Publications by authors named "Brian S Hooker"

22 Publications

  • Page 1 of 1

Analysis of health outcomes in vaccinated and unvaccinated children: Developmental delays, asthma, ear infections and gastrointestinal disorders.

SAGE Open Med 2020 27;8:2050312120925344. Epub 2020 May 27.

Institute of Medical and Scientific Inquiry, Santa Fe, NM, USA.

Objective: The aim of this study was to compare the health of vaccinated versus unvaccinated pediatric populations.

Methods: Using data from three medical practices in the United States with children born between November 2005 and June 2015, vaccinated children were compared to unvaccinated children during the first year of life for later incidence of developmental delays, asthma, ear infections and gastrointestinal disorders. All diagnoses utilized International Classification of Diseases-9 and International Classification of Diseases-10 codes through medical chart review. Subjects were a minimum of 3 years of age, stratified based on medical practice, year of birth and gender and compared using a logistic regression model.

Results: Vaccination before 1 year of age was associated with increased odds of developmental delays (OR = 2.18, 95% CI 1.47-3.24), asthma (OR = 4.49, 95% CI 2.04-9.88) and ear infections (OR = 2.13, 95% CI 1.63-2.78). In a quartile analysis, subjects were grouped by number of vaccine doses received in the first year of life. Higher odds ratios were observed in Quartiles 3 and 4 (where more vaccine doses were received) for all four health conditions considered, as compared to Quartile 1. In a temporal analysis, developmental delays showed a linear increase as the age cut-offs increased from 6 to 12 to 18 to 24 months of age (ORs = 1.95, 2.18, 2.92 and 3.51, respectively). Slightly higher ORs were also observed for all four health conditions when time permitted for a diagnosis was extended from ⩾ 3 years of age to ⩾ 5 years of age.

Conclusion: In this study, which only allowed for the calculation of unadjusted observational associations, higher ORs were observed within the vaccinated versus unvaccinated group for developmental delays, asthma and ear infections. Further study is necessary to understand the full spectrum of health effects associated with childhood vaccination.
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http://dx.doi.org/10.1177/2050312120925344DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7268563PMC
May 2020

Systematic Assessment of Research on Autism Spectrum Disorder (ASD) and Mercury Reveals Conflicts of Interest and the Need for Transparency in Autism Research.

Sci Eng Ethics 2017 12;23(6):1691-1718

Institute of Chronic Illnesses, Inc, 14 Redgate Court, Silver Spring, MD, 20905, USA.

Historically, entities with a vested interest in a product that critics have suggested is harmful have consistently used research to back their claims that the product is safe. Prominent examples are: tobacco, lead, bisphenol A, and atrazine. Research literature indicates that about 80-90% of studies with industry affiliation found no harm from the product, while only about 10-20% of studies without industry affiliation found no harm. In parallel to other historical debates, recent studies examining a possible relationship between mercury (Hg) exposure and autism spectrum disorder (ASD) show a similar dichotomy. Studies sponsored and supported by industry or entities with an apparent conflict of interest have most often shown no evidence of harm or no "consistent" evidence of harm, while studies without such affiliations report positive evidence of a Hg/autism association. The potentially causal relationship between Hg exposure and ASD differs from other toxic products since there is a broad coalition of entities for whom a conflict of interest arises. These include influential governmental public health entities, the pharmaceutical industry, and even the coal burning industry. This review includes a systematic literature search of original studies on the potential relationship between Hg and ASD from 1999 to August 2015, finding that of the studies with public health and/or industry affiliation, 86% reported no relationship between Hg and ASD. However, among studies without public health and/or industry affiliation, only 21% find no relationship between Hg and ASD. The discrepancy in these results suggests a bias indicative of a conflict of interest.
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http://dx.doi.org/10.1007/s11948-017-9983-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5705731PMC
December 2017

Influenza Vaccination in the First Trimester of Pregnancy and Risk of Autism Spectrum Disorder.

Authors:
Brian S Hooker

JAMA Pediatr 2017 06;171(6):600

Simpson University, Redding, California.

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http://dx.doi.org/10.1001/jamapediatrics.2017.0734DOI Listing
June 2017

Thimerosal-Preserved Hepatitis B Vaccine and Hyperkinetic Syndrome of Childhood.

Brain Sci 2016 Mar 15;6(1). Epub 2016 Mar 15.

Institute of Chronic Illnesses, Inc., Silver Spring, MD 20905, USA.

(1) BACKGROUND: Hyperkinetic syndrome of childhood (HKSoC) is an International Classification of Diseases, Ninth Revision, Clinical Modification (ICD-9) category in which the majority of the children are also diagnosed under the Diagnostic and Statistical Manual of Mental Disorders, 4th Edition, Text Revision (DSM-IV-TR), where the umbrella term is "Attention-Deficit and Disruptive Behavior Disorders". The diagnostic criteria for HKSoC are developmentally inappropriate inattention, hyperactivity, and impulsivity. Some studies have implicated mercury (Hg) exposure as a risk factor. (2) METHODS: This hypothesis testing study; using the Vaccine Safety Datalink; assessed the toxicological effects of bolus exposure to organic-Hg from Thimerosal-containing vaccines (TCVs) by examining the relationship between Thimerosal-preserved hepatitis B vaccines (TM-HepB) given at varying levels and at specific intervals in the first six months after birth and the risk of a child being diagnosed with HKSoC. (3) RESULTS: Children diagnosed with HKSoC were significantly more likely to be exposed to increased organic-Hg from TM-HepB doses given within the first month (odds ratio = 1.45; 95% confidence interval (CI) = 1.30-1.62); within the first two months (odds ratio = 1.43; 95% CI = 1.28-1.59); and within the first six months (odds ratio = 4.51; 95% CI = 3.04-6.71) than controls. (4) CONCLUSION: The results indicate that increasing organic-Hg exposure from TCVs heightens the risk of a HKSoC diagnosis.
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http://dx.doi.org/10.3390/brainsci6010009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4810179PMC
March 2016

A Prospective Longitudinal Assessment of Medical Records for Diagnostic Substitution among Subjects Diagnosed with a Pervasive Developmental Disorder in the United States.

Front Pediatr 2015 12;3:85. Epub 2015 Oct 12.

The Institute of Chronic Illnesses, Inc , Silver Spring, MD , USA.

Background: Previously, investigators suggested that diagnostic substitution from other diagnoses, e.g., mental retardation (MR) and/or cerebral palsy (CP) to pervasive developmental disorder (PDD) is a driving factor behind increases in autism. This study evaluated potential diagnostic substitution among subjects diagnosed with PDD vs. MR or CP by examining birth characteristic overlap.

Methods: SAS(®) and StatsDirect software examined medical records for subjects within the Vaccine Safety Datalink database who were Health Maintenance Organization-enrolled from birth until diagnosed with an International Classification of Disease, 9th revision (ICD-9) outcome of PDD (299.xx, n = 84), CP (343.xx, n = 300), or MR (317.xx, 318.xx, or 319.xx, n = 51).

Results: Subjects with PDD had significantly (p < 0.01) increased: male/female ratio (PDD = 5.5 vs. CP = 1.5 or MR = 1.3), mean age of initial diagnosis in years (PDD = 3.13 vs. CP = 1.09 or MR = 1.62), mean gestational age in weeks at birth (PDD = 38.73 vs. CP = 36.20 or MR = 34.84), mean birth weight in grams (PDD = 3,368 vs. CP = 2,767 or MR = 2,406), and mean Appearance-Pulse-Grimace-Activity-Respiration scores at 1 min (PDD = 7.82 vs. CP = 6.37 or MR = 6.76) and 5 min (PDD = 8.77 vs. CP = 7.92 or MR = 8.04), as compared to subjects diagnosed with CP or MR.

Conclusion: This study suggests diagnostic substitution cannot fully explain increased PDD prevalence during the 1990s within the United States.
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http://dx.doi.org/10.3389/fped.2015.00085DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4600915PMC
November 2015

A longitudinal cohort study of the relationship between Thimerosal-containing hepatitis B vaccination and specific delays in development in the United States: Assessment of attributable risk and lifetime care costs.

J Epidemiol Glob Health 2016 06 9;6(2):105-18. Epub 2015 Jul 9.

Institute of Chronic Illnesses, Inc, Silver Spring, MD, USA.

Epidemiological evidence suggests a link between mercury (Hg) exposure from Thimerosal-containing vaccines and specific delays in development. A hypothesis-testing longitudinal cohort study (n=49,835) using medical records in the Vaccine Safety Datalink (VSD) was undertaken to evaluate the relationship between exposure to Hg from Thimerosal-containing hepatitis B vaccines (T-HBVs) administered at specific intervals in the first 6months of life and specific delays in development [International Classification of Disease, 9th revision (ICD-9): 315.xx] among children born between 1991 and 1994 and continuously enrolled from birth for at least 5.81years. Infants receiving increased Hg doses from T-HBVs administered within the first month, the first 2months, and the first 6months of life were significantly more likely to be diagnosed with specific delays in development than infants receiving no Hg doses from T-HBVs. During the decade in which T-HBVs were routinely recommended and administered to US infants (1991-2001), an estimated 0.5-1million additional US children were diagnosed with specific delays in development as a consequence of 25μg or 37.5μg organic Hg from T-HBVs administered within the first 6months of life. The resulting lifetime costs to the United States may exceed $1 trillion.
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http://dx.doi.org/10.1016/j.jegh.2015.06.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7320444PMC
June 2016

Thimerosal exposure and increased risk for diagnosed tic disorder in the United States: a case-control study.

Interdiscip Toxicol 2015 Jun;8(2):68-76

Institute of Chronic Illnesses, Inc., 14 Redgate Ct, Silver Spring, MD, USA.

A hypothesis testing, case-control study evaluated automated medical records for exposure to organic-Hg from Thimerosal-containing hepatitis B vaccines (TM-HepB) administered at specific intervals in the first six-months-of-life among cases diagnosed with a tic disorder (TD) or cerebral degeneration (CD) (an outcome not biologically plausibly linked to TM exposure) in comparison to controls; both cases and controls were continuously enrolled from birth (born from 1991-2000) within the Vaccine Safety Datalink (VSD) database. TD cases were significantly more likely than controls to have received increased organic-Hg from TM-HepB administered within the first month-of-life (odds ratio (OR)=1.59, p<0.00001), first two-months-of-life (OR=1.59, p<0.00001), and first six-months-of-life (OR=2.97, p<0.00001). Male TD cases were significantly more likely than male controls to have received increased organic-Hg from TM-HepB administered within the first month-of-life (OR =1.65, p<0.0001), first two-months-of-life (OR=1.64, p<0.0001), and first six months-of-life (OR=2.47, p<0.05), where as female TD were significantly more likely than female controls to have received increased organic-Hg from TM-HepB administered within the first six-months-of-life (OR=4.97, p<0.05). By contrast, CD cases were no more likely than controls to have received increased organic-Hg exposure from TM-HepB administered at any period studied within the first six-months-of-life. Although routine childhood vaccination is considered an important public health tool to combat infectious diseases, the present study associates increasing organic-Hg exposure from TM-HepB and the subsequent risk of a TD diagnosis.
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http://dx.doi.org/10.1515/intox-2015-0011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4961900PMC
June 2015

Thimerosal: clinical, epidemiologic and biochemical studies.

Clin Chim Acta 2015 Apr 21;444:212-20. Epub 2015 Feb 21.

Institute of Chronic Illnesses, Inc., 14 Redgate Ct., Silver Spring, MD 20905, USA. Electronic address:

Introduction: Thimerosal (or Thiomersal) is a trade name for an organomercurial compound (sodium ethyl-mercury (Hg) thiosalicylate) that is 49.55% Hg by weight, which rapidly decomposes in aqueous saline solutions into ethyl-Hg hydroxide and ethyl-Hg chloride. Developed in 1927, it has been and is still being used as a preservative in some cosmetics, topical pharmaceuticals, and biological drug products, including vaccines. Concerns have been voiced about its use because it is toxic to human cells. Although it is banned in several countries, it continues to be added to some vaccines in the United States and many vaccines in the developing world.

Discussion: This critical review focuses on the clinical, epidemiological, and biochemical studies of adverse effects from Thimerosal in developing humans. This review will include research that examines fetal, infant, and childhood death; birth defects; neurodevelopmental testing deficits in children; and neurodevelopmental disorders (attention deficit/hyperactivity disorder, autism spectrum disorder, tic disorder, and specific developmental delays). The review will also look at the research that examined the outcomes of acute accidental ethyl-Hg poisoning in humans. The studies that examine the underlying biochemical insights into the neuronal cellular damage will also be explored.

Conclusion: The culmination of the research that examines the effects of Thimerosal in humans indicates that it is a poison at minute levels with a plethora of deleterious consequences, even at the levels currently administered in vaccines.
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http://dx.doi.org/10.1016/j.cca.2015.02.030DOI Listing
April 2015

Thimerosal-containing hepatitis B vaccination and the risk for diagnosed specific delays in development in the United States: a case-control study in the vaccine safety datalink.

N Am J Med Sci 2014 Oct;6(10):519-31

Institute of Chronic Illnesses, Inc, Silver Spring, Maryland, USA.

Background: Within the first 3 years of life, the brain develops rapidly. Its development is characterized by critical developmental periods for speech, vision, hearing, language, balance, etc.; and alteration in any of the processes occurring in those critical periods can lead to specific delays in development.

Aims: The present study evaluated the potential toxic effects of organic-mercury exposure from Thimerosal (49.55% mercury by weight) in childhood vaccines and its hypothesized possible relationship with specific delays in development.

Materials And Methods: A hypothesis testing case-control study was undertaken to evaluate the relationship between exposure to Thimerosal-containing hepatitis B vaccines administered at specific intervals in the first 6 months among cases diagnosed with specific delays in development and controls born between 1991-2000, utilizing data in the Vaccine Safety Datalink database.

Results: Cases were significantly more likely than controls to have received increased organic-mercury from Thimerosal-containing hepatitis B vaccine administered in the first, second, and sixth month of life.

Conclusion: Though routine childhood vaccination may be an important public health tool to reduce the morbidity and mortality associated with infectious diseases, the present study supports an association between increasing organic-mercury exposure from Thimerosal-containing childhood vaccines and the subsequent risk of specific delays in development among males and females.
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http://dx.doi.org/10.4103/1947-2714.143284DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4215490PMC
October 2014

A dose-response relationship between organic mercury exposure from thimerosal-containing vaccines and neurodevelopmental disorders.

Int J Environ Res Public Health 2014 Sep 5;11(9):9156-70. Epub 2014 Sep 5.

Institute of Chronic Illnesses, Inc., 14 Redgate Ct., Silver Spring, MD 20905, USA.

A hypothesis testing case-control study evaluated concerns about the toxic effects of organic-mercury (Hg) exposure from thimerosal-containing (49.55% Hg by weight) vaccines on the risk of neurodevelopmental disorders (NDs). Automated medical records were examined to identify cases and controls enrolled from their date-of-birth (1991-2000) in the Vaccine Safety Datalink (VSD) project. ND cases were diagnosed with pervasive developmental disorder (PDD), specific developmental delay, tic disorder or hyperkinetic syndrome of childhood. In addition, putative non-thimerosal-related outcomes of febrile seizure, failure to thrive and cerebral degenerations were examined. The cumulative total dose of Hg exposure from thimerosal-containing hepatitis B vaccine (T-HBV) administered within the first six months of life was calculated. On a per microgram of organic-Hg basis, PDD (odds ratio (OR) = 1.054), specific developmental delay (OR = 1.035), tic disorder (OR = 1.034) and hyperkinetic syndrome of childhood (OR = 1.05) cases were significantly more likely than controls to receive increased organic-Hg exposure. By contrast, none of the non-thimerosal related outcomes were significantly more likely than the controls to have received increased organic-Hg exposure. Routine childhood vaccination may be an important public health tool to reduce infectious disease-associated morbidity/mortality, but the present study significantly associates organic-Hg exposure from T-HBV with an increased risk of an ND diagnosis.
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http://dx.doi.org/10.3390/ijerph110909156DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4199012PMC
September 2014

An Evaluation of the Effect of Increasing Parental Age on the Phenotypic Severity of Autism Spectrum Disorder.

J Child Neurol 2014 Aug 27. Epub 2014 Aug 27.

The Institute of Chronic Illnesses, Inc, Silver Spring, MD, USA

It was recently postulated that because increased genetic load and increased parental age are both purportedly associated with the risk to develop an autism spectrum disorder, there must be a linkage between increasing genetic load and increasing parental age in autism spectrum disorder pathogenesis. The present study examined the hypothesis that if increased genetic load from increasing paternal age is important to autism spectrum disorder pathogenesis, then there should be a significant relationship between increasing parental age and increasing autism spectrum disorder phenotypic severity. Outpatient clinical records were retrospectively examined to identify a consecutive cohort of subjects diagnosed with an autism spectrum disorder (n = 351). Increasing autism spectrum disorder phenotypic severity was found not to be associated with increasing maternal/paternal age. The present study failed to support the hypothesis that increasing parental age was associated with increasing autism spectrum disorder phenotypic severity, but future studies should examine the relationship between genetic mutations in subjects diagnosed with an autism spectrum disorder and increasing parental age.
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http://dx.doi.org/10.1177/0883073814541478DOI Listing
August 2014

Measles-mumps-rubella vaccination timing and autism among young African American boys: a reanalysis of CDC data.

Authors:
Brian S Hooker

Transl Neurodegener 2014 8;3:16. Epub 2014 Aug 8.

Simpson University, Redding, CA, USA.

Background: A significant number of children diagnosed with autism spectrum disorder suffer a loss of previously-acquired skills, suggesting neurodegeneration or a type of progressive encephalopathy with an etiological basis occurring after birth. The purpose of this study is to investigate the effectof the age at which children got their first Measles-Mumps-Rubella (MMR) vaccine on autism incidence. This is a reanalysis of the data set, obtained from the U.S. Centers for Disease Control and Protection (CDC), used for the Destefano et al. 2004 publication on the timing of the first MMR vaccine and autism diagnoses.

Methods: The author embarked on the present study to evaluate whether a relationship exists between child age when the first MMR vaccine was administered among cases diagnosed with autism and controls born between 1986 through 1993 among school children in metropolitan Atlanta. The Pearson's chi-squared method was used to assess relative risks of receiving an autism diagnosis within the total cohort as well as among different race and gender categories.

Results: When comparing cases and controls receiving their first MMR vaccine before and after 36 months of age, there was a statistically significant increase in autism cases specifically among African American males who received the first MMR prior to 36 months of age. Relative risks for males in general and African American males were 1.69 (p=0.0138) and 3.36 (p=0.0019), respectively. Additionally, African American males showed an odds ratio of 1.73 (p=0.0200) for autism cases in children receiving their first MMR vaccine prior to 24 months of age versus 24 months of age and thereafter.

Conclusions: The present study provides new epidemiologic evidence showing that African American males receiving the MMR vaccine prior to 24 months of age or 36 months of age are more likely to receive an autism diagnosis.
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http://dx.doi.org/10.1186/2047-9158-3-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4128611PMC
August 2014

A two-phase study evaluating the relationship between Thimerosal-containing vaccine administration and the risk for an autism spectrum disorder diagnosis in the United States.

Transl Neurodegener 2013 Dec 19;2(1):25. Epub 2013 Dec 19.

The Institute of Chronic Illnesses Inc, 14 Redgate Ct, Silver Spring, MD, USA.

Background: Autism spectrum disorder (ASD) is defined by standardized criteria of qualitative impairments in social interaction, qualitative impairments in communication, and restricted and stereotyped patterns of behavior, interests, and activities. A significant number of children diagnosed with ASD suffer a loss of previously-acquired skills, which is suggestive of neurodegeneration or a type of progressive encephalopathy with an etiological pathogenic basis occurring after birth. To date, the etiology of ASD remains under debate, however, many studies suggest toxicity, especially from mercury (Hg), in individuals diagnosed with an ASD. The present study evaluated concerns about the toxic effects of organic-Hg exposure from Thimerosal (49.55% Hg by weight) in childhood vaccines by conducting a two-phased (hypothesis generating/hypothesis testing) study with documented exposure to varying levels of Thimerosal from vaccinations.

Methods: A hypothesis generating cohort study was undertaken to evaluate the relationship between exposure to organic-Hg from a Thimerosal-containing Diphtheria-Tetanus-acellular-Pertussis (DTaP) vaccine in comparison to a Thimerosal-free DTaP vaccine administered, from 1998 through 2000, for the risk of ASD as reported in the Vaccine Adverse Event Reporting System (VAERS) database (phase I). A hypothesis testing case-control study was undertaken to evaluate the relationship between organic-Hg exposure from Thimerosal-containing hepatitis B vaccines administered at specific intervals in the first six months of life among cases diagnosed with an ASD and controls born between 1991 through 1999 in the Vaccine Safety Datalink (VSD) database (phase II).

Results: In phase I, it was observed that there was a significantly increased risk ratio for the incidence of ASD reported following the Thimerosal-containing DTaP vaccine in comparison to the Thimerosal-free DTaP vaccine. In phase II, it was observed that cases diagnosed with an ASD were significantly more likely than controls to receive increased organic-Hg from Thimerosal-containing hepatitis B vaccine administered within the first, second, and sixth month of life.

Conclusions: Routine childhood vaccination is an important public health tool to reduce the morbidity and mortality associated with infectious diseases, but the present study provides new epidemiological evidence supporting an association between increasing organic-Hg exposure from Thimerosal-containing childhood vaccines and the subsequent risk of an ASD diagnosis.
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http://dx.doi.org/10.1186/2047-9158-2-25DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3878266PMC
December 2013

An analysis pipeline for the inference of protein-protein interaction networks.

Int J Data Min Bioinform 2009 ;3(4):409-30

Computational Sciences and Mathematics Division, Pacific Northwest National Laboratory (US Department of Energy), Richland, WA 99352, USA.

We present a platform for the reconstruction of protein-protein interaction networks inferred from Mass Spectrometry (MS) bait-prey data. The Software Environment for Biological Network Inference (SEBINI), an environment for the deployment of network inference algorithms that use high-throughput data, forms the platform core. Among the many algorithms available in SEBINI is the Bayesian Estimator of Probabilities of Protein-Protein Associations (BEPro3) algorithm, which is used to infer interaction networks from such MS affinity isolation data. Also, the pipeline incorporates the Collective Analysis of Biological Interaction Networks (CABIN) software. We have thus created a structured workflow for protein-protein network inference and supplemental analysis.
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http://dx.doi.org/10.1504/ijdmb.2009.029204DOI Listing
March 2010

A general system for studying protein-protein interactions in Gram-negative bacteria.

J Proteome Res 2008 Aug 1;7(8):3319-28. Epub 2008 Jul 1.

Biosciences Division, Chemical Sciences Division, Computer Science and Mathematics Division, and Physical Sciences Directorate, Oak Ridge National Laboratory, Oak Ridge, TN 37831, USA.

One of the most promising methods for large-scale studies of protein interactions is isolation of an affinity-tagged protein with its in vivo interaction partners, followed by mass spectrometric identification of the copurified proteins. Previous studies have generated affinity-tagged proteins using genetic tools or cloning systems that are specific to a particular organism. To enable protein-protein interaction studies across a wider range of Gram-negative bacteria, we have developed a methodology based on expression of affinity-tagged "bait" proteins from a medium copy-number plasmid. This construct is based on a broad-host-range vector backbone (pBBR1MCS5). The vector has been modified to incorporate the Gateway DEST vector recombination region, to facilitate cloning and expression of fusion proteins bearing a variety of affinity, fluorescent, or other tags. We demonstrate this methodology by characterizing interactions among subunits of the DNA-dependent RNA polymerase complex in two metabolically versatile Gram-negative microbial species of environmental interest, Rhodopseudomonas palustris CGA010 and Shewanella oneidensis MR-1. Results compared favorably with those for both plasmid and chromosomally encoded affinity-tagged fusion proteins expressed in a model organism, Escherichia coli.
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http://dx.doi.org/10.1021/pr8001832DOI Listing
August 2008

Methods for mapping of interaction networks involving membrane proteins.

Biochem Biophys Res Commun 2007 Nov 19;363(3):457-61. Epub 2007 Sep 19.

Pacific Northwest National Laboratory, Richland, Washington, USA.

Nearly one-third of all genes in various organisms encode membrane-associated proteins that participate in numerous protein-protein interactions important to the processes of life. However, membrane protein interactions pose significant challenges due to the need to solubilize membranes without disrupting protein-protein interactions. Traditionally, analysis of isolated protein complexes by high-resolution 2D gel electrophoresis has been the main method used to obtain an overall picture of proteome constituents and interactions. However, this method is time consuming, labor intensive, detects only abundant proteins and is limited with respect to the coverage required to elucidate large interaction networks. In this review, we discuss the application of various methods to elucidate interactions involving membrane proteins. These techniques include methods for the direct isolation of single complexes or interactors as well as methods for characterization of entire subcellular and cellular interactomes.
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http://dx.doi.org/10.1016/j.bbrc.2007.09.031DOI Listing
November 2007

Statistically inferring protein-protein associations with affinity isolation LC-MS/MS assays.

J Proteome Res 2007 Sep 11;6(9):3788-95. Epub 2007 Aug 11.

Clemson University, 237 Barre Hall, Clemson, South Carolina 29634-0313, Pacific Northwest National Laboratory, P.O. Box 999, Richland, Washington 99352, USA.

Affinity isolation of protein complexes followed by protein identification by LC-MS/MS is an increasingly popular approach for mapping protein interactions. However, systematic and random assay errors from multiple sources must be considered to confidently infer authentic protein-protein interactions. To address this issue, we developed a general, robust statistical method for inferring authentic interactions from protein prey-by-bait frequency tables using a binomial-based likelihood ratio test (LRT) coupled with Bayes' Odds estimation. We then applied our LRT-Bayes' algorithm experimentally using data from protein complexes isolated from Rhodopseudomonas palustris. Our algorithm, in conjunction with the experimental protocol, inferred with high confidence authentic interacting proteins from abundant, stable complexes, but few or no authentic interactions for lower-abundance complexes. The algorithm can discriminate against a background of prey proteins that are detected in association with a large number of baits as an artifact of the measurement. We conclude that the experimental protocol including the LRT-Bayes' algorithm produces results with high confidence but moderate sensitivity. We also found that Monte Carlo simulation is a feasible tool for checking modeling assumptions, estimating parameters, and evaluating the significance of results in protein association studies.
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http://dx.doi.org/10.1021/pr0701106DOI Listing
September 2007

Optimization of Acidothermus cellulolyticus endoglucanase (E1) production in transgenic tobacco plants by transcriptional, post-transcription and post-translational modification.

Transgenic Res 2005 Oct;14(5):627-43

Chemical and Biological Processing Development Group, Process Science and Engineering Division, Pacific Northwest National Laboratory, Richland, WA 99352, USA.

An attempt was made to obtain a high-level production of intact Acidothermus cellulolyticus endoglucanase (E1) in transgenic tobacco plants. The E1 expression was examined under the control of the constitutive and strong Mac promoter or light-inducible tomato Rubisco small sub-unit (RbcS-3C) promoter with its original or Alfalfa Mosaic Virus (AMV) RNA4 5'-untranslated leader (UTL) and targeted to different sub-cellular compartments via transit peptides. The transit peptides included native E1, endoplasmic reticulum, vacuole, apoplast, and chloroplast. E1 expression and its stability in transgenic plants were determined via E1 activity, protein immunoblotting, and RNA gel-blotting analyses. Effects of sub-cellular compartments on E1 production and its stability were determined in transgenic tobacco plants carrying one of six transgene expression vectors, where the E1 was under the control of Mac promoter, mannopine synthase transcription terminator, and one of the five transit peptides. Transgenic tobacco plants with an apoplastic transit peptide had the highest average E1 activity and protein accumulation, which was about 0.25% of total leaf soluble proteins estimated via E1 specific activity and protein gel blots. Intercellular fluid analyses confirmed that E1 signal peptide functioned properly in tobacco cells to secret E1 protein into the apoplast. By replacing RbcS-3C UTL with AMV RNA4 UTL E1 production was enhanced more than twofold, while it was less effective than the mannopine synthase UTL. It was observed that RbcS-3C promoter was more favorable for E1 expression in transgenic plants than the Mac promoter. E1 activity in dried tobacco seeds stored one year at room temperature was 45% higher than that observed immediately after harvesting, suggesting that E1 protein can be stored at room temperature for a long period. E1 stability in different sub-cellular compartments and the optimal combination of promoter, 5'-UTL, and sub-cellular compartmentation for heterologous protein production in transgenic plants are discussed.
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http://dx.doi.org/10.1007/s11248-005-5695-5DOI Listing
October 2005

Simple protein complex purification and identification method for high-throughput mapping of protein interaction networks.

J Proteome Res 2005 Mar-Apr;4(2):268-74

Pacific Northwest National Laboratory, 902 Battelle Blvd., P.O. Box 999, Richland, Washington 99354, USA.

Most current methods for purification and identification of protein complexes use endogenous expression of affinity-tagged bait, tandem affinity tag purification of protein complexes followed by specific elution of complexes from beads, and gel separation and in-gel digestion prior to mass spectrometric analysis of protein interactors. We propose a single affinity tag in vitro pull-down assay with denaturing elution, trypsin digestion in organic solvent, and LC-ESI MS/MS protein identification using SEQUEST analysis. Our method is simple and easy to scale-up and automate, making it suitable for high-throughput mapping of protein interaction networks and functional proteomics.
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http://dx.doi.org/10.1021/pr049847aDOI Listing
July 2005

Overexpression of multi-heme C-type cytochromes.

Biotechniques 2005 Feb;38(2):297-9

Pacific Northwest National Laboratory, Richland, WA, USA.

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http://dx.doi.org/10.2144/05382PT01DOI Listing
February 2005

Expression of functional human coagulation factor XIII A-domain in plant cell suspensions and whole plants.

Protein Expr Purif 2004 Sep;37(1):89-96

Battelle Memorial Institute, 902 Battelle Blvd., Richland, WA 99352, USA.

Coagulation factor XIII, a zymogen present in blood as a tetramer (A2B2) of A- and B-domains, is one of the components of many "wound sealants" which are proposed for use or currently in use as effective hemostatic agents, sealants, and tissue adhesives in surgery. After activation by alpha-thrombin cleavage, coagulation factor XIII A-domain, a transglutaminase, is formed and catalyzes the covalent cross-linking of the alpha- and gamma-chains of linear fibrin to form homopolymers, which can quickly stop bleeding. We have successfully expressed the A-domain of factor XIII in both plant cell cultures and whole plants. Transgenic plant cell culture allows a rapid method for testing production feasibility while expression in whole plants demonstrates an economic production system for recombinant human plasma-based proteins. The expressed factor XIII A-domain had a similar size as that of human plasma-derived factor XIII. Crude plant extract containing recombinant factor XIII A-domain showed transglutaminase activity with monodansylcadaverine and casein as substrates and cross-linking activity in the presence of linear fibrin. The expression of factor XIII A-domain was not affected by plant leaf position.
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http://dx.doi.org/10.1016/j.pep.2004.04.022DOI Listing
September 2004