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Find video protocols related to scientific articles indexed in Pubmed.
A two-stage multiplex method for quantitative analysis of botulinum neurotoxins type a, B, e, and f by maldi-tof mass spectrometry.
Anal. Chem.
PUBLISHED: 10-20-2014
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In this publication, we report on the development of a quantitative enzymatic method for the detection of four botulinum neurotoxin (BoNT) serotypes responsible for human botulism by MALDI-TOF mass spectrometry. Factors that might affect the linearity and dynamic range for detection of BoNT cleavage products were initially examined, including the amount of peptide substrate and internal standard, the timing of cleavage reaction, and the components in the reaction solution. It was found that a long incubation time produced sensitive results, but was not capable of determining higher toxin concentrations, whereas a short incubation time was less sensitive so that lower toxin concentrations were not detected. In order to overcome these limitations, a two-stage analysis strategy was applied. The first stage analysis involved a short incubation period (e.g., 30 min). If no toxin was detected at this stage, the cleavage reaction was allowed to continue and the samples were analyzed at a second time point (4 h), so that toxin levels lower than 1 mouse LD50 or 55 attomoles per milliliter (55 amol/mL) could be quantified. By combining the results from two-stage quantification, 4 or 5 orders of magnitude in dynamic range were achieved for the detection of the serotypes of BoNT/A, BoNT/B, BoNT/E, or BoNT/F. The effect of multiplexing the assay by mixing substrates for different BoNT serotypes into a single reaction was also investigated in order to reduce the numbers of the cleavage reactions and save valuable clinical samples.
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Determinants of Butyrylcholinesterase Inhibition Among Agricultural Pesticide Handlers in Washington State: An Update.
Ann Occup Hyg
PUBLISHED: 09-28-2014
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Organophosphate (OP) and N-methyl-carbamate (CB) insecticides are used widely in agriculture to manage insect pests of economic importance. Agricultural workers are more likely to suffer exposure because of the widespread use of OP/CBs in agriculture, and pesticide-related illnesses among handlers may be more severe when compared to other farm workers. The goal of this study was to identify occupational and personal characteristics associated with butyrylcholinesterase (BuChE) inhibition in participants recruited from the Washington State Cholinesterase Monitoring Program from 2006 to 2011.
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Historical and Current Perspectives on Clostridium botulinum Diversity.
Res. Microbiol.
PUBLISHED: 06-22-2014
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For nearly one hundred years, researchers have attempted to categorize botulinum neurotoxin-producing clostridia and the toxins that they produce according to biochemical characterizations, serological comparisons, and genetic analyses. Throughout this period the bacteria and their toxins have defied such attempts at categorization. Below is a description of both historic and current C. botulinum strain and neurotoxin information that illustrates how each new finding has significantly added to the knowledge of the botulinum neurotoxin-containing clostridia and their diversity.
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Three enzymatically active neurotoxins of Clostridium botulinum strain Af84: BoNT/A2, /F4, and /F5.
Anal. Chem.
PUBLISHED: 03-13-2014
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Botulinum neurotoxins (BoNTs) are produced by various species of clostridia and are potent neurotoxins which cause the disease botulism, by cleaving proteins needed for successful nerve transmission. There are currently seven confirmed serotypes of BoNTs, labeled A-G, and toxin-producing clostridia typically only produce one serotype of BoNT. There are a few strains (bivalent strains) which are known to produce more than one serotype of BoNT, producing either both BoNT/A and /B, BoNT/A and /F, or BoNT/B and /F, designated as Ab, Ba, Af, or Bf. Recently, it was reported that Clostridium botulinum strain Af84 has three neurotoxin gene clusters: bont/A2, bont/F4, and bont/F5. This was the first report of a clostridial organism containing more than two neurotoxin gene clusters. Using a mass spectrometry based proteomics approach, we report here that all three neurotoxins, BoNT/A2, /F4, and /F5, are produced by C. botulinum Af84. Label free MS(E) quantification of the three toxins indicated that toxin composition is 88% BoNT/A2, 1% BoNT/F4, and 11% BoNT/F5. The enzymatic activity of all three neurotoxins was assessed by examining the enzymatic activity of the neurotoxins upon peptide substrates, which mimic the toxins' natural targets, and monitoring cleavage of the substrates by mass spectrometry. We determined that all three neurotoxins are enzymatically active. This is the first report of three enzymatically active neurotoxins produced in a single strain of Clostridium botulinum.
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Centers for disease control and prevention expert panel meetings on prevention and treatment of anthrax in adults.
Emerging Infect. Dis.
PUBLISHED: 01-23-2014
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The Centers for Disease Control and Prevention convened panels of anthrax experts to review and update guidelines for anthrax postexposure prophylaxis and treatment. The panels included civilian and military anthrax experts and clinicians with experience treating anthrax patients. Specialties represented included internal medicine, pediatrics, obstetrics, infectious disease, emergency medicine, critical care, pulmonology, hematology, and nephrology. Panelists discussed recent patients with systemic anthrax; reviews of published, unpublished, and proprietary data regarding antimicrobial drugs and anthrax antitoxins; and critical care measures of potential benefit to patients with anthrax. This article updates antimicrobial postexposure prophylaxis and antimicrobial and antitoxin treatment options and describes potentially beneficial critical care measures for persons with anthrax, including clinical procedures for infected nonpregnant adults. Changes from previous guidelines include an expanded discussion of critical care and clinical procedures and additional antimicrobial choices, including preferred antimicrobial drug treatment for possible anthrax meningitis.
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A literature review of laboratory-acquired brucellosis.
J. Clin. Microbiol.
PUBLISHED: 07-03-2013
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Brucellosis is a bacterial zoonotic disease which has been associated with laboratory-acquired infections. No recent reviews have addressed the characteristics of laboratory-acquired brucellosis (LAB). English-language literature was reviewed to identify reports of laboratory exposures to Brucella spp. and LAB cases between 1982 and 2007. Evaluation of 28 case reports identified 167 potentially exposed laboratory workers, of whom 71 had LAB. Nine reports were identified that summarized an additional 186 cases of LAB. Only 18 (11%) exposures were due to laboratory accidents, 147 (88%) exposures were due to aerosolization of organisms during routine identification activities, and the circumstances of 2 (1%) exposures were unknown. Brucella melitensis was the causative agent in 80% (135/167) of the exposures. Workers with high-risk exposures were 9.3 times more likely to develop LAB than workers with low-risk exposures (95% confidence interval [CI], 3.0 to 38.6; P < 0.0001); they were also 0.009 times likelier to develop LAB if they took antimicrobial PEP than if they did not (95% CI, 0 to 0.042; P < 0.0001). The median incubation period in case and summary reports was 8 weeks (range 1 to 40 weeks). Antimicrobial PEP is effective in preventing LAB. The incubation period may be used to identify appropriate serological and symptom surveillance time frames for exposed laboratory workers.
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Genetic diversity within Clostridium botulinum serotypes, botulinum neurotoxin gene clusters and toxin subtypes.
Curr. Top. Microbiol. Immunol.
PUBLISHED: 04-05-2013
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Clostridium botulinum is a species of spore-forming anaerobic bacteria defined by the expression of any one or two of seven serologically distinct botulinum neurotoxins (BoNTs) designated BoNT/A-G. This Gram-positive bacterium was first identified in 1897 and since then the paralyzing and lethal effects of its toxin have resulted in the recognition of different forms of the intoxication known as food-borne, infant, or wound botulism. Early microbiological and biochemical characterization of C. botulinum isolates revealed that the bacteria within the species had different characteristics and expressed different toxin types. To organize the variable bacterial traits within the species, Group I-IV designations were created. Interestingly, it was observed that isolates within different Groups could express the same toxin type and conversely a single Group could express different toxin types. This discordant phylogeny between the toxin and the host bacteria indicated that horizontal gene transfer of the toxin was responsible for the variation observed within the species. The recent availability of multiple C. botulinum genomic sequences has offered the ability to bioinformatically analyze the locations of the bont genes, the composition of their toxin gene clusters, and the genes flanking these regions to understand their variation. Comparison of the genomic sequences representing multiple serotypes indicates that the bont genes are not in random locations. Instead the analyses revealed specific regions where the toxin genes occur within the genomes representing serotype A, B, C, E, and F C. botulinum strains and C. butyricum type E strains. The genomic analyses have provided evidence of horizontal gene transfer, site-specific insertion, and recombination events. These events have contributed to the variation observed among the neurotoxins, the toxin gene clusters and the bacteria that contain them, and has supported the historical microbiological, and biochemical characterization of the Group classification within the species.
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Discovery of a novel enzymatic cleavage site for botulinum neurotoxin F5.
FEBS Lett.
PUBLISHED: 10-20-2011
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Botulinum neurotoxins (BoNTs) cause botulism by cleaving proteins necessary for nerve transmission. There are seven serotypes of BoNT, A-G, characterized by their response to antisera. Many serotypes are further distinguished into differing subtypes based on amino acid sequence, some of which result in functional differences. Our laboratory previously reported that all tested subtypes within each serotype have the same site of enzymatic activity. Recently, three new subtypes of BoNT/F; /F3, /F4, and /F5, were reported. Here, we report that BoNT/F5 cleaves substrate synaptobrevin-2 in a different location than the other BoNT/F subtypes, between (54)L and (55)E. This is the first report of cleavage of synaptobrevin-2 in this location.
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Analysis of Clostridium botulinum serotype E strains by using multilocus sequence typing, amplified fragment length polymorphism, variable-number tandem-repeat analysis, and botulinum neurotoxin gene sequencing.
Appl. Environ. Microbiol.
PUBLISHED: 10-14-2011
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A total of 41 Clostridium botulinum serotype E strains from different geographic regions, including Canada, Denmark, Finland, France, Greenland, Japan, and the United States, were compared by multilocus sequence typing (MLST), amplified fragment length polymorphism (AFLP) analysis, variable-number tandem-repeat (VNTR) analysis, and botulinum neurotoxin (bont) E gene sequencing. The strains, representing environmental, food-borne, and infant botulism samples collected from 1932 to 2007, were analyzed to compare serotype E strains from different geographic regions and types of botulism and to determine whether each of the strains contained the transposon-associated recombinase rarA, involved with bont/E insertion. MLST examination using 15 genes clustered the strains into several clades, with most members within a cluster sharing the same BoNT/E subtype (BoNT/E1, E2, E3, or E6). Sequencing of the bont/E gene identified two new variants (E7, E8) that showed regions of recombination with other E subtypes. The AFLP dendrogram clustered the 41 strains similarly to the MLST dendrogram. Strains that could not be differentiated by AFLP, MLST, or bont gene sequencing were further examined using three VNTR regions. Both intact and split rarA genes were amplified by PCR in each of the strains, and their identities were confirmed in 11 strains by amplicon sequencing. The findings suggest that (i) the C. botulinum serotype E strains result from the targeted insertion of the bont/E gene into genetically conserved bacteria and (ii) recombination events (not random mutations) within bont/E result in toxin variants or subtypes within strains.
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Extraction and inhibition of enzymatic activity of botulinum neurotoxins /B1, /B2, /B3, /B4, and /B5 by a panel of monoclonal anti-BoNT/B antibodies.
BMC Biochem.
PUBLISHED: 08-08-2011
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Botulism is caused by botulinum neurotoxins (BoNTs), extremely toxic proteins which can induce respiratory failure leading to long-term intensive care or death. Treatment for botulism includes administration of antitoxins, which must be administered early in the course of the intoxication; therefore, rapid determination of human exposure to BoNT is an important public health goal. In previous work, our laboratory reported on Endopep-MS, a mass spectrometry-based activity method for detecting and differentiating BoNT/A, /B, /E, and /F in clinical samples. We also demonstrated that antibody-capture is effective for purification and concentration of BoNTs from complex matrices such as clinical samples. However, some antibodies inhibit or neutralize the enzymatic activity of BoNT, so the choice of antibody for toxin extraction is critical.
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Habits, routines, and roles of graduate students: effects of hurricane ike.
Occup Ther Health Care
PUBLISHED: 07-28-2011
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ABSTRACT Disasters such as a major hurricane are likely to disrupt individuals habits, routines, and roles. The purpose of this qualitative collective case study was to explore the extent to which masters students habits, routines, and role participation were affected by Hurricane Ike during the transition from academic work to Level II Fieldwork placement. Three masters level occupational therapy students who experienced the hurricane while attending school were recruited for the study and were administered a qualitative interview and the Role Checklist. On the basis of the interview, emerging themes with subthemes were Temporal Aspects-preparation, storm, immediate poststorm, and recovery/rebuilding; Effects of Storm on Occupational Performance-loss of personal space, lack of leisure participation, changes in habits, and loss of routines; and Personal Outcomes-areas of transformation and changes in roles. As noted by the Role Checklist, some new roles were assumed by the participants following the storm, while some prehurricane roles were not resumed posthurricane. Implications for occupational therapy for individuals affected by disasters are highlighted including the importance of role participation and impact upon occupational performance.
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Epidemiology and investigation of melioidosis, Southern Arizona.
Emerging Infect. Dis.
PUBLISHED: 07-19-2011
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Burkholderia pseudomallei is a bacterium endemic to Southeast Asia and northern Australia, but it has not been found to occur endemically in the United States. We report an ostensibly autochthonous case of melioidosis in the United States. Despite an extensive investigation, the source of exposure was not identified.
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Burkholderia pseudomallei infection in a child with cystic fibrosis: acquisition in the Western Hemisphere.
Chest
PUBLISHED: 07-07-2011
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Melioidosis, an infection caused by the bacterium Burkholderia pseudomallei, is endemic to Southeast Asia and northern Australia but is only very rarely seen in patients in the United States. We report pulmonary B pseudomallei infection in a young girl with cystic fibrosis (CF) who had never traveled to Asia or Australia. Biochemical and epidemiologic investigation determined Aruba as the likely site of disease acquisition. This report highlights the ability of patients with CF to acquire this organism outside of Southeast Asia and describes an aggressive treatment regimen that has kept this patient culture-negative for the organism over a long period of time.
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Alcohol, drugs, and links to sexual risk behaviors among a sample of Virginia college students.
J Drug Educ
PUBLISHED: 06-17-2011
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This project was significant in that it administered the National College Health Risk Behavior Survey (NCHRBS), a health risk assessment, to a sample of students at three public universities in Virginia. Virginia was never included in the original or subsequent nationwide assessments using this instrument. This health risk assessment is comprehensive, easy to administer, and free. The NCHRBS assesses risk behaviors in six categories including: (1) behaviors that contribute to unintentional and intentional injuries; (2) tobacco use; (3) alcohol and other drug use; (4) sexual behaviors that contribute to unintended pregnancy and STDs, including the HIV infection; (5) unhealthy dietary behaviors; and (6) physical inactivity. This article focuses on student responses to questions about alcohol and other drugs and sexual behaviors linked to the use of these substances. It provided baseline data on health risk behaviors of college students which can help determine wellness/health education course objectives and health promotion programming and services provided to the students within the universities. In addition, this project provided protocol to expand use of the survey statewide.
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Different substrate recognition requirements for cleavage of synaptobrevin-2 by Clostridium baratii and Clostridium botulinum type F neurotoxins.
Appl. Environ. Microbiol.
PUBLISHED: 12-17-2010
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Botulinum neurotoxins (BoNTs) cause botulism, which can be fatal if it is untreated. BoNTs cleave proteins necessary for nerve transmission, resulting in paralysis. The in vivo protein target has been reported for all seven serotypes of BoNT, i.e., serotypes A to G. Knowledge of the cleavage sites has led to the development of several assays to detect BoNT based on its ability to cleave a peptide substrate derived from its in vivo protein target. Most serotypes of BoNT can be subdivided into subtypes, and previously, we demonstrated that three of the currently known subtypes of BoNT/F cleave a peptide substrate, a shortened version of synaptobrevin-2, between Q58 and K59. However, our research indicated that Clostridium baratii type F toxin did not cleave this peptide. In this study, we detail experiments demonstrating that Clostridium baratii type F toxin cleaves recombinant synaptobrevin-2 in the same location as that cleaved by proteolytic F toxin. In addition, we demonstrate that Clostridium baratii type F toxin can cleave a peptide substrate based on the sequence of synaptobrevin-2. This peptide substrate is an N-terminal extension of the original peptide substrate used for detection of other BoNT/F toxins and can be used to detect four of the currently known BoNT/F subtypes by mass spectrometry.
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Identification of mild cognitive impairments in cancer survivors.
Occup Ther Health Care
PUBLISHED: 12-06-2010
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ABSTRACT Changes in cognitive functioning are a frequent complaint of persons diagnosed and treated for cancer. The purposes of this study were to explore the feasibility of the use of the Montreal Cognitive Assessment (MoCA) for identifying mild cognitive impairment in persons who are cancer survivors as well as begin to identify the prevalence of mild cognitive impairment in cancer survivors as identified by the MoCA. Thirty-eight cancer survivors participated in this study, and 14 scored below the cutoff score of 26 on the MoCA, which indicated mild cognitive impairment. These results indicate assessment of cognitive changes in cancer patients and survivors should be part of the occupational therapy evaluation and that the MoCA is a feasible tool for such use.
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Antimicrobial-resistant nocardia isolates, United States, 1995-2004.
Clin. Infect. Dis.
PUBLISHED: 11-08-2010
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We conducted a 10-year retrospective evaluation of the epidemiology and identification of Nocardia isolates submitted to the Centers for Disease Control and Prevention for antimicrobial susceptibility testing. The species most commonly identified were N. nova (28%), N. brasiliensis (14%), and N. farcinica (14%). Of 765 isolates submitted, 61% were resistant to sulfamethoxazole and 42% were resistant to trimethoprim-sulfamethoxazole.
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International Conference on Emerging Infectious Diseases, 2010.
Emerging Infect. Dis.
PUBLISHED: 10-30-2010
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The seventh International Conference on Emerging Infectious Diseases (ICEID) was held in Atlanta, Georgia, USA, July 11-14, 2010. The conference goal was to bring together public health professionals to encourage exchange of scientific and public health information on global emerging infectious disease issues. The conference was organized by the Centers for Disease Control and Prevention (CDC), American Society for Microbiology, the Council of State and Territorial Epidemiologists, the Association of Public Health Laboratories, and the World Health Organization; additional support was provided by 40 other multidisciplinary public health partners.
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Purification of a recombinant heavy chain fragment C vaccine candidate against botulinum serotype C neurotoxin [rBoNTC(H(c))] expressed in Pichia pastoris.
Protein Expr. Purif.
PUBLISHED: 07-15-2010
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A purification process for the manufacture of a recombinant C-terminus heavy chain fragment from botulinum neurotoxin serotype C [rBoNTC(H(c))], a potential vaccine candidate, has been defined and successfully scaled-up. The rBoNTC(H(c)) was produced intracellularly in Pichia pastoris X-33 using a three step fermentation process, i.e., glycerol batch phase, a glycerol fed-batch phase to achieve high cell densities, followed by a methanol induction phase. The rBoNTC(H(c)) was captured from the soluble protein fraction of cell lysate using hydrophobic charge induction chromatography (HCIC; MEP HyperCel™), and then further purified using a CM 650M ion exchange chromatography step followed by a polishing step using HCIC once again. Method development at the bench scale was achieved using 5-100mL columns and the process was performed at the pilot scale using 0.6-1.6L columns in preparation for technology transfer to cGMP manufacturing. The process yielded approximately 2.5 g of rBoNTC(H(c))/kg wet cell weight (WCW) at the bench scale and 1.6 g rBoNTC(H(c))/kg WCW at the pilot scale. The purified rBoNTC(H(c)) was stable for at least 3 months at 5 and -80°C as determined by reverse phase-HPLC and SDS-PAGE and was stable for 24 months at -80 °C based on mouse potency bioassay. N-Terminal amino acid sequencing confirmed that the N-terminus of the purified rBoNTC(H(c)) was intact.
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Extraction of BoNT/A, /B, /E, and /F with a single, high affinity monoclonal antibody for detection of botulinum neurotoxin by Endopep-MS.
PLoS ONE
PUBLISHED: 05-19-2010
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Botulinum neurotoxins (BoNTs) are extremely potent toxins that are capable of causing respiratory failure leading to long-term intensive care or death. The best treatment for botulism includes serotype-specific antitoxins, which are most effective when administered early in the course of the intoxication. Early confirmation of human exposure to any serotype of BoNT is an important public health goal. In previous work, we focused on developing Endopep-MS, a mass spectrometry-based endopeptidase method for detecting and differentiating the seven serotypes (BoNT/A-G) in buffer and BoNT/A, /B, /E, and /F (the four serotypes that commonly affect humans) in clinical samples. We have previously reported the success of antibody-capture to purify and concentrate BoNTs from complex matrices, such as clinical samples. However, to check for any one of the four serotypes of BoNT/A, /B, /E, or /F, each sample is split into 4 aliquots, and tested for the specific serotypes separately. The discovery of a unique monoclonal antibody that recognizes all four serotypes of BoNT/A, /B, /E and /F allows us to perform simultaneous detection of all of them. When applied in conjunction with the Endopep-MS assay, the detection limit for each serotype of BoNT with this multi-specific monoclonal antibody is similar to that obtained when using other serotype-specific antibodies.
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Universal and specific quantitative detection of botulinum neurotoxin genes.
BMC Microbiol.
PUBLISHED: 04-20-2010
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Clostridium botulinum, an obligate anaerobic spore-forming bacterium, produces seven antigenic variants of botulinum toxin that are distinguished serologically and termed "serotypes". Botulinum toxin blocks the release of acetylcholine at neuromuscular junctions resulting in flaccid paralysis. The potential lethality of the disease warrants a fast and accurate means of diagnosing suspected instances of food contamination or human intoxication. Currently, the Food and Drug Administration (FDA)-accepted assay to detect and type botulinum neurotoxins (BoNTs) is the mouse protection bioassay. While specific and sensitive, this assay requires the use of laboratory animals, may take up to four days to achieve a diagnosis, and is unsuitable for high-throughput analysis. We report here a two-step PCR assay that identifies all toxin types, that achieves the specificity of the mouse bioassay while surpassing it in equivalent sensitivity, that has capability for high-throughput analysis, and that provides quantitative results within hours. The first step of our assay consists of a conventional PCR that detects the presence of C. botulinum regardless of the neurotoxin type. The second step uses quantitative PCR (qPCR) technology to determine the specific serotype of the neurotoxin.
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Investigation of an apparent outbreak of Rhodococcus equi bacteremia.
Diagn. Microbiol. Infect. Dis.
PUBLISHED: 01-13-2010
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During January to April 2007, hospital staff reported 3 patients with Rhodococcus equi bloodstream infections. Isolates were analyzed at the Centers for Disease Control and Prevention, Atlanta, GA, to confirm identification and to assess strain relatedness; 2 were R. equi but genetically distinct, and 1 was identified as Gordonia polyisoprenivorans. Rapid reference laboratory support prevented an unnecessary outbreak investigation.
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The gene CBO0515 from Clostridium botulinum strain Hall A encodes the rare enzyme N5-(carboxyethyl) ornithine synthase, EC 1.5.1.24.
J. Bacteriol.
PUBLISHED: 11-20-2009
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Sequencing of the genome of Clostridium botulinum strain Hall A revealed a gene (CBO0515), whose putative amino acid sequence was suggestive of the rare enzyme N(5)-(1-carboxyethyl) ornithine synthase. To test this hypothesis, CBO0515 has been cloned, and the encoded polypeptide was purified and characterized. This unusual gene appears to be confined to proteolytic strains assigned to group 1 of C. botulinum.
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Recombination and insertion events involving the botulinum neurotoxin complex genes in Clostridium botulinum types A, B, E and F and Clostridium butyricum type E strains.
BMC Biol.
PUBLISHED: 09-10-2009
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Clostridium botulinum is a taxonomic designation for at least four diverse species that are defined by the expression of one (monovalent) or two (bivalent) of seven different C. botulinum neurotoxins (BoNTs, A-G). The four species have been classified as C. botulinum Groups I-IV. The presence of bont genes in strains representing the different Groups is probably the result of horizontal transfer of the toxin operons between the species.
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Epigenetic modulation of the retinoid X receptor alpha by green tea in the azoxymethane-Apc Min/+ mouse model of intestinal cancer.
Mol. Carcinog.
PUBLISHED: 04-21-2009
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We investigated the possible mechanisms of inhibition of colorectal carcinogenesis by green tea (GT) in azoxymethane-treated (AOM) Apc(Min/+) mice. Mice received water or a 0.6% (w/v) solution of GT as the only source of beverage. GT treatment commenced at the 8th week of age and lasted for 8 wk. The treatment caused a statistically significant reduction in the number of newly formed tumors (28%, P < 0.05). Immunohistochemical analysis showed that GT decreased the levels of beta-catenin and its downstream target cyclin D1. To probe a mechanism, we further investigated the expression of retinoic X receptor alpha (RXR alpha) in AOM/Apc(Min/+) tumors. Our results show that RXR alpha is selectively downregulated in AOM/Apc(Min/+) mouse intestinal tumors. In contrast, other retinoic receptors including retinoic acid receptor alpha (RAR alpha), RAR beta, RXR beta, and RXR gamma were all expressed in Apc(Min/+) adenomas. Furthermore, our results show that RXR alpha downregulation is an early event in colorectal carcinogenesis and is independent of beta-catenin expression. GT significantly increased the protein levels of RXR alpha. In addition, RT-PCR analysis showed that GT induced a similar increase in the levels of RXR alpha mRNA. Genomic bisulfite treatment of colonic DNA followed by pyrosequencing of 24 CpG sites in the promoter region of RXR alpha gene showed a significant decrease in CpG methylation with GT treatment. The results suggest that a low concentration of GT is sufficient to desilence RXR alpha and inhibit intestinal tumorigenesis in the Apc(Min/+) mouse.
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Production of catalytically inactive BoNT/A1 holoprotein and comparison with BoNT/A1 subunit vaccines against toxin subtypes A1, A2, and A3.
Vaccine
PUBLISHED: 04-20-2009
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A recombinant, catalytically inactive Clostridium botulinum neurotoxin A1 holoprotein (ciBoNT/A1 HP) was constructed by introducing amino acid substitutions H223A, E224A, and H227A in the active site to ablate proteolytic activity. ciBoNT/A1 HP was produced in the yeast Pichia pastoris and the purified product was evaluated as a vaccine candidate by comparison against recombinant BoNT/A1 LC, LC-belt, LC-H(n), and H(c) antigens and a LC-H(n)+H(c) combination in mouse potency and efficacy bioassays when challenged with BoNT/A subtypes /A1, /A2, and /A3. A single dose of ciBoNT/A1 HP provided equivalent or greater protective immunity, not only against the homologous toxin, but also against two distinct toxin subtypes with significant amino acid divergence. Only the LC-H(n)+H(c) combination provided comparable protection against /A1; however, it was less effective against subtypes /A2 and /A3. Differences in protective immunity diminished after multiple vaccinations with either ciBoNT/A1 HP or BoNT/A1 H(c), and the survival rates were more comparable at the toxin levels used to challenge.
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Extraction and inhibition of enzymatic activity of botulinum neurotoxins/A1, /A2, and /A3 by a panel of monoclonal anti-BoNT/A antibodies.
PLoS ONE
PUBLISHED: 03-25-2009
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Botulinum neurotoxins (BoNTs) are extremely potent toxins that are capable of causing death or respiratory failure leading to long-term intensive care. Treatment includes serotype-specific antitoxins, which must be administered early in the course of the intoxication. Rapidly determining human exposure to BoNT is an important public health goal. In previous work, our laboratory focused on developing Endopep-MS, a mass spectrometry-based endopeptidase method for detecting and differentiating BoNT/A-G serotypes in buffer and BoNT/A, /B, /E, and /F in clinical samples. We have previously reported the effectiveness of antibody-capture to purify and concentrate BoNTs from complex matrices, such as clinical samples. Because some antibodies inhibit or neutralize the activity of BoNT, the choice of antibody with which to extract the toxin is critical. In this work, we evaluated a panel of 16 anti-BoNT/A monoclonal antibodies (mAbs) for their ability to inhibit the in vitro activity of BoNT/A1, /A2, and /A3 complex as well as the recombinant LC of A1. We also evaluated the same antibody panel for the ability to extract BoNT/A1, /A2, and /A3. Among the mAbs, there were significant differences in extraction efficiency, ability to extract BoNT/A subtypes, and inhibitory effect on BoNT catalytic activity. The mAbs binding the C-terminal portion of the BoNT/A heavy chain had optimal properties for use in the Endopep-MS assay.
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Engagement in occupation and adaptation to low vision.
Occup Ther Health Care
PUBLISHED: 01-01-2009
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The purpose of this grounded theory study was to explore how engagement in occupation affects the adaptation process for older women with visual impairment. Data were collected and analyzed for seven participants using a constant comparative method. Nine categories emerged and were grounded in the data. A theoretical model was developed with the core category of adaptation. The interactions of participants visual impairment, concurrent issues, threats to performance, losses, getting help, methods of doing, abilities, integration of losses and abilities, and adaptation. For these participants, the adaptation process was aided by getting help and finding methods of doing their preferred occupations.
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Zoonotic infections among employees from Great Smoky Mountains and Rocky Mountain National Parks, 2008-2009.
Vector Borne Zoonotic Dis.
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U.S. National Park Service employees may have prolonged exposure to wildlife and arthropods, placing them at increased risk of infection with endemic zoonoses. To evaluate possible zoonotic risks present at both Great Smoky Mountains (GRSM) and Rocky Mountain (ROMO) National Parks, we assessed park employees for baseline seroprevalence to specific zoonotic pathogens, followed by evaluation of incident infections over a 1-year study period. Park personnel showed evidence of prior infection with a variety of zoonotic agents, including California serogroup bunyaviruses (31.9%), Bartonella henselae (26.7%), spotted fever group rickettsiae (22.2%), Toxoplasma gondii (11.1%), Anaplasma phagocytophilum (8.1%), Brucella spp. (8.9%), flaviviruses (2.2%), and Bacillus anthracis (1.5%). Over a 1-year study period, we detected incident infections with leptospirosis (5.7%), B. henselae (5.7%), spotted fever group rickettsiae (1.5%), T. gondii (1.5%), B. anthracis (1.5%), and La Crosse virus (1.5%) in staff members at GRSM, and with spotted fever group rickettsiae (8.5%) and B. henselae (4.3%) in staff at ROMO. The risk of any incident infection was greater for employees who worked as resource managers (OR 7.4; 95% CI 1.4,37.5; p=0.02), and as law enforcement rangers/rescue crew (OR 6.5; 95% CI 1.1,36.5; p=0.03), relative to those who worked primarily in administration or management. The results of this study increase our understanding of the pathogens circulating within both parks, and can be used to inform the development of effective guidelines and interventions to increase visitor and staff awareness and help prevent exposure to zoonotic agents.
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De novo subtype and strain identification of botulinum neurotoxin type B through toxin proteomics.
Anal Bioanal Chem
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Botulinum neurotoxins (BoNTs) cause the disease botulism, which can be lethal if untreated. There are seven known serotypes of BoNT, A-G, defined by their response to antisera. Many serotypes are distinguished into differing subtypes based on amino acid sequence, and many subtypes are further differentiated into toxin variants. Previous work in our laboratory described the use of a proteomics approach to distinguish subtype BoNT/A1 from BoNT/A2 where BoNT identities were confirmed after searching data against a database containing protein sequences of all known BoNT/A subtypes. We now describe here a similar approach to differentiate subtypes BoNT/B1, /B2, /B3, /B4, and /B5. Additionally, to identify new subtypes or hitherto unpublished amino acid substitutions, we created an amino acid substitution database covering every possible amino acid change. We used this database to differentiate multiple toxin variants within subtypes of BoNT/B1 and B2. More importantly, with our amino acid substitution database, we were able to identify a novel BoNT/B subtype, designated here as BoNT/B7. These techniques allow for subtype and strain level identification of both known and unknown BoNT/B rapidly with no DNA required.
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