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author:("Li, tingling")
1.  Near real-time adverse drug reaction surveillance within population-based health networks: methodology considerations for data accrual 
Purpose
This study describes practical considerations for implementation of near real-time medical product safety surveillance in a distributed health data network.
Methods
We conducted pilot active safety surveillance comparing generic divalproex sodium to historical branded product at 4 health plans from April – October 2009. Outcomes reported are all-cause emergency room (ER) visits and fractures. One retrospective data extract was completed (1/2002–6/2008), followed by seven prospective monthly extracts (1/2008–11/2009). To evaluate delays in claims processing, we used three analytic approaches: near real-time sequential analysis, sequential analysis with 1.5 month delay, and nonsequential (using final retrospective data). Sequential analyses used the maximized sequential probability ratio test. Procedural and logistical barriers to active surveillance were documented.
Results
We identified 6,586 new users of generic divalproex sodium and 43,960 new users of the branded product. Quality control methods identified 16 extract errors, which were corrected. Near real-time extracts captured 87.5% of ER visits and 50.0% of fractures, which improved to 98.3% and 68.7% respectively with 1.5 month delay. We did not identify signals for either outcome regardless of extract timeframe; slight differences in the test statistic and relative risk estimates were found.
Conclusions
Near real-time sequential safety surveillance is feasible, but several barriers warrant attention. Data quality review of each data extract was necessary. Although signal detection was not affected by delay in analysis, when using a historical control group differential accrual between exposure and outcomes may theoretically bias near real-time risk estimates towards the null, causing failure to detect a signal.
doi:10.1002/pds.3412
PMCID: PMC3644310  PMID: 23401239
Drug Surveillance; Prospective Analysis; Analytic Methods
2.  Outcomes After Periodic Use of Inhaled Corticosteroids in Children 
Background
Many children with persistent asthma use inhaled corticosteroids on a periodic basis. Clinical trials in adults suggest that periodic use of inhaled corticosteroids may be effective for patients with mild persistent asthma. However, scant information exists on the clinical outcomes of children with asthma who are using inhaled corticosteroids on a periodic basis in real-world settings.
Objective
This prospective cohort study compared clinical outcomes during a 12-month follow-up period between children with persistent asthma whose parents believed that they were supposed to use inhaled steroids either (a) periodically or (b) daily year-round at the start of the period. The clinical outcomes studied were (1) asthma-related emergency department (ED) visits or hospitalizations, (2) uncontrolled asthma based on health care and medication use, and (3) outpatient visits for asthma.
Patients and methods
The study population included children with persistent asthma from two health plans whose parents reported that they were using inhaled corticosteroids during a baseline telephone interview. The interviews collected information on whether the children’s parents believed they were supposed to use inhaled corticosteroids on a periodic or daily basis, as well as baseline asthma symptom status, sociodemographic, and behavioral variables. We used computerized databases to identify clinical events for each child during the 12 months after their baseline interview. Uncontrolled asthma was defined as any asthma-related ED visit or hospitalization, two or more oral steroid prescription fills, or four or more beta-agonists canisters filled during the 12-month period. We compared these outcomes between the periodic versus daily users of inhaled corticosteroids using logistic regression analyses. We conducted both (1) a traditional logistic regression analysis in which we adjusted for selection bias by including covariates such as age, asthma physical status, sociodemographic and behavioral variables, and history of asthma-related health care use during the year before interview and (2) an analysis using propensity scores to more fully adjust for selection bias.
Results
Of a total of 476 children in the study, 55% of parents believed their children were supposed to be using inhaled corticosteroids on a periodic basis and 45% believed their children were supposed to be using them daily year-round based on the baseline parent interview. At baseline, periodic inhaled corticosteroid users had less severe asthma than daily users based on several measures including better asthma physical status scores on the Children’s Health Survey for Asthma (mean 87 ± 16.0 vs. 81 ± 17.4, p = < 0.0001). During the year before the baseline interview, periodic users compared with daily users were less likely to have an ED visit or hospitalization (10% vs. 23%, p = 0.0001) and less likely to have had five or more albuterol prescription fills (13% vs. 31%, p < 0.0001). During the follow-up year, those who believed inhaled steroids were for periodic use were less likely than those who believed inhaled steroids were for daily use to have an ED visit or hospitalization for asthma (OR 0.36, 95% CI: 0.18–0.73), even after adjusting for baseline asthma status and other covariates. Similarly, those who believed inhaled steroids were for periodic use were less likely to have uncontrolled asthma, OR 0.38 (95% CI: 0.24–0.62). Analyses using propensity score adjustment yielded similar results to the logistic regression analyses.
Conclusion
Children whose parents believed they were supposed to use inhaled corticosteroids on a periodic basis had less severe asthma at baseline than those whose parents believed they were supposed to be using them daily. Periodic users were less likely than daily users to have adverse asthma outcomes during 1-year follow-up. This suggests that clinicians may be applying appropriate selection criteria by choosing patients with less severe asthma for periodic inhaled corticosteroid regimens.
doi:10.1080/02770900802468517
PMCID: PMC4004094  PMID: 19544175
asthma; periodic inhaled corticosteroids; children
3.  On weighting approaches for missing data 
We review the class of inverse probability weighting (IPW) approaches for the analysis of missing data under various missing data patterns and mechanisms. The IPW methods rely on the intuitive idea of creating a pseudo-population of weighted copies of the complete cases to remove selection bias introduced by the missing data. However, different weighting approaches are required depending on the missing data pattern and mechanism. We begin with a uniform missing data pattern (i.e., a scalar missing indicator indicating whether or not the full data is observed) to motivate the approach. We then generalize to more complex settings. Our goal is to provide a conceptual overview of existing IPW approaches and illustrate the connections and differences among these approaches.
doi:10.1177/0962280211403597
PMCID: PMC3998729  PMID: 21705435
missing data; inverse probability weighting; missing at random; missing not at random; monotone missing; non-monotone missing
4.  Automated Detection and Classification of Type 1 Versus Type 2 Diabetes Using Electronic Health Record Data 
Diabetes Care  2013;36(4):914-921.
OBJECTIVE
To create surveillance algorithms to detect diabetes and classify type 1 versus type 2 diabetes using structured electronic health record (EHR) data.
RESEARCH DESIGN AND METHODS
We extracted 4 years of data from the EHR of a large, multisite, multispecialty ambulatory practice serving ∼700,000 patients. We flagged possible cases of diabetes using laboratory test results, diagnosis codes, and prescriptions. We assessed the sensitivity and positive predictive value of novel combinations of these data to classify type 1 versus type 2 diabetes among 210 individuals. We applied an optimized algorithm to a live, prospective, EHR-based surveillance system and reviewed 100 additional cases for validation.
RESULTS
The diabetes algorithm flagged 43,177 patients. All criteria contributed unique cases: 78% had diabetes diagnosis codes, 66% fulfilled laboratory criteria, and 46% had suggestive prescriptions. The sensitivity and positive predictive value of ICD-9 codes for type 1 diabetes were 26% (95% CI 12–49) and 94% (83–100) for type 1 codes alone; 90% (81–95) and 57% (33–86) for two or more type 1 codes plus any number of type 2 codes. An optimized algorithm incorporating the ratio of type 1 versus type 2 codes, plasma C-peptide and autoantibody levels, and suggestive prescriptions flagged 66 of 66 (100% [96–100]) patients with type 1 diabetes. On validation, the optimized algorithm correctly classified 35 of 36 patients with type 1 diabetes (raw sensitivity, 97% [87–100], population-weighted sensitivity, 65% [36–100], and positive predictive value, 88% [78–98]).
CONCLUSIONS
Algorithms applied to EHR data detect more cases of diabetes than claims codes and reasonably discriminate between type 1 and type 2 diabetes.
doi:10.2337/dc12-0964
PMCID: PMC3609529  PMID: 23193215
5.  Complete Genome Sequence of Mycobacterium avium subsp. paratuberculosis, Isolated from Human Breast Milk 
Genome Announcements  2014;2(1):e01252-13.
Mycobacterium avium subsp. paratuberculosis is the etiologic agent of Johne’s disease in ruminants and has also been associated with human Crohn’s disease. We report the complete genome sequence of M. avium subsp. paratuberculosis, isolated from the breast milk of a Crohn’s disease patient. This sequence has high identity with characterized strains recovered from cattle.
doi:10.1128/genomeA.01252-13
PMCID: PMC3916490  PMID: 24503996
6.  Evaluation of novel oral vaccine candidates and validation of a caprine model of Johne's disease 
Johne's disease (JD) caused by Mycobacterium avium subspecies paratuberculosis (MAP) is a major threat to the dairy industry and possibly some cases of Crohn's disease in humans. A MAP vaccine that reduced of clinical disease and/or reduced fecal shedding would aid in the control of JD. The objectives of this study were (1) to evaluate the efficacy of 5 attenuated strains of MAP as vaccine candidates compared to a commercial control vaccine using the protocol proposed by the Johne's Disease Integrated Program (JDIP) Animal Model Standardization Committee (AMSC), and (2) to validate the AMSC Johne's disease goat challenge model. Eighty goat kids were vaccinated orally twice at 8 and 10 weeks of age with an experimental vaccine or once subcutaneously at 8 weeks with Silirum® (Zoetis), or a sham control oral vaccine at 8 and 10 weeks. Kids were challenged orally with a total of approximately 1.44 × 109 CFU divided in two consecutive daily doses using MAP ATCC-700535 (K10-like bovine isolate). All kids were necropsied at 13 months post-challenge. Results indicated that the AMSC goat challenge model is a highly efficient and valid model for JD challenge studies. None of the experimental or control vaccines evaluated prevented MAP infection or eliminated fecal shedding, although the 329 vaccine lowered the incidence of infection, fecal shedding, tissue colonization and reduced lesion scores, but less than the control vaccine. Based on our results the relative performance ranking of the experimental live-attenuated vaccines evaluated, the 329 vaccine was the best performer, followed by the 318 vaccine, then 316 vaccine, 315 vaccine and finally the 319 vaccine was the worst performer. The subcutaneously injected control vaccine outperformed the orally-delivered mutant vaccine candidates. Two vaccines (329 and 318) do reduce presence of JD gross and microscopic lesions, slow progression of disease, and one vaccine (329) reduced fecal shedding and tissue colonization.
doi:10.3389/fcimb.2014.00026
PMCID: PMC3941644  PMID: 24624365
Mycobacterium avium subsp paratuberculosis; vaccine efficacy; mutant vaccines; diagnostic tests; goats
7.  Sensitivity analysis for causal inference using inverse probability weighting 
Evaluation of impact of potential uncontrolled confounding is an important component for causal inference based on observational studies. In this article, we introduce a general framework of sensitivity analysis that is based on inverse probability weighting. We propose a general methodology that allows both non-parametric and parametric analyses, which are driven by two parameters that govern the magnitude of the variation of the multiplicative errors of the propensity score and their correlations with the potential outcomes. We also introduce a specific parametric model that offers a mechanistic view on how the uncontrolled confounding may bias the inference through these parameters. Our method can be readily applied to both binary and continuous outcomes and depends on the covariates only through the propensity score that can be estimated by any parametric or non-parametric method. We illustrate our method with two medical data sets.
doi:10.1002/bimj.201100042
PMCID: PMC3777387  PMID: 21770046
Causal inference; Inverse probability weighting; Propensity score; Sensitivity analysis; Uncontrolled confounding
8.  Effect of Vitamin D and Inhaled Corticosteroid Treatment on Lung Function in Children 
Rationale: Low vitamin D levels are associated with asthma and decreased airway responsiveness. Treatment with inhaled corticosteroids improves airway responsiveness and asthma control.
Objectives: To assess the effect of vitamin D levels on prebronchodilator FEV1, bronchodilator response, and responsiveness to methacholine (PC20, provocative concentration of methacholine producing a 20% decline in FEV1) in patients with asthma treated with inhaled corticosteroids.
Methods: We measured 25-hydroxyvitamin D levels in the serum of children with persistent asthma at the time of enrollment in the Childhood Asthma Management Program. We divided subjects into the vitamin D sufficiency (>30 ng/ml), insufficiency (20–30 ng/ml), and deficiency (<20 ng/ml) groups. Covariates included age, treatment, sex, body mass index, race, history of emergency department visits, hospitalizations, and season that vitamin D specimen was drawn. Our main outcome measures were change in prebronchodilator FEV1, bronchodilator response, and PC20 from enrollment to 8–12 months.
Measurements and Main Results: Of the 1,024 subjects, 663 (65%) were vitamin D sufficient, 260 (25%) were insufficient, and 101 (10%) were deficient. Vitamin D–deficient subjects were more likely to be older, African American, and have a higher body mass index compared with the vitamin D–sufficient and insufficient subjects. In the inhaled corticosteroid treatment group, prebronchodilator FEV1 increased from randomization to 12 months by 140 ml in the vitamin D–deficient group and prebronchodilator FEV1 increased by 330 ml in the vitamin D insufficiency group and by 290 ml in the vitamin D sufficiency group (P = 0.0072), in adjusted models.
Conclusions: In children with asthma treated with inhaled corticosteroids, vitamin D deficiency is associated with poorer lung function than in children with vitamin D insufficiency or sufficiency.
doi:10.1164/rccm.201202-0351OC
PMCID: PMC3480528  PMID: 22798322
asthma; vitamin D; lung function; forced expiratory volume; children
9.  Guillain-Barré Syndrome, Influenza Vaccination, and Antecedent Respiratory and Gastrointestinal Infections: A Case-Centered Analysis in the Vaccine Safety Datalink, 2009–2011 
PLoS ONE  2013;8(6):e67185.
Background
Guillain-Barré Syndrome (GBS) can be triggered by gastrointestinal or respiratory infections, including influenza. During the 2009 influenza A (H1N1) pandemic in the United States, monovalent inactivated influenza vaccine (MIV) availability coincided with high rates of wildtype influenza infections. Several prior studies suggested an elevated GBS risk following MIV, but adjustment for antecedent infection was limited.
Methods
We identified patients enrolled in health plans participating in the Vaccine Safety Datalink and diagnosed with GBS from July 2009 through June 2011. Medical records of GBS cases with 2009–10 MIV, 2010–11 trivalent inactivated influenza vaccine (TIV), and/or a medically-attended respiratory or gastrointestinal infection in the 1 through 141 days prior to GBS diagnosis were reviewed and classified according to Brighton Collaboration criteria for diagnostic certainty. Using a case-centered design, logistic regression models adjusted for patient-level time-varying sources of confounding, including seasonal vaccinations and infections in GBS cases and population-level controls.
Results
Eighteen confirmed GBS cases received vaccination in the 6 weeks preceding onset, among 1.27 million 2009–10 MIV recipients and 2.80 million 2010–11 TIV recipients. Forty-four confirmed GBS cases had infection in the 6 weeks preceding onset, among 3.77 million patients diagnosed with medically-attended infection. The observed-versus-expected odds that 2009–10 MIV/2010–11 TIV was received in the 6 weeks preceding GBS onset was odds ratio = 1.54, 95% confidence interval (CI), 0.59–3.99; risk difference = 0.93 per million doses, 95% CI, −0.71–5.16. The association between GBS and medically-attended infection was: odds ratio = 7.73, 95% CI, 3.60–16.61; risk difference = 11.62 per million infected patients, 95% CI, 4.49–26.94. These findings were consistent in sensitivity analyses using alternative infection definitions and risk intervals for prior vaccination shorter than 6 weeks.
Conclusions
After adjusting for antecedent infections, we found no evidence for an elevated GBS risk following 2009–10 MIV/2010–11 TIV influenza vaccines. However, the association between GBS and antecedent infection was strongly elevated.
doi:10.1371/journal.pone.0067185
PMCID: PMC3694016  PMID: 23840621
10.  Development of a Pharmacogenetic Predictive Test in asthma: proof of concept 
Pharmacogenetics and genomics  2010;20(2):86-93.
Objective
To assess the feasibility of developing a Combined Clinical and Pharmacogenetic Predictive Test, comprised of multiple single nucleotide polymorphisms (SNPs) that is associated with poor bronchodilator response (BDR).
Methods
We genotyped SNPs that tagged the whole genome of the parents and children in the Childhood Asthma Management Program (CAMP) and implemented an algorithm using a family-based association test that ranked SNPs by statistical power. The top eight SNPs that were associated with BDR comprised the Pharmacogenetic Predictive Test. The Clinical Predictive Test was comprised of baseline forced expiratory volume in 1 s (FEV1). We evaluated these predictive tests and a Combined Clinical and Pharmacogenetic Predictive Test in three distinct populations: the children of the CAMP trial and two additional clinical trial populations of asthma. Our outcome measure was poor BDR, defined as BDR of less than 20th percentile in each population. BDR was calculated as the percent difference between the prebronchodilator and postbronchodilator (two puffs of albuterol at 180 μg/puff) FEV1 value. To assess the predictive ability of the test, the corresponding area under the receiver operating characteristic curves (AUROCs) were calculated for each population.
Results
The AUROC values for the Clinical Predictive Test alone were not significantly different from 0.50, the AUROC of a random classifier. Our Combined Clinical and Pharmacogenetic Predictive Test comprised of genetic polymorphisms in addition to FEV1 predicted poor BDR with an AUROC of 0.65 in the CAMP children (n= 422) and 0.60 (n= 475) and 0.63 (n= 235) in the two independent populations. Both the Combined Clinical and Pharmacogenetic Predictive Test and the Pharmacogenetic Predictive Test were significantly more accurate than the Clinical Predictive Test (AUROC between 0.44 and 0.55) in each of the populations.
Conclusion
Our finding that genetic polymorphisms with a clinical trait are associated with BDR suggests that there is promise in using multiple genetic polymorphisms simultaneously to predict which asthmatics are likely to respond poorly to bronchodilators.
doi:10.1097/FPC.0b013e32833428d0
PMCID: PMC3654515  PMID: 20032818
asthma; bronchodilator response; personalized medicine; pharmacogenetic test; predictive medicine
11.  Comparative safety of infliximab and etanercept on the risk of serious infections – Does the association vary by patient characteristics? 
Purpose
Infliximab, a chimeric monoclonal anti-TNFα antibody, has been found to increase the risk of serious infections compared with the TNF receptor fusion protein etanercept in some studies. It is unclear whether the risk varies by patient characteristics. We conducted a study to address this question.
Methods
We identified members of Kaiser Permanente Northern California who initiated infliximab (n=793) or etanercept (n=2,692) in 1997–2007. Using a Cox model, we estimated the propensity score-adjusted hazard ratio (HR) and 95% confidence interval (CI) of serious infections requiring hospitalization or opportunistic infections comparing infliximab with etanercept following treatment initiation. We tested whether the adjusted HR differed by age, sex, race/ethnicity, body mass index, and smoking status.
Results
The crude incidence rate of serious infections per 100 person-years was 5.4 (95% CI: 3.8, 7.5) in patients <65 years and 16.0 (10.4, 23.4) in patients ≥65 years during the first three months following treatment initiation. Compared with etanercept, the adjusted HR during this period was elevated for infliximab in patients <65 years (HR 3.01; 95% CI: 1.49, 6.07), but not in those ≥65 years (HR 0.94; 0.41, 2.13). Findings did not suggest that the HR varied by other patient characteristics examined.
Conclusions
An increased risk of serious infections associated with infliximab relative to etanercept did not appear to be modified by patients’ sex, race/ethnicity, body mass index, or smoking status. There was an indication that the increased risk might be limited to patients <65 years. Additional studies are warranted to verify or refute this finding.
doi:10.1002/pds.3238
PMCID: PMC3330193  PMID: 22411435
Anti-TNF agents; Database; Pharmacoepidemiology; Propensity score; Serious infections
12.  Metabolic engineering of Escherichia coli for high-specificity production of isoprenol and prenol as next generation of biofuels 
Background
The isopentenols, including isoprenol and prenol, are excellent alternative fuels. However, they are not compounds largely accumulated in natural organism. The need for the next generation of biofuels with better physical and chemical properties impels us to develop biosynthetic routes for the production of isoprenol and prenol from renewable sugar. In this study, we use the heterogenous mevalonate-dependent (MVA) isoprenoid pathway for the synthesis of isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) intermediates, and then convert IPP and DMAPP to isoprenol and prenol, respectively.
Results
A mevalonate titer of 1.7 g/L was obtained by constructing an efficient MVA upper pathway in engineered E. coli. Different phosphatases and pyrophosphatases were investigated for their abilities in hydrolyzing the IPP and DMAPP. Consequently, ADP-ribose pyrophosphatase was found to be an efficient IPP and DMAPP hydrolase. Moreover, ADP-ribose pyrophosphatase from Bacillus subtilis (BsNudF) exhibited a equivalent substrate specificity towards IPP and DMAPP, while ADP-ribose pyrophosphatase from E. coli (EcNudF) presented a high substrate preference for DMAPP. Without the expression of any phosphatases or pyrophosphatases, a background level of isopentenols was synthesized. When the endogenous pyrophosphatase genes (EcNudF and yggV) that were capable of enhancing the hydrolyzation of the IPP and DMAPP were knocked out, the background level of isopentenols was still obtained. Maybe the synthesized IPP and DMAPP were hydrolyzed by some unknown hydrolases of E. coli. Finally, 1.3 g/L single isoprenol was obtained by blocking the conversion of IPP to DMAPP and employing the BsNudF, and 0.2 g/L ~80% prenol was produced by employing the EcNudF. A maximal yield of 12% was achieved in both isoprenol and prenol producing strains.
Conclusions
To the best of our knowledge, this is the first successful report on high-specificity production of isoprenol and prenol by microbial fermentation. Over 1.3 g/L isoprenol achieved in shake-flask experiments represents a quite encouraging titer of higher alcohols. In addition, the substrate specificities of ADP-ribose pyrophosphatases were determined and successfully applied for the high-specificity synthesis of isoprenol and prenol. Altogether, this work presents a promising strategy for high-specificity production of two excellent biofuels, isoprenol and prenol.
doi:10.1186/1754-6834-6-57
PMCID: PMC3654967  PMID: 23618128
Isoprenol; Prenol; Metabolic engineering; Escherichia coli; Biofuel
13.  Electrochemiluminescence energy transfer-promoted ultrasensitive immunoassay using near-infrared-emitting CdSeTe/CdS/ZnS quantum dots and gold nanorods 
Scientific Reports  2013;3:1529.
The marriage of energy transfer with electrochemiluminescence has produced a new technology named electrochemiluminescence energy transfer (ECL-ET), which can realize effective and sensitive detection of biomolecules. To obtain optimal ECL-ET efficiency, perfect energy overlapped donor/acceptor pair is of great importance. Herein, we present a sensitive ECL-ET based immunosensor for the detection of tumor markers, using energy tunable CdSeTe/CdS/ZnS double shell quantum dots (QDs) and gold nanorods (GNRs) as the donor and acceptor, respectively. Firstly a facile microwave-assisted strategy for the synthesis of green- to near-infrared-emitting CdSeTe/CdS/ZnS QDs with time- and component-tunable photoluminescence was proposed. And, on the basis of the adjustable optical properties of both CdSeTe/CdS/ZnS QDs and GNRs, excellent overlap between donor emission and acceptor absorption can be obtained to ensure effective ECL-ET quenching, thus improving the sensing sensitivity. This method represents a novel approach for versatile detection of biomolecules at low concentrations.
doi:10.1038/srep01529
PMCID: PMC3607123  PMID: 23524874
14.  Quadratic semiparametric Von Mises calculus 
Metrika  2009;69(2-3):227-247.
We discuss a new method of estimation of parameters in semiparametric and nonparametric models. The method is based on U-statistics constructed from quadratic influence functions. The latter extend ordinary linear influence functions of the parameter of interest as defined in semiparametric theory, and represent second order derivatives of this parameter. For parameters for which the matching cannot be perfect the method leads to a bias-variance trade-off, and results in estimators that converge at a slower than n–1/2-rate. In a number of examples the resulting rate can be shown to be optimal. We are particularly interested in estimating parameters in models with a nuisance parameter of high dimension or low regularity, where the parameter of interest cannot be estimated at n–1/2-rate.
doi:10.1007/s00184-008-0214-3
PMCID: PMC3475538  PMID: 23087487
Von Mises calculus; Semiparametric models; Missing data; Tangent space; Influence function; Rate of convergence
15.  Boosting the free fatty acid synthesis of Escherichia coli by expression of a cytosolic Acinetobacter baylyi thioesterase 
Background
Thioesterases remove the fatty acyl moiety from the fatty acyl-acyl carrier proteins (ACPs), releasing them as free fatty acids (FFAs), which can be further used to produce a variety of fatty acid-based biofuels, such as biodiesel, fatty alcohols and alkanes. Thioesterases play a key role in the regulation of the fatty acid synthesis in Escherichia coli. Therefore, exploring more promising thioesterases will contribute to the development of industrial microbial lipids production.
Results
We cloned and expressed a cytosolic Acinetobacter baylyi thioesterase (‘AcTesA) in E. coli by deleting its leader sequence. Protein sequence alignment, structure modeling and site-directed mutagenesis demonstrated that Ser10, Gly48, Asn77, Asp158 and His161 residues composed the active centre of ‘AcTesA. The engineered strain that overexpressed ‘AcTesA achieved a FFAs titer of up to 501.2 mg/L in shake flask, in contrast to only 20.5 mg/L obtained in wild-type E. coli, demonstrating that the expression of ‘AcTesA indeed boosted the synthesis of FFAs. The ‘AcTesA exhibited a substrate preference towards the C8-C16 acyl groups, with C14:0, C16:1, C12:0 and C8:0 FFAs being the top four components. Optimization of expression level of ‘AcTesA made the FFAs production increase to 551.3 mg/L. The FFAs production further increased to 716.1 mg/L by optimization of the culture medium. Fed-batch fermentation was also carried out to evaluate the FFAs production in a scaleable process. Finally, 3.6 g/L FFAs were accumulated within 48 h, and a maximal FFAs yield of 6.1% was achieved in 12–16 h post induction.
Conclusions
For the first time, an A. baylyi thioesterase was cloned and solubly expressed in the cytosol of E. coli. This leaderless thioesterase (‘AcTesA) was found to be capable of enhancing the FFAs production of E. coli. Without detailed optimization of the strain and fermentation, the finally achieved 3.6 g/L FFAs is encouraging. In addition, ‘AcTesA exhibited different substrate specificity from other thioesterases previously reported, and can be used to supply the fatty acid-based biofuels with high quality of FFAs. Altogether, this study provides a promising thioesterase for FFAs production, and is of great importance in enriching the library of useful thioesterases.
doi:10.1186/1754-6834-5-76
PMCID: PMC3524773  PMID: 23057831
Thioesterase; Acinetobacter baylyi; Escherichia coli; Free fatty acid; Substrate specificity; Active-site residues
16.  Purification and characterization of a novel plant lectin from Pinellia ternata with antineoplastic activity 
SpringerPlus  2012;1:13.
A novel Pinellia ternata lectin was purified from the bulbs of a Chinese herb Pinellia ternata using a combination of hydrophobic chromatography and DEAE-ion exchange chromatography. The lectin was found to be a homodimer of 12093.3 Da subunits as determined by gel filtration and MS. Biochemical characterization of the lectin revealed the existence of a glycoprotein, which contains 3.22% neutral sugars. The N-terminal 10-amino acid sequence of the lectin, QGVNISGQVK, has not been reported for other lectins. The lectin had a special agglutinating activity with mouse erythrocytes at a minimum concentration of 8.0 ug/ml. The lectin was stable in the pH range of pH 5–12 and temperatures up to 80°C for 30 min. The results of MTT experiment showed that the lectin had significant effect towards tumor cells, the maximum inhibition of cell proliferation with Sarcoma 180, HeLa and K562 cell line were 85.2%, 74.6% and 59.4% respectively. Experimental therapy in vivo also showed that PTL apparently inhibited transplanted tumor in mice. Flow cytometric analysis demonstrated that PTL inhibited the proliferation of Sarcoma 180 in a time- and dose-dependent manner through inhibiting the transition of G1/S and subsequently inducing G0/G1 cell cycle arrest. Thus, Pinellia ternata lectin displays a high potential for antitumor activity.
doi:10.1186/2193-1801-1-13
PMCID: PMC3725870  PMID: 23961344
Lectin; Pinellia ternata; Antineoplastic activity; Purification
17.  Propensity Score-based Sensitivity Analysis Method for Uncontrolled Confounding 
American Journal of Epidemiology  2011;174(3):345-353.
The authors developed a sensitivity analysis method to address the issue of uncontrolled confounding in observational studies. In this method, the authors use a 1-dimensional function of the propensity score, which they refer to as the sensitivity function (SF), to quantify the hidden bias due to unmeasured confounders. The propensity score is defined as the conditional probability of being treated given the measured covariates. Then the authors construct SF-corrected inverse-probability-weighted estimators to draw inference on the causal treatment effect. This approach allows analysts to conduct a comprehensive sensitivity analysis in a straightforward manner by varying sensitivity assumptions on both the functional form and the coefficients in the 1-dimensional SF. Furthermore, 1-dimensional continuous functions can be well approximated by low-order polynomial structures (e.g., linear, quadratic). Therefore, even if the imposed SF is practically certain to be incorrect, one can still hope to obtain valuable information on treatment effects by conducting a comprehensive sensitivity analysis using polynomial SFs with varying orders and coefficients. The authors demonstrate the new method by implementing it in an asthma study which evaluates the effect of clinician prescription patterns regarding inhaled corticosteroids for children with persistent asthma on selected clinical outcomes.
doi:10.1093/aje/kwr096
PMCID: PMC3202161  PMID: 21659349
confounding factors (epidemiology); inverse probability weighting; propensity score; sensitivity analysis; sensitivity function; uncontrolled confounding
18.  Higher Order Inference On A Treatment Effect Under Low Regularity Conditions 
Statistics & probability letters  2011;81(7):821-828.
We describe a novel approach to nonparametric point and interval estimation of a treatment effect in the presence of many continuous confounders. We show the problem can be reduced to that of point and interval estimation of the expected conditional covariance between treatment and response given the confounders. Our estimators are higher order U-statistics. The approach applies equally to the regular case where the expected conditional covariance is root-n estimable and to the irregular case where slower non-parametric rates prevail.
doi:10.1016/j.spl.2011.02.030
PMCID: PMC3088168  PMID: 21552339
Minimax; U-statistics; Influence functions; Nonparametric; Semi-parametric; Robust Inference
19.  A note on overadjustment in inverse probability weighted estimation 
Biometrika  2010;97(4):997-1001.
Summary
Standardized means, commonly used in observational studies in epidemiology to adjust for potential confounders, are equal to inverse probability weighted means with inverse weights equal to the empirical propensity scores. More refined standardization corresponds with empirical propensity scores computed under more flexible models. Unnecessary standardization induces efficiency loss. However, according to the theory of inverse probability weighted estimation, propensity scores estimated under more flexible models induce improvement in the precision of inverse probability weighted means. This apparent contradiction is clarified by explicitly stating the assumptions under which the improvement in precision is attained.
doi:10.1093/biomet/asq049
PMCID: PMC3371719  PMID: 22822256
Causal inference; Propensity score; Standardized mean
20.  Predicted Interval Plots (PIPS): A Graphical Tool for Data Monitoring of Clinical Trials 
Group sequential designs are often used in clinical trials to evaluate efficacy and/or futility. Many methods have been developed for different types of endpoints and scenarios. However, few of these methods convey information regarding effect sizes (e.g., treatment differences) and none uses prediction to convey information regarding potential effect size estimates and associated precision, with trial continuation. To address these limitations, Evans et al. (2007) proposed to use prediction and predicted intervals as a flexible and practical tool for quantitative monitoring of clinical trials. In this article, we reaffirm the importance and usefulness of this innovative approach and introduce a graphical summary, predicted interval plots (PIPS), to display the information obtained in the prediction process in a straightforward yet comprehensive manner. We outline the construction of PIPS and apply this method in two examples. The results and the interpretations of the PIPS are discussed.
doi:10.1198/sbr.2009.0041
PMCID: PMC3059316  PMID: 21423789
Clinical trials; Data monitoring; Interim analyses; Prediction; Predicted intervals
21.  Energy transfer dyads based on Nile Red 
Tetrahedron letters  2009;50(47):6442-6445.
This project was initiated to develop energy transfer dyads emitting in the 600 – 700 nm region by using Nile Red acceptors. Thus fluorescein- and BODIPY-based donors were linked to these acceptors via alkynes or triazoles. The product dyads (1 – 5) have energy transfer efficiencies of 77 – 97% in organic media.
doi:10.1016/j.tetlet.2009.08.130
PMCID: PMC2770174  PMID: 20160911
fluorophores; microwave reactions; Sonogashira coupling
22.  Repeatability of Response to Asthma Medications 
Background
Pharmacogenetic studies of drug response in asthma assume that patients respond consistently to a treatment but that treatment response varies across patients, however, no formal studies have demonstrated this.
Objective
To determine the repeatability of commonly used outcomes for treatment response to asthma medications: bronchodilator response, forced expiratory volume in 1 second (FEV1), and provocative concentration of methacholine producing a 20% decline in FEV1 (PC20).
Methods
The Childhood Asthma Management Program (CAMP) was a multi-center clinical trial of children randomized to receiving budesonide, nedocromil, or placebo. We determined the intraclass correlation coefficient (ICC) for each outcome over repeated visits over four years in CAMP using mixed effects regression models. We adjusted for the covariates: age, race/ethnicity, height, family income, parental education, and symptom score. We incorporated each outcome for each child as repeated outcome measurements and stratified by treatment group.
Results
The ICC for bronchodilator response was 0.31 in the budesonide group, 0.35 in the nedocromil group, and 0.40 in the placebo group, after adjusting for covariates. The ICC for FEV1 was 0.71 in the budesonide group, 0.60 in the nedocromil group, and 0.69 in the placebo group, after adjusting for covariates. The ICC for PC20 was 0.67 in the budesonide and placebo groups and 0.73 in the nedocromil group, after adjusting for covariates.
Conclusion
The within treatment group repeatability of FEV1 and PC20 are high; thus these phenotypes are heritable. FEV1 and PC20 may be better phenotypes than bronchodilator response for studies of treatment response in asthma.
doi:10.1016/j.jaci.2008.10.015
PMCID: PMC2980870  PMID: 19064281
asthma; drug response; heritability; bronchodilator; pharmacogenetics
23.  Functionalization Of The 4,4-Difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) Core 
The new BODIPY systems 1 and 2 were prepared then used as substrates to explore SNAr and F–B displacement reactions. Chloride was easily displaced from 1 by a piperidine/ester, methyl magnesium bromide selectively displaced fluoride, and cyanide could attack both sites. System 2 readily added soft nucleophiles to the electrophilic carbon atoms, providing a new method for bioconjugation of BODIPYs to proteins while also introducing a 19F probe.
doi:10.1016/j.bmcl.2007.10.103
PMCID: PMC2430868  PMID: 18037291
24.  The YebC Family Protein PA0964 Negatively Regulates the Pseudomonas aeruginosa Quinolone Signal System and Pyocyanin Production ▿  
Journal of Bacteriology  2008;190(18):6217-6227.
Bacterial pathogenicity is often manifested by the expression of various cell-associated and secreted virulence factors, such as exoenzymes, protease, and toxins. In Pseudomonas aeruginosa, the expression of virulence genes is coordinately controlled by the global regulatory quorum-sensing systems, which includes the las and rhl systems as well as the Pseudomonas quinolone signal (PQS) system. Phenazine compounds are among the virulence factors under the control of both the rhl and PQS systems. In this study, regulation of the phzA1B1C1D1E1 (phzA1) operon, which is involved in phenazine synthesis, was investigated. In an initial study of inducing conditions, we observed that phzA1 was induced by subinhibitory concentrations of tetracycline. Screening of 13,000 mutants revealed 32 genes that altered phzA1 expression in the presence of subinhibitory tetracycline concentrations. Among them, the gene PA0964, designated pmpR (pqsR-mediated PQS regulator), has been identified as a novel regulator of the PQS system. It belongs to a large group of widespread conserved hypothetical proteins with unknown function, the YebC protein family (Pfam family DUF28). It negatively regulates the quorum-sensing response regulator pqsR of the PQS system by binding at its promoter region. Alongside phzA1 expression and phenazine and pyocyanin production, a set of virulence factors genes controlled by both rhl and the PQS were shown to be modulated by PmpR. Swarming motility and biofilm formation were also significantly affected. The results added another layer of regulation in the rather complex quorum-sensing systems in P. aeruginosa and demonstrated a clear functional clue for the YebC family proteins.
doi:10.1128/JB.00428-08
PMCID: PMC2546791  PMID: 18641136
25.  Comparative analysis of Mycobacterium avium subsp. paratuberculosis isolates from cattle, sheep and goats by short sequence repeat and pulsed-field gel electrophoresis typing 
BMC Microbiology  2008;8:204.
Background
Mycobacterium avium subsp. paratuberculosis (Map) causes the chronic enteritis called paratuberculosis mainly in cattle, sheep and goats. Evidences that point out an association between Map and Crohn's Disease in humans are increasing. Strain differentiation among Map isolates has proved to be difficult and has limited the study of the molecular epidemiology of paratuberculosis. In order to asses the usefulness of the PCR based short sequence repeat (SSR) analysis of locus 1 and locus 8 in the epidemiological tracing of paratuberculosis strains we here compare for the first time the results of SSR and SnaBI-SpeI pulsed-field gel electrophoresis (PFGE) typing methods in a set of 268 Map isolates from different hosts (cattle, sheep, goats, bison, deer and wild boar).
Results
A total of nineteen different multi-locus SSR (SSR1_SSR8) types were identified amongst the 268 isolates compared to the 37 multiplex profiles differentiated by the SnaBI-SpeI PFGE. SSR type 7_4 was the predominant genotype (51.2% of all isolates and 54.3% of cattle isolates), but combined with PFGE results the abundance of the most prevalent genotype (7_4&{2-1}) dropped down to 37.7%. SSR types 7_3 and 14_3 were significantly spread amongst isolates recovered from small ruminants. The comparison of SSR1_SSR8 and SnaBI-SpeI PFGE typing of these isolates has shown that both methods perform at similar discriminatory level. These were 0.691 and 0.693, respectively for SSR and PFGE as indicated Simpson's Index of Diversity, and 0.82 when calculated for combined SSR and PFGE genotypes. Overall, SSR1_SSR8 analysis seemed to detect higher levels of within-farm strain diversity and seemed to give higher year-related information. Combination of both typing methods revealed 20 multi-type farms out of the 33 bovine farms studied with more than one isolate.
Conclusion
The particular SSR and PFGE typing approaches described here are in general agreement but they showed some discrepancies that might reflect differing evolutionary processes of Map strains. Both methods are able to reciprocally complement their results and neither should be replaced with the other if sufficient material and time is available. Overall, the results of our comparative analyses suggest that, based on current methodologies available, a combined approach that includes SSR and PFGE seems to provide the highest level of discrimination for Map strain typing with meaningful epidemiological information.
doi:10.1186/1471-2180-8-204
PMCID: PMC2605457  PMID: 19032737

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