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  • 1
    Language: English
    In: FEMS Immunology and Medical Microbiology, 1999, Vol.26(2), pp.159-166
    Description: To purchase or authenticate to the full-text of this article, please visit this link: http://dx.doi.org/10.1111/j.1574-695X.1999.tb01384.x Byline: Richard J. Karalus (a), Timothy F. Murphy (a) Keywords: Bacterial vaccine; Outer membrane protein; Protein purification; Haemophilus influenzae Abstract: Abstract Outer membrane protein P6 is a promising vaccine antigen with potential to prevent infections caused by non-typeable Haemophilus influenzae. A convenient and reliable method for the purification of P6 and an assessment of the purity, yield, protein structure, antigenicity and immunogenicity of the purified protein are described. The method begins with intact H. influenzae and utilizes a series of incubations and centrifugations using a single buffer to remove all cell components with the exception of the peptidoglycan to which the P6 is associated. P6 is dissociated from the complex with heat and the insoluble peptidoglycan is removed by centrifugation. The procedure yields highly purified P6. Contamination with lipooligosaccharide is less than 0.025 endotoxin U per [mu]g P6. The yield of P6 is approximately 2 mg of P6 per l H. influenzae culture. The purified P6 retains both the secondary and tertiary structure as measured by circular dichroism and analysis with monoclonal antibodies. The purified P6 is immunogenic in animals. A convenient method for purifying P6 which retains antigenicity and immunogenicity will be an important tool for future studies of the vaccine potential of P6. Author Affiliation: (a)Department of Microbiology, State University of New York at Buffalo, Buffalo, NY, USA (b) Division of Infectious Diseases of the Department of Medicine, State University of New York at Buffalo, Buffalo, NY, USA (c) Department of Veterans Affairs, Western New York Healthcare System (151), 3495 Bailey Avenue, Buffalo, NY 14215, USA Article History: Received 2 August 1999, Accepted 13 August 1999 Article note: (*) Corresponding author. Tel.: +1 (716) 829-2173; Fax: +1 (716) 862-6526, E-mail address: murphyt@acsu.buffalo.edu
    Keywords: Bacterial Vaccine ; Outer Membrane Protein ; Protein Purification ; Haemophilus Influenzae ; Medicine ; Biology
    ISSN: 0928-8244
    E-ISSN: 1574-695X
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  • 2
    Language: English
    In: PLoS ONE, 2011, Vol.6(3), p.e17585
    Description: Yersinia pestis , the causative agent of plague, has caused several pandemics throughout history and remains endemic in the rodent populations of the western United States. More recently, Y. pestis is one of several bacterial pathogens considered to be a potential agent of bioterrorism. Thus, elucidating potential mechanisms of survival and persistence in the environment would be important in the event of an intentional release of the organism. One such mechanism is entry into the viable but non-culturable (VBNC) state, as has been demonstrated for several other bacterial pathogens. In this study, we showed that Y. pestis became nonculturable by normal laboratory methods after 21 days in a low-temperature tap water microcosm. We further show evidence that, after the loss of culturability, the cells remained viable by using a variety of criteria, including cellular membrane integrity, uptake and incorporation of radiolabeled amino acids, and protection of genomic DNA from DNase I digestion. Additionally, we identified morphological and ultrastructural characteristics of Y. pestis VBNC cells, such as cell rounding and large periplasmic spaces, by electron microscopy, which are consistent with entry into the VBNC state in other bacteria. Finally, we demonstrated resuscitation of a small number of the non-culturable cells. This study provides compelling evidence that Y. pestis persists in a low-temperature tap water microcosm in a viable state yet is unable to be cultured under normal laboratory conditions, which may prove useful in risk assessment and remediation efforts, particularly in the event of an intentional release of this organism.
    Keywords: Research Article ; Biology ; Microbiology
    E-ISSN: 1932-6203
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  • 3
    Language: English
    In: Journal of visualized experiments : JoVE, 15 May 2012(63), pp.e4202
    Description: Traditional and emerging pathogens such as Enterohemorrhagic Escherichia coli (EHEC), Yersinia pestis, or prion-based diseases are of significant concern for governments, industries and medical professionals worldwide. For example, EHECs, combined with Shigella, are responsible for the deaths of approximately 325,000 children each year and are particularly prevalent in the developing world where laboratory-based identification, common in the United States, is unavailable (1). The development and distribution of low cost, field-based, point-of-care tools to aid in the rapid identification and/or diagnosis of pathogens or disease markers could dramatically alter disease progression and patient prognosis. We have developed a tool to isolate nucleic acids and proteins from a sample by solid-phase extraction (SPE) without electricity or associated laboratory equipment (2). The isolated macromolecules can be used for diagnosis either in a forward lab or using field-based point-of-care platforms. Importantly, this method provides for the direct comparison of nucleic acid and protein data from an un-split sample, offering a confidence through corroboration of genomic and proteomic analysis. Our isolation tool utilizes the industry standard for solid-phase nucleic acid isolation, the BOOM technology, which isolates nucleic acids from a chaotropic salt solution, usually guanidine isothiocyanate, through binding to silica-based particles or filters (3). CUBRC's proprietary solid-phase extraction chemistry is used to purify protein from chaotropic salt solutions, in this case, from the waste or flow-thru following nucleic acid isolation(4). By packaging well-characterized chemistries into a small, inexpensive and simple platform, we have generated a portable system for nucleic acid and protein extraction that can be performed under a variety of conditions. The isolated nucleic acids are stable and can be transported to a position where power is available for PCR amplification while the protein content can immediately be analyzed by hand held or other immunological-based assays. The rapid identification of disease markers in the field could significantly alter the patient's outcome by directing the proper course of treatment at an earlier stage of disease progression. The tool and method described are suitable for use with virtually any infectious agent and offer the user the redundancy of multi-macromolecule type analyses while simultaneously reducing their logistical burden.
    Keywords: Chemical Fractionation -- Methods ; Nucleic Acids -- Isolation & Purification ; Proteins -- Isolation & Purification
    E-ISSN: 1940-087X
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  • 4
    Language: English
    In: Journal of Nucleic Acids, Annual, 2014
    Description: Large-scale genomics projects are identifying biomarkers to detect human disease. B. pseudomallei and B. mallei are two closely related select agents that cause melioidosis and glanders. Accurate characterization of metagenomic samples is dependent on accurate measurements of genetic variation between isolates with resolution down to strain level. Often single biomarker sensitivity is augmented by use of multiple or panels of biomarkers. In parallel with single biomarker validation, advances in DNA sequencing enable analysis of entire genomes in a single run: population-sequencing. Potentially, direct sequencing could be used to analyze an entire genome to serve as the biomarker for genome identification. However, genome variation and population diversity complicate use of direct sequencing, as well as differences caused by sample preparation protocols including sequencing artifacts and mistakes. As part of a Department of Homeland Security program in bacterial forensics, we examined how to implement whole genome sequencing (WGS) analysis as a judicially defensible forensic method for attributing microbial sample relatedness; and also to determine the strengths and limitations of whole genome sequence analysis in a forensics context. Herein, we demonstrate use of sequencing to provide genetic characterization of populations: direct sequencing of populations.
    Keywords: Burkholderia -- Identification And Classification ; Burkholderia -- Genetic Aspects ; Burkholderia -- Research ; Dna Sequencing -- Usage ; Microbial Forensics -- Research
    ISSN: 2090-021X
    Source: Cengage Learning, Inc.
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  • 5
    In: Infection and Immunity, 2002, Vol. 70(4), p.1889
    Description: Many pathogens produce one or more superoxide dismutases (SODs), enzymes involved in the detoxification of endogenous and exogenous reactive oxygen species that are encountered during the infection process. One detectable cytoplasmic SOD was identified in the human mucosal pathogen Moraxella catarrhalis, and the gene responsible for the SOD activity, sodA, was isolated from a recent pediatric clinical isolate (strain 7169). Sequence analysis of the cloned M. catarrhalis 7169 DNA fragment revealed an open reading frame of 618 bp encoding a polypeptide of 205 amino acids with 48 to 67% identity to known bacterial manganese-cofactored SODs. An isogenic M. catarrhalis sodA mutant was constructed in strain 7169 by allelic exchange. In contrast to the wild-type 7169, the 7169::sodK20 mutant was severely attenuated for aerobic growth, even in rich medium containing supplemental amino acids, and exhibited extreme sensitivity to the redox-active agent methyl viologen. The ability of recombinant SodA to rescue the aerobic growth defects of E. coli QC774, a sodA sodB-deficient mutant, demonstrated the functional expression of SOD activity by cloned M. catarrhalis sodA. Indirect SOD detection assays were used to visualize both native and recombinant SodA activity in bacterial lysates. This study demonstrates that M. catarrhalis SodA plays a critical role in the detoxification of endogenous, metabolically produced oxygen radicals. In addition, the outer membrane protein (OMP) profile of 7169::sodK20 was consistent with iron starvation in spite of growth under iron-replete conditions. This novel observation indicates that M. catarrhalis strains lacking SodA constitutively express immunogenic OMPs previously described as iron repressible, and this potentially attenuated mutant strain may be an attractive vaccine candidate.
    Keywords: Moraxella Catarrhalis ; Moraxella Catarrhalis ; Reactive Oxygen Species ; Superoxide Dismutase ; Iron ; Outer Membranes ; Membrane Proteins ; Vaccines ; Nucleotide Sequence ; Gene Expression ; Reactive Oxygen Species ; Superoxide Dismutase ; Iron ; Outer Membranes ; Membrane Proteins ; Vaccines ; Nucleotide Sequence ; Gene Expression ; Soda Protein ; Soda Protein ; Gene Regulation ; RNA and Ribosomes ; Soda Protein;
    ISSN: 0019-9567
    ISSN: 00199567
    E-ISSN: 10985522
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  • 6
    In: Infection and Immunity, 2001, Vol. 69(2), p.773
    ISSN: 0019-9567
    ISSN: 00199567
    E-ISSN: 10985522
    Source: American Society of Microbiology
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  • 7
    In: Journal of Nucleic Acids, 2013, Vol.2013, 13 pages
    Description: Large-scale genomics projects are identifying biomarkers to detect human disease. and are two closely related select agents that cause melioidosis and glanders. Accurate characterization of metagenomic samples is dependent on accurate measurements of genetic variation between isolates with resolution down to strain level. Often single biomarker sensitivity is augmented by use of multiple or panels of biomarkers. In parallel with single biomarker validation, advances in DNA sequencing enable analysis of entire genomes in a single run: population-sequencing. Potentially, direct sequencing could be used to analyze an entire genome to serve as the biomarker for genome identification. However, genome variation and population diversity complicate use of direct sequencing, as well as differences caused by sample preparation protocols including sequencing artifacts and mistakes. As part of a Department of Homeland Security program in bacterial forensics, we examined how to implement whole genome sequencing (WGS) analysis as a judicially defensible forensic method for attributing microbial sample relatedness; and also to determine the strengths and limitations of whole genome sequence analysis in a forensics context. Herein, we demonstrate use of sequencing to provide genetic characterization of populations: direct sequencing of populations.
    Keywords: Anatomy & Physiology;
    ISSN: 2090-0201
    E-ISSN: 2090-021X
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  • 8
    In: FEMS Immunology & Medical Microbiology, 1999, Vol. 26(2), pp.159-166
    Description: Outer membrane protein P6 is a promising vaccine antigen with potential to prevent infections caused by non-typeable Haemophilus influenzae . A convenient and reliable method for the purification of P6 and an assessment of the purity, yield, protein structure, antigenicity and immunogenicity of the purified protein are described. The method begins with intact H. influenzae and utilizes a series of incubations and centrifugations using a single buffer to remove all cell components with the exception of the peptidoglycan to which the P6 is associated. P6 is dissociated from the complex with heat and the insoluble peptidoglycan is removed by centrifugation. The procedure yields highly purified P6. Contamination with lipooligosaccharide is less than 0.025 endotoxin U per µg P6. The yield of P6 is approximately 2 mg of P6 per l H. influenzae culture. The purified P6 retains both the secondary and tertiary structure as measured by circular dichroism and analysis with monoclonal antibodies. The purified P6 is immunogenic in animals. A convenient method for purifying P6 which retains antigenicity and immunogenicity will be an important tool for future studies of the vaccine potential of P6.
    Keywords: Bacterial Vaccine ; Outer Membrane Protein ; Protein Purification ; 〈Kwd〉〈Italic〉Haemophilus Influenzae〈/Italic〉〈/Kwd〉
    ISSN: 0928-8244
    E-ISSN: 1574-695X
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  • 9
    Language: English
    In: Spectroscopy, June, 2006, Vol.21(6), p.20(8)
    ISSN: 0887-6703
    Source: Cengage Learning, Inc.
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  • 10
    Language: English
    In: LC-GC North America, Feb, 2006, Vol.24(2), p.170(10)
    ISSN: 1527-5949
    Source: Cengage Learning, Inc.
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