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1.  Hypertension and chronic kidney disease: controversies in pathogenesis and treatment 
The relationship between hypertension and chronic kidney disease (CKD) has long been the subject of controversy. The pathogenetic mechanisms of nephropathy in non-diabetic individuals with hypertension, as well as optimal hypertension treatment targets in populations with nephropathy remain important clinical concerns. This manuscript reviews breakthroughs in molecular genetics that have clarified the complex relationship between hypertension and kidney disease, answering the question of which factor comes first. An overview of the potential roles that hyperuricemia plays in the pathogenesis of hypertension and CKD and current blood pressure treatment guidelines in populations with CKD are discussed. The ongoing National Institutes of Health-sponsored Systolic Blood Pressure Intervention Trial (SPRINT) is underway to help answer these important questions. Enrollment of 9,250 hypertensive SPRINT participants will be completed in 2013; important results on ideal blood pressure control targets for reducing nephropathy progression, cardiovascular disease end-points, and preserving cognitive function are expected. As such, many of the controversial aspects of hypertension management will likely be clarified in the near future.
PMCID: PMC4030753  PMID: 23538309
APOL1; blood pressure control; chronic kidney disease; FSGS; hypertension; uric acid
2.  Discovery of a Novel Enzymatic Cleavage Site for Botulinum Neurotoxin F5 
Febs Letters  2011;586(2):109-115.
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 54L and 55E. This is the first report of cleavage of synaptobrevin-2 in this location.
PMCID: PMC3263758  PMID: 22172278
3.  De novo subtype and strain identification of botulinum neurotoxin type B through toxin proteomics 
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.
FigureIdentification of an existing or new BoNT/B can be accomplished through MS/MS analysis of digestion fragments of the protein.
PMCID: PMC3309144  PMID: 22395449
Botulinum neurotoxin; Botulism; Mass spectrometry; Proteomics
4.  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 Biochemistry  2011;12:58.
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.
In this work, we evaluated 24 anti-BoNT/B monoclonal antibodies (mAbs) for their ability to inhibit the in vitro activity of BoNT/B1, /B2, /B3, /B4, and /B5 and to extract those toxins. Among the mAbs, there were significant differences in ability to extract BoNT/B subtypes and inhibitory effect on BoNT catalytic activity. Some of the mAbs tested enhanced the in vitro light chain activity of BoNT/B, suggesting that BoNT/B may undergo conformational change upon binding some mAbs.
In addition to determining in vitro inhibition abilities of a panel of mAbs against BoNT/B1-/B5, this work has determined B12.2 and 2B18.2 to be the best mAbs for sample preparation before Endopep-MS. These mAb characterizations also have the potential to assist with mechanistic studies of BoNT/B protection and treatment, which is important for studying alternative therapeutics for botulism.
PMCID: PMC3250939  PMID: 22085466
5.  Different Substrate Recognition Requirements for Cleavage of Synaptobrevin-2 by Clostridium baratii and Clostridium botulinum Type F Neurotoxins▿  
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.
PMCID: PMC3067225  PMID: 21169446
6.  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  2010;5(8):e12237.
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.
PMCID: PMC2923190  PMID: 20808925
7.  Mass Spectrometric Analysis of Multiple Pertussis Toxins and Toxoids 
Bordetella pertussis (Bp) is the causative agent of pertussis, a vaccine preventable disease occurring primarily in children. In recent years, there has been increased reporting of pertussis. Current pertussis vaccines are acellular and consist of Bp proteins including the major virulence factor pertussis toxin (Ptx), a 5-subunit exotoxin. Variation in Ptx subunit amino acid (AA) sequence could possibly affect the immune response. A blind comparative mass spectrometric (MS) analysis of commercially available Ptx as well as the chemically modified toxoid (Ptxd) from licensed vaccines was performed to assess peptide sequence and AA coverage variability as well as relative amounts of Ptx subunits. Qualitatively, there are similarities among the various sources based on AA percent coverages and MS/MS fragmentation profiles. Additionally, based on a label-free mass spectrometry-based quantification method there is differential relative abundance of the subunits among the sources.
PMCID: PMC2874995  PMID: 20508854
8.  Kinetics of Lethal Factor and Poly-d-Glutamic Acid Antigenemia during Inhalation Anthrax in Rhesus Macaques ▿  
Infection and Immunity  2009;77(8):3432-3441.
Systemic anthrax manifests as toxemia, rapidly disseminating septicemia, immune collapse, and death. Virulence factors include the anti-phagocytic γ-linked poly-d-glutamic acid (PGA) capsule and two binary toxins, complexes of protective antigen (PA) with lethal factor (LF) and edema factor. We report the characterization of LF, PA, and PGA levels during the course of inhalation anthrax in five rhesus macaques. We describe bacteremia, blood differentials, and detection of the PA gene (pagA) by PCR analysis of the blood as confirmation of infection. For four of five animals tested, LF exhibited a triphasic kinetic profile. LF levels (mean ± standard error [SE] between animals) were low at 24 h postchallenge (0.03 ± 1.82 ng/ml), increased at 48 h to 39.53 ± 0.12 ng/ml (phase 1), declined at 72 h to 13.31 ± 0.24 ng/ml (phase 2), and increased at 96 h (82.78 ± 2.01 ng/ml) and 120 h (185.12 ± 5.68 ng/ml; phase 3). The fifth animal had an extended phase 2. PGA levels were triphasic; they were nondetectable at 24 h, increased at 48 h (2,037 ± 2 ng/ml), declined at 72 h (14 ± 0.2 ng/ml), and then increased at 96 h (3,401 ± 8 ng/ml) and 120 h (6,004 ± 187 ng/ml). Bacteremia was also triphasic: positive at 48 h, negative at 72 h, and positive at euthanasia. Blood neutrophils increased from preexposure (34.4% ± 0.13%) to 48 h (75.6% ± 0.08%) and declined at 72 h (62.4% ± 0.05%). The 72-h declines may establish a “go/no go” turning point in infection, after which systemic bacteremia ensues and the host's condition deteriorates. This study emphasizes the value of LF detection as a tool for early diagnosis of inhalation anthrax before the onset of fulminant systemic infection.
PMCID: PMC2715684  PMID: 19506008
9.  Extraction and Inhibition of Enzymatic Activity of Botulinum Neurotoxins/A1, /A2, and /A3 by a Panel of Monoclonal Anti-BoNT/A Antibodies 
PLoS ONE  2009;4(4):e5355.
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.
PMCID: PMC2670495  PMID: 19399171
10.  Differentiation of Streptococcus pneumoniae Conjunctivitis Outbreak Isolates by Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry▿  
Applied and Environmental Microbiology  2008;74(19):5891-5897.
Streptococcus pneumoniae (pneumococcus [Pnc]) is a causative agent of many infectious diseases, including pneumonia, septicemia, otitis media, and conjunctivitis. There have been documented conjunctivitis outbreaks in which nontypeable (NT), nonencapsulated Pnc has been identified as the etiological agent. The use of mass spectrometry to comparatively and differentially analyze protein and peptide profiles of whole-cell microorganisms remains somewhat uncharted. In this report, we discuss a comparative proteomic analysis between NT S. pneumoniae conjunctivitis outbreak strains (cPnc) and other known typeable or NT pneumococcal and streptococcal isolates (including Pnc TIGR4 and R6, Streptococcus oralis, Streptococcus mitis, Streptococcus pseudopneumoniae, and Streptococcus pyogenes) and nonstreptococcal isolates (including Escherichia coli, Enterococcus faecalis, and Staphylococcus aureus) as controls. cPnc cells and controls were grown to mid-log phase, harvested, and subsequently treated with a 10% trifluoroacetic acid-sinapinic acid matrix mixture. Protein and peptide fragments of the whole-cell bacterial isolate-matrix combinations ranging in size from 2 to 14 kDa were evaluated by matrix-assisted laser desorption ionization-time of flight mass spectrometry. Additionally Random Forest analytical tools and dendrogramic representations (Genesis) suggested similarities and clustered the isolates into distinct clonal groups, respectively. Also, a peak list of protein and peptide masses was obtained and compared to a known Pnc protein mass library, in which a peptide common and unique to cPnc isolates was tentatively identified. Information gained from this study will lead to the identification and validation of proteins that are commonly and exclusively expressed in cPnc strains which could potentially be used as a biomarker in the rapid diagnosis of pneumococcal conjunctivitis.
PMCID: PMC2565961  PMID: 18708515
11.  Urinary Perchlorate and Thyroid Hormone Levels in Adolescent and Adult Men and Women Living in the United States 
Environmental Health Perspectives  2006;114(12):1865-1871.
Perchlorate is commonly found in the environment and known to inhibit thyroid function at high doses. Assessing the potential effect of low-level exposure to perchlorate on thyroid function is an area of ongoing research.
We evaluated the potential relationship between urinary levels of perchlorate and serum levels of thyroid stimulating hormone (TSH) and total thyroxine (T4) in 2,299 men and women, ≥ 12 years of age, participating in the National Health and Nutrition Examination Survey (NHANES) during 2001–2002.
We used multiple regression models of T4 and TSH that included perchlorate and covariates known to be or likely to be associated with T4 or TSH levels: age, race/ethnicity, body mass index, estrogen use, menopausal status, pregnancy status, premenarche status, serum C-reactive protein, serum albumin, serum cotinine, hours of fasting, urinary thiocyanate, urinary nitrate, and selected medication groups.
Perchlorate was not a significant predictor of T4 or TSH levels in men. For women overall, perchlorate was a significant predictor of both T4 and TSH. For women with urinary iodine < 100 μg/L, perchlorate was a significant negative predictor of T4 (p < 0.0001) and a positive predictor of TSH (p = 0.001). For women with urinary iodine ≥ 100 μg/L, perchlorate was a significant positive predictor of TSH (p = 0.025) but not T4 (p = 0.550).
These associations of perchlorate with T4 and TSH are coherent in direction and independent of other variables known to affect thyroid function, but are present at perchlorate exposure levels that were unanticipated based on previous studies.
PMCID: PMC1764147  PMID: 17185277
exposure; iodine; NHANES; perchlorate; thyroid; thyroxine; TSH
12.  Trends in the Exposure of Nonsmokers in the U.S. Population to Secondhand Smoke: 1988–2002 
Environmental Health Perspectives  2006;114(6):853-858.
The objective of this study was to describe the exposure of nonsmokers in the U.S. population to secondhand smoke (SHS) using serum cotinine concentrations measured over a period of 14 years, from October 1988 through December 2002. This study consists of a series of National Health and Nutrition Examination Surveys (NHANES) measuring serum cotinine as an index of SHS exposure of participants. Study participants were individuals representative of the U.S. civilian, noninstitutionalized population, ≥ 4 years of age. We analyzed serum cotinine and interview data from NHANES obtained during surveys conducted during four distinct time periods. Our results document a substantial decline of approximately 70% in serum cotinine concentrations in non-smokers during this period. This decrease was reflected in all groups within the population regardless of age, sex, or race/ethnicity. The large decrease that we observed in serum cotinine concentrations suggests a substantial reduction in the exposure of the U.S. population to SHS during the 1990s. The exposure of nonsmokers to SHS represents an important public health concern. Our findings suggest that recent public health efforts to reduce such exposures have had an important effect, although children and non-Hispanic black nonsmokers show relatively higher levels of serum cotinine.
PMCID: PMC1480505  PMID: 16759984
biomarker; cotinine; environmental tobacco smoke; ETS; health and nutrition examination survey; NHANES; secondhand smoke; SHS; tandem mass spectrometry
14.  Urinary Creatinine Concentrations in the U.S. Population: Implications for Urinary Biologic Monitoring Measurements 
Environmental Health Perspectives  2004;113(2):192-200.
Biologic monitoring (i.e., biomonitoring) is used to assess human exposures to environmental and workplace chemicals. Urinary biomonitoring data typically are adjusted to a constant creatinine concentration to correct for variable dilutions among spot samples. Traditionally, this approach has been used in population groups without much diversity. The inclusion of multiple demographic groups in studies using biomonitoring for exposure assessment has increased the variability in the urinary creatinine levels in these study populations. Our objectives were to document the normal range of urinary creatinine concentrations among various demographic groups, evaluate the impact that variations in creatinine concentrations can have on classifying exposure status of individuals in epidemiologic studies, and recommend an approach using multiple regression to adjust for variations in creatinine in multivariate analyses. We performed a weighted multivariate analysis of urinary creatinine concentrations in 22,245 participants of the Third National Health and Nutrition Examination Survey (1988–1994) and established reference ranges (10th–90th percentiles) for each demographic and age category. Significant predictors of urinary creatinine concentration included age group, sex, race/ethnicity, body mass index, and fat-free mass. Time of day that urine samples were collected made a small but statistically significant difference in creatinine concentrations. For an individual, the creatinine-adjusted concentration of an analyte should be compared with a “reference” range derived from persons in a similar demographic group (e.g., children with children, adults with adults). For multiple regression analysis of population groups, we recommend that the analyte concentration (unadjusted for creatinine) should be included in the analysis with urinary creatinine added as a separate independent variable. This approach allows the urinary analyte concentration to be appropriately adjusted for urinary creatinine and the statistical significance of other variables in the model to be independent of effects of creatinine concentration.
PMCID: PMC1277864  PMID: 15687057
biomonitoring; creatinine; creatinine adjustment; urine
15.  Concentrations of dialkyl phosphate metabolites of organophosphorus pesticides in the U.S. population. 
Environmental Health Perspectives  2004;112(2):186-200.
We report population-based concentrations, stratified by age, sex, and racial/ethnic groups, of dialkyl phosphate (DAP) metabolites of multiple organophosphorus pesticides. We measured dimethylphosphate (DMP), dimethylthiophosphate (DMTP), dimethyldithiophosphate (DMDTP), diethylphosphate (DEP), diethylthiophosphate (DETP), and diethyldithiophosphate (DEDTP) concentrations in 1,949 urine samples collected in U.S. residents 6-59 years of age during 1999 and 2000 as a part of the ongoing National Health and Nutrition Examination Survey (NHANES). We detected each DAP metabolite in more than 50% of the samples, with DEP being detected most frequently (71%) at a limit of detection of 0.2 microg/L. The geometric means for the metabolites detected in more than 60% of the samples were 1.85 microg/L for DMTP and 1.04 microg/L for DEP. The 95th percentiles for each metabolite were DMP, 13 microg/L; DMTP, 46 microg/L; DMDTP, 19 micro g/L; DEP, 13 microg/L; DETP, 2.2 microg/L; and DEDTP, 0.87 microg/L. We determined the molar sums of the dimethyl-containing and diethyl-containing metabolites; their geometric mean concentrations were 49.4 and 10.5 nmol/L, respectively, and their 95th percentiles were 583 and 108 nmol/L, respectively. These data are also presented as creatinine-adjusted concentrations. Multivariate analyses showed concentrations of DAPs in children 6-11 years of age that were consistently significantly higher than in adults and often higher than in adolescents. Although the concentrations between sexes and among racial/ethnic groups varied, no significant differences were observed. These data will be important in evaluating the impact of organophosphorus pesticide exposure in the U.S. population and the effectiveness of regulatory actions.
PMCID: PMC1241828  PMID: 14754573
16.  Measurement of p-nitrophenol in the urine of residents whose homes were contaminated with methyl parathion. 
Environmental Health Perspectives  2002;110(Suppl 6):1085-1091.
During the last several years, illegal commercial application of methyl parathion (MP) in domestic settings in several U.S. Southeastern and Midwestern States has affected largely inner-city residents. As part of a multiagency response involving the U.S. Environmental Protection Agency (U.S. EPA), the Agency for Toxic Substances and Disease Registry (ATSDR), and state and local health departments, our laboratory developed a rapid, high-throughput, selective method for quantifying p-nitrophenol (PNP), a biomarker of MP exposure, using isotope dilution high-performance liquid chromatography-tandem mass spectrometry. We measured PNP in approximately 16,000 samples collected from residents of seven different states. Using this method, we were able to receive sample batches from each state; prepare, analyze, and quantify the samples for PNP; verify the results; and report the data to the health departments and ATSDR in about 48 hr. These data indicate that many residents had urinary PNP concentrations well in excess of those of the general U.S. population. In fact, their urinary PNP concentrations were more consistent with those seen in occupational settings or in poisoning cases. Although these data, when coupled with other MP metabolite data, suggest that many residents with the highest concentrations of urinary PNP had significant exposure to MP, they do not unequivocally rule out exposure to PNP resulting from environmental degradation of MP. Even with their limitations, these data were used with the assumption that all PNP was derived from MP exposure, which enabled the U.S. EPA and ATSDR to develop a comprehensive, biologically driven response that was protective of human health, especially susceptible populations, and included clinical evaluations, outreach activities, community education, integrated pest management, and decontamination of homes.
PMCID: PMC1241298  PMID: 12634145

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