide, as galactosides bearing β-(1 → 6) glycosidic linkages showed dissociation constants six- and seven-fold higher than those involving β-(1 → 4) and β-(1 → 3) linkages, respectively. Moreover, docking experiments revealed the presence of hydrogen bond interactions between the N-acetyl group of the glucosaminopyranose moiety of the evaluated galactosides and specific amino acid residues of Gal-1, relevant for galectin-glycan affinity. Noticeably, the binding free energies (ΔG) derived from the molecular docking were in good agreement with experimental values determined by ITC measurements (ΔG), evidencing a good correlation between theoretical and experimental approaches, which validates the in silico simulations and constitutes an important tool for the rational design of future optimized ligands.
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http://dx.doi.org/10.1016/j.bmc.2021.116309DOI Listing
August 2021

A biotechnological tool for glycoprotein desialylation based on immobilized neuraminidase from .

Biochem Biophys Rep 2021 Jul 2;26:100940. Epub 2021 Mar 2.

Laboratorio de Bioquímica, Departamento de Biociencias, Facultad de Química, UdelaR, Gral. Flores, 2124, Montevideo, Uruguay.

Background: Sialic acids are widely distributed in nature and have biological relevance owing to their varied structural and functional roles. Immobilized neuraminidase can selectively remove terminal N-acetyl neuraminic acid from glycoproteins without altering the protein backbone while it can be easily removed from the reaction mixture avoiding sample contamination. This enables the evaluation of changes in glycoprotein performance upon desialylation.

Methods: Neuraminidase was immobilized onto agarose activated with cyanate ester groups and further used for desialylation of model glycoproteins, a lysate from tumour cells and tumour cells. Desialylation process was analysed by lectin binding assay, determination of sialyl-Tn or flow cytometry.

Results: neuraminidase was immobilized with 91 % yield and expressed activity yield was of 41%. It was effective in the desialylation of bovine fetal serum fetuin, bovine lactoferrin and ovine submaxilar mucin. A decrease in sialic-specific SNA lectin recognition of 83% and 53 % was observed for fetuin and lactoferrin with a concomitant increase in galactose specific ECA and PNA lectin recognition. Likewise, a decrease in the recognition of a specific antibody (82%) upon mucin desialylation was observed. Moreover, desialylation of a protein lysate from the sialic acid-rich cell line TA3/Ha was also possible leading to a decrease in 47 % in SNA recognition. Immobilized neuraminidase kept 100% of its initial activity upon five desialylation cycles.

Conclusions: Immobilized neuraminidase is an interesting as well as a robust biotechnological tool for enzymatic desialylation purposes.

General Significance: Immobilized neuraminidase would contribute to understand the role of sialic acid in biological processes.
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http://dx.doi.org/10.1016/j.bbrep.2021.100940DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7937660PMC
July 2021

Immobilized peptide-N-glycosidase F onto magnetic nanoparticles: A biotechnological tool for protein deglycosylation under native conditions.

Biotechnol Appl Biochem 2021 Jan 12. Epub 2021 Jan 12.

Laboratorio de Bioquímica, Departamento de Biociencias, Facultad de Química, UdelaR, Gral. Flores 2124, Montevideo, Uruguay.

The elucidation of glycans biological function is essential to understand their role in biological processes, both normal and pathological. Immobilized glycoenzymes are excellent tools for this purpose as they can selectively release glycans from glycoproteins without altering their backbone. They can be easily removed from the reaction mixture avoiding their interference in subsequent experiments. Here, we describe the immobilization of peptide-N-glycosidase F (PNGase F) onto silica magnetic nanoparticles with immobilization yields of 86% and activity yields of 12%. Immobilized PNGase F showed higher thermal stability than its soluble counterpart, and could be reused for at least seven deglycosylation cycles. It was efficient in the deglycosylation of several glycoproteins (ribonuclease B, bovine fetuin, and ovalbumin) and a protein lysate from the parasite Fasciola hepatica under native conditions, with similar performance to that of the soluble enzyme. Successful deglycosylation was evidenced by a decrease in specific lectin recognition of the glycoproteins (40%-80%). Moreover, deglycosylated F. hepatica lysate allowed us to confirm the role of parasite N-glycans in the inhibition of the lipopolysaccharide-induced maturation of dendritic cells. Immobilized PNGase F probed to be a robust biotechnological tool for deglycosylation of glycoproteins and complex biological samples under native conditions.
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http://dx.doi.org/10.1002/bab.2099DOI Listing
January 2021

Enzymatic synthesis of non-natural trisaccharides and galactosides; Insights of their interaction with galectins as a function of their structure.

Carbohydr Res 2019 Jan 3;472:1-15. Epub 2018 Nov 3.

Laboratorio de Bioquímica, Departamento de Biociencias, Facultad de Química, UdelaR, Gral. Flores, 2124, Montevideo, Uruguay. Electronic address:

Galectins are a family of carbohydrate-recognizing proteins that by interacting with specific glycoepitopes can mediate important biological processes, including immune cell homeostasis and activation of tolerogenic circuits. Among the different members of this family, Galectin 1 and 3 have shown pro-tumorigenic effects, being overexpressed in numerous neoplasic diseases, proving to be relevant in tumor immune escape, tumor progression and resistance to drug-induced apoptosis. Thus, generation of specific glycosides that could inhibit their pro-tumorigenic ability by blocking their carbohydrate recognition domain is one of the current major challenges in the field. Considering that galectin-ligand binding strength is closely related to the ligand structure, analysis of this relationship provides valuable information for rational design of high-affinity ligands that could work as effective galectin inhibitors. Taking profit of the ability of glycosidases to catalyze transglycosylation reactions we achieved the enzymatic synthesis of β-d-Galp-(1 → 6)-β-d-Galp-(1 → 4)-d-Glcp(2), a mixture of β-d-Galp-(1 → 6)-β-d-Glcp-(1 → 4)-d-Glcp(5) and β-d-Galp-(1 → 3)-β-d-Glcp-(1 → 4)-d-Glcp(6), and finally benzyl β-d-galactopyranoside (9), with reaction yields between 16 and 27%. All the galactosides were purified, and characterized using H and C nuclear magnetic resonance spectroscopy. Docking results performed between the synthesized compounds and human Galectin 1 (hGal-1) and human Galectin 3 (hGal-3) showed that the replacement of a glucose moiety linked to the terminal galactose with a galactose moiety, decreases the affinity for these galectins. Moreover, regarding the interglycosidic bond the most favorable β-Gal linkage seems to be β(1 → 4) followed by β(1 → 3) and β(1 → 6) for hGal-1, and β(1 → 4) followed by β(1 → 6) and β(1 → 3) for hGal-3. These results were in accordance with the IC50 values obtained with in vitro solid phase inhibition assays. Therefore, docking results obtained in this work proved to be a very good approximation for predicting binding affinity of novel galactosides.
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http://dx.doi.org/10.1016/j.carres.2018.10.011DOI Listing
January 2019

Immobilization of β-galactosidase and α-mannosidase onto magnetic nanoparticles: A strategy for increasing the potentiality of valuable glycomic tools for glycosylation analysis and biological role determination of glycoconjugates.

Enzyme Microb Technol 2018 Oct 31;117:45-55. Epub 2018 May 31.

Laboratorio de Bioquímica, Departamento de Biociencias, Facultad de Química, UdelaR, Gral. Flores 2124, Montevideo, Uruguay. Electronic address:

Glycans present in biological glycoconjugates have several structural and functional roles. Elucidation of glycan structure and biological function is critical to understand their role in physiological and pathogenic process, enabling the development of diagnostic methods and disease treatment. Immobilized glycosidases are powerful tools for glycan analysis, as they are able to remove specific carbohydrates without altering the protein structure. Here we describe the individual immobilization of Aspergillus oryzae β-galactosidase and Canavalia ensiformis α-mannosidase onto agarose and silica magnetic nanoparticles activated with cyanate ester groups. High immobilization yields (70-90%) were achieved, keeping above 60% of its original activity. Immobilized glycosidases were effective in the selective deglycosylation of model glycoproteins and a Fasciola hepatica lysate, evidenced by a decrease in specific lectin recognition of 40-50% after enzymatic deglycosylation. Immobilized glycosidases were reused for several deglycosylation cycles without loss of effectiveness. Their use was extended to the elucidation of the glycan role of native glycoconjugates. A decrease in the recognition of lactoferrin treated with α-mannosidase by a C-type lectin receptor, DC-SIGN was found. Also the specific deglycosylation of a F. hepatica lysate demonstrated the relevance of mannosylated glycans in the induction of Th2/Treg immune responses during the infection. Our results show successful immobilization of specific glycosidases in nano-supports and validate their utility to identify glycans biological functions.
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http://dx.doi.org/10.1016/j.enzmictec.2018.05.012DOI Listing
October 2018

Fasciola hepatica glycoconjugates immuneregulate dendritic cells through the Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin inducing T cell anergy.

Sci Rep 2017 Apr 24;7:46748. Epub 2017 Apr 24.

Laboratorio de Inmunomodulación y Desarrollo de Vacunas, Departamento de Inmunobiología, Facultad de Medicina, Universidad de La República, Montevideo, Uruguay.

Dendritic cell-specific ICAM-3 grabbing non-integrin (DC-SIGN) expressed on a variety of DCs, is a C-type lectin receptor that recognizes glycans on a diverse range of pathogens, including parasites. The interaction of DC-SIGN with pathogens triggers specific signaling events that modulate DC-maturation and activity and regulate T-cell activation by DCs. In this work we evaluate whether F. hepatica glycans can immune modulate DCs via DC-SIGN. We demonstrate that DC-SIGN interacts with F. hepatica glycoconjugates through mannose and fucose residues. We also show that mannose is present in high-mannose structures, hybrid and trimannosyl N-glycans with terminal GlcNAc. Furthermore, we demonstrate that F. hepatica glycans induce DC-SIGN triggering leading to a strong production of TLR-induced IL-10 and IL-27p28. In addition, parasite glycans induced regulatory DCs via DC-SIGN that decrease allogeneic T cell proliferation, via the induction of anergic/regulatory T cells, highlighting the role of DC-SIGN in the regulation of innate and adaptive immune responses by F. hepatica. Our data confirm the immunomodulatory properties of DC-SIGN triggered by pathogen-derived glycans and contribute to the identification of immunomodulatory glyans of helminths that might eventually be useful for the design of vaccines against fasciolosis.
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http://dx.doi.org/10.1038/srep46748DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5402274PMC
April 2017

Synthesis of Oligosaccharides Derived from Lactulose (OsLu) Using Soluble and Immobilized Aspergillus oryzae β-Galactosidase.

Front Bioeng Biotechnol 2016 7;4:21. Epub 2016 Mar 7.

Departamento Producción y Caracterización de Nuevos Alimentos, Instituto de Investigación en Ciencias de la Alimentación (CIAL, CSIC-UAM) , Madrid , Spain.

β-Galactosidase from Aspergillus oryzae offers a high yield for the synthesis of oligosaccharides derived from lactulose (OsLu) by transgalactosylation. Oligosaccharides with degree of polymerization (DP) ≥ 3 have shown to possess higher in vitro bifidogenic effect than di- and tetrasaccharides. Thus, in this work, an optimization of reaction conditions affecting the specific selectivity of A. oryzae β-galactosidase for synthesis of OsLu has been carried out to enhance OsLu with DP ≥ 3 production. Assays with β-galactosidase immobilized onto a glutaraldehyde-agarose support were also carried out with the aim of making the process cost-effective and industrially viable. Optimal conditions with both soluble and immobilized enzyme for the synthesis of OsLu with DP ≥ 3 were 50 °C, pH 6.5, 450 g/L of lactulose, and 8 U/mL of enzyme, reaching yields of ca. 50% (w/v) of total OsLu and ca. 20% (w/v) of OsLu with DP 3, being 6'-galactosyl-lactulose the major one, after a short reaction time. Selective formation of disaccharides, however, was favored at 60 °C, pH 4.5, 450 g/L of lactulose and 8 U/mL of enzyme. Immobilization increased the enzymatic stability to temperature changes and allowed to reuse the enzyme. We can conclude that the use, under determined optimal conditions, of the A. oryzae β-galactosidase immobilized on a support of glutaraldehyde-agarose constitutes an efficient and cost-effective alternative to the use of soluble β-galactosidases for the synthesis of prebiotic OsLu mixtures.
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http://dx.doi.org/10.3389/fbioe.2016.00021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4780266PMC
March 2016

Glycans from Fasciola hepatica Modulate the Host Immune Response and TLR-Induced Maturation of Dendritic Cells.

PLoS Negl Trop Dis 2015 Dec 31;9(12):e0004234. Epub 2015 Dec 31.

Laboratory of Immunomodulation and Vaccine Development, Departamento de Inmunobiología, Facultad de Medicina, UdelaR, Montevideo, Uruguay.

Helminths express various carbohydrate-containing glycoconjugates on their surface, and they release glycan-rich excretion/secretion products that can be very important in their life cycles, infection and pathology. Recent evidence suggests that parasite glycoconjugates could play a role in the evasion of the immune response, leading to a modified Th2-polarized immune response that favors parasite survival in the host. Nevertheless, there is limited information about the nature or function of glycans produced by the trematode Fasciola hepatica, the causative agent of fasciolosis. In this paper, we investigate whether glycosylated molecules from F. hepatica participate in the modulation of host immunity. We also focus on dendritic cells, since they are an important target of immune-modulation by helminths, affecting their activity or function. Our results indicate that glycans from F. hepatica promote the production of IL-4 and IL-10, suppressing IFNγ production. During infection, this parasite is able to induce a semi-mature phenotype of DCs expressing low levels of MHCII and secrete IL-10. Furthermore, we show that parasite glycoconjugates mediate the modulation of LPS-induced maturation of DCs since their oxidation restores the capacity of LPS-treated DCs to secrete high levels of the pro-inflammatory cytokines IL-6 and IL-12/23p40 and low levels of the anti-inflammatory cytokine IL-10. Inhibition assays using carbohydrates suggest that the immune-modulation is mediated, at least in part, by the recognition of a mannose specific-CLR that signals by recruiting the phosphatase Php2. The results presented here contribute to the understanding of the role of parasite glycosylated molecules in the modulation of the host immunity and might be useful in the design of vaccines against fasciolosis.
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http://dx.doi.org/10.1371/journal.pntd.0004234DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4697847PMC
December 2015

Comparative analysis of lentiviral vectors and modular protein nanovectors for traumatic brain injury gene therapy.

Mol Ther Methods Clin Dev 2014 15;1:14047. Epub 2014 Oct 15.

Neuroinflammation and Gene Therapy Laboratory, Institut Pasteur de Montevideo , Montevideo, Uruguay ; Departmento de Histología y Embriología, Facultad de Medicina, UDELAR , Montevideo, Uruguay.

Traumatic brain injury (TBI) remains as one of the leading causes of mortality and morbidity worldwide and there are no effective treatments currently available. Gene therapy applications have emerged as important alternatives for the treatment of diverse nervous system injuries. New strategies are evolving with the notion that each particular pathological condition may require a specific vector. Moreover, the lack of detailed comparative studies between different vectors under similar conditions hampers the selection of an ideal vector for a given pathological condition. The potential use of lentiviral vectors versus several modular protein-based nanovectors was compared using a controlled cortical impact model of TBI under the same gene therapy conditions. We show that variables such as protein/DNA ratio, incubation volume, and presence of serum or chloroquine in the transfection medium impact on both nanovector formation and transfection efficiency in vitro. While lentiviral vectors showed GFP protein 1 day after TBI and increased expression at 14 days, nanovectors showed stable and lower GFP transgene expression from 1 to 14 days. No toxicity after TBI by any of the vectors was observed as determined by resulting levels of IL-1β or using neurological sticky tape test. In fact, both vector types induced functional improvement per se.
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http://dx.doi.org/10.1038/mtm.2014.47DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4362363PMC
May 2015

Enzymatic generation of chitooligosaccharides from chitosan using soluble and immobilized glycosyltransferase (Branchzyme).

J Agric Food Chem 2013 Oct 16;61(43):10360-7. Epub 2013 Oct 16.

Departamento Bioactividad y Análisis de Alimentos, Instituto de Investigación en Ciencias de la Alimentación, CIAL (CSIC-UAM), CEI (UAM+CSIC) , Nicolás Cabrera, 9, 28049, Madrid, Spain.

Chitooligosaccharides possessing remarkable biological properties can be obtained by enzymatic hydrolysis of chitin. In this work, the chitosanase activity of soluble and immobilized glycosyltransferase (Branchzyme) toward chitosan and biochemical characterization are described for the first time. This enzyme was found to be homotetrameric with a molecular weight of 256 kDa, an isoelectric point of 5.3, and an optimal temperature range of between 50 and 60 °C. It was covalently immobilized to glutaraldehyde-agarose with protein and activity immobilization yields of 67% and 17%, respectively. Immobilization improved enzyme stability, increasing its half-life 5-fold, and allowed enzyme reuse for at least 25 consecutive cycles. The chitosanase activity of Branchzyme on chitosan was similar for the soluble and immobilized forms. The reaction mixture was constituted by chitooligosaccharides with degrees of polymerization of between 2 and 20, with a higher concentration having degrees of polymerization of 3-8.
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http://dx.doi.org/10.1021/jf403321rDOI Listing
October 2013

Enzymatic synthesis of 2-aminoethyl β-D-galactopyranoside catalyzed by Aspergillus oryzae β-galactosidase.

Carbohydr Res 2013 Mar 19;368:104-10. Epub 2012 Dec 19.

Cátedra de Bioquímica, Dpto. de Biociencias, Facultad de Química, UdelaR, Gral. Flores 2124, CC 1157, Montevideo, Uruguay.

Glycosidases provide a powerful resource for in vitro synthesis of novel anomerically pure glycosides. Generation of new low molecular weight galactosides is of interest since they are potential galectin inhibitors. Galectins are molecular targets for cancer therapy and thus their inhibitors are potential antitumor agents. Here we report the enzymatic synthesis and structural characterization of 2-aminoethyl β-D-galactopyranoside. Critical parameters for transgalactosylation using either soluble or immobilized enzyme were investigated and optimized for the galactoside synthesis. We found that 0.2 M lactose, and 0.5 M 2-aminoethanol at 50 °C for 30 min were the optimal conditions for synthesis. 2-Aminoethanol proved to be an enzyme inhibitor, fitting a mixed inhibition model with inhibition constants, K(ic)=0.31±0.04 M and K(iu)=0.604±0.035 M.
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http://dx.doi.org/10.1016/j.carres.2012.12.009DOI Listing
March 2013

Characterization of galactosyl derivatives obtained by transgalactosylation of lactose and different polyols using immobilized beta-galactosidase from Aspergillus oryzae.

J Agric Food Chem 2009 Dec;57(23):11302-7

Departamento de Biociencias, Facultad de Química, Universidad de la República, Montevideo, Uruguay.

The synthesis of novel galactosides is interesting because of their important role in several biological processes. Their properties greatly depend upon the configuration and type of galactoside. Therefore, to study biological activity, it is essential to elucidate the structure of the products. Glycosidases are capable of catalyzing glycosidic linkages with absolute stereoselectivity of the anomeric center. We report the enzymatic synthesis of galactosyl-ethylene glycol, galactosyl-glycerol, and galactosyl-erythritol by immobilized beta-galactosidase from Aspegillus oryzae. The obtained galactosides were isolated and fully characterized by an extensive nuclear magnetic resonance (NMR) study. Complete structure elucidation and full proton and carbon assignments were carried out using 1D ((1)H and (13)C) and 2D (gCOSY, TOCSY, multiplicity-edited gHSQC, and gHMBC) NMR experiments. The beta-galactosidase from A. oryzae showed a strong preference for primary alcohols. For galactosyl-glycerol and galactosyl-erythritol, this preference generated one and two chiral centers, respectively, and a mixture of stereoisomers was obtained as a consequence.
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http://dx.doi.org/10.1021/jf901834kDOI Listing
December 2009

Covalent immobilization of tobacco-etch-virus NIa protease: a useful tool for cleavage of the histidine tag of recombinant proteins.

Biotechnol Appl Biochem 2009 May 29;53(Pt 3):165-74. Epub 2009 May 29.

Centro de Biologia Molecular Estrutural, Departamento de Bioquímica, Universidade Federal de Santa Catarina, 88040-900 Florianópolis, SC, Brazil.

Addition of tags [such as His (histidine) tags] is extremely helpful for the affinity purification of recombinant proteins. In several cases, these tags must be removed before performing functional and structural studies. The enzyme most frequently used to cleave tags of recombinant proteins is the TEV-protease (tobacco-etch-virus NIa protease). The continuous production of this enzyme in soluble form is quite an expensive process and not easily accessible to many laboratories. Thus an interesting alternative is the use of TEV-protease in an immobilized form, which may be reutilized several times. The main objective of the present study was to obtain a TEV-protease in an immobilized form, by covalent immobilization on to solid supports through selective use of different amino acid residues, lysine or cysteine. High protein immobilization yields (75-97%) were obtained with both strategies. The TEV-protease immobilized through its exposed cysteine thiol groups maintained its ability for cleaving a 20 kDa substrate. While the activity of the immobilized TEV-protease maintained only 30% of the activity of the enzyme in soluble form, its stability at 4 degrees C was improved three times. Moreover, this enzyme could be reutilized in at least five cycles of cleavage without loss of performance. The present results indicate that the use of a TEV-protease in an immobilized form is a potentially useful tool for the cleavage of His tags of recombinant proteins and may be useful for reducing the cost of the total process of cleavage.
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http://dx.doi.org/10.1042/BA20080063DOI Listing
May 2009
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