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Babetta L Marrone

Publications and source records attributed to Babetta L Marrone.

9 recordsLinked to original sources
PubMed2006

Proteomic analysis of beryllium-induced genotoxicity in an Escherichia coli mutant model system.

Beryllium is the second lightest metal, has a high melting point and high strength-to-weight ratio, and is chemically stable. These unique chemical characteristics make beryllium metal an ideal choice as a component material for a wide variety of applications in aerospace, defense, nuclear weapons, and industry. However, inhalation of beryllium dust or fumes induces significant health effects, including chronic beryllium disease and lung cancer. In this study, the mutagenicity of beryllium sulfate (BeSO(4)) and the comutagenicity of beryllium with a known mutagen 1-methyl-3-nitro-1-nitrosoguanidine (MNNG) were evaluated using a forward mutant detection system developed in Escherichia coli. In this system, BeSO(4) was shown to be weakly mutagenic alone and significantly enhanced the mutagenicity of MNNG up to 3.5-fold over MNNG alone. Based on these results a proteomic study was conducted to identify the proteins regulated by BeSO(4). Using the techniques of 2-DE and oMALDI-TOF MS, we successfully identified 32 proteins being differentially regulated by beryllium and/or MNNG in the E. coli test system. This is the first study to describe the proteins regulated by beryllium in vitro, and the results suggest several potential pathways for the focus of further research into the mechanisms underlying beryllium-induced genotoxicity.

Beryllium↗
PubMed2005

Probing the kinetics of SYTOX Orange stain binding to double-stranded DNA with implications for DNA analysis.

Rapid binding kinetics of SYTOX Orange stain with double-stranded DNA (dsDNA) was revealed on the DNA fragment sizing flow cytometer. We demonstrated for the first time that the dye molecules could be adsorbed onto the capillary surface and native DNA fragments can be dynamically stained while passing through the capillary. High-quality burst size distribution histograms were obtained for DNA samples analyzed immediately after staining, dilution, or mixing. These observations indicated that rapid interactions exist between SYTOX Orange dye molecules and dsDNA. A stopped-flow fluorescence apparatus was set up to capture the fast association traces of intercalating dyes binding to dsDNA. Kinetic equations were derived to fit the association curves for determination of association rates and to model the dynamic staining, dilution, and mixing processes of DNA samples stained with intercalating dyes. The measured association rates for both SYTOX Orange and PicoGreen stains intercalating into dsDNA were on the order of 10(8) M-1 s-1, suggesting a diffusion-controlled process. Simulations indicate that reequilibration can be reached in seconds upon staining, dilution, or mixing. Insight into the kinetics of DNA binding dyes will help implement efficient sample-handling practices in DNA analysis, including DNA fragment sizing flow cytometry.

Binding Sites↗
PubMed2005

Fingerprinting of single viral genomes.

We demonstrate the use of technology developed for optical mapping to acquire DNA fingerprints from single genomes for the purpose of discrimination and identification of bacteria and viruses. Single genome fingerprinting (SGF) provides not only the size but also the order of the restriction fragments, which adds another dimension to the information that can be used for discrimination. Analysis of single organisms may eliminate the need to culture cells and thereby significantly reduce analysis time. In addition, samples containing mixtures of several organisms can be analyzed. For analysis, cells are embedded in an agarose matrix, lysed, and processed to yield intact DNA. The DNA is then deposited on a derivatized glass substrate. The elongated genome is digested with a restriction enzyme and stained with the intercalating dye YOYO-1. DNA is then quantitatively imaged with a fluorescence microscope and the fragments are sized to an accuracy >or=90% by their fluorescence intensity and contour length. Single genome fingerprints were obtained from pure samples of adenovirus, from bacteriophages lambda and T4 GT7, and from a mixture of the three viral genomes. SGF will enable the fingerprinting of uncultured and unamplified samples and allow rapid identification of microorganisms with applications in forensics, medicine, public health, and environmental microbiology.

Calibration↗
PubMed2004

Microsphere-based duplexed immunoassay for influenza virus typing by flow cytometry.

We have developed a rapid, duplexed microsphere-based immunoassay for the characterization of influenza virus types that has the potential to overcome many of the limitations of current detection methods. The assay uses microspheres of two sizes, each coupled to an influenza type A- or type B-specific monoclonal antibody (MAb), to capture influenza viruses in the sample. A cocktail of fluorescently labeled, influenza-specific polyclonal antibodies then binds the captured viruses. The sandwich complexes are measured using a multiparameter flow cytometer. The assay can distinguish between influenza types A and B in a single reaction with good reproducibility and high sensitivity. Detection sensitivity is much higher than that of commercially available influenza diagnosis quick kits: the FLU OIA (Thermo Biostar) kit and the Directigen Flu A+B kit (Becton Dickinson). The multiplexing capabilities of the current assay, which are not possible with enzyme-linked immunosorbent assay (ELISA) and the commercially available kits, reduce sample handling and consume fewer costly reagents. This assay represents a more efficient and sensitive method of characterizing influenza types. With inclusion of influenza subtype-specific antibodies as capture antibodies, this microsphere-based immunoassay can be expanded to differentiate among influenza types and subtypes in a single reaction to improve world-wide influenza surveillance.

Antibodies, Monoclonal↗
PubMed2004

Performance assessment of DNA fragment sizing by high-sensitivity flow cytometry and pulsed-field gel electrophoresis.

The sizing of restriction fragments is the chief analytical technique utilized in the production of DNA fingerprints. Few techniques have been able to compete with pulsed-field gel electrophoresis (PFGE), which is capable of discriminating among bacteria at species and strain levels by resolving restriction fragments. However, an ultrasensitive flow cytometer (FCM) developed in our lab has also demonstrated the ability to discriminate bacteria at species and strain levels. The abilities of FCM warrant a quantitative parallel comparison with PFGE to assess and evaluate the accuracy and precision of DNA fragment sizing by both techniques. Replicate samples of Staphylococcus aureus Mu50 were analyzed along with two clinical S. aureus isolates. The absolute fragment sizing accuracy was determined for PFGE (5% +/- 2%) and FCM (4% +/- 4%), with sequence-predicted Mu50 SmaI fragment sizes used as a reference. Precision was determined by simple arithmetic methods (relative standard deviation for PFGE [RSD(PFGE) ] = 3% +/- 2% and RSD(FCM) = 1.2% +/- 0.8%) as well as by the use of dendrograms derived from Dice coefficient-unweighted pair group method with arithmetic averages (UPGMA) and Pearson-UPGMA analyses. All quantitative measures of PFGE and FCM precision were equivalent, within error. The precision of both methods was not limited by any single sample preparation or analysis step that was tracked in this study. Additionally, we determined that the curve-based clustering of fingerprint data provided a more informative and useful assessment than did traditional band-based methods.

Bacteriological Techniques↗
PubMed2003

Public health. Building microbial forensics as a response to bioterrorism.

Combating bioterrorism is a challenge to all of us. To be proactive, the U.S. Government has formalized the discipline of "microbial forensics" to deter and attribute perpetrators of such acts. This Policy Forum describes the foundations of the microbial forensics program: the creation of a national bioforensics laboratory, a partnership laboratory network, and a peer-consensus scientific working group and the promulgation of quality assurance guidelines.

Advisory Committees↗
PubMed2003

Potential binding modes of beryllium with the class II major histocompatibility complex HLA-DP: a combined theoretical and structural database study.

In an effort to understand the molecular basis of chronic beryllium disease (CBD), a study of the chemical relationship between beryllium, antigen, and the major histocompatibility complex II, HLA-DP, was undertaken. A homology model of the HLA-DP protein was developed. An analysis of the sequences of HLA-DPB1 and HLA-DPA1 alleles most common among CBD patients revealed several carboxylate rich regions in the peptide-binding cleft. These regions contain many hard Lewis base sites that may provide bonding opportunities for beryllium, a hard Lewis acid. Quantum chemistry calculations and structural database results support the presence of beryllium clusters, bridged by carboxylate, hydroxo, and/or oxo ligands, in the HLA-DP binding cleft. These results strongly suggest that beryllium clusters are an integral part of the antigen, and may even act solely as antigen. This work provides an initial model for thinking about beryllium interactions with proteins relevant to CBD and other metal-induced diseases.

Amino Acid Sequence↗
PubMed2002

Evaluation of different nucleic acid stains for sensitive double-stranded DNA analysis with capillary electrophoretic separation.

This paper outlines the first use of SYTOX Orange, SYTO 82 and SYTO 25 nucleic acid stains for on-column staining of double-stranded DNA (dsDNA) fragments separated by capillary electrophoresis (CE). Low-viscosity, replaceable poly(vinylpyrrolidone) (PVP) polymer solution was used as the sieving matrix on an uncoated fused-silica capillary. The effects of PVP concentration, electric field strength, and incorporated nucleic acid stain concentrations on separation efficiency were examined for a wide range of DNA fragment sizes. Our study was focused on using nucleic acid stains efficiently excitable at a wavelength of 532 nm. Among the five tested nucleic acid stains, SYTOX Orange stain was shown to have the best sensitivity for dsDNA detection by CE. About a 500-fold lower detection limit was obtained compared to commonly used ethidium bromide and propidium iodide. SYTOX Orange stain also provided a wide linear dynamic range for direct DNA quantitation with on-line CE detection. Use of SYTOX Orange stain can greatly improve the measurement of DNA fragments by CE, which will enable an expanded set of applications in genomics and diagnostics.

Coloring Agents↗