Publications by authors named "Claudio Rivetti"

37 Publications

Actin-Resistant DNase1L2 as a Potential Therapeutics for CF Lung Disease.

Biomolecules 2021 Mar 10;11(3). Epub 2021 Mar 10.

Department of Chemistry, Life Sciences and Environmental Sustainability, University of Parma, 43124 Parma, Italy.

In cystic fibrosis (CF), the accumulation of viscous lung secretions rich in DNA and actin is a major cause of chronic inflammation and recurrent infections leading to airway obstruction. Mucolytic therapy based on recombinant human DNase1 reduces CF mucus viscosity and promotes airway clearance. However, the marked susceptibility to actin inhibition of this enzyme prompts the research of alternative treatments that could overcome this limitation. Within the human DNase repertoire, DNase1L2 is ideally suited for this purpose because it exhibits metal-dependent endonuclease activity on plasmid DNA in a broad range of pH with acidic optimum and is minimally inhibited by actin. When tested on CF artificial mucus enriched with actin, submicromolar concentrations of DNase1L2 reduces mucus viscosity by 50% in a few seconds. Inspection of superimposed model structures of DNase1 and DNase1L2 highlights differences at the actin-binding interface that justify the increased resistance of DNase1L2 toward actin inhibition. Furthermore, a PEGylated form of the enzyme with preserved enzymatic activity was obtained, showing interesting results in terms of activity. This work represents an effort toward the exploitation of natural DNase variants as promising alternatives to DNase1 for the treatment of CF lung disease.
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http://dx.doi.org/10.3390/biom11030410DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8002113PMC
March 2021

In vitro characterization and in vivo comparison of the pulmonary outcomes of Poractant alfa and Calsurf in ventilated preterm rabbits.

PLoS One 2020 13;15(3):e0230229. Epub 2020 Mar 13.

Departments of Pediatrics and Neonatology, Children's Hospital of Fudan University, Shanghai, China.

Poractant alfa and Calsurf are two natural surfactants widely used in China for the treatment of neonatal respiratory distress syndrome, which are extracted from porcine and calf lungs, respectively. The purpose of this experimental study was to compare their in vitro characteristics and in vivo effects in the improvement of pulmonary function and protection of lung injury. The biophysical properties, ultrastructure, and lipid composition of both surfactant preparations were respectively analysed in vitro by means of Langmuir-Blodgett trough (LBT), atomic force microscopy (AFM), and liquid-chromatography mass-spectrometry (LC-MS). Then, as core pharmacological activity, both head-to-head (100 and 200 mg/kg for both surfactants) and licensed dose comparisons (70 mg/kg Calsurf vs. 200 mg/kg Poractant alfa) between the two surfactants were conducted as prophylaxis in preterm rabbits with primary surfactant deficiency, assessing survival time and rate and dynamic compliance of the respiratory system (Cdyn). Intrapulmonary surfactant pools, morphometric volume density as alveolar expansion (Vv), and lung injury scores were determined post mortem. AFM and LC-MS analysis revealed qualitative differences in the ultrastructure as well as in the lipid composition of both preparations. Calsurf showed a longer plateau region of the LBT isotherm and lower film compressibility. In vivo, both surfactant preparations improved Cdyn at any dose, although maximum benefits in terms of Vv and intrapulmonary surfactant pools were seen with the 200 mg/kg dose in both surfactants. The group of animals treated with 200 mg/kg of Poractant alfa showed a prolonged survival time and rate compared to untreated but ventilated controls, and significantly ameliorated lung injury compared to Calsurf at any dose, including 200 mg/kg. The overall outcomes suggest the pulmonary effects to be dose dependent for both preparations. The group of animals treated with 200 mg/kg of Poractant alfa showed a significant reduction of mortality. Compared to Calsurf, Poractant alfa exerted better effects if licensed doses were compared, which requires further investigation.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0230229PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7069639PMC
June 2020

Functional characterization of the type I toxin Lpt from Lactobacillus rhamnosus by fluorescence and atomic force microscopy.

Sci Rep 2019 10 23;9(1):15208. Epub 2019 Oct 23.

Department of Food and Drug, University of Parma, 43124, Parma, Italy.

Lpt is a 29 amino acid long type I toxin identified in the plasmid DNA of wild Lactobacillus rhamnosus strains isolated from food. We previously reported that transcription of the encoding gene was upregulated under nutritional starvation conditions mimicking cheese ripening environment. The heterologous expression of the Lpt peptide in E. coli resulted in cell growth inhibition, nucleoid condensation and compromised integrity of the cell membrane. Fusion of the Lpt peptide with the fluorescent protein mCherry allowed to visualize the accumulation of the peptide into the membrane, while mutagenesis experiments showed that either the insertion of a negatively charged amino acid into the hydrophobic α-helix or deletion of the hydrophilic C-terminal region, leads to a non-toxic peptide. AFM imaging of Lpt expressing E. coli cells has revealed the presence of surface defects that are compatible with the loss of portions of the outer membrane bilayer. This observation provides support for the so-called "carpet" model, by which the Lpt peptide is supposed to destabilize the phospholipid packing through a detergent-like mechanism leading to the removal of small patches of bilayer through micellization.
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http://dx.doi.org/10.1038/s41598-019-51523-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6811638PMC
October 2019

Cytotoxic activity of copper(ii), nickel(ii) and platinum(ii) thiosemicarbazone derivatives: interaction with DNA and the H2A histone peptide.

Metallomics 2019 10;11(10):1729-1742

Department of Chemistry, Life Sciences and Environmental Sustainability, University of Parma, Parco Area delle Scienze 11/a, 43124 Parma, Italy.

Metal complexes still represent promising pharmacological tools in the development of new anticancer drugs. Bis(citronellalthiosemicarbazonate)nickel(ii) is a metal compound extremely effective against leukemic and NCS cancer cell lines. Preliminary experiments performed with this compound and with its Cu(ii) and Pt(ii) analogues evidenced alterations, detectable by comet assay, in the DNA of treated U937 cells. In addition, [Cu(tcitr)2] and [Pt(tcitr)2] were also able to induce gene mutations and produce frameshift events. To gain further insights into the mechanism of action of these metal compounds, we carried out a multidisciplinary study to investigate whether their biological activity can be ascribed to the direct interaction with DNA or with chromatin. The DNA interaction was investigated by means of CD and UV-Vis spectroscopic techniques and by AFM, whereas the chromatin interaction was studied by analyzing the effects of the compounds on the structure of a peptide that mimicks the potential metal binding site in the "C-tail" region of histone H2A by means of NMR, CD, UV-Vis and MS. The intensities of the effects induced by the metal compounds on the peptide follow the order [Ni(tcitr)2] > [Pt(tcitr)2] ≫ [Cu(tcitr)2]. From the AFM data, a remarkable DNA compaction was observed in the presence of [Pt(tcitr)2], while [Ni(tcitr)2] causes the formation of large interlaced DNA aggregates.
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http://dx.doi.org/10.1039/c9mt00166bDOI Listing
October 2019

Identification and first characterization of DinJ-YafQ toxin-antitoxin systems in Lactobacillus species of biotechnological interest.

Sci Rep 2019 05 21;9(1):7645. Epub 2019 May 21.

Department of Food and Drug, University of Parma, 43124, Parma, Italy.

DinJ-YafQ is a type II TA system comprising the ribosome-dependent RNase YafQ toxin and the DinJ antitoxin protein. Although the module has been extensively characterized in Escherichia coli, little information is available for homologous systems in lactic acid bacteria. In this study, we employed bioinformatics tools to identify DinJ-YafQ systems in Lactobacillus casei, Lactobacillus paracasei and Lactobacillus rhamnosus species, commonly used in biotechnological processes. Among a total of nineteen systems found, two TA modules from Lactobacillus paracasei and two modules from Lactobacillus rhamnosus wild strains were isolated and their activity was verified by growth assays in Escherichia coli either in liquid and solid media. The RNase activity of the YafQ toxins was verified in vivo by probing mRNA dynamics and metabolism with single-cell Thioflavin T fluorescence. Our findings demonstrate that, albeit DinJ-YafQ TA systems are widely distributed in lactic acid bacteria, only few are fully functional, while others have lost toxicity even though they maintain high sequence identity with wild type YafQ and a likely functional antitoxin protein.
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http://dx.doi.org/10.1038/s41598-019-44094-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6529426PMC
May 2019

Analysis of single, cisplatin-induced DNA bends by atomic force microscopy and simulations.

J Mol Recognit 2018 10 3;31(10):e2731. Epub 2018 Jun 3.

Department of Physics, Wake Forest University, Winston-Salem, NC, USA.

Bent DNA, or DNA that is locally more flexible, is a recognition motif for many DNA binding proteins. These DNA conformational properties can thus influence many cellular processes, such as replication, transcription, and DNA repair. The importance of these DNA conformational properties is juxtaposed to the experimental difficulty to accurately determine small bends, locally more flexible DNA, or a combination of both (bends with increased flexibility). In essence, many current bulk methods use average quantities, such as the average end-to-end distance, to extract DNA conformational properties; they cannot access the additional information that is contained in the end-to-end distance distributions. We developed a method that exploits this additional information to determine DNA conformational parameters. The method is based on matching end-to-end distance distributions obtained experimentally by atomic force microscopy imaging to distributions obtained from simulations. We applied this method to investigate cisplatin GG biadducts. We found that cisplatin induces a bend angle of 36° and softens the DNA locally around the bend.
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http://dx.doi.org/10.1002/jmr.2731DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6168373PMC
October 2018

Toward the identification of a type I toxin-antitoxin system in the plasmid DNA of dairy Lactobacillus rhamnosus.

Sci Rep 2017 09 21;7(1):12051. Epub 2017 Sep 21.

Department of Food and Drug, University of Parma, 43124, Parma, Italy.

Plasmids carry genes that give bacteria beneficial traits and allow them to survive in competitive environments. In many cases, they also harbor toxin-antitoxin (TA) systems necessary for plasmid maintenance. TA systems are generally characterized by a stable "toxin", a protein or peptide capable of killing the cell upon plasmid loss and by an unstable "antitoxin", a protein or a non-coding RNA that inhibits toxin activity. Here we report data toward the identification of a RNA-regulated TA system in the plasmid DNA of L. rhamnosus isolated from cheese. The proposed TA system comprises two convergently transcribed RNAs: a toxin RNA encoding a 29 amino acid peptide named Lpt and an antitoxin non-coding RNA. Both toxin and antitoxin RNAs resulted upregulated under conditions mimicking cheese ripening. The toxicity of the Lpt peptide was demonstrated in E. coli by cloning the Lpt ORF under the control of an inducible promoter. Bioinformatics screening of the bacterial nucleotide database, shows that regions homologous to the Lpt TA locus are widely distributed in the Lactobacillus genus, particularly within the L. casei group, suggesting a relevant role of TA systems in plasmid maintenance of cheese microbiota.
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http://dx.doi.org/10.1038/s41598-017-12218-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5608710PMC
September 2017

Physiological, Biochemical, and Biophysical Characterization of the Lung-Lavaged Spontaneously-Breathing Rabbit as a Model for Respiratory Distress Syndrome.

PLoS One 2017 6;12(1):e0169190. Epub 2017 Jan 6.

Chiesi Farmaceutici, R&D Department, Parma, Italy.

Nasal continuous positive airway pressure (nCPAP) is a widely accepted technique of non-invasive respiratory support in spontaneously-breathing premature infants with respiratory distress syndrome (RDS). Surfactant administration techniques compatible with nCPAP ventilation strategy are actively investigated. Our aim is to set up and validate a respiratory distress animal model that can be managed on nCPAP suitable for surfactant administration techniques studies. Surfactant depletion was induced by bronchoalveolar lavages (BALs) on 18 adult rabbits. Full depletion was assessed by surfactant component analysis on the BALs samples. Animals were randomized into two groups: Control group (nCPAP only) and InSurE group, consisting of a bolus of surfactant (Poractant alfa, 200 mg/kg) followed by nCPAP. Arterial blood gases were monitored until animal sacrifice, 3 hours post treatment. Lung mechanics were evaluated just before and after BALs, at the time of treatment, and at the end of the procedure. Surfactant phospholipids and protein analysis as well as surface tension measurements on sequential BALs confirmed the efficacy of the surfactant depletion procedure. The InSurE group showed a significant improvement of blood oxygenation and lung mechanics. On the contrary, no signs of recovery were appreciated in animals treated with just nCPAP. The surfactant-depleted adult rabbit RDS model proved to be a valuable and efficient preclinical tool for mimicking the clinical scenario of preterm infants affected by mild/moderate RDS who spontaneously breathe and do not require mechanical ventilation. This population is of particular interest as potential target for the non-invasive administration of surfactant.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0169190PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5217971PMC
August 2017

Study of DNA binding and bending by Bacillus subtilis GabR, a PLP-dependent transcription factor.

Biochim Biophys Acta Gen Subj 2017 Jan 14;1861(1 Pt A):3474-3489. Epub 2016 Sep 14.

Dipartimento di Bioscienze, Università degli Studi di Parma, Parma, Italy. Electronic address:

Background: GabR is a transcriptional regulator belonging to the MocR/GabR family, characterized by a N-terminal wHTH DNA-binding domain and a C-terminal effector binding and/or oligomerization domain, structurally homologous to aminotransferases (ATs). In the presence of γ-aminobutyrate (GABA) and pyridoxal 5'-phosphate (PLP), GabR activates the transcription of gabT and gabD genes involved in GABA metabolism.

Methods: Here we report a biochemical and atomic force microscopy characterization of Bacillus subtilis GabR in complex with DNA. Complexes were assembled in vitro to study their stoichiometry, stability and conformation.

Results: The fractional occupancy of the GabR cognate site suggests that GabR binds as a dimer with Kd of 10nM. Upon binding GabR bends the DNA by 80° as measured by anomalous electrophoretic mobility. With GABA we observed a decrease in affinity and conformational rearrangements compatible with a less compact nucleo-protein complex but no changes of the DNA bending angle. By employing promoter and GabR mutants we found that basic residues of the positively charged groove on the surface of the AT domain affect DNA affinity.

Conclusions: The present data extend current understanding of the GabR-DNA interaction and the effect of GABA and PLP. A model for the GabR-DNA complex, corroborated by a docking simulation, is proposed.

General Significance: Characterization of the GabR DNA binding mode highlights the key role of DNA bending and interactions with bases outside the canonical direct repeats, and might be of general relevance for the action mechanism of MocR transcription factors.
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http://dx.doi.org/10.1016/j.bbagen.2016.09.013DOI Listing
January 2017

Metal-responsive promoter DNA compaction by the ferric uptake regulator.

Nat Commun 2016 08 25;7:12593. Epub 2016 Aug 25.

Department of Pharmacy and Biotechnology (FaBiT), University of Bologna, 40126 Bologna, Italy.

Short-range DNA looping has been proposed to affect promoter activity in many bacterial species and operator configurations, but only few examples have been experimentally investigated in molecular detail. Here we present evidence for a metal-responsive DNA condensation mechanism controlled by the Helicobacter pylori ferric uptake regulator (Fur), an orthologue of the widespread Fur family of prokaryotic metal-dependent regulators. H. pylori Fur represses the transcription of the essential arsRS acid acclimation operon through iron-responsive oligomerization and DNA compaction, encasing the arsR transcriptional start site in a repressive macromolecular complex. A second metal-dependent regulator NikR functions as nickel-dependent anti-repressor at this promoter, antagonizing the binding of Fur to the operator elements responsible for the DNA condensation. The results allow unifying H. pylori metal ion homeostasis and acid acclimation in a mechanistically coherent model, and demonstrate, for the first time, the existence of a selective metal-responsive DNA compaction mechanism controlling bacterial transcriptional regulation.
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http://dx.doi.org/10.1038/ncomms12593DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5007355PMC
August 2016

New insights into the regulatory mechanisms of ppGpp and DksA on Escherichia coli RNA polymerase-promoter complex.

Nucleic Acids Res 2015 May 27;43(10):5249-62. Epub 2015 Apr 27.

Dipartimento di Bioscienze, Università degli Studi di Parma, Parma, Italy

The stringent response modulators, guanosine tetraphosphate (ppGpp) and protein DksA, bind RNA polymerase (RNAP) and regulate gene expression to adapt bacteria to different environmental conditions. Here, we use Atomic Force Microscopy and in vitro transcription assays to study the effects of these modulators on the conformation and stability of the open promoter complex (RPo) formed at the rrnA P1, rrnB P1, its discriminator (dis) variant and λ pR promoters. In the absence of modulators, RPo formed at these promoters show different extents of DNA wrapping which correlate with the position of UP elements. Addition of the modulators affects both DNA wrapping and RPo stability in a promoter-dependent manner. Overall, the results obtained under different conditions of ppGpp, DksA and initiating nucleotides (iNTPs) indicate that ppGpp allosterically prevents the conformational changes associated with an extended DNA wrapping that leads to RPo stabilization, while DksA interferes directly with nucleotide positioning into the RNAP active site. At the iNTPs-sensitive rRNA promoters ppGpp and DksA display an independent inhibitory effect, while at the iNTPs-insensitive pR promoter DksA reduces the effect of ppGpp in accordance with their antagonistic role.
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http://dx.doi.org/10.1093/nar/gkv391DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4446441PMC
May 2015

Titanium dioxide nanoparticles promote arrhythmias via a direct interaction with rat cardiac tissue.

Part Fibre Toxicol 2014 Dec 9;11:63. Epub 2014 Dec 9.

Department of Life Sciences, University of Parma, Parma, Italy.

Background: In light of recent developments in nanotechnologies, interest is growing to better comprehend the interaction of nanoparticles with body tissues, in particular within the cardiovascular system. Attention has recently focused on the link between environmental pollution and cardiovascular diseases. Nanoparticles <50 nm in size are known to pass the alveolar-pulmonary barrier, enter into bloodstream and induce inflammation, but the direct pathogenic mechanisms still need to be evaluated. We thus focused our attention on titanium dioxide (TiO₂) nanoparticles, the most diffuse nanomaterial in polluted environments and one generally considered inert for the human body.

Methods: We conducted functional studies on isolated adult rat cardiomyocytes exposed acutely in vitro to TiO₂ and on healthy rats administered a single dose of 2 mg/Kg TiO₂ NPs via the trachea. Transmission electron microscopy was used to verify the actual presence of TiO₂ nanoparticles within cardiac tissue, toxicological assays were used to assess lipid peroxidation and DNA tissue damage, and an in silico method was used to model the effect on action potential.

Results: Ventricular myocytes exposed in vitro to TiO₂ had significantly reduced action potential duration, impairment of sarcomere shortening and decreased stability of resting membrane potential. In vivo, a single intra-tracheal administration of saline solution containing TiO₂ nanoparticles increased cardiac conduction velocity and tissue excitability, resulting in an enhanced propensity for inducible arrhythmias. Computational modeling of ventricular action potential indicated that a membrane leakage could account for the nanoparticle-induced effects measured on real cardiomyocytes.

Conclusions: Acute exposure to TiO₂ nanoparticles acutely alters cardiac excitability and increases the likelihood of arrhythmic events.
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http://dx.doi.org/10.1186/s12989-014-0063-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4349471PMC
December 2014

Unravelling mechanisms behind the biological activity of bis(S-citronellalthiosemicarbazonato)nickel(II).

Metallomics 2014 Apr;6(4):783-92

Dipartimento di Bioscienze, Università di Parma, Parco Area delle Scienze 11A, 43124 Parma, Italy.

Bis(S-citronellalthiosemicarbazonato)nickel(II), [Ni(tcitr)2], is a compound that inhibits proliferation of tumour line U937 by inducing a G2/M block and leading the cancer cells to apoptosis. This nickel derivative shows no activity on non proliferating healthy cells. In this paper we report our studies on the action mechanisms of [Ni(tcitr)2]. Apoptosis in U937 cells exposed to [Ni(tcitr)2] takes place through activation of caspase-9, and therefore through an intrinsic triggering mechanism. Given the DNA damage observed in the Comet assay, the mutagenic activity of the metal complex was tested, including with the Ames test, micronuclei and DNA damage recovery, but neither mutagenicity nor recovery were detected. Nickel-complex-DNA interactions were analyzed by direct action of the compound on plasmidic and linear DNA by UV-vis and CD spectroscopy, gel electrophoresis and Atomic Force Microscopy. These experiments reveal that [Ni(tcitr)2] does not cause DNA breaks and does not intercalate, but significantly alters the DNA conformation creating knot-like structures and hairpins.
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http://dx.doi.org/10.1039/c3mt00345kDOI Listing
April 2014

Epifluorescence and atomic force microscopy: Two innovative applications for studying phage-host interactions in Lactobacillus helveticus.

J Microbiol Methods 2012 Jan 15;88(1):41-6. Epub 2011 Oct 15.

Agriculture Research Council, Fodder and Dairy Productions Research Centre (CRA-FLC), via Lombardo 11, 26900 Lodi, Italy.

Bacteriophages attacking lactic acid bacteria (LAB) still represent a crucial problem in industrial dairy fermentations. The consequences of a phage infection against LAB can lead to fermentation delay, alteration of the product quality and, in most severe cases, the product loss. Phage particles enumeration and phage-host interactions are normally evaluated by conventional plaque count assays, but, in many cases, these methods can be unsuccessful. Bacteriophages of Lactobacillus helveticus, a LAB species widely used as dairy starter or probiotic cultures, are often unable to form lysis plaques, thus impairing their enumeration by plate assay. In this study, we used epifluorescence microscopy to enumerate L. helveticus phage particles from phage-infected cultures and Atomic Force Microscopy (AFM) to visualize both phages and bacteria during the different stages of the lytic cycle. Preliminary, we tested the sensitivity of phage counting by epifluorescence microscopy. To this end, phage particles of ΦAQ113, a lytic phage of L. helveticus isolated from a whey starter culture, were stained by SYBR Green I and enumerated by epifluorescence microscopy. Values obtained by the microscopic method were 10 times higher than plate counts, with a lowest sensitivity limit of ≥6log phage/ml. The interaction of phage ΦAQ113 with its host cell L. helveticus Lh1405 was imaged by AFM after 0, 2 and 5h from phage-host adsorption. The lytic cycle was followed by epifluorescence microscopy counting and the concomitant cell wall changes were visualized by AFM imaging. Our results showed that these two methods can be combined for a reliable phage enumeration and for studying phage and host morphology during infection processes, thus giving a complete overview of phage-host interactions in L. helveticus strains involved in dairy productions.
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http://dx.doi.org/10.1016/j.mimet.2011.10.006DOI Listing
January 2012

Genetic analysis and morphological identification of pilus-like structures in members of the genus Bifidobacterium.

Microb Cell Fact 2011 Aug 30;10 Suppl 1:S16. Epub 2011 Aug 30.

Laboratory of Probiogenomics, Department of Genetics, Biology of Microorganisms, Anthropology and Evolution, University of Parma, Italy.

Background: Cell surface pili in Gram positive bacteria have been reported to orchestrate the colonization of host tissues, evasion of immunity and the development of biofilms. So far, little if any information is available on the presence of pilus-like structures in human gut commensals like bifidobacteria.

Results And Discussion: In this report, Atomic Force Microscopy (AFM) of various bifidobacterial strains belonging to Bifidobacterium bifidum, Bifidobacterium longum subsp. longum, Bifidobacterium dentium, Bifidobacterium adolescentis and Bifidobacterium animalis subsp. lactis revealed the existence of appendages resembling pilus-like structures. Interestingly, these microorganisms harbour two to six predicted pilus gene clusters in their genome, with each organized in an operon encompassing the major pilin subunit-encoding gene (designated fimA or fimP) together with one or two minor pilin subunit-encoding genes (designated as fimB and/or fimQ), and a gene encoding a sortase enzyme (strA). Quantitative Real Time (qRT)-PCR analysis and RT-PCR experiments revealed a polycistronic mRNA, encompassing the fimA/P and fimB/Q genes, which are differentially expressed upon cultivation of bifidobacteria on various glycans.
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http://dx.doi.org/10.1186/1475-2859-10-S1-S16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3231923PMC
August 2011

DNA contour length measurements as a tool for the structural analysis of DNA and nucleoprotein complexes.

Authors:
Claudio Rivetti

Methods Mol Biol 2011 ;749:235-54

Department of Biochemistry and Molecular Biology, University of Parma, Parma, Italy.

The atomic force microscope (AFM) is a widely used tool to image DNA and nucleoprotein complexes at the molecular level. This is because the AFM is relatively easy to operate, has the capability to image biomolecules under aqueous solutions, and, most importantly, can image mesoscopic macromolecular structures that are too complex to be studied by X-ray or NMR and too small to be visualized with the optical microscope. Although there are many AFM studies about the structure and the physical properties of DNA, only in few cases a rigorous method has been applied to analyze AFM images. This chapter describes procedures to prepare DNA and nucleoprotein complexes for AFM imaging and methods used to carry out simple image measurements to obtain structural data. In particular, methods to measure DNA contour length and the volume of free or DNA-bound proteins are presented and discussed.
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http://dx.doi.org/10.1007/978-1-61779-142-0_17DOI Listing
December 2011

Lactococcal phage p2 ORF35-Sak3 is an ATPase involved in DNA recombination and AbiK mechanism.

Mol Microbiol 2011 Apr 15;80(1):102-16. Epub 2011 Feb 15.

Architecture et Fonction des Macromolécules Biologiques, UMR 6098 CNRS and Universités d'Aix-Marseille I & II, Campus de Luminy, case 932, 13288 Marseille cedex 09, France.

Virulent phages of the Siphoviridae family are responsible for milk fermentation failures worldwide. Here, we report the characterization of the product of the early expressed gene orf35 from Lactococcus lactis phage p2 (936 group). ORF35(p2), also named Sak3, is involved in the sensitivity of phage p2 to the antiviral abortive infection mechanism AbiK. The localization of its gene upstream of a gene coding for a single-strand binding protein as well as its membership to a superfamily of single-strand annealing proteins (SSAPs) suggested a possible role in homologous recombination. Electron microscopy showed that purified ORF35(p2) form a hexameric ring-like structure that is often found in proteins with a conserved RecA nucleotide-binding core. Gel shift assays and surface plasmon resonance data demonstrated that ORF35(p2) interacts preferentially with single-stranded DNA with nanomolar affinity. Atomic force microscopy showed also that it preferentially binds to sticky DNA substrates over blunt ends. In addition, in vitro assays demonstrated that ORF35(p2) is able to anneal complementary strands. Sak3 also stimulates Escherichia coli RecA-mediated homologous recombination. Remarkably, Sak3 was shown to possess an ATPase activity that is required for RecA stimulation. Collectively, our results demonstrate that ORF35(p2) is a novel SSAP stimulating homologous recombination.
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http://dx.doi.org/10.1111/j.1365-2958.2011.07561.xDOI Listing
April 2011

Deciphering the function of lactococcal phage ul36 Sak domains.

J Struct Biol 2010 Jun 29;170(3):462-9. Epub 2009 Dec 29.

Architecture et Fonction des Macromolécules Biologiques, UMR 6098 CNRS and Universités d'Aix-Marseille I & II, Campus de Luminy, case 932, 13288 Marseille Cedex 09, France.

Virulent phages are responsible for milk fermentation failures in the dairy industry, due to their ability to infect starter cultures containing strains of Lactococcus lactis. Single-strand annealing proteins (SSAPs) have been found in several lactococcal phages, among which Sak in the phage ul36. Sak has been recently shown to be a functional homolog of the human protein RAD52, involved in homologous recombination. A comparison between full-length Sak and its N- and C-terminal domains was carried out to elucidate functional characteristics of each domain. We performed HPLC-SEC, AFM and SPR experiments to evaluate oligomerization states and compare the affinities to DNA. We have shown that the N-terminal domain (1-171) is essential and sufficient for oligomerization and binding to DNA, while the C-terminal domain (172-252) does not bind DNA nor oligomerize. Modelisation of Sak N-terminal domain suggests that DNA may bind a positively charged crevice that runs external to the ring. Annealing and stimulation of RecA strand exchange indicate that only the N-terminal domain is capable of single-strand annealing and both domains do not stimulate the RecA strand exchange reaction. We propose that Sak N-terminus is involved in DNA binding and annealing while the C-terminus may serve to contact Sak partners.
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http://dx.doi.org/10.1016/j.jsb.2009.12.021DOI Listing
June 2010

Structure and function of phage p2 ORF34(p2), a new type of single-stranded DNA binding protein.

Mol Microbiol 2009 Sep 25;73(6):1156-70. Epub 2009 Aug 25.

Architecture et Fonction des Macromolécules Biologiques, UMR 6098 CNRS and Universités d'Aix-Marseille I and II, Campus de Luminy, Marseille Cedex 09, France.

Lactococcus lactis, a Gram-positive bacterium widely used by the dairy industry, is subject to infection by a diverse population of virulent phages, predominantly by those of the 936 group, including the siphovirus phage p2. Confronted with the negative impact of phage infection on milk fermentation, the study of the biology of lactococcal provides insight from applied and fundamental perspectives. We decided to characterize the product of the orf34 gene from lactococcus phage p2, which was considered as a candidate single-stranded DNA binding protein (SSB) due to its localization downstream of a gene coding for a single-strand annealing protein. Two-dimensional gel electrophoresis showed that ORF34(p2) is expressed in large amounts during the early phases of phage infection, suggesting an important role in this process. Gel-shift assays, surface plasmon resonance and atomic force microscopy demonstrated that ORF34(p2) interacts with single-strand DNA with nanomolar affinity. We also determined the crystal structure of ORF34(p2) and showed that it bears a variation of the typical oligonucleotide/oligosaccharide binding-fold of SSBs. Finally, we found that ORF34(p2) is able to stimulate Escherichia coli RecA-mediated homologous recombination. The specific structural and biochemical properties that distinguish ORF34(p2) from other SSB proteins are discussed.
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http://dx.doi.org/10.1111/j.1365-2958.2009.06844.xDOI Listing
September 2009

A simple and optimized length estimator for digitized DNA contours.

Authors:
Claudio Rivetti

Cytometry A 2009 Oct;75(10):854-61

Department of Biochemistry and Molecular Biology, University of Parma, Parma 43100, Italy.

The determination of the contour length of DNA imaged by either electron microscopy or atomic force microscopy is frequently required for investigating the physical properties of nucleic acids. Nevertheless, these measurements are often carried out with methods that are not optimized for the curvilinear shape of DNA or are too complex to be of practical use. The aim of this study is to provide a method for the contour length measurements of DNA that is accurate, practical, and computationally simple. Computer simulated DNA fragments were used as experimental benchmarks in order to compute the coefficients a and b of the (n(e), n(o))-characterization [L(n(e),n(o)) = an(e) + bn(o)] so as to minimize the error of the measurements. The data show that, at variance with straight lines, a DNA length estimator depends on both the DNA flexibility and the image resolution, but it is independent of the DNA contour length. A table with DNA estimators to be used for length measurements of digitized contours obtained under commonly used imaging conditions is provided. Although the method has been developed using DNA as a benchmark, its applicability can be extended to other polymers as well as to other imaging techniques.
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http://dx.doi.org/10.1002/cyto.a.20781DOI Listing
October 2009

Sequence-dependent upstream DNA-RNA polymerase interactions in the open complex with lambdaPR and lambdaPRM promoters and implications for the mechanism of promoter interference.

J Mol Biol 2009 Jan 24;385(3):748-60. Epub 2008 Nov 24.

Department of Biochemistry and Molecular Biology, University of Parma, Viale G. P. Usberti 23/A, 43100 Parma, Italy.

Upstream interactions of Escherichia coli RNA polymerase (RNAP) in an open promoter complex (RPo) formed at the P(R) and P(RM) promoters of bacteriophage lambda have been studied by atomic force microscopy. We demonstrate that the previously described 30-nm DNA compaction observed upon RPo formation at P(R) [Rivetti, C., Guthold, M. & Bustamante, C. (1999). Wrapping of DNA around the E. coli RNA polymerase open promoter complex. EMBO J., 18, 4464-4475.] is a consequence of the specific interaction of the RNAP with two AT-rich sequence determinants positioned from -36 to -59 and from -80 to -100. Likewise, RPos formed at P(RM) showed a specific contact between RNAP and the upstream DNA sequence. We further demonstrate that this interaction, which results in DNA wrapping against the polymerase surface, is mediated by the C-terminal domains of alpha-subunits (carboxy-terminal domain). Substitution of these AT-rich sequences with heterologous DNA reduces DNA wrapping but has only a small effect on the activity of the P(R) promoter. We find, however, that the frequency of DNA templates with both P(R) and P(RM) occupied by an RNAP significantly increases upon loss of DNA wrapping. These results suggest that alpha carboxy-terminal domain interactions with upstream DNA can also play a role in regulating the expression of closely spaced promoters. Finally, a model for a possible mechanism of promoter interference between P(R) and P(RM) is proposed.
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http://dx.doi.org/10.1016/j.jmb.2008.11.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4565456PMC
January 2009

Specificity of the TraA-DNA interaction in the regulation of the pPD1-encoded sex pheromone response in Enterococcus faecalis.

J Mol Biol 2008 Jul 29;380(5):932-45. Epub 2008 May 29.

Department of Biochemistry and Molecular Biology, University of Parma, Viale G.P. Usberti 23/A, 43100 Parma, Italy.

The Enterococcus faecalis conjugative plasmid pPD1 encodes proteins responsible for the mating response to the sex pheromone cPD1 secreted by a recipient cell. This response involves the respectively negative and positive determinants traA and traE, the pheromone-inhibitor determinant ipd and structural genes participating in the conjugation process. TraA is capable of binding to key sites within the regulatory gene cluster. The binding of TraA to cognate sites is modulated by the pheromone (cPD1) and the pheromone-inhibitor (iPD1) peptides. Using atomic force microscopy and classic biochemical techniques, we mapped and characterized the TraA-DNA interactions within the pPD1 regulatory gene cluster and the role of TraA in the transcription regulation of the sex pheromone response. A previous report showed that TraA binds to three adjacent sites (tab1, tab2 and tab3) located upstream of the ipd promoter region. Here, we provide direct evidence for such interactions and show that TraA alone or in the presence of iPD1 inhibits ipd transcription by preferentially binding to tab1, whereas in the presence of saturating cPD1, the overall binding to the tab sites decreases, TraA preferentially binds to tab3 and the ipd repression is relieved. Moreover, TraA alone or in the presence of iPD1 binds to two non-adjacent sites within the ipd terminators T1 and T2, an interaction that is also relieved in the presence of cPD1. The binding of TraA to the termination region of ipd may play an important role in controlling traE and traF expression via a transcriptional read-through mechanism already postulated for the pAD1 plasmid. TraA may also regulate its own expression by binding to a site in the proximity of the traA promoter, which has been relocated 200 bp downstream of the ipd gene. A model for the TraA-mediated regulation of the pPD1-encoded sex pheromone response is presented.
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http://dx.doi.org/10.1016/j.jmb.2008.05.058DOI Listing
July 2008

Patterned gallium surfaces as molecular mirrors.

Biosens Bioelectron 2007 Sep 28;23(2):290-4. Epub 2007 Jun 28.

Dipartimento Scientifico e Tecnologico, Università di Verona, 37134 Verona, Italy.

An entirely new means of printing molecular information on a planar film, involving casting nanoscale impressions of the template protein molecules in molten gallium, is presented here for the first time. The metallic imprints not only replicate the shape and size of the proteins used as template. They also show specific binding for the template species. Such a simple approach to the creation of antibody-like properties in metallic mirrors can lead to applications in separations, microfluidic devices, and the development of new optical and electronic sensors, and will be of interest to chemists, materials scientists, analytical specialists, and electronic engineers.
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http://dx.doi.org/10.1016/j.bios.2007.06.001DOI Listing
September 2007

The neutrophil-activating Dps protein of Helicobacter pylori, HP-NAP, adopts a mechanism different from Escherichia coli Dps to bind and condense DNA.

Nucleic Acids Res 2007 19;35(7):2247-56. Epub 2007 Mar 19.

C.N.R. Institute of Molecular Biology and Pathology, Department of Biochemical Sciences A. Rossi-Fanelli, University of Rome La Sapienza, Rome, Italy.

The Helicobacter pylori neutrophil-activating protein (HP-NAP), a member of the Dps family, is a fundamental virulence factor involved in H.pylori-associated disease. Dps proteins protect bacterial DNA from oxidizing radicals generated by the Fenton reaction and also from various other damaging agents. DNA protection has a chemical component based on the highly conserved ferroxidase activity of Dps proteins, and a physical one based on the capacity of those Dps proteins that contain a positively charged N-terminus to bind and condense DNA. HP-NAP does not possess a positively charged N-terminus but, unlike the other members of the family, is characterized by a positively charged protein surface. To establish whether this distinctive property could be exploited to bind DNA, gel shift, fluorescence quenching and atomic force microscopy (AFM) experiments were performed over the pH range 6.5-8.5. HP-NAP does not self-aggregate in contrast to Escherichia coli Dps, but is able to bind and even condense DNA at slightly acid pH values. The DNA condensation capacity acts in concert with the ferritin-like activity and could be used to advantage by H.pylori to survive during host-infection and other stress challenges. A model for DNA binding/condensation is proposed that accounts for all the experimental observations.
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http://dx.doi.org/10.1093/nar/gkm077DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1874666PMC
June 2007

Upstream promoter sequences and alphaCTD mediate stable DNA wrapping within the RNA polymerase-promoter open complex.

EMBO Rep 2007 Mar 9;8(3):271-8. Epub 2007 Feb 9.

Department of Biochemistry and Molecular Biology, University of Parma, Viale G.P. Usberti 23/A, 43100 Parma, Italy.

We show that the extent of stable DNA wrapping by Escherichia coli RNA polymerase (RNAP) in the RNAP-promoter open complex depends on the sequence of the promoter and, in particular, on the sequence of the upstream region of the promoter. We further show that the extent of stable DNA wrapping depends on the presence of the RNAP alpha-subunit carboxy-terminal domain and on the presence and length of the RNAP alpha-subunit interdomain linker. Our results indicate that the extensive stable DNA wrapping observed previously in the RNAP-promoter open complex at the lambda P(R) promoter is not a general feature of RNAP-promoter open complexes.
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http://dx.doi.org/10.1038/sj.embor.7400888DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1808028PMC
March 2007

Conformation-sensitive antibodies against alzheimer amyloid-beta by immunization with a thioredoxin-constrained B-cell epitope peptide.

J Biol Chem 2007 Apr 31;282(15):11436-45. Epub 2007 Jan 31.

Department of Biochemistry and Molecular Biology, Chiesi Farmaceutici, University of Parma, 43100 Parma, Italy.

Immunotherapy against the amyloid-beta (Abeta) peptide is a valuable potential treatment for Alzheimer disease (AD). An ideal antigen should be soluble and nontoxic, avoid the C-terminally located T-cell epitope of Abeta, and yet be capable of eliciting antibodies that recognize Abeta fibrils and neurotoxic Abeta oligomers but not the physiological monomeric species of Abeta. We have described here the construction and immunological characterization of a recombinant antigen with these features obtained by tandem multimerization of the immunodominant B-cell epitope peptide Abeta1-15 (Abeta15) within the active site loop of bacterial thioredoxin (Trx). Chimeric Trx(Abeta15)n polypeptides bearing one, four, or eight copies of Abeta15 were constructed and injected into mice in combination with alum, an adjuvant approved for human use. All three polypeptides were found to be immunogenic, yet eliciting antibodies with distinct recognition specificities. The anti-Trx(Abeta15)4 antibody, in particular, recognized Abeta42 fibrils and oligomers but not monomers and exhibited the same kind of conformational selectivity against transthyretin, an amyloidogenic protein unrelated in sequence to Abeta. We have also demonstrated that anti-Trx(Abeta15)4, which binds to human AD plaques, markedly reduces Abeta pathology in transgenic AD mice. The data indicate that a conformational epitope shared by oligomers and fibrils can be mimicked by a thioredoxin-constrained Abeta fragment repeat and identify Trx(Abeta15)4 as a promising new tool for AD immunotherapy.
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http://dx.doi.org/10.1074/jbc.M609690200DOI Listing
April 2007

DNA condensation and cell transfection properties of guanidinium calixarenes: dependence on macrocycle lipophilicity, size, and conformation.

J Am Chem Soc 2006 Nov;128(45):14528-36

Dipartimento di Chimica Organica e Industriale, Università degli Studi, Viale G. P. Usberti 17/A, 43100 Parma, Italy.

Calix[n]arenes functionalized with guanidinium groups at the upper rim and alkyl chains at the lower rim bind to DNA, condense it, and in some cases, promote cell transfection depending on their structure and lipophilicity. Atomic force microscopy (AFM) studies indicate that upon DNA binding the hydrophobic association of the lipophilic chains of cone guanidinium calix[4]arenes drives the formation of intramolecular DNA condensates, characterized by DNA loops emerging from a dense core. Furthermore, hexyl and octyl chains confer to these calixarenes cell transfection capabilities. Conversely, larger and conformationally mobile calix[6]- and calix[8]arene methoxy derivatives form intermolecular aggregates characterized by "gorgonlike" structures composed of multiple plectomenes. These adducts, in which interstrand connections are dominated by electrostatic interactions, fail to promote cell transfection. Finally, calix[4]arenes in a 1,3-alternate conformation show an intermediate behavior because they condense DNA, but the process is driven by charge-charge interactions.
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http://dx.doi.org/10.1021/ja0634425DOI Listing
November 2006

Collision events between RNA polymerases in convergent transcription studied by atomic force microscopy.

Nucleic Acids Res 2006 29;34(19):5416-25. Epub 2006 Sep 29.

Department of Oral Biology, University of Leeds, Leeds LS2 9LU, UK.

Atomic force microscopy (AFM) has been used to image, at single molecule resolution, transcription events by Escherichia coli RNA polymerase (RNAP) on a linear DNA template with two convergently aligned lambda(pr) promoters. For the first time experimentally, the outcome of collision events during convergent transcription by two identical RNAP has been studied. Measurement of the positions of the RNAP on the DNA, allows distinction of open promoter complexes (OPCs) and elongating complexes (EC) and collided complexes (CC). This discontinuous time-course enables subsequent analysis of collision events where both RNAP remain bound on the DNA. After collision, the elongating RNAP has caused the other (usually stalled) RNAP to back-track along the template. The final positions of the two RNAP indicate that these are collisions between an EC and a stalled EC (SEC) or OPC (previously referred to as sitting-ducks). Interestingly, the distances between the two RNAP show that they are not always at closest approach after 'collision' has caused their arrest.
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http://dx.doi.org/10.1093/nar/gkl668DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1636470PMC
December 2006

Structural and functional properties of lengsin, a pseudo-glutamine synthetase in the transparent human lens.

Biochem Biophys Res Commun 2006 Nov 25;350(2):424-9. Epub 2006 Sep 25.

Dipartimento di Biochimica e Biologia Molecolare, Università di Parma, Viale G.P. Usberti 23/A, I-43100 Parma, Italy.

Lengsin (LGS) is an abundant transcript in the human lens, encoding a predicted polypeptide similar to glutamine synthetase (GS). We show that a major alternatively spliced product of LGS codes for a 57kDa polypeptide that assembles into a catalytically inactive dodecamer, cross-reacts with anti-GS antibodies, and is expressed at high levels in transparent, but not cataractous, human lenses. Based on this characteristic oligomeric organization, preferential expression in the transparent lens, and amyloid-beta association previously reported for GS, a potential chaperone-like role of LGS has been investigated. We find that LGS has six binding sites for the hydrophobic surface probe bis-ANS and relieves cellular toxicity caused by amyloid-beta expression in a folding-impaired yeast mutant. While documenting the structural similarity between LGS and prokaryotic GS-I, the data rule out any involvement of lengsin in glutamine biosynthesis and suggest an unrelated role that may be important for lens homeostasis and transparency.
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http://dx.doi.org/10.1016/j.bbrc.2006.09.062DOI Listing
November 2006

DNA condensation and self-aggregation of Escherichia coli Dps are coupled phenomena related to the properties of the N-terminus.

Nucleic Acids Res 2004 8;32(19):5935-44. Epub 2004 Nov 8.

C.N.R. Institute of Molecular Biology and Pathology, Department of Biochemical Sciences A. Rossi-Fanelli, University of Rome La Sapienza, 00185 Rome, Italy.

Escherichia coli Dps (DNA-binding proteins from starved cells) is the prototype of a DNA-protecting protein family expressed by bacteria under nutritional and oxidative stress. The role of the lysine-rich and highly mobile Dps N-terminus in DNA protection has been investigated by comparing the self-aggregation and DNA-condensation capacity of wild-type Dps and two N-terminal deletion mutants, DpsDelta8 and DpsDelta18, lacking two or all three lysine residues, respectively. Gel mobility and atomic force microscopy imaging showed that at pH 6.3, both wild type and DpsDelta8 self-aggregate, leading to formation of oligomers of variable size, and condense DNA with formation of large Dps-DNA complexes. Conversely, DpsDelta18 does not self-aggregate and binds DNA without causing condensation. At pH 8.2, DpsDelta8 and DpsDelta18 neither self-aggregate nor cause DNA condensation, a behavior also displayed by wild-type Dps at pH 8.7. Thus, Dps self-aggregation and Dps-driven DNA condensation are parallel phenomena that reflect the properties of the N-terminus. DNA protection against the toxic action of Fe(II) and H2O2 is not affected by the N-terminal deletions either in vitro or in vivo, in accordance with the different structural basis of this property.
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http://dx.doi.org/10.1093/nar/gkh915DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC528800PMC
December 2004