Publications by authors named "Furio Pezzetti"

81 Publications

Phenytoin and gingival mucosa: A molecular investigation.

Int J Immunopathol Pharmacol 2019 Jan-Dec;33:2058738419828259

Department of Medical Sciences, University of Ferrara, Ferrara, Italy.

Several distinct classes of drugs, such as anticonvulsants, immunosuppressants, and calcium channel blockers, caused gingival overgrowth. One of the main drugs associated with the gingival overgrowth is the anti-epileptic such as phenytoin, which affects gingival tissues by altering extracellular matrix metabolism. In our study, we evaluate the effect of phenytoin, a drug whose active substance is phenytoin, on gingival fibroblasts of healthy volunteers. Gene expression of 29 genes was investigated in gingival fibroblasts' cell culture treated with phenytoin compared with untreated cells. Among the studied genes, only 13 genes (CXCL5, CXCL10, CCR1, CCR3, CCR5, CCR6, IL-1A, IL-1B, IL-5, IL-7, IL-6R, BMP-2, and TNFSF-10) were statistically significant. All but one gene resulted downregulated after 24 h of treatment with phenytoin. BPM2 was the only, although weakly, up-expressed gene. Probably, we have not highlighted overexpression of the other inflammatory molecules because the study was performed on healthy people. Many studies show that phenytoin induces the overexpression of these cytokines but, probably, in our study, the drug does not have the same effect because we used gingival fibroblasts of healthy people.
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http://dx.doi.org/10.1177/2058738419828259DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6822187PMC
March 2020

Possible effect of SNAIL family transcriptional repressor 1 polymorphisms in non-syndromic cleft lip with or without cleft palate.

Clin Oral Investig 2018 Sep 27;22(7):2535-2541. Epub 2018 Jan 27.

Department of Experimental, Diagnostic and Specialty Medicine, University of Bologna, Via Belmeloro, 8, 40126, Bologna, Italy.

Objective: Orofacial development is a complex process subjected to failure impairing. Indeed, the cleft of the lip and/or of the palate is among the most frequent inborn malformations. The JARID2 gene has been suggested to be involved in non-syndromic cleft lip with or without cleft palate (nsCL/P) etiology. JARID2 interacts with the polycomb repressive complex 2 (PRC2) in regulating the expression patterns of developmental genes by modifying the chromatin state.

Materials And Methods: Genes coding for the PRC2 components, as well as other genes active in cell differentiation and embryonic development, were selected for a family-based association study to verify their involvement in nsCL/P. A total of 632 families from Italy and Asia participated to the study.

Results: Evidence of allelic association was found with polymorphisms of SNAI1; in particular, the rs16995010-G allele was undertransmitted to the nsCL/P cases [P = 0.004, odds ratio = 0.69 (95% C.I. 0.54-0.89)]. However, the adjusted significance value corrected for all the performed tests was P = 0.051.

Conclusions: The findings emerging by the present study suggest for the first time an involvement of SNAI1 in the nsCL/P onset.

Clinical Relevance: Interestingly, SNAI1 is known to promote epithelial to mesenchymal transition by repressing E-cadherin expression, but it needs an intact PRC2 to act this function. Alterations of this process could contribute to the complex etiology of nsCL/P.
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http://dx.doi.org/10.1007/s00784-018-2350-0DOI Listing
September 2018

Role of the MIR146A polymorphism in the origin and progression of oral squamous cell carcinoma.

Eur J Oral Sci 2014 Jun 10;122(3):198-201. Epub 2014 Mar 10.

Department of Experimental, Diagnostic and Specialty Medicine, University of Bologna, Bologna, Italy.

Gene expression and cell behavior are regulated by several factors, including small non-coding RNAs. MicroRNAs affecting cell growth, differentiation, and apoptosis are thought to play an important role in tumorigenesis. The levels of miR-146 appear to be associated with cancer development and progression, including that of oral squamous cell carcinoma. The aim of this investigation was to ascertain whether the single nucleotide polymorphism, rs2910164, mapping in the MIR146A gene, has a role in oral squamous cell carcinoma progression. A genetic association study was performed with a sample set of 346 oral squamous cell carcinomas collected in Italy. Our data indicate that the rs2910164 polymorphism is not associated with tumor development. However, a slight increase in the frequency of the variant allele was observed in Stage II tumors. Further investigations are needed to verify a possible role of the variant allele or rs2910164 in oral squamous cell carcinoma progression.
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http://dx.doi.org/10.1111/eos.12121DOI Listing
June 2014

Evaluation of gene expression in MG63 human osteoblastlike cells exposed to tantalum powder by microarray technology.

Int J Periodontics Restorative Dent 2011 Jul-Aug;31(4):e17-28

Orthopedic Clinic, University of Ferrara, Ferrara, Italy.

Conventional orthopedic implants are composed from titanium. To improve some characteristics (ie, volumetric porosity, modulus of elasticity, frictional modulus), a new porous tantalum biomaterial has been developed and its biocompatibility reported. By using DNA microarrays containing 20,000 genes, several genes whose expression were significantly up- or down-regulated were identified in an osteoblastlike cell line (MG63) cultured with tantalum powder (TP). The differentially expressed genes cover a broad range of functional activities: signaling transduction; transcription; cell cycle regulation, proliferation, and apoptosis; and cytoskeleton formation. To the authors' knowledge, the data reported represent the first genetic portrait of TP.
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February 2012

New evidence for the role of cystathionine beta-synthase in non-syndromic cleft lip with or without cleft palate.

Eur J Oral Sci 2011 Jun 5;119(3):193-7. Epub 2011 May 5.

Department of Histology, Embryology and Applied Biology, Centre of Molecular Genetics, CARISBO Foundation, University of Bologna, Italy.

Orofacial clefts have a multifactorial aetiology encompassing both genetic and environmental components. While there is wide agreement on the importance of both genetic and nutritional factors, genetic influence in particular has not been well defined. As genetic variants in folate and homocysteine metabolism have been reported to influence the risk of orofacial clefts, an Italian cleft lip with or without cleft palate (CL/P) data set was enrolled for an analysis based on family association to test betaine-homocysteine methyltransferase (BHMT and BHMT2) and cystathionine beta-synthase (CBS) variants. No significant level of association was found between BHMT and BHMT2 variants, while evidence of an allelic association with CL/P was found for the single nucleotide polymorphism rs4920037, mapping at the CBS gene. A log-linear approach indicated that the best genetic model takes into account both mother and child genotypes. This suggests that human orofacial development is influenced by CBS genotypes that possibly operate through intergenerational fetal-maternal interaction.
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http://dx.doi.org/10.1111/j.1600-0722.2011.00824.xDOI Listing
June 2011

No evidence of HAND2 involvement in nonsyndromic cleft lip with or without cleft palate.

Clin Oral Investig 2012 Apr 24;16(2):619-23. Epub 2011 Mar 24.

Department of Histology, Embryology and Applied Biology, Centre of Molecular Genetics, University of Bologna, Via Belmeloro 8, 40126 Bologna, Italy.

Craniofacial morphogenesis is determined by multistep processes involving signalling molecules and transcription factors, which are organised into highly coordinated pathways. Derailment from this intricate network can lead to congenital malformations. Cells migrate from neural crests to populate different structures, such as branchial arches, involved in embryonal orofacial development. The EDN1 pathway is involved in branchial arch development. Gene knockout and knockdown experiments on EDN1 or its downstream effector dHAND resulted in mice that were characterised by craniofacial defects and cleft palate. Our aim was to evaluate whether the transcription factor HAND2 could be implicated in non-syndromic cleft lip with or without cleft palate (CL/P) aetiology. A sample study composed of 39 multiplex Italian pedigrees was enrolled to test linkage between two microsatellite flanking HAND2 locus and CL/P. No evidence of linkage between HAND2 and CL/P was obtained. Indeed, formal levels of exclusion were obtained with different inheritance models. Investigation results did not support a role of HAND2 in CL/P aetiology. Nevertheless a minor contribute of the gene in clefting could not be ruled out.
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http://dx.doi.org/10.1007/s00784-011-0539-6DOI Listing
April 2012

Diazepam effects on non-syndromic cleft lip with or without palate: epidemiological studies, clinical findings, genes and extracellular matrix.

Expert Opin Drug Saf 2011 Jan 20;10(1):23-33. Epub 2010 Jul 20.

Department of Experimental Medicine and Biochemical Sciences, Section of Histology and Embryology, Faculty of Medicine, University of Perugia, via del Giochetto, Perugia, Italy.

Importance Of The Field: This review analyses international studies investigating the combined genetic and environmental causes of cleft lip with or without cleft palate (CL/P) and describes successes and limitations in identifying underlying genetic and environmental factors. CL/P, the most common congenital facial malformation, is a major public health burden in terms of medical costs and emotional stress to patients and families. Because genetic and environmental factors determine risk of occurrence, CL/P has a complex, multifactor aetiology.

Areas Covered In This Review: English language reports from 1980 to 2010 were searched for in Medline, PubMed, Science Citation Index, textbooks and review articles on drugs and pregnancy. Key words were diazepam or benzodiazepine(s) combined with cleft lip, cleft palate, oral malformations, prenatal exposure, GABA, gene expression and extracellular matrix.

What The Reader Will Gain: This review presents an updated assessment of the mutagenic and genotoxic effects of diazepam (DZ), one of the most commonly used benzodiazepines, on CL/P occurrence.

Take Home Message: Data are divergent; more studies are needed for an in-depth picture of the effects of DZ during gestation on the child's development, particularly on orofacial clefts.
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http://dx.doi.org/10.1517/14740338.2010.506478DOI Listing
January 2011

New insights in collagen turnover in orofacial cleft patients.

Cleft Palate Craniofac J 2010 Jul;47(4):393-9

Department of Human Morphology and Biomedical Sciences–Città Study, Extracellular Matrix Laboratory, University of Milan, Italy.

Objective: We aimed to characterize the fibroblast phenotype of patients by analyzing gene and protein expression of cleft lip and/or cleft palate fibroblasts in relation to collagen turnover and extracellular matrix remodeling.

Patients: Human palatal fibroblasts were obtained from three healthy subjects without cleft lip and/or cleft palate and from three subjects with nonsyndromic cleft lip and/or cleft palate. Collagen turnover-related gene and protein expression were analyzed by real-time polymerase chain reaction, Western and dot blots, and sodium dodecyl sulfate zymography.

Results: Cleft lip and/or cleft palate fibroblasts, compared with controls, displayed a down-regulation of collagens type I and III messenger RNA (p < .0001 and p < .001, respectively) but an opposite tendency to increase protein levels. Cleft lip and/or cleft palate cells had higher lysyl hydroxylase-2b messenger RNA levels expressed in relation to collagen type I messenger RNA, down-regulated matrix metalloproteinase-1, tissue inhibitor of matrix metalloproteinase-1, and Secreted Protein Acidic and Rich in Cysteine messenger RNA (p < .0001 and p < .01, respectively). Pro-matrix metalloproteinase-1 tended to decrease, and pro-matrix metalloproteinase-2 and -9 were down-regulated (p < .01, p < .05, respectively), as was Secreted Protein Acidic and Rich in Cysteine protein expression (p < .05).

Conclusions: Our results suggest that the cleft lip and/or cleft palate fibroblast phenotype is characterized by a tendency toward interstitial collagen deposition due to posttranslational modifications, such as decreased collagen degradation by matrix metalloproteinases and increased collagen cross-links. These findings may contribute to the knowledge of the cleft lip and/or cleft palate fibroblast phenotype and may be useful to the surgeon when considering the potential wound contraction and subsequent undesired scarring in cleft lip and/or cleft palate ocurring after the surgical closure of a cleft palate.
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http://dx.doi.org/10.1597/07-196.1DOI Listing
July 2010

Expression and association data strongly support JARID2 involvement in nonsyndromic cleft lip with or without cleft palate.

Hum Mutat 2010 Jul;31(7):794-800

Department of Histology, Embryology and Applied Biology, Centre of Molecular Genetics, University of Bologna, Via Belmeloro, 8, 40126 Bologna, Italy.

Nonsyndromic cleft lip with or without cleft palate (CL/P) affects approximately 1 in 1,000 births. Genetic studies have provided evidence for the role of several genes and candidate loci in clefting; however, conflicting results have frequently been obtained and much have to be done to unravel the complex genetics of CL/P. In the present investigation we have focused on the candidate region in 6p23, a region that have been found linked to CL/P in several investigations, in the attempt to find out the susceptibility gene provisionally named OFC1. Gene expression experiments in mice embryo of positional candidate genes revealed that JARID2 was highly and specifically expressed in epithelial cells in merging palatal shelves. A family-based linkage disequilibrium study confirmed the pivotal role of JARID2 in orofacial development and strongly supports a role for this gene in CL/P etiology (multiallelic haplotype test P=6 x 10(-5)). Understanding the molecular role of JARID2 within facial development may offer additional information to further unravel the complex genetics of CL/P.
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http://dx.doi.org/10.1002/humu.21266DOI Listing
July 2010

Effects of pulsed electromagnetic fields on human osteoblastlike cells (MG-63): a pilot study.

Clin Orthop Relat Res 2010 Aug 13;468(8):2260-77. Epub 2010 Apr 13.

Istituto di Clinica Ortopedica Università di Ferrara, Corso Giovecca 203, 44100 Ferrara, Italy.

Background: Although pulsed electromagnetic fields (PEMFs) are used to treat delayed unions and nonunions, their mechanisms of action are not completely clear. However, PEMFs are known to affect the expression of certain genes.

Questions/purposes: We asked (1) whether PEMFs affect gene expression in human osteoblastlike cells (MG63) in vitro, and (2) whether and to what extent stimulation by PEMFs induce cell proliferation and differentiation in MG-63 cultures.

Methods: We cultured two groups of MG63 cells. One group was treated with PEMFs for 18 hours whereas the second was maintained in the same culture condition without PEMFs (control). Gene expression was evaluated throughout cDNA microarray analysis containing 19,000 genes spanning a substantial fraction of the human genome.

Results: PEMFs induced the upregulation of important genes related to bone formation (HOXA10, AKT1), genes at the transductional level (CALM1, P2RX7), genes for cytoskeletal components (FN1, VCL), and collagenous (COL1A2) and noncollagenous (SPARC) matrix components. However, PEMF induced downregulation of genes related to the degradation of extracellular matrix (MMP-11, DUSP4).

Conclusions And Clinical Relevance: PEMFs appear to induce cell proliferation and differentiation. Furthermore, PEMFs promote extracellular matrix production and mineralization while decreasing matrix degradation and absorption. Our data suggest specific mechanisms of the observed clinical effect of PEMFs, and thus specific approaches for use in regenerative medicine.
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http://dx.doi.org/10.1007/s11999-010-1341-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2895828PMC
August 2010

Anorganic bovine bone (Bio-Oss) regulates miRNA of osteoblast-like cells.

Int J Periodontics Restorative Dent 2010 Feb;30(1):83-7

Institute of Histology, University of Bologna and Center of Molecular Genetics, CARISBO Foundation, Bologna, Italy.

Bio-Oss (Geistlich) is composed of an organic bovine bone and has been widely used in several bone regeneration procedures during oral surgery. However, how this biomaterial enhances osteoblast activity to promote bone formation is not completely understood. MicroRNAs (miRNAs) represent a class of small, functional, noncoding RNAs of 19 to 23 nucleotides that regulate the transcription of messenger RNAs (mRNAs) in proteins. In this study, the miRNA microarray technique was used to investigate translation regulation in an osteoblast-like cell line (MG63) exposed to Bio-Oss. Nine up-regulated miRNAs (mir-423, mir-492, mir-191, mir-23a, mir-377, mir-494, mir-214, mir-193b, mir-320) and 4 down-regulated miRNAs (mir-27a, mir-24, mir-188, let-7c) were identified. Because each miRNA regulates 100 mRNAs, only mRNAs related to bone formation were analyzed. The vast majority of detected mRNAs are down-regulated, including some homeobox genes (genes that regulate the morphogenesis of an entire segment of the body), such as noggin and EN1. An indirect positive effect was demonstrated on bone morphogenetic protein-4. To the authors' knowledge, the data reported here are the first on translation regulation in osteoblasts exposed to Bio-Oss. This study may be relevant in better understanding the molecular mechanism of bone regeneration and used as a potential tool for analyzing the combined use of cytokines.
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February 2010

PerioGlas® Acts on Human Stem Cells Isolated from Peripheral Blood.

Dent Res J (Isfahan) 2010 ;7(1):28-34

Assistant Professor, Orthopedic Clinic, University of Ferrara, Ferrara, Italy.

Background: PerioGlas® (PG) is an alloplastic material used for grafting periodontal osseous defects since 1995. In animal models, it has been proven that PG achieves histologically good repair of sur-gically created defects. In clinical trials, PG was effective as an adjunct to conventional surgery in the treatment of intrabony defects. Because the molecular events due to PG that are able to alter osteob-last activity to promote bone formation are poorly understood, we investigated the expression of os-teoblastic related genes in mesenchymal stem cells exposed to PG.

Methods: The expression levels of bone related genes like RUNX2, SP7, SPP1, COL1A1, COL3A1, BGLAP, ALPL, and FOSL1 and mesenchymal stem cells marker (CD105) were analyzed, using real time reverse transcription-polymerase chain reaction. Pearson's chi-square (χ(2)) test was used to detect markers with significant differences in gene expression.

Results: PG caused induction of osteoblast transcriptional factor (like RUNX2), bone related genes osteopontin (SPP1), osteocalcin (BGLAP) and alkaline phosphatase (ALPL). All had statistical sig-nificant P values (< 0.05).

Conclusion: PG has a differentiation effect on mesenchymal stem cells derived from peripheral blood. The obtained results can be relevant to better understanding of the molecular mechanism of bone regeneration and as a model for comparing other materials with similar clinical effects.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3065339PMC
July 2011

Human cleft lip and palate fibroblasts and normal nicotine-treated fibroblasts show altered in vitro expressions of genes related to molecular signaling pathways and extracellular matrix metabolism.

J Cell Physiol 2010 Mar;222(3):748-56

Department of Experimental Medicine and Biochemical Sciences, University of Perugia, via del Giochetto, 06100 Perugia, Italy.

Nonsyndromic cleft lip with or without cleft palate (CLP) is a frequent craniofacial malformation caused by both genetic and environmental factors. Maternal smoking during pregnancy is a known risk factor, due to the teratogenic role of nicotine. To assess and compare the impact of CLP and nicotine, we studied the quantitative expression of genes involved in signaling pathways and extracellular matrix (ECM) metabolism in human normal nicotine-treated (NicN) and CLP fibroblasts compared to normal control (CTRL) cells. Palatal fibroblast cultures from seven CLP children and seven age-matched CTRL subjects were established and subconfluent cells incubated for 24 h without (CTRL and CLP fibroblasts) or with (NicN fibroblasts) 0.6 mM nicotine. Gene expressions were analyzed by real-time quantitative PCR. For the first time, a regulated cholinergic signaling in our human fibroblasts in vitro was demonstrated. Members of TGF-beta, retinoic acid (RA), and GABA-ergic signaling systems were also differently regulated. Among the ECM genes, fibronectin, syndecan, integrin alpha2, and MMP13 genes were concordantly modulated, while integrin beta5, and decorin genes were discordantly modulated. Interestingly, nicotine treatment regulated gene expressions of CD44 and CLPTM1, two candidate genes for CLP. Our findings show a positive association between nicotine treatment and CLP phenotype. Results suggest that nicotine deranges normal palate development, which might contribute to the development of a CLP malformative phenotype, through the impairment of some important signaling systems and ECM composition.
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http://dx.doi.org/10.1002/jcp.22006DOI Listing
March 2010

A comparison between genetic portraits of normal osteoblasts and osteosarcoma cell lines.

Indian J Dent Res 2009 Jan-Mar;20(1):52-9

Institute of Histology, University of Bologna, Bologna, Italy.

Background: Osteosarcoma (OS) is the most frequent malignant bone tumor occurring in young patients in the first two decades of life. Metastases are the cause of 90% of cancer deaths for patients with OS. OS of the jaw is rare and aggressive malignancy constitutes approximately 5-13% of all cases of skeletal OS. Chemotherapy plus surgery are the first choice for treatment.

Aims: Because OS cell lines (OCLs) should share a common pathway with primary OS and new drugs are screened in in vitro systems, new insight about the genetic profiling of OCLs is of paramount importance to a better understanding of the molecular mechanism of this rare tumor and detecting a potential target for specific therapy.

Materials And Methods: The SAOS2 and TE85 cell lines were analysed using DNA microarrays containing 19,000 genes. Several genes in which expression was significantly differentially expressed in OCLs vs. normal osteoblast (NO) were detected.

Results: The differentially expressed genes cover a broad range of functional activities: (a) cell cycle regulation, (b) cell differentiation, (c) apoptosis, and (d) immunity.

Conclusion: The reported data can be relevant to a better understanding of the biology of OS and as a model for comparing the effect of drugs used in OS treatment.
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http://dx.doi.org/10.4103/0970-9290.49069DOI Listing
July 2009

Diphenylhydantoin plays a role in gene expression related to cytoskeleton and protein adhesion in human normal palate fibroblasts.

Pathology 2009 ;41(3):261-8

Histology, Embryology and Applied Biology Department, University of Bologna, Bologna, Italy.

Aims: Morphogenetic processes during palate development are related to extracellular matrix composition. The cell-extracellular matrix relation plays a role in cell activity and in gene expression. We studied the effect of diphenylhydantoin, a teratogen known to induce cleft palate in human newborns, on extracellular matrix production. We investigated whether diphenylhydantoin treatment caused any differences in glycosaminoglycans, collagen synthesis and gene expression in human normal palate fibroblasts.

Methods: Human palate fibroblasts were maintained for 24 hours in serum-free 199 medium containing 5 microg/mL (3)H-glucosamine or (3)H proline hydrochloride. Collagen and glycosaminoglycan classes were then measured using biochemical methods, gene expression with microarray analysis and cytoskeleton components with immunofluorescent antibodies and computer analysis.

Results: In normal fibroblasts diphenylhydantoin reduced collagen and glycosaminoglycan synthesis with a marked effect on sulphated glycosaminoglycans. There were also substantial decreases in tubulin, vimentin and alpha-actin staining and an increase of vinculin compared to controls. Diphenylhydantoin acted on several genes related to the synthesis of cytoskeleton and adhesion membrane proteins. It inhibited caderin, caveolin, RTK and alpha-actin, and increased nectin, cytoplasmatic FRG vinculin, ITGA, ITGB extracellular matrix ligand and EDG2 gene expression. DNA binding gene expression, which plays a role in cell growth and senescence, was activated.

Conclusions: Since cell activity is dependent on the cell morphology and extracellular matrix composition, these findings indicate that in human normal palate fibroblasts diphenylhydantoin can modify cytoskeletal components and extracellular matrix-cell adhesion, with consequent effects on gene expression. These changes might be related to anomalous palate development.
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http://dx.doi.org/10.1080/00313020902756899DOI Listing
August 2009

Gene expression, cytoskeletal changes and extracellular matrix synthesis in human osteoblasts treated with cyclosporin A.

Biomed Pharmacother 2009 Nov 27;63(9):619-26. Epub 2008 Dec 27.

Department of Human Morphology, University of Milan, Milan, Italy.

Cyclosporin A (CyA) is an immunosuppressive agent used to prevent allograft rejection, but unfortunately it causes adverse effects such as bone diseases, osteoporosis and osteomalacia. These pathologies involve an imbalance between synthesis, degradation and mineralization of extracellular matrix. CyA can modify extracellular matrix components such as glycosaminoglycans (GAG) and collagen fibers. In addition, normal cell activity is dependent on cell morphology and substrate cell attachment. We treated normal human osteoblasts with CyA and analyzed: (i) gene expression by a microarray method; (ii) extracellular GAG and collagen after (3)H-glucosamine and Western blot analysis; and (iii) cytoskeletal changes, using actin and tubulin fluorescent antibodies. CyA increased intra- and extracellular GAG and extracellular GAG classes such as hyaluronic acid, chondroitin sulphate, and dermatan sulphate; there was no noteworthy effect on heparan sulphate and the ratio of non-sulphated to sulphated GAG. In osteoblast cultures the drug reduced cytoskeletal actin, while tubulin did not change. In vivo the osteoblasts showed morphological changes with different extracellular matrix synthesis. Microarray analysis indicated the inhibition of gene pathways related to Wnt signaling molecules, and the cytoskeletal and focal adhesion cascade. In in vitro human osteoblasts CyA modified gene expression related to cytoskeletal pattern organization and cell morphology. Since in bone pathologies osteoblasts show different morphology related to cell size, these data suggest that in vivo osteoblast different functions could be dependent on alteration of osteoblast differentiation.
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http://dx.doi.org/10.1016/j.biopha.2008.12.001DOI Listing
November 2009

Patterns of some extracellular matrix gene expression are similar in cells from cleft lip-palate patients and in human palatal fibroblasts exposed to diazepam in culture.

Toxicology 2009 Mar 9;257(1-2):10-6. Epub 2008 Dec 9.

Department of Experimental Medicine and Biochemical Science, University of Perugia, Italy.

Prenatal exposure to diazepam, a prototype sedative drug that belongs to Benzodiazepines, can lead to orofacial clefting in human newborns. By using real-time PCR, in the present study we investigated whether diazepam elicits gene expression alterations in extracellular matrix (ECM) components, growth factors and gamma-aminobutyric acid receptor (GABRB3), implicated in the coordinate regulation of palate development. Palate fibroblasts were treated with diazepam (Dz-N fibroblasts) and compared to cleft lip-palate (CLP) fibroblasts obtained from patients with no known exposure to diazepam or other teratogens. Untreated fibroblasts from non-CLP patients were used as control. The results showed significant convergences in gene expression pattern of collagens, fibromodulin, vitronectin, tenascin C, integrins and metalloprotease MMP13 between Dz-N and CLP fibroblasts. Among the growth factors, constitutive Fibroblast Growth Factor 2 (FGF2) was greatly enhanced in Dz-N and CLP fibroblasts and associated with a higher reduction of FGF receptor. Transforming Growth Factor beta 3 (TGFbeta(3)) resulted up-regulated in CLP fibroblasts and decreased in Dz-N fibroblasts. We found phenotypic differences exhibited by Dz-N and CLP fibroblasts in GABRB3 gene regulation, so further studies are necessary to determine whether GABAergic system could be involved in the development of diazepam mediated CLP phenotype. Taken together the results elucidate the molecular mechanisms underlying possible toxicology effects induced by diazepam. Counselling of women on the safety of diazepam exposure is clinically important, also for the forensic consequences.
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http://dx.doi.org/10.1016/j.tox.2008.12.002DOI Listing
March 2009

Genes causing clefting syndromes as candidates for non-syndromic cleft lip with or without cleft palate: a family-based association study.

Eur J Oral Sci 2008 Dec;116(6):507-11

Department of Histology, Embryology and Applied Biology, Centre of Molecular Genetics, University of Bologna, Bologna, Italy.

Clefts of the orofacial region are among the most common congenital defects, caused by abnormal facial development during gestation. Non-syndromic cleft lip with or without cleft palate (NSCLP) is a complex trait most probably caused by multiple interacting loci, with possible additional environmental factors. As facial clefts form part of more than 300 syndromes, one strategy for identifying the genetic causes of NSCLP could be to study candidate genes responsible for clefting syndromes. Three genes were selected for this investigation: TP63, which codes for the tumour protein p63 and causes Ectrodactyly-Ectodermal dysplasia-orofacial Cleft syndrome; JAG2, a downstream gene of TP63; and MID1, which is responsible for Opitz syndrome. A linkage disequilibrium investigation was performed with intragenic single nucleotide polymorphisms on each of these genes in a sample study of 239 patients/parents trios. Evidence which suggests that JAG2 and MID1 may play a role in NSCLP was obtained.
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http://dx.doi.org/10.1111/j.1600-0722.2008.00574.xDOI Listing
December 2008

Anorganic bovine bone and a silicate-based synthetic bone activate different microRNAs.

J Oral Sci 2008 Sep;50(3):301-7

Department of Histology, Embryology and Applied Biology, University of Bologna and Center of Molecular Genetics, CARISBO Foundation, Bologna, Italy.

Bio-Oss (BO), composed of anorganic bovine bone, is widely used in several bone regeneration procedures in oral surgery. PerioGlas (PG) is an alloplastic material that has been used for grafting of periodontal osseous defects since the 1990s. However, how these biomaterials alter osteoblast activity to promote bone formation is poorly understood. We attempted to address this question by using microRNA microarray techniques to investigate differences in translational regulation in osteoblasts exposed to BO and PG. By using miRNA microarrays containing 329 probes designed from human miRNA sequences, we investigated miRNAs whose expression was significantly modified in an osteoblast-like cell line (MG-63) cultured with BO vs PG. Three up-regulated miRNAs (mir-337, mir-200b, mir-377) and 4 down-regulated miRNAs (mir-130a, mir-214, mir-27a, mir-93) were identified. Our results indicated that BO and PG act on different miRNAs. Globally, PG causes activation of bone-forming signaling, whereas BO also activates cartilage-related pathways.
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http://dx.doi.org/10.2334/josnusd.50.301DOI Listing
September 2008

Titanium acts on osteoblast translational process.

J Oral Implantol 2008 ;34(4):190-5

Institute of Histology, University of Bologna and Center of Molecular Genetics, Cassa di Risparmio di Bologna Foundation, Bologna, Italy.

Titanium is a highly biocompatible material and very osteogenic in vivo. However, how titanium regulates osteoblast activity to promote bone formation is incompletely characterized. We, therefore, attempted to get more information by using microRNA (miRNA) microarray techniques to investigate translation regulation in osteoblasts grown on titanium disks. The miRNA oligonucleotide microarray provides a novel method to carry out genome-wide miRNA profiling in human samples. By using miRNA microarrays containing 329 probes designed from the human miRNA sequence, several miRNA were identified in osteoblast-like cell line (MG 63) grown on titanium disks. There were 13 upregulated miRNAs (ie, mir-23a, mir-222, mir-523, mir-22, mir-23b, mir-143, mir-377, mir-24, mir-422b, mir-26a, mir-29a, mir-17-5p, mir-182) and 2 down-regulated miRNAs (ie, mir-187, mir-339). The data reported are, to our knowledge, the first study on translation regulation in osteoblasts exposed to titanium. The data can be relevant to understand better the molecular mechanism of osteoblast activation and as a model for comparing other materials with similar clinical effects.
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http://dx.doi.org/10.1563/0.869.1DOI Listing
November 2008

Displayed correlation between gene expression profiles and submicroscopic alterations in response to cetuximab, gefitinib and EGF in human colon cancer cell lines.

BMC Cancer 2008 Aug 8;8:227. Epub 2008 Aug 8.

Dipartimento di Istologia, Embriologia e Biologia Applicata, Università di Bologna, Via Belmeloro 8, 40126 Bologna, Italy.

Background: EGFR is frequently overexpressed in colon cancer. We characterized HT-29 and Caco-2, human colon cancer cell lines, untreated and treated with cetuximab or gefitinib alone and in combination with EGF.

Methods: Cell growth was determined using a variation on the MTT assay. Cell-cycle analysis was conducted by flow cytometry. Immunohistochemistry was performed to evaluate EGFR expression and scanning electron microscopy (SEM) evidenced the ultrastructural morphology. Gene expression profiling was performed using hybridization of the microarray Ocimum Pan Human 40 K array A.

Results: Caco-2 and HT-29 were respectively 66.25 and 59.24 % in G0/G1. They maintained this level of cell cycle distribution after treatment, suggesting a predominantly differentiated state. Treatment of Caco-2 with EGF or the two EGFR inhibitors produced a significant reduction in their viability. SEM clearly showed morphological cellular transformations in the direction of cellular death in both cell lines treated with EGFR inhibitors. HT-29 and Caco-2 displayed an important reduction of the microvilli (which also lose their erect position in Caco-2), possibly invalidating microvilli absorption function. HT-29 treated with cetuximab lost their boundary contacts and showed filipodi; when treated with gefitinib, they showed some vesicles: generally membrane reshaping is evident. Both cell lines showed a similar behavior in terms of on/off switched genes upon treatment with cetuximab. The gefitinib global gene expression pattern was different for the 2 cell lines; gefitinib treatment induced more changes, but directly correlated with EGF treatment. In cetuximab or gefitinib plus EGF treatments there was possible summation of the morphological effects: cells seemed more weakly affected by the transformation towards apoptosis. The genes appeared to be less stimulated than for single drug cases.

Conclusion: This is the first study to have systematically investigated the effect of cetuximab or gefitinib, alone and in combination with EGF, on human colon cancer cell lines. The EGFR inhibitors have a weaker effect in the presence of EGF that binds EGFR. Cetuximab treatment showed an expression pattern that inversely correlates with EGF treatment. We found interesting cyto-morphological features closely relating to gene expression profile. Both drugs have an effect on differentiation towards cellular death.
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http://dx.doi.org/10.1186/1471-2407-8-227DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2528013PMC
August 2008

PerioGlas regulates osteoblast RNA interfering.

J Prosthodont 2008 Oct 20;17(7):522-6. Epub 2008 Jun 20.

Institute of Histology, University of Bologna and Center of Molecular Genetics, CARISBO Foundation, Bologna, Italy.

Purpose: PerioGlas (PG) is an alloplastic material that has been used for grafting periodontal osseous defects since the 1990s. In animal models, it has been proven that PG achieves histologically good repairs of surgically created defects. In clinical trials, PG is effective as an adjunct to conventional surgery in the treatment of intrabony defects; however, how PG alters osteoblast activity to promote bone formation is poorly understood. We therefore attempted to address this question by using microRNA (miRNA) microarray techniques to investigate the translation process in osteoblasts exposed to PG.

Materials And Methods: By using miRNA microarrays containing 329 probes designed from human miRNA sequences, we identified several miRNA whose expression was significantly modified in osteoblast-like cell lines (MG-63) cultured with PG.

Results: There were ten up-regulated miRNA (mir-337, mir-377, mir-9, mir-516, mir-515-3p, mir-496, mir-200b, mir-489, mir-25, mir-423) and two down-regulated miRNA (mir-26a, mir-30d).

Conclusion: PG acts on miRNAs, which in turn regulate several messengers. Among them there are mRNAs related to bone formation and skeletal and cartilage development. The vast majority of detected genes are down-regulated, and some are homeobox genes like NOG, EN1, and CHRD. Other down-regulated genes are receptors (like GHRHR) and extracellular matrix proteins (like COMP). Although the exact mechanism of PG action on osteoblasts is still incompletely understood, these data demonstrate that PG has not only an osteoconductive effect, but also regulates bone formation.
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http://dx.doi.org/10.1111/j.1532-849X.2008.00331.xDOI Listing
October 2008

Comparison between genetic portraits of osteoblasts derived from primary cultures and osteoblasts obtained from human pulpar stem cells.

J Craniofac Surg 2008 May;19(3):616-25; discussion 626-7

Department of DMCCC, Section of Maxillofacial Surgery, University of Ferrara, Ferrara, Italy.

Harvesting bone for autologous grafting is a daily problem encountered by craniofacial and oral surgeons. Stem cells derived from human dental pulp are able to differentiate in osteoblasts and are a potential source of autologous bone produced in vitro. However, as stem cells are characterized by self-renewing and commitment in several cellular subtypes (ie, pluripotential differentiation), some concerns may arise as regards their potential uncontrolled proliferation. To screen the behavior of osteoblasts derived from human pulpar stem cells (ODHPSCs), we used microarray techniques to identify genes that are differently regulated in ODHPSC in comparison to normal osteoblasts (NOs). Osteoblasts derived from human pulpar stem cells were obtained from human dental pulp, and cells were selected using a cytometer. The cell profile was c-kit+/CD34+/STRO-1+/CD45-. These cells were capable of differentiation of osteoblasts in vitro. By using DNA microarrays containing 19,200 genes, we identified in ODHPSC some genes whose expression was significantly up- and downregulated compared to NO. The differentially expressed genes have different functional activities: (a) cell differentiation, (b) developmental maturation, (c) cell adhesion, and (d) production of cytoskeleton elements. Thus, some molecular differences exist between NO and ODHPSC, although the previously considered histologic parameters show a normal phenotype.
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http://dx.doi.org/10.1097/SCS.0b013e31816aabc8DOI Listing
May 2008

Investigation of MYH14 as a candidate gene in cleft lip with or without cleft palate.

Eur J Oral Sci 2008 Jun;116(3):287-90

Department of Histology, Embryology and Applied Biology, Centre of Molecular Genetics CARISBO Foundation, University of Bologna, Via Belmeloro, Bologna, Italy.

Clefts of the orofacial region are among the most common facial defects and are caused by abnormal facial development during gestation. Cleft lip with or without cleft palate (CL/P) is a birth defect with a complex etiology resulting from a mixture of genetic and environmental factors. In the present study we considered myosin 14 (MYH14) as a candidate gene for CL/P. This gene codes for the heavy chain of non-muscle myosin IIC (NMMHC-IIC), maps in the OFC3 region, and shares significant homology with myosin 9, a gene that our group has recently seen to be involved in CL/P. A linkage disequilibrium investigation was conducted with six single nucleotide polymorphisms in MYH14 and a sample of 239 CL/P nonsyndromic patients and their parents. Our family-based investigation provided no evidence of association between MYH14 and CL/P alleles. These data do not support the involvement of MYH14 in CL/P among the Italian population.
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http://dx.doi.org/10.1111/j.1600-0722.2008.00534.xDOI Listing
June 2008

Medpor regulates osteoblast's microRNAs.

Biomed Mater Eng 2008 ;18(2):91-7

Institute of Histology, University of Bologna and Center of Molecular Genetics, CARISBO Foundation, Bologna, Italy.

Porous polyethylene (PP or Medpor) is an alloplastic material worldwide used for craniofacial reconstruction. Although several clinical studies are available, there is a lack as regard the genetic effects. Because PP is always fixed on bone and the mechanism by which PP acts on osteoblasts is unknown, we therefore attempted to address this question by using microRNA microarray techniques to investigate the translation regulation in osteoblasts exposed to PP. The miRNA oligonucleotide microarray provides a novel method to carry out genome-wide microRNA profiling in human samples. By using miRNA microarrays containing 329 probe designed from Human miRNA sequence, we identified in osteoblast-like cells line (MG-63) cultured with Medpor (Porex Corporation, Fairburn, Georgia, USA) several miRNA which expression is significantly modified. We identified 16 up-regulated miRNA (i.e. mir-337, mir-515-3p, mir-377, mir-153, mir-367, mir-152, let-7b, mir-92, mir-155, mir-424, mir-148b, mir-368, mir-18b, mir-520d, mir-20b, mir-128a) and 2 down-regulated miRNA (i.e. mir-143, mir-32). The data reported are, to our knowledge, the first study on translation regulation in osteoblasts exposed to PP. They can be relevant to better understand the molecular mechanism of bone regeneration and as a model for comparing other materials with similar clinical effects.
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July 2008

Comparison between osteoblasts derived from human dental pulp stem cells and osteosarcoma cell lines.

Cell Biol Int 2008 Jul 4;32(7):733-8. Epub 2008 Mar 4.

Centre of Molecular Genetics, CARISBO Foundation, Institute of Histology and General Embryology, School of Medicine, University of Bologna, Bologna, Italy.

Stem cells derived from human dental pulp are able to differentiate into osteoblasts and are a potential source of autologous bone. The aim of this study was to compare genes differentially expressed in osteoblastoids from human dental pulp (OHDP) to osteosarcoma cells (OCs). Human dental pulp was extracted and immersed in a digestive solution. Cells were cultured and selected using c-kit, CD34, CD45 and STRO-1 antibodies. In parallel, two OCs (i.e., SAOS2 and TE85) were cultured. RNA was extracted from different populations of cells and cDNA was used for the hybridisation of human 19.2K DNA microarrays. We identified several differences in gene expression between OHDP and OCs. Some down-regulated OHDP genes, such as RUNX1, MAP4K4 and PRDM2, are involved in bone development, cell motility and transcript regulation. Gene expression in OHDP is significantly different from that in OCs, suggesting differences in cell function and activity between these cells.
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http://dx.doi.org/10.1016/j.cellbi.2008.02.003DOI Listing
July 2008

Anatase nanosurface regulates microRNAs.

J Craniofac Surg 2008 Mar;19(2):328-33

Institute of Histology, University of Bologna and Center of Molecular Genetics, CARISBO Foundation, Bologna, Italy.

Titanium is the criterion standard among materials used for prosthetic devices because of its good mechanical and chemical properties. When exposed to oxygen, titanium becomes an oxide that is biocompatible and able to induce osseointegration. There are 3 allotropic forms of titanium dioxide: brookite, rutile, and anatase. Anatase can be prepared as a colloidal suspension and then used to coat surfaces. Anatase coating (AC) can potentially have specific biologic effects and specifically induce bone formation. To get more information as regards the osteogenic effect of AC, we used microRNA (miRNA) microarray techniques to investigate translation regulation in osteoblasts exposed to AC. Transduction, transcription, and translation are the 3 levels of regulation of cell activity. Recently, a new type of translation regulation has been identified: RNA interference. RNA interference is a process in which miRNA (i.e., noncoding RNAs of 19-23 nucleotides) can induce sequence-specific mRNA degradation and/or translational repression. The human genome encodes a few hundred miRNAs that can posttranscriptionally repress thousands of genes. miRNA oligonucleotide microarray provides a novel method of carrying out genome-wide miRNA profiling in human samples. By using miRNA microarrays containing 329 probes designed from human miRNA sequences, we identified in osteoblast-like cell line (MG-63) cultured with AC several miRNA whose expression had been significantly modified. The data reported constitute, to our knowledge, the first study on translation regulation in osteoblasts exposed to AC. They can be relevant to a better understanding of the molecular mechanism of bone regeneration and as a model for comparing other materials with similar clinical effects.
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http://dx.doi.org/10.1097/SCS.0b013e3181534ab3DOI Listing
March 2008

Peptide-15 changes miRNA expression in osteoblast-like cells.

Implant Dent 2008 Mar;17(1):100-8

Institute of Histology, University of Bologna and Center of Molecular Genetics, CARISBO Foundation, Bologna, Italy.

Purpose: Peptide-15 (P-15) is an analog of the cell-binding domain of collagen. P-15 has been shown to facilitate physiological process in a way similar to collagen, to serve as anchorage for cells, and to promote the binding, migration, and differentiation of cells. However, how P-15 alters osteoblast activity to promote bone formation is poorly understood. We therefore attempted to address this question by using microarray techniques to investigate the microRNA (miRNA) expression in osteoblasts exposed to P-15.

Materials: The miRNA oligonucleotide microarray provides a novel method to carry out genome-wide miRNA profiling in human samples. By using miRNA microarrays containing 329 probe designed from human miRNA sequence, we identified in osteoblast-like cells line (MG-63) cul-tured with P-15 several miRNA whose expression is significantly modified.

Results: We identified 11 up-regulated miRNA (i.e., mir-337, mir-15b, mir-377, mir-100, mir-148a, mir-125a, mir-199a, mir-221, mir-let-7d, mir-92, mir-23b) and six down-regulated miRNA (i.e., mir-422a, mir-19a, mir-224, mir-145, mir-22, mir-29a).

Conclusion: The data reported are, to our knowledge, the first on translation regulation in osteoblasts exposed to P-15. They can be relevant to better understand the molecular mechanism of bone regeneration and can serve as a model for comparing other materials with similar clinical effects.
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http://dx.doi.org/10.1097/ID.0b013e318166d182DOI Listing
March 2008

Zirconium oxide regulates RNA interfering of osteoblast-like cells.

J Mater Sci Mater Med 2008 Jun 6;19(6):2471-6. Epub 2008 Feb 6.

Institute of Histology, University of Bologna and Center of Molecular Genetics, CARISBO Foundation, Bologna, Italy.

Zirconium oxide (ZO) has outstanding mechanical properties, high biocompatibility and high resistance to scratching. Since dental implants are made with ZO and the genetic effects of ZO on osteoblasts are incompletely understood, we used microRNA microarray techniques to investigate the translation process in osteoblasts exposed to ZO. By using miRNA microarrays containing 329 probes designed from Human miRNA sequences, we identified in osteoblast-like cells line (MG-63) cultured on ZO disks several miRNA whose expression was significantly modified. The most notable regulated genes acting on osteoblasts are: NOG, SHOX, IGF1, BMP1 and FGFR1. The data reported below represent the first study on translation regulation in osteoblasts exposed to zirconium and one in which the effect of ZO on bone formation has been detected.
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http://dx.doi.org/10.1007/s10856-008-3386-5DOI Listing
June 2008

Short-period effects of zirconia and titanium on osteoblast microRNAs.

Clin Implant Dent Relat Res 2008 Sep 30;10(3):200-5. Epub 2008 Jan 30.

Institute of Histology, University of Bologna and Center of Molecular Genetics, CARISBO Foundation, Bologna, Italy.

Background: MicroRNAs (miRNAs) are a class of small, functional, noncoding RNAs of 19 to 23 nucleotides which induce degradation of specific messenger RNAs (mRNAs), thus controlling the translational process (ie, synthesis of proteins from mRNAs). In addition, mRNAs regulate the promoter of specific miRNAs activating an autoregulatory feedback loop.

Purpose: Titanium and zirconium dioxide ceramics (ZDCs) are used to make dental implants. Because the molecular mechanism by which ZDC and Ti act on osteoblasts is incompletely understood, we attempted to get more information by comparing the effect of ZDC and Ti on osteoblast miRNAs.

Materials And Methods: By using miRNA microarray technique, we identified in osteoblast-like cell line (MG63) grown on grade 3 Ti and ZDC disks several miRNAs whose expression was modified. We collected mRNAs after 24 hours of cell culturing to better understand molecular events related to early bone healing around inserted implants. An mRNA microarray technique was then performed as a control.

Results: There were six up- and four down-regulated miRNAs. Because every miRNA regulates hundreds of genes, we focused only on those related to bone formation. Among them, the most notable are BMP4 and 7, which are both up-regulated in osteoblasts cultured on Ti disks.

Conclusion: The detected miRNAs differentially expressed in osteoblast-like cells grown on ZDC versus Ti act on a limited number of miRNAs and bone-related genes. The most notable are BMP4 and 7, which are more expressed in osteoblasts exposed to Ti surface. Consequently, we suggest that Ti surfaces could provide some advantages to immediate load implantology.
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http://dx.doi.org/10.1111/j.1708-8208.2007.00078.xDOI Listing
September 2008