Publications by authors named "Alessandra Castegna"

48 Publications

Differential Expression of ADP/ATP Carriers as a Biomarker of Metabolic Remodeling and Survival in Kidney Cancers.

Biomolecules 2020 Dec 30;11(1). Epub 2020 Dec 30.

Department of Biosciences, Biotechnologies, Biopharmaceutics, University "Aldo Moro" of Bari, Via E. Orabona, 4, 70125 Bari, Italy.

ADP/ATP carriers (AACs) are mitochondrial transport proteins playing a strategic role in maintaining the respiratory chain activity, fueling the cell with ATP, and also regulating mitochondrial apoptosis. To understand if AACs might represent a new molecular target for cancer treatment, we evaluated AAC expression levels in cancer/normal tissue pairs available on the Tissue Cancer Genome Atlas database (TCGA), observing that AACs are dysregulated in most of the available samples. It was observed that at least two AACs showed a significant differential expression in all the available kidney cancer/normal tissue pairs. Thus, we investigated AAC expression in the corresponding kidney non-cancer (HK2)/cancer (RCC-Shaw and CaKi-1) cell lines, grown in complete medium or serum starvation, for investigating how metabolic alteration induced by different growth conditions might influence AAC expression and resistance to mitochondrial apoptosis initiators, such as "staurosporine" or the AAC highly selective inhibitor "carboxyatractyloside". Our analyses showed that AAC2 and AAC3 transcripts are more expressed than AAC1 in all the investigated kidney cell lines grown in complete medium, whereas serum starvation causes an increase of at least two AAC transcripts in kidney cancer cell lines compared to non-cancer cells. However, the total AAC protein content is decreased in the investigated cancer cell lines, above all in the serum-free medium. The observed decrease in AAC protein content might be responsible for the decrease of OXPHOS activity and for the observed lowered sensitivity to mitochondrial apoptosis induced by staurosporine or carboxyatractyloside. Notably, the cumulative probability of the survival of kidney cancer patients seriously decreases with the decrease of AAC1 expression in KIRC and KIRP tissues making AAC1 a possible new biomarker of metabolic remodeling and survival in kidney cancers.
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http://dx.doi.org/10.3390/biom11010038DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7824283PMC
December 2020

Editorial: Metabolism Meets Function: Untangling the Cross-Talk Between Signaling and Metabolism.

Front Oncol 2020 20;10:607511. Epub 2020 Oct 20.

Department of Molecular Biotechnology and Health Science, Molecular Biotechnology Center, University of Torino, Torino, Italy.

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http://dx.doi.org/10.3389/fonc.2020.607511DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7607004PMC
October 2020

Glufosinate constrains synchronous and metachronous metastasis by promoting anti-tumor macrophages.

EMBO Mol Med 2020 10 4;12(10):e11210. Epub 2020 Sep 4.

Laboratory of Tumor Inflammation and Angiogenesis, Center for Cancer Biology (CCB), VIB, Leuven, Belgium.

Glutamine synthetase (GS) generates glutamine from glutamate and controls the release of inflammatory mediators. In macrophages, GS activity, driven by IL10, associates to the acquisition of M2-like functions. Conditional deletion of GS in macrophages inhibits metastasis by boosting the formation of anti-tumor, M1-like, tumor-associated macrophages (TAMs). From this basis, we evaluated the pharmacological potential of GS inhibitors in targeting metastasis, identifying glufosinate as a specific human GS inhibitor. Glufosinate was tested in both cultured macrophages and on mice bearing metastatic lung, skin and breast cancer. We found that glufosinate rewires macrophages toward an M1-like phenotype both at the primary tumor and metastatic site, countering immunosuppression and promoting vessel sprouting. This was also accompanied to a reduction in cancer cell intravasation and extravasation, leading to synchronous and metachronous metastasis growth inhibition, but no effects on primary tumor growth. Glufosinate treatment was well-tolerated, without liver and brain toxicity, nor hematopoietic defects. These results identify GS as a druggable enzyme to rewire macrophage functions and highlight the potential of targeting metabolic checkpoints in macrophages to treat cancer metastasis.
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http://dx.doi.org/10.15252/emmm.201911210DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7539200PMC
October 2020

Targeting monoamine oxidase to dampen NLRP3 inflammasome activation in inflammation.

Cell Mol Immunol 2020 Apr 28. Epub 2020 Apr 28.

Department of Biomedical Sciences, University of Padova, 35131, Padova, Italy.

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http://dx.doi.org/10.1038/s41423-020-0441-8DOI Listing
April 2020

Pharmacological targets of metabolism in disease: Opportunities from macrophages.

Pharmacol Ther 2020 06 6;210:107521. Epub 2020 Mar 6.

Department of Biomedical Sciences, University of Padua, Italy; Fondazione Città della Speranza, Istituto di Ricerca Pediatrica, Padua, Italy. Electronic address:

From advances in the knowledge of the immune system, it is emerging that the specialized functions displayed by macrophages during the course of an immune response are supported by specific and dynamically-connected metabolic programs. The study of immunometabolism is demonstrating that metabolic adaptations play a critical role in modulating inflammation and, conversely, inflammation deeply influences the acquisition of specific metabolic settings.This strict connection has been proven to be crucial for the execution of defined immune functional programs and it is now under investigation with respect to several human disorders, such as diabetes, sepsis, cancer, and autoimmunity. The abnormal remodelling of the metabolic pathways in macrophages is now emerging as both marker of disease and potential target of therapeutic intervention. By focusing on key pathological conditions, namely obesity and diabetes, rheumatoid arthritis, atherosclerosis and cancer, we will review the metabolic targets suitable for therapeutic intervention in macrophages. In addition, we will discuss the major obstacles and challenges related to the development of therapeutic strategies for a pharmacological targeting of macrophage's metabolism.
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http://dx.doi.org/10.1016/j.pharmthera.2020.107521DOI Listing
June 2020

Impact of Immunometabolism on Cancer Metastasis: A Focus on T Cells and Macrophages.

Cold Spring Harb Perspect Med 2020 09 1;10(9). Epub 2020 Sep 1.

Laboratory of Tumor Inflammation and Angiogenesis, Center for Cancer Biology, VIB, Leuven B3000, Belgium.

Despite improved treatment options, cancer remains the leading cause of morbidity and mortality worldwide, with 90% of this mortality correlated to the development of metastasis. Since metastasis has such an impact on treatment success, disease outcome, and global health, it is important to understand the different steps and factors playing key roles in this process, how these factors relate to immune cell function and how we can target metabolic processes at different steps of metastasis in order to improve cancer treatment and patient prognosis. Recent insights in immunometabolism direct to promising therapeutic targets for cancer treatment, however, the specific contribution of metabolism on antitumor immunity in different metastatic niches warrant further investigation. Here, we provide an overview of what is so far known in the field of immunometabolism at different steps of the metastatic cascade, and what may represent the next steps forward. Focusing on metabolic checkpoints in order to translate these findings from in vitro and mouse studies to the clinic has the potential to revolutionize cancer immunotherapy and greatly improve patient prognosis.
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http://dx.doi.org/10.1101/cshperspect.a037044DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7461771PMC
September 2020

The Metabolic Signature of Macrophage Responses.

Front Immunol 2019 3;10:1462. Epub 2019 Jul 3.

Department of Biosciences, Biotechnologies and Biopharmaceutics, University of Bari, Bari, Italy.

Macrophages are a heterogeneous population of immune cells playing several and diverse functions in homeostatic and immune responses. The broad spectrum of macrophage functions depends on both heterogeneity and plasticity of these cells, which are highly specialized in sensing the microenvironment and modify their properties accordingly. Although it is clear that macrophage phenotypes are difficult to categorize and should be seen as plastic and adaptable, they can be simplified into two extremes: a pro-inflammatory (M1) and an anti-inflammatory/pro-resolving (M2) profile. Based on this definition, M1 macrophages are able to start and sustain inflammatory responses, secreting pro-inflammatory cytokines, activating endothelial cells, and inducing the recruitment of other immune cells into the inflamed tissue; on the other hand, M2 macrophages promote the resolution of inflammation, phagocytose apoptotic cells, drive collagen deposition, coordinate tissue integrity, and release anti-inflammatory mediators. Dramatic switches in cell metabolism accompany these phenotypic and functional changes of macrophages. In particular, M1 macrophages rely mainly on glycolysis and present two breaks on the TCA cycle that result in accumulation of itaconate (a microbicide compound) and succinate. Excess of succinate leads to Hypoxia Inducible Factor 1α (HIF1α) stabilization that, in turn, activates the transcription of glycolytic genes, thus sustaining the glycolytic metabolism of M1 macrophages. On the contrary, M2 cells are more dependent on oxidative phosphorylation (OXPHOS), their TCA cycle is intact and provides the substrates for the complexes of the electron transport chain (ETC). Moreover, pro- and anti-inflammatory macrophages are characterized by specific pathways that regulate the metabolism of lipids and amino acids and affect their responses. All these metabolic adaptations are functional to support macrophage activities as well as to sustain their polarization in specific contexts. The aim of this review is to discuss recent findings linking macrophage functions and metabolism.
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http://dx.doi.org/10.3389/fimmu.2019.01462DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6618143PMC
October 2020

The Crowded Crosstalk between Cancer Cells and Stromal Microenvironment in Gynecological Malignancies: Biological Pathways and Therapeutic Implication.

Int J Mol Sci 2019 May 15;20(10). Epub 2019 May 15.

Department of Biomedical and Human Oncological Science, 2nd Unit of Obstetrics and Gynecology, University of Bari "Aldo Moro", Piazza G. Cesare, 11-Policlinico 70124 Bari, Italy.

The tumor microenvironment plays a pillar role in the progression and the distance dissemination of cancer cells in the main malignancies affecting women-epithelial ovarian cancer, endometrial cancer and cervical cancer. Their acquires specific properties thanks to intense crosstalk between stromal and cancer cells, leading to a vicious circle. Fibroblasts, pericytes, lymphocytes and tumor associated-macrophages orchestrate most of the biological pathways. In epithelial ovarian cancer, high rates of activated pericytes determine a poorer prognosis, defining a common signature promoting ovarian cancer proliferation, local invasion and distant spread. Mesenchymal cells also release chemokines and cytokines under hormonal influence, such as estrogens that drive most of the endometrial cancers. Interestingly, the architecture of the cervical cancer is shaped by the synergy of high-risk Human Papilloma Virus oncoproteins and the activity of stromal estrogen receptor α. Lymphocytes represent a shield against cancer cells but some cell subpopulation could lead to immunosuppression, tumor growth and dissemination. Cytotoxic tumor infiltrating lymphocytes can be eluded by over-adapted cancer cells in a scenario of immune-tolerance driven by T-regulatory cells. Therefore, the tumor microenvironment has a high translational potential offering many targets for biological and immunological therapies.
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http://dx.doi.org/10.3390/ijms20102401DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6567055PMC
May 2019

Molecular identification and functional characterization of a novel glutamate transporter in yeast and plant mitochondria.

Biochim Biophys Acta Bioenerg 2018 11 8;1859(11):1249-1258. Epub 2018 Aug 8.

Laboratory of Biochemistry and Molecular Biology, Department of Biosciences, Biotechnologies and Biopharmaceutics, University of Bari Aldo Moro, Bari, Italy; CNR Institute of Biomembranes, Bioenergetics and Molecular Biotechnologies (IBIOM), Bari, Italy. Electronic address:

The genome of Saccharomyces cerevisiae encodes 35 members of the mitochondrial carrier family (MCF) and 58 MCF members are coded by the genome of Arabidopsis thaliana, most of which have been functionally characterized. Here two members of this family, Ymc2p from S. cerevisiae and BOU from Arabidopsis, have been thoroughly characterized. These proteins were overproduced in bacteria and reconstituted into liposomes. Their transport properties and kinetic parameters demonstrate that Ymc2p and BOU transport glutamate, and to a much lesser extent L-homocysteinesulfinate, but not other amino acids and many other tested metabolites. Transport catalyzed by both carriers was saturable, inhibited by mercuric chloride and dependent on the proton gradient across the proteoliposomal membrane. The growth phenotype of S. cerevisiae cells lacking the genes ymc2 and agc1, which encodes the only other S. cerevisiae carrier capable to transport glutamate besides aspartate, was fully complemented by expressing Ymc2p, Agc1p or BOU. Mitochondrial extracts derived from ymc2Δagc1Δ cells, reconstituted into liposomes, exhibited no glutamate transport at variance with wild-type, ymc2Δ and agc1Δ cells, showing that S. cerevisiae cells grown in the presence of acetate do not contain additional mitochondrial transporters for glutamate besides Ymc2p and Agc1p. Furthermore, mitochondria isolated from wild-type, ymc2Δ and agc1Δ strains, but not from the double mutant ymc2Δagc1Δ strain, swell in isosmotic ammonium glutamate showing that glutamate is transported by Ymc2p and Agc1p together with a H. It is proposed that the function of Ymc2p and BOU is to transport glutamate across the mitochondrial inner membrane and thereby play a role in intermediary metabolism, C1 metabolism and mitochondrial protein synthesis.
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http://dx.doi.org/10.1016/j.bbabio.2018.08.001DOI Listing
November 2018

Monoamine oxidase-dependent histamine catabolism accounts for post-ischemic cardiac redox imbalance and injury.

Biochim Biophys Acta Mol Basis Dis 2018 09 25;1864(9 Pt B):3050-3059. Epub 2018 Jun 25.

Department of Biomedical Sciences, University of Padova, Italy; Venetian Institute of Molecular Medicine (VIMM), Padova, Italy. Electronic address:

Monoamine oxidase (MAO), a mitochondrial enzyme that oxidizes biogenic amines generating hydrogen peroxide, is a major source of oxidative stress in cardiac injury. However, the molecular mechanisms underlying its overactivation in pathological conditions are still poorly characterized. Here, we investigated whether the enhanced MAO-dependent hydrogen peroxide production can be due to increased substrate availability using a metabolomic profiling method. We identified N-methylhistamine -the main catabolite of histamine- as an important substrate fueling MAO in Langendorff mouse hearts, directly perfused with a buffer containing hydrogen peroxide or subjected to ischemia/reperfusion protocol. Indeed, when these hearts were pretreated with the MAO inhibitor pargyline we observed N-methylhistamine accumulation along with reduced oxidative stress. Next, we showed that synaptic terminals are the major source of N-methylhistamine. Indeed, in vivo sympathectomy caused a decrease of N-methylhistamine levels, which was associated with a marked protection in post-ischemic reperfused hearts. As far as the mechanism is concerned, we demonstrate that exogenous histamine is transported into isolated cardiomyocytes and triggers a rise in the levels of reactive oxygen species (ROS). Once again, pargyline pretreatment induced intracellular accumulation of N-methylhistamine along with decrease in ROS levels. These findings uncover a receptor-independent mechanism for histamine in cardiomyocytes. In summary, our study reveals a novel and important pathophysiological causative link between MAO activation and histamine availability during pathophysiological conditions such as oxidative stress/cardiac injury.
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http://dx.doi.org/10.1016/j.bbadis.2018.06.018DOI Listing
September 2018

Glutamine Synthetase: Localization Dictates Outcome.

Genes (Basel) 2018 Feb 19;9(2). Epub 2018 Feb 19.

Hematology Unit, National Cancer Research Center, Istituto Tumori 'Giovanni Paolo II', Bari 70124, Italy.

Glutamine synthetase (GS) is the adenosine triphosphate (ATP)-dependent enzyme that catalyses the synthesis of glutamine by condensing ammonium to glutamate. In the circulatory system, glutamine carries ammonia from muscle and brain to the kidney and liver. In brain reduction of GS activity has been suggested as a mechanism mediating neurotoxicity in neurodegenerative disorders. In cancer, the delicate balance between glutamine synthesis and catabolism is a critical event. In vitro evidence, confirmed in vivo in some cases, suggests that reduced GS activity in cancer cells associates with a more invasive and aggressive phenotype. However, GS is known to be highly expressed in cells of the tumor microenvironment, such as fibroblasts, adipocytes and immune cells, and their ability to synthesize glutamine is responsible for the acquisition of protumoral phenotypes. This has opened a new window into the complex scenario of the tumor microenvironment, in which the balance of glutamine consumption versus glutamine synthesis influences cellular function. Since GS expression responds to glutamine starvation, a lower glutamine synthesizing power due to the absence of GS in cancer cells might apply a metabolic pressure on stromal cells. This event might push stroma towards a GS-high/protumoral phenotype. When referred to stromal cells, GS expression might acquire a 'bad' significance to the point that GS inhibition might be considered a conceivable strategy against cancer metastasis.
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http://dx.doi.org/10.3390/genes9020108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5852604PMC
February 2018

SLC25A10 biallelic mutations in intractable epileptic encephalopathy with complex I deficiency.

Hum Mol Genet 2018 02;27(3):499-504

Department of Biosciences, Biotechnology and Biopharmaceutics, University of Bari, 70125 Bari, Italy.

Mitochondrial diseases are a plethora of inherited neuromuscular disorders sharing defects in mitochondrial respiration, but largely different from one another for genetic basis and pathogenic mechanism. Whole exome sequencing was performed in a familiar trio (trio-WES) with a child affected by severe epileptic encephalopathy associated with respiratory complex I deficiency and mitochondrial DNA depletion in skeletal muscle. By trio-WES we identified biallelic mutations in SLC25A10, a nuclear gene encoding a member of the mitochondrial carrier family. Genetic and functional analyses conducted on patient fibroblasts showed that SLC25A10 mutations are associated with reduction in RNA quantity and aberrant RNA splicing, and to absence of SLC25A10 protein and its transporting function. The yeast SLC25A10 ortholog knockout strain showed defects in mitochondrial respiration and mitochondrial DNA content, similarly to what observed in the patient skeletal muscle, and growth susceptibility to oxidative stress. Albeit patient fibroblasts were depleted in the main antioxidant molecules NADPH and glutathione, transport assays demonstrated that SLC25A10 is unable to transport glutathione. Here, we report the first recessive mutations of SLC25A10 associated to an inherited severe mitochondrial neurodegenerative disorder. We propose that SLC25A10 loss-of-function causes pathological disarrangements in respiratory-demanding conditions and oxidative stress vulnerability.
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http://dx.doi.org/10.1093/hmg/ddx419DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5886107PMC
February 2018

Metabolism and TAM functions-it takes two to tango.

FEBS J 2018 02 8;285(4):700-716. Epub 2017 Nov 8.

Hematology Unit, National Cancer Research Center, Istituto Tumori 'Giovanni Paolo II', Bari, Italy.

From the evidence on clinical studies and experimental mouse models we now know that tumor-associated macrophages (TAMs) sustain tumor development in many different ways. They play a role in angiogenesis, tumor cell invasion, and metastasis formation. Additionally, TAMs interfere with natural killer and T-cell antitumoral activities, producing an immune-suppressive environment that protects tumor cell growth. This indicates that the tumoricidal activity of macrophages within the tumor microenviroment is lost due to an imbalance of the regulatory mechanisms underpinning these cells' function. Since metabolism is emerging as a major modulator of macrophage function, metabolic changes in response to signals coming from cancer or other immune cells might promote this imbalance, enhancing the tumorigenic activities of TAMs. In this review we describe the novel, most recent findings on how metabolism shapes TAM functions or conversely, how TAMs influence the activity of other cells through metabolic mechanisms. The complete elucidation of the metabolic switches between pro- and antitumoral properties of macrophages, now still in its infancy, is destined to provide scientists with new instruments not only to understand but also to combat cancer.
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http://dx.doi.org/10.1111/febs.14295DOI Listing
February 2018

Pharmacologic or Genetic Targeting of Glutamine Synthetase Skews Macrophages toward an M1-like Phenotype and Inhibits Tumor Metastasis.

Cell Rep 2017 08;20(7):1654-1666

Department of Biosciences, Biotechnologies and Biopharmaceutics, University of Bari, 70125 Bari, Italy; Hematology Unit, National Cancer Research Center, Istituto Tumori "Giovanni Paolo II," 70124 Bari, Italy. Electronic address:

Glutamine-synthetase (GS), the glutamine-synthesizing enzyme from glutamate, controls important events, including the release of inflammatory mediators, mammalian target of rapamycin (mTOR) activation, and autophagy. However, its role in macrophages remains elusive. We report that pharmacologic inhibition of GS skews M2-polarized macrophages toward the M1-like phenotype, characterized by reduced intracellular glutamine and increased succinate with enhanced glucose flux through glycolysis, which could be partly related to HIF1α activation. As a result of these metabolic changes and HIF1α accumulation, GS-inhibited macrophages display an increased capacity to induce T cell recruitment, reduced T cell suppressive potential, and an impaired ability to foster endothelial cell branching or cancer cell motility. Genetic deletion of macrophagic GS in tumor-bearing mice promotes tumor vessel pruning, vascular normalization, accumulation of cytotoxic T cells, and metastasis inhibition. These data identify GS activity as mediator of the proangiogenic, immunosuppressive, and pro-metastatic function of M2-like macrophages and highlight the possibility of targeting this enzyme in the treatment of cancer metastasis.
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http://dx.doi.org/10.1016/j.celrep.2017.07.054DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5575233PMC
August 2017

Down-regulation of the mitochondrial aspartate-glutamate carrier isoform 1 AGC1 inhibits proliferation and N-acetylaspartate synthesis in Neuro2A cells.

Biochim Biophys Acta Mol Basis Dis 2017 06 21;1863(6):1422-1435. Epub 2017 Feb 21.

Institute of Biomembranes and Bioenergetics, Consiglio Nazionale delle Ricerche, Bari 70126, Italy. Electronic address:

The mitochondrial aspartate-glutamate carrier isoform 1 (AGC1) catalyzes a Ca-stimulated export of aspartate to the cytosol in exchange for glutamate, and is a key component of the malate-aspartate shuttle which transfers NADH reducing equivalents from the cytosol to mitochondria. By sustaining the complete glucose oxidation, AGC1 is thought to be important in providing energy for cells, in particular in the CNS and muscle where this protein is mainly expressed. Defects in the AGC1 gene cause AGC1 deficiency, an infantile encephalopathy with delayed myelination and reduced brain N-acetylaspartate (NAA) levels, the precursor of myelin synthesis in the CNS. Here, we show that undifferentiated Neuro2A cells with down-regulated AGC1 display a significant proliferation deficit associated with reduced mitochondrial respiration, and are unable to synthesize NAA properly. In the presence of high glutamine oxidation, cells with reduced AGC1 restore cell proliferation, although oxidative stress increases and NAA synthesis deficit persists. Our data suggest that the cellular energetic deficit due to AGC1 impairment is associated with inappropriate aspartate levels to support neuronal proliferation when glutamine is not used as metabolic substrate, and we propose that delayed myelination in AGC1 deficiency patients could be attributable, at least in part, to neuronal loss combined with lack of NAA synthesis occurring during the nervous system development.
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http://dx.doi.org/10.1016/j.bbadis.2017.02.022DOI Listing
June 2017

SLC25A26 overexpression impairs cell function via mtDNA hypermethylation and rewiring of methyl metabolism.

FEBS J 2017 03 21;284(6):967-984. Epub 2017 Feb 21.

Department of Biosciences, Biotechnologies and Biopharmaceutics, University of Bari 'Aldo Moro', Italy.

Cancer cells down-regulate different genes to give them a selective advantage in invasiveness and/or metastasis. The SLC25A26 gene encodes the mitochondrial carrier that catalyzes the import of S-adenosylmethionine (SAM) into the mitochondrial matrix, required for mitochondrial methylation processes, and is down-regulated in cervical cancer cells. In this study we show that SLC25A26 is down-regulated due to gene promoter hypermethylation, as a mechanism to promote cell survival and proliferation. Furthermore, overexpression of SLC25A26 in CaSki cells increases mitochondrial SAM availability and promotes hypermethylation of mitochondrial DNA, leading to decreased expression of key respiratory complex subunits, reduction of mitochondrial ATP and release of cytochrome c. In addition, increased SAM transport into mitochondria leads to impairment of the methionine cycle with accumulation of homocysteine at the expense of glutathione, which is strongly reduced. All these events concur to arrest the cell cycle in the S phase, induce apoptosis and enhance chemosensitivity of SAM carrier-overexpressing CaSki cells to cisplatin.
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http://dx.doi.org/10.1111/febs.14028DOI Listing
March 2017

Blockade of Glutamine Synthetase Enhances Inflammatory Response in Microglial Cells.

Antioxid Redox Signal 2017 03 2;26(8):351-363. Epub 2016 Nov 2.

1 Department of Biosciences, Biotechnologies and Biopharmaceutics, University of Bari , Bari, Italy .

Aims: Microglial cells are brain-resident macrophages engaged in surveillance and maintained in a constant state of relative inactivity. However, their involvement in autoimmune diseases indicates that in pathological conditions microglia gain an inflammatory phenotype. The mechanisms underlying this change in the microglial phenotype are still unclear. Since metabolism is an important modulator of immune cell function, we focused our attention on glutamine synthetase (GS), a modulator of the response to lipopolysaccharide (LPS) activation in other cell types, which is expressed by microglia.

Results: GS inhibition enhances release of inflammatory mediators of LPS-activated microglia in vitro, leading to perturbation of the redox balance and decreased viability of cocultured neurons. GS inhibition also decreases insulin-mediated glucose uptake in microglia. In vivo, microglia-specific GS ablation enhances expression of inflammatory markers upon LPS treatment. In the spinal cords from experimental autoimmune encephalomyelitis (EAE), GS expression levels and glutamine/glutamate ratios are reduced.

Innovation: Recently, metabolism has been highlighted as mediator of immune cell function through the discovery of mechanisms that (behind these metabolic changes) modulate the inflammatory response. The present study shows for the first time a metabolic mechanism mediating microglial response to a proinflammatory stimulus, pointing to GS activity as a master modulator of immune cell function and thus unraveling a potential therapeutic target.

Conclusions: Our study highlights a new role of GS in modulating immune response in microglia, providing insights into the pathogenic mechanisms associated with inflammation and new strategies of therapeutic intervention. Antioxid. Redox Signal. 26, 351-363.
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http://dx.doi.org/10.1089/ars.2016.6715DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5346956PMC
March 2017

Mitochondrial carriers in inflammation induced by bacterial endotoxin and cytokines.

Biol Chem 2017 03;398(3):303-317

Significant metabolic changes occur in the shift from resting to activated cellular status in inflammation. Thus, changes in expression of a large number of genes and extensive metabolic reprogramming gives rise to acquisition of new functions (e.g. production of cytokines, intermediates for biosynthesis, lipid mediators, PGE, ROS and NO). In this context, mitochondrial carriers, which catalyse the transport of solute across mitochondrial membrane, change their expression to transport mitochondrially produced molecules, among which citrate and succinate, to be used as intracellular signalling molecules in inflammation. This review summarises the mitochondrial carriers studied so far that are, directly or indirectly, involved in inflammation.
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http://dx.doi.org/10.1515/hsz-2016-0260DOI Listing
March 2017

The Effects of Chronic Lifelong Activation of the AHR Pathway by Industrial Chemical Pollutants on Female Human Reproduction.

PLoS One 2016 23;11(3):e0152181. Epub 2016 Mar 23.

Unit of Pathophysiology of Human Reproduction and Gametes Cryopreservation, Dept. of General Surgery, Gynecology, Obstetrics and Anesthesiology, University Hospital of Bari, Consorziale, Policlinico. piazza Giulio Cesare 11, 70124, Bari, Italy.

Environmental chemicals, such as heavy metals, affect female reproductive function. A biological sensor of the signals of many toxic chemical compounds seems to be the aryl hydrocarbon receptor (AHR). Previous studies demonstrated the environmental of heavy metals in Taranto city (Italy), an area that has been influenced by anthropogenic factors such as industrial activities and waste treatments since 1986. However, the impact of these elements on female fertility in this geographic area has never been analyzed. Thus, in the present study, we evaluated the AHR pathway, sex steroid receptor pattern and apoptotic process in granulosa cells (GCs) retrieved from 30 women, born and living in Taranto, and 30 women who are living in non-contaminated areas (control group), who were undergoing in vitro fertilization (IVF) protocol. In follicular fluids (FFs) of both groups the toxic and essential heavy metals, such as chromiun (Cr), Manganese (Mn), iron (Fe), cobalt (Co), nickel (Ni), copper (Cu), zinc (Zn), cadmium (Cd) and lead (Pb), were also analyzed. Higher levels of Cr, Fe, Zn and Pb were found in the FFs of the women from Taranto as compared to the control group, as were the levels of AHR and AHR-dependent cytochrome P450 1A1 and 1B1; while CYP19A1 expression was decreased. The anti-apoptotic process found in the GCs of women fromTaranto was associated with the highest levels of progesterone receptor membrane component 1 (PGRMC1), a novel progesterone receptor, the expression of which is subjected to AHR activated by its highest affinity ligands (e.g., dioxins) or indirectly by other environmental pollutants, such as heavy metals. In conclusion, decreased production of estradiol and decreased number of retrieved mature oocytes found in women from Taranto could be due to chronic exposure to heavy metals, in particular to Cr and Pb.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0152181PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4805276PMC
August 2016

Clinical implications from proteomic studies in neurodegenerative diseases: lessons from mitochondrial proteins.

Expert Rev Proteomics 2016 ;13(3):259-74

b Department of Biosciences, Biotechnologies and Biopharmaceutics , University of Bari 'Aldo Moro' , Bari , Italy.

Mitochondria play a key role in eukaryotic cells, being mediators of energy, biosynthetic and regulatory requirements of these cells. Emerging proteomics techniques have allowed scientists to obtain the differentially expressed proteome or the proteomic redox status in mitochondria. This has unmasked the diversity of proteins with respect to subcellular location, expression and interactions. Mitochondria have become a research 'hot spot' in subcellular proteomics, leading to identification of candidate clinical targets in neurodegenerative diseases in which mitochondria are known to play pathological roles. The extensive efforts to rapidly obtain differentially expressed proteomes and unravel the redox proteomic status in mitochondria have yielded clinical insights into the neuropathological mechanisms of disease, identification of disease early stage and evaluation of disease progression. Although current technical limitations hamper full exploitation of the mitochondrial proteome in neurosciences, future advances are predicted to provide identification of specific therapeutic targets for neurodegenerative disorders.
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http://dx.doi.org/10.1586/14789450.2016.1149470DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4949485PMC
December 2016

The mitochondrial side of epigenetics.

Physiol Genomics 2015 Aug 2;47(8):299-307. Epub 2015 Jun 2.

Department of Sciences, University of Basilicata, Potenza, Italy.

The bidirectional cross talk between nuclear and mitochondrial DNA is essential for cellular homeostasis and proper functioning. Mitochondria depend on nuclear contribution for much of their functionality, but their activities have been recently recognized to control nuclear gene expression as well as cell function in many different ways. Epigenetic mechanisms, which tune gene expression in response to environmental stimuli, are key regulatory events at the interplay between mitochondrial and nuclear interactions. Emerging findings indicate that epigenetic factors can be targets or instruments of mitochondrial-nuclear cross talk. Additionally, the growing interest into mtDNA epigenetic modifications opens new avenues into the interaction mechanisms between mitochondria and nucleus. In this review we summarize the points of mitochondrial and nuclear reciprocal control involving epigenetic factors, focusing on the role of mitochondrial genome and metabolism in shaping epigenetic modulation of gene expression. The relevance of the new findings on the methylation of mtDNA is also highlighted as a new frontier in the complex scenario of mitochondrial-nuclear communication.
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http://dx.doi.org/10.1152/physiolgenomics.00096.2014DOI Listing
August 2015

Acetylation of human mitochondrial citrate carrier modulates mitochondrial citrate/malate exchange activity to sustain NADPH production during macrophage activation.

Biochim Biophys Acta 2015 Aug 24;1847(8):729-38. Epub 2015 Apr 24.

Department of Biosciences, Biotechnologies and Biopharmaceutics, University of Bari "Aldo Moro", Bari, Italy; Center of Excellence in Comparative Genomics, University of Bari "Aldo Moro", Bari, Italy. Electronic address:

The mitochondrial citrate-malate exchanger (CIC), a known target of acetylation, is up-regulated in activated immune cells and plays a key role in the production of inflammatory mediators. However, the role of acetylation in CIC activity is elusive. We show that CIC is acetylated in activated primary human macrophages and U937 cells and the level of acetylation is higher in glucose-deprived compared to normal glucose medium. Acetylation enhances CIC transport activity, leading to a higher citrate efflux from mitochondria in exchange with malate. Cytosolic citrate levels do not increase upon activation of cells grown in deprived compared to normal glucose media, indicating that citrate, transported from mitochondria at higher rates from acetylated CIC, is consumed at higher rates. Malate levels in the cytosol are lower in activated cells grown in glucose-deprived compared to normal glucose medium, indicating that this TCA intermediate is rapidly recycled back into the cytosol where it is used by the malic enzyme. Additionally, in activated cells CIC inhibition increases the NADP+/NADPH ratio in glucose-deprived cells; this ratio is unchanged in glucose-rich grown cells due to the activity of the pentose phosphate pathway. Consistently, the NADPH-producing isocitrate dehydrogenase level is higher in activated glucose-deprived as compared to glucose rich cells. These results demonstrate that, in the absence of glucose, activated macrophages increase CIC acetylation to enhance citrate efflux from mitochondria not only to produce inflammatory mediators but also to meet the NADPH demand through the actions of isocitrate dehydrogenase and malic enzyme.
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http://dx.doi.org/10.1016/j.bbabio.2015.04.009DOI Listing
August 2015

Glutamine synthetase desensitizes differentiated adipocytes to proinflammatory stimuli by raising intracellular glutamine levels.

FEBS Lett 2014 Dec 20;588(24):4807-14. Epub 2014 Nov 20.

Department of Biosciences, Biotechnologies and Biopharmaceutics, University of Bari, Bari, Italy. Electronic address:

The role of glutamine synthetase (GS) during adipocyte differentiation is unclear. Here, we assess the impact of GS on the adipocytic response to a proinflammatory challenge at different differentiation stages. GS expression at the late stages of differentiation desensitized mature adipocytes to bacterial lipopolysaccharide (LPS) by increasing intracellular glutamine levels. Furthermore, LPS-activated mature adipocytes were unable to produce inflammatory mediators; LPS sensitivity was rescued following GS inhibition and the associated drop in intracellular glutamine levels. The ability of adipocytes to differentially respond to LPS during differentiation negatively correlates to GS expression and intracellular glutamine levels. Hence, modulation of intracellular glutamine levels by GS expression represents an endogenous mechanism through which mature adipocytes control the inflammatory response.
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http://dx.doi.org/10.1016/j.febslet.2014.11.015DOI Listing
December 2014

Hyperhomocysteinemia: related genetic diseases and congenital defects, abnormal DNA methylation and newborn screening issues.

Mol Genet Metab 2014 Sep-Oct;113(1-2):27-33. Epub 2014 Jul 21.

Department of Translational Medical Sciences, Section of Pediatrics, Federico II University, Naples, Italy.

Homocysteine, a sulfur-containing amino acid derived from the methionine metabolism, is located at the branch point of two pathways of the methionine cycle, i.e. remethylation and transsulfuration. Gene abnormalities in the enzymes catalyzing reactions in both pathways lead to hyperhomocysteinemia. Hyperhomocysteinemia is associated with increased risk for congenital disorders, including neural tube closure defects, heart defects, cleft lip/palate, Down syndrome, and multi-system abnormalities in adults. Since hyperhomocysteinemia is known to affect the extent of DNA methylation, it is likely that abnormal DNA methylation during embryogenesis, may be a pathogenic factor for these congenital disorders. In this review we highlight the importance of homocysteinemia by describing the genes encoding for enzymes of homocysteine metabolism relevant to the clinical practice, especially cystathionine-β-synthase and methylenetetrahydrofolate reductase mutations, and the impairment of related metabolites levels. Moreover, a possible correlation between hyperhomocysteine and congenital disorders through the involvement of abnormal DNA methylation during embryogenesis is discussed. Finally, the relevance of present and future diagnostic tools such as tandem mass spectrometry and next generation sequencing in newborn screening is highlighted.
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http://dx.doi.org/10.1016/j.ymgme.2014.07.016DOI Listing
June 2015

UCP2 transports C4 metabolites out of mitochondria, regulating glucose and glutamine oxidation.

Proc Natl Acad Sci U S A 2014 Jan 6;111(3):960-5. Epub 2014 Jan 6.

Department of Biosciences, Biotechnologies, and Biopharmaceutics and Center of Excellence in Comparative Genomics, University of Bari, 70125 Bari, Italy.

Uncoupling protein 2 (UCP2) is involved in various physiological and pathological processes such as insulin secretion, stem cell differentiation, cancer, and aging. However, its biochemical and physiological function is still under debate. Here we show that UCP2 is a metabolite transporter that regulates substrate oxidation in mitochondria. To shed light on its biochemical role, we first studied the effects of its silencing on the mitochondrial oxidation of glucose and glutamine. Compared with wild-type, UCP2-silenced human hepatocellular carcinoma (HepG2) cells, grown in the presence of glucose, showed a higher inner mitochondrial membrane potential and ATP:ADP ratio associated with a lower lactate release. Opposite results were obtained in the presence of glutamine instead of glucose. UCP2 reconstituted in lipid vesicles catalyzed the exchange of malate, oxaloacetate, and aspartate for phosphate plus a proton from opposite sides of the membrane. The higher levels of citric acid cycle intermediates found in the mitochondria of siUCP2-HepG2 cells compared with those found in wild-type cells in addition to the transport data indicate that, by exporting C4 compounds out of mitochondria, UCP2 limits the oxidation of acetyl-CoA-producing substrates such as glucose and enhances glutaminolysis, preventing the mitochondrial accumulation of C4 metabolites derived from glutamine. Our work reveals a unique regulatory mechanism in cell bioenergetics and provokes a substantial reconsideration of the physiological and pathological functions ascribed to UCP2 based on its purported uncoupling properties.
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http://dx.doi.org/10.1073/pnas.1317400111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3903233PMC
January 2014

The Saccharomyces cerevisiae gene YPR011c encodes a mitochondrial transporter of adenosine 5'-phosphosulfate and 3'-phospho-adenosine 5'-phosphosulfate.

Biochim Biophys Acta 2014 Feb 1;1837(2):326-34. Epub 2013 Dec 1.

Department of Biosciences, Biotechnologies and Biopharmaceutics, University of Bari, via Orabona 4, 70125 Bari, Italy; Center of Excellence in Comparative Genomics, University of Bari, Italy. Electronic address:

The genome of Saccharomyces cerevisiae contains 35 members of the mitochondrial carrier family, nearly all of which have been functionally characterized. In this study, the identification of the mitochondrial carrier for adenosine 5'-phosphosulfate (APS) is described. The corresponding gene (YPR011c) was overexpressed in bacteria. The purified protein was reconstituted into phospholipid vesicles and its transport properties and kinetic parameters were characterized. It transported APS, 3'-phospho-adenosine 5'-phosphosulfate, sulfate and phosphate almost exclusively by a counter-exchange mechanism. Transport was saturable and inhibited by bongkrekic acid and other inhibitors. To investigate the physiological significance of this carrier in S. cerevisiae, mutants were subjected to thermal shock at 45°C in the presence of sulfate and in the absence of methionine. At 45°C cells lacking YPR011c, engineered cells (in which APS is produced only in mitochondria) and more so the latter cells, in which the exit of mitochondrial APS is prevented by the absence of YPR011cp, were less thermotolerant. Moreover, at the same temperature all these cells contained less methionine and total glutathione than wild-type cells. Our results show that S. cerevisiae mitochondria are equipped with a transporter for APS and that YPR011cp-mediated mitochondrial transport of APS occurs in S. cerevisiae under thermal stress conditions.
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http://dx.doi.org/10.1016/j.bbabio.2013.11.013DOI Listing
February 2014

Mitochondrial DNA methylation as a next-generation biomarker and diagnostic tool.

Mol Genet Metab 2013 Sep-Oct;110(1-2):25-34. Epub 2013 Jul 19.

Department of Biosciences, Biotechnology and Pharmacological Sciences, University of Bari, via Orabona 4, 70125 Bari, Italy.

Recent expansion of our knowledge on epigenetic changes strongly suggests that not only nuclear DNA (nDNA), but also mitochondrial DNA (mtDNA) may be subjected to epigenetic modifications related to disease development, environmental exposure, drug treatment and aging. Thus, mtDNA methylation is attracting increasing attention as a potential biomarker for the detection and diagnosis of diseases and the understanding of cellular behavior in particular conditions. In this paper we review the current advances in mtDNA methylation studies with particular attention to the evidences of mtDNA methylation changes in diseases and physiological conditions so far investigated. Technological advances for the analysis of epigenetic variations are promising tools to provide insights into methylation of mtDNA with similar resolution levels as those reached for nDNA. However, many aspects related to mtDNA methylation are still unclear. More studies are needed to understand whether and how changes in mtDNA methylation patterns, global and gene specific, are associated to diseases or risk factors.
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http://dx.doi.org/10.1016/j.ymgme.2013.07.012DOI Listing
March 2014

Identification of mitochondrial coenzyme a transporters from maize and Arabidopsis.

Plant Physiol 2013 Jun 16;162(2):581-8. Epub 2013 Apr 16.

Microbiology and Cell Science Department , University of Florida, Gainesville, Florida 32611, USA.

Plants make coenzyme A (CoA) in the cytoplasm but use it for reactions in mitochondria, chloroplasts, and peroxisomes, implying that these organelles have CoA transporters. A plant peroxisomal CoA transporter is already known, but plant mitochondrial or chloroplastic CoA transporters are not. Mitochondrial CoA transporters belonging to the mitochondrial carrier family, however, have been identified in yeast (Saccharomyces cerevisiae; Leu-5p) and mammals (SLC25A42). Comparative genomic analysis indicated that angiosperms have two distinct homologs of these mitochondrial CoA transporters, whereas nonflowering plants have only one. The homologs from maize (Zea mays; GRMZM2G161299 and GRMZM2G420119) and Arabidopsis (Arabidopsis thaliana; At1g14560 and At4g26180) all complemented the growth defect of the yeast leu5Δ mitochondrial CoA carrier mutant and substantially restored its mitochondrial CoA level, confirming that these proteins have CoA transport activity. Dual-import assays with purified pea (Pisum sativum) mitochondria and chloroplasts, and subcellular localization of green fluorescent protein fusions in transiently transformed tobacco (Nicotiana tabacum) Bright Yellow-2 cells, showed that the maize and Arabidopsis proteins are targeted to mitochondria. Consistent with the ubiquitous importance of CoA, the maize and Arabidopsis mitochondrial CoA transporter genes are expressed at similar levels throughout the plant. These data show that representatives of both monocotyledons and eudicotyledons have twin, mitochondrially located mitochondrial carrier family carriers for CoA. The highly conserved nature of these carriers makes possible their reliable annotation in other angiosperm genomes.
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http://dx.doi.org/10.1104/pp.113.218081DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3668054PMC
June 2013

Identification of mitochondrial thiamin diphosphate carriers from Arabidopsis and maize.

Funct Integr Genomics 2012 Jun 18;12(2):317-26. Epub 2012 Mar 18.

Horticultural Sciences Department, University of Florida, Gainesville, FL 32611, USA.

It is currently held that thiamin is made in chloroplasts and converted in the cytosol to the active cofactor thiamin diphosphate (ThDP), and that mitochondria and plastids import ThDP. The organellar transporters that mediate ThDP import in plants have not been identified. Comparative genomic analysis indicated that two members of the mitochondrial carrier family (MCF) in Arabidopsis (At5g48970 and At3g21390) and two in maize (GRMZM2G118515 and GRMZM2G124911) are related to the ThDP carriers of animals and Saccharomyces cerevisiae. Expression of each of these plant proteins in a S. cerevisiae ThDP carrier (TPC1) null mutant complemented the growth defect on fermentable carbon sources and restored the level of mitochondrial ThDP and the activity of the mitochondrial ThDP-dependent enzyme acetolactate synthase. The plant proteins were targeted to mitochondria as judged by dual import assays with purified pea mitochondria and chloroplasts, and by microscopic analysis of the subcellular localization of green fluorescent protein fusions in transiently transformed tobacco suspension cells. Both maize genes were shown to be expressed throughout the plant, which is consistent with the known ubiquity of mitochondrial ThDP-dependent enzymes. Collectively, these data establish that plants have mitochondrially located MCF carriers for ThDP, and indicate that these carriers are highly evolutionarily conserved. Our data provide a firm basis to propagate the functional annotation of mitochondrial ThDP carriers to other angiosperm genomes.
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http://dx.doi.org/10.1007/s10142-012-0273-4DOI Listing
June 2012

Impairment of methyl cycle affects mitochondrial methyl availability and glutathione level in Down's syndrome.

Mol Genet Metab 2011 Mar 9;102(3):378-82. Epub 2010 Dec 9.

Department of Chemistry, University of Basilicata, 85100 Potenza, Italy.

In Down's syndrome there is evidence that increased gene expression coding for specific cystathionine beta-synthase translates directly into biochemical aberrations, which result in a biochemical and metabolic imbalance of the methyl status. This event is destined to impact mitochondrial function since methylation is a necessary event in mitochondria and relies on the availability and uptake of the methyl donor S-adenosylmethionine. Indeed mitochondrial dysfunctions have been widely described in Down's syndrome, but they have never been correlated to a possible mitochondrial methyl unbalance. In the present study we find that the mitochondrial levels of S-adenosylmethionine are reduced in Down's syndrome compared to control cells demonstrating the effect of the methyl unbalance on mitochondria. The possible role of methylation in mitochondria is discussed and some preliminary results on a possible methylation target are presented.
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http://dx.doi.org/10.1016/j.ymgme.2010.11.166DOI Listing
March 2011