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1.  Clinical and Laboratory Findings of the First Imported Case of Middle East Respiratory Syndrome Coronavirus to the United States 
The first US case of Middle East respiratory syndrome coronavirus was confirmed in May 2014 in a 65-year-old physician who worked in Saudi Arabia and presented to an Indiana hospital on illness day 11. He had bilateral pneumonia and recovered fully.
Background. The Middle East respiratory syndrome coronavirus (MERS-CoV) was discovered September 2012 in the Kingdom of Saudi Arabia (KSA). The first US case of MERS-CoV was confirmed on 2 May 2014.
Methods. We summarize the clinical symptoms and signs, laboratory and radiologic findings, and MERS-CoV–specific tests.
Results. The patient is a 65-year-old physician who worked in a hospital in KSA where MERS-CoV patients were treated. His illness onset included malaise, myalgias, and low-grade fever. He flew to the United States on day of illness (DOI) 7. His first respiratory symptom, a dry cough, developed on DOI 10. On DOI 11, he presented to an Indiana hospital as dyspneic, hypoxic, and with a right lower lobe infiltrate on chest radiography. On DOI 12, his serum tested positive by real-time reverse transcription polymerase chain reaction (rRT-PCR) for MERS-CoV and showed high MERS-CoV antibody titers, whereas his nasopharyngeal swab was rRT-PCR negative. Expectorated sputum was rRT-PCR positive the following day, with a high viral load (5.31 × 106 copies/mL). He was treated with antibiotics, intravenous immunoglobulin, and oxygen by nasal cannula. He was discharged on DOI 22. The genome sequence was similar (>99%) to other known MERS-CoV sequences, clustering with those from KSA from June to July 2013.
Conclusions. This patient had a prolonged nonspecific prodromal illness before developing respiratory symptoms. Both sera and sputum were rRT-PCR positive when nasopharyngeal specimens were negative. US clinicians must be vigilant for MERS-CoV in patients with febrile and/or respiratory illness with recent travel to the Arabian Peninsula, especially among healthcare workers.
PMCID: PMC4650772  PMID: 25100864
Middle East respiratory syndrome coronavirus; viral pneumonia
2.  Laboratory Investigation and Phylogenetic Analysis of an Imported Middle East Respiratory Syndrome Coronavirus Case in Greece 
PLoS ONE  2015;10(4):e0125809.
Rapid and reliable laboratory diagnosis of persons suspected of Middle East respiratory syndrome coronavirus (MERS-CoV) infection is important for timely implementation of infection control practices and disease management. In addition, monitoring molecular changes in the virus can help elucidate chains of transmission and identify mutations that might influence virus transmission efficiency. This was illustrated by a recent laboratory investigation we conducted on an imported MERS-CoV case in Greece. Two oropharyngeal swab specimens were collected on the 1st and 2nd day of patient hospitalization and tested using two real-time RT-PCR (rRT-PCR) assays targeting the UpE and Orf-1a regions of the MERS-CoV genome and RT-PCR and partial sequencing of RNA-dependent RNA polymerase and nucleocapsid genes. Serum specimens were also collected and serological test were performed. Results from the first swab sample were inconclusive while the second swab was strongly positive for MERS-CoV RNA by rRT-PCR and confirmed positive by RT-PCR and partial gene sequencing. Positive serologic test results further confirmed MERS-CoV infection. Full-length nucleocapsid and spike gene coding sequences were later obtained from the positive swab sample. Phylogenetic analysis revealed that the virus was closely related to recent human-derived MERS-CoV strains obtained in Jeddah and Makkah, Saudi Arabia, in April 2014 and dromedary camels in Saudi Arabia and Qatar. These findings were consistent with the patient’s history. We also identified a unique amino acid substitution in the spike receptor binding domain that may have implications for receptor binding efficiency. Our initial inconclusive rRT-PCR results highlight the importance of collecting multiple specimens from suspect MERS-CoV cases and particularly specimens from the lower respiratory tract.
PMCID: PMC4412533  PMID: 25919137
3.  Pre-pregnancy cytogenetic analysis of general couples in eastern China 
Scientific Reports  2014;4:7224.
The aim of this study was to investigate the contribution of chromosomal anomalies and the frequency of particular types of aberrations in general couples preparing for pregnancy and make recommendations for pregnancy on the basis of the medical literature. A total of 6,198 general couples were included in the present study. The karyotypes were generated from the peripheral blood lymphocyte cultures and the cytogenetic analysis was performed using G-banding. In 12,396 cases, chromosomal anomalies were detected in 59 cases (0.48%, 59/12,396). Among of them, the frequency of translocation was 0.35% (n = 43). Sex chromosomal anomalies accounted for 0.07% (n = 9), including Klinefelter syndrome (KS) (n = 4), Turner syndrome (TS) (n = 4), and XYY syndrome (n = 1). The others, including inversions (n = 6) and deletion (n = 1), accounted for 0.06%. Our study indicates that clinically important chromosomal defects are present at a remarkable frequency in the general couples in eastern China, suggesting pre-pregnancy cytogenetic analysis should be routinely performed among general couples in this area so that informed decision can be made, which will help to improve the quality of the pregnancy.
PMCID: PMC4245518  PMID: 25428275
4.  Unusual High Oxygen Reduction Performance in All-Carbon Electrocatalysts 
Scientific Reports  2014;4:6289.
Carbon-based electrocatalysts are more durable and cost-effective than noble materials for the oxygen reduction reaction (ORR), which is an important process in energy conversion technologies. Heteroatoms are considered responsible for the excellent ORR performance in many carbon-based electrocatalysts. But whether an all-carbon electrocatalyst can effectively reduce oxygen is unknown. We subtly engineered the interfaces between planar graphene sheets and curved carbon nanotubes (G-CNT) and gained a remarkable activity/selectivity for ORR (larger current, and n = 3.86, ~93% hydroxide + ~7% peroxide). This performance is close to that of Pt; and the durability is much better than Pt. We further demonstrate the application of this G-CNT hybrid as an all-carbon cathode catalyst for lithium oxygen batteries.We speculate that the high ORR activity of this G-CNT hybrid stems from the localized charge separation at the interface of the graphene and carbon nanotube, which results from the tunneling electron transfer due to the Fermi level mismatch on the planar and curved sp2 surfaces. Our result represents a conceptual breakthrough and pioneers the new avenues towards practical all-carbon electrocatalysis.
PMCID: PMC4164027  PMID: 25189141
5.  Sirtuin 1 Regulates Hepatitis B Virus Transcription and Replication by Targeting Transcription Factor AP-1 
Journal of Virology  2014;88(5):2442-2451.
Chronic hepatitis B virus (HBV) infection is a major risk factor for liver cirrhosis and hepatocellular carcinoma. Nevertheless, the molecular mechanism of HBV replication remains elusive. SIRT1 is a class III histone deacetylase that is a structure component of the HBV cccDNA minichromosome. In this study, we found by using microarray-based gene expression profiling analysis that SIRT1 was upregulated in HBV-expressing cells. Gene silencing of SIRT1 significantly inhibited HBV DNA replicative intermediates, 3.5-kb mRNA, and core protein levels. In contrast, the overexpression of SIRT1 augmented HBV replication. Furthermore, SIRT1 enhanced the activity of HBV core promoter by targeting transcription factor AP-1. The c-Jun subunit of AP-1 was bound to the HBV core promoter region, as demonstrated by using a chromatin immunoprecipitation assay. Mutation of AP-1 binding site or knockdown of AP-1 abolished the effect of SIRT1 on HBV replication. Finally, SIRT1 inhibitor sirtinol also suppressed the HBV DNA replicative intermediate, as well as 3.5-kb mRNA. Our study identified a novel host factor, SIRT1, which may facilitate HBV replication in hepatocytes. These data suggest a rationale for the use of SIRT1 inhibitor in the treatment of HBV infection.
PMCID: PMC3958108  PMID: 24335313
6.  Family Cluster of Middle East Respiratory Syndrome Coronavirus Infections, Tunisia, 2013 
Emerging Infectious Diseases  2014;20(9):1527-1530.
In 2013 in Tunisia, 3 persons in 1 family were infected with Middle East respiratory syndrome coronavirus (MERS-CoV). The index case-patient’s respiratory tract samples were negative for MERS-CoV by reverse transcription PCR, but diagnosis was retrospectively confirmed by PCR of serum. Sequences clustered with those from Saudi Arabia and United Arab Emirates.
PMCID: PMC4178422  PMID: 25148113
MERS coronavirus; ARDS; infection; ICU; viruses; Tunisia; MERS-CoV; Middle East respiratory syndrome; family cluster
7.  Molecular Detection of Adenoviruses, Rhabdoviruses, and Paramyxoviruses in Bats from Kenya 
We screened 217 bats of at least 20 species from 17 locations in Kenya during July and August of 2006 for the presence of adenovirus, rhabdovirus, and paramyxovirus nucleic acids using generic reverse transcription polymerase chain reaction (RT-PCR) and PCR assays. Of 217 bat fecal swabs examined, 4 bats were adenovirus DNA-positive, 11 bats were paramyxovirus RNA-positive, and 2 bats were rhabdovirus RNA-positive. Three bats were coinfected by two different viruses. By sequence comparison and phylogenetic analysis, the Kenya bat paramyxoviruses and rhabdoviruses from this study may represent novel viral lineages within their respective families; the Kenya bat adenoviruses could not be confirmed as novel, because the same region sequences from other known bat adenovirus genomes for comparison were lacking. Our study adds to previous evidence that bats carry diverse, potentially zoonotic viruses and may be coinfected with more than one virus.
PMCID: PMC4125246  PMID: 24865685
8.  A SNaPshot assay for the rapid and simple detection of hepatitis B virus genotypes 
Molecular Medicine Reports  2014;10(3):1245-1251.
A simple technique for the identification of common genotypes of the hepatitis B virus (HBV) remains to be identified. The present study was conducted to establish such a methodology. Four plasmids of genotypes A-D and 123 clinical serum specimens of HBV-infected patients were genotyped. HBV genotypes would be detected successfully when the HBV genotype reached a viral load of 1 × 103 copies/ml or the BC genotype mixed samples reached a 5% level. The lower limit of detection of HBV DNA in serum specimens was determined to be 2.14×102 IU/ml. The assay sensitivity and specificity were 100% and the consistency was demonstrated to reach as high as 90.24 and 100% compared with that of the DNA sequencing and cloning. The frequencies of the genotypes B, C, BC, BD and BCD were found to be 65.0, 23.6, 7.3, 3.3 and 0.8%, respectively. The accuracy of detection of the mixed infections was also higher using the rapid and simple SNaPshot method compared with that achieved with the DNA sequencing methods. The results of the present study indicated that the SNaPshot technique accurately distinguishes the HBV genotypes A-D and is able to be readily applied as a monitoring tool in HBV prognosis and treatment.
PMCID: PMC4121402  PMID: 25018054
SNaPshot assay; hepatitis B virus; genotypes
9.  Towards ultrahigh volumetric capacitance: graphene derived highly dense but porous carbons for supercapacitors 
Scientific Reports  2013;3:2975.
A small volumetric capacitance resulting from a low packing density is one of the major limitations for novel nanocarbons finding real applications in commercial electrochemical energy storage devices. Here we report a carbon with a density of 1.58 g cm−3, 70% of the density of graphite, constructed of compactly interlinked graphene nanosheets, which is produced by an evaporation-induced drying of a graphene hydrogel. Such a carbon balances two seemingly incompatible characteristics: a porous microstructure and a high density, and therefore has a volumetric capacitance for electrochemical capacitors (ECs) up to 376 F cm−3, which is the highest value so far reported for carbon materials in an aqueous electrolyte. More promising, the carbon is conductive and moldable, and thus could be used directly as a well-shaped electrode sheet for the assembly of a supercapacitor device free of any additives, resulting in device-level high energy density ECs.
PMCID: PMC3797987  PMID: 24131954
10.  New World Bats Harbor Diverse Influenza A Viruses 
PLoS Pathogens  2013;9(10):e1003657.
Aquatic birds harbor diverse influenza A viruses and are a major viral reservoir in nature. The recent discovery of influenza viruses of a new H17N10 subtype in Central American fruit bats suggests that other New World species may similarly carry divergent influenza viruses. Using consensus degenerate RT-PCR, we identified a novel influenza A virus, designated as H18N11, in a flat-faced fruit bat (Artibeus planirostris) from Peru. Serologic studies with the recombinant H18 protein indicated that several Peruvian bat species were infected by this virus. Phylogenetic analyses demonstrate that, in some gene segments, New World bats harbor more influenza virus genetic diversity than all other mammalian and avian species combined, indicative of a long-standing host-virus association. Structural and functional analyses of the hemagglutinin and neuraminidase indicate that sialic acid is not a ligand for virus attachment nor a substrate for release, suggesting a unique mode of influenza A virus attachment and activation of membrane fusion for entry into host cells. Taken together, these findings indicate that bats constitute a potentially important and likely ancient reservoir for a diverse pool of influenza viruses.
Author Summary
Previous studies indicated that a novel influenza A virus (H17N10) was circulating in fruit bats from Guatemala (Central America). Herein, we investigated whether similar viruses are present in bat species from South America. Analysis of rectal swabs from bats sampled in the Amazon rainforest region of Peru identified another new influenza A virus from bats that is phylogenetically distinct from the one identified in Guatemala. The genes that encode the surface proteins of the new virus from the flat-faced fruit bat were designated as new subtype H18N11. Serologic testing of blood samples from several species of Peruvian bats indicated a high prevalence of antibodies to the surface proteins. Phylogenetic analyses demonstrate that bat populations from Central and South America maintain as much influenza virus genetic diversity in some gene segments as all other mammalian and avian species combined. The crystal structures of the hemagglutinin and neuraminidase proteins indicate that sialic acid is not a receptor for virus attachment nor a substrate for release, suggesting a novel mechanism of influenza A virus attachment and activation of membrane fusion for entry into host cells. In summary, our findings indicate that bats constitute a potentially important reservoir for influenza viruses.
PMCID: PMC3794996  PMID: 24130481
11.  Ampulla dilation with different sized balloons to remove common bile duct stones 
AIM: To assess the outcomes of ampulla dilation with different sized balloons to remove common bile duct (CBD) stones.
METHODS: Patients (n = 208) were divided into five groups based on the largest CBD stone size of < 5, 6-8, 8-12, 12-14, and > 14 mm. Patients underwent limited endoscopic sphincterotomy (EST) alone or limited EST followed by endoscopic papillary balloon dilation with 8, 10, 12 and 14 mm balloons, such that the size of each balloon did not exceed the size of the CBD. Short- and long-term outcomes, such as post-endoscopic retrograde cholangiopancreatography (ERCP) pancreatitis, perforation, bleeding, and pneumobilia were compared among the five groups.
RESULTS: The overall rate of successful stone removal in all groups was 100%, and all patients were cured. Eight (3.85%) patients had post-ERCP pancreatitis, none had perforations, and 6 (2.9%) had bleeding requiring transfusion. There were no significant differences in early complication rates among the five groups. We observed significant correlations between increased balloon size and the short- and long-term rates of post-ERCP pneumobilia. Post-ERCP pancreatitis and bleeding correlated significantly with age, with post-ERCP pancreatitis occurring more frequently in patients aged < 60 years, and bleeding occurring more frequently in patients aged > 70 years. We observed a significant correlation between patient age and the diameter of the largest CBD stone, with stones > 12 mm occurring more frequently in patients > 60 years old.
CONCLUSION: Choosing a balloon size based on the largest stone diameter is safe and effective for removing CBD stones. Balloon size should not exceed 15 mm.
PMCID: PMC3574888  PMID: 23431070
Endoscopic papillary balloon dilation; Endoscopic sphincterotomy; Common bile duct stone; Endoscopic retrograde cholangiopancreatography; Pancreatitis
12.  Information Visualization Techniques in Bioinformatics during the Postgenomic Era 
Drug discovery today. Biosilico  2004;2(6):237-245.
Information visualization techniques, which take advantage of the bandwidth of human vision, are powerful tools for organizing and analyzing a large amount of data. In the postgenomic era, information visualization tools are indispensable for biomedical research. This paper aims to present an overview of current applications of information visualization techniques in bioinformatics for visualizing different types of biological data, such as from genomics, proteomics, expression profiling and structural studies. Finally, we discuss the challenges of information visualization in bioinformatics related to dealing with more complex biological information in the emerging fields of systems biology and systems medicine.
PMCID: PMC2957900  PMID: 20976032
Information visualization; Bioinformatics
13.  Visualizing Information across Multidimensional Post-Genomic Structured and Textual Databases 
Bioinformatics (Oxford, England)  2004;21(8):1659-1667.
Visualizing relations among biological information to facilitate understanding is crucial to biological research during the post-genomic era. Although different systems have been developed to view gene-phenotype relations for specific databases, very few have been designed specifically as a general flexible tool for visualizing multidimensional genotypic and phenotypic information together. Our goal is to develop a method for visualizing multidimensional genotypic and phenotypic information and a model that unifies different biological databases in order to present the integrated knowledge using a uniform interface.
We developed a novel, flexible and generalizable visualization tool, called PhenoGenesviewer (PGviewer), which in this paper was used to display gene-phenotype relations from a human-curated database (OMIM) and from an automatic method using a Natural Language Processing tool called BioMedLEE. Data obtained from multiple databases were first integrated into a uniform structure and then organized by PGviewer. PGviewer provides a flexible query interface that allows dynamic selection and ordering of any desired dimension in the databases. Based on users’ queries, results can be visualized using hierarchical expandable trees that present views specified by users according to their research interests. We believe that this method, which allows users to dynamically organize and visualize multiple dimensions, is a potentially powerful and promising tool that should substantially facilitate biological research.
PMCID: PMC2901923  PMID: 15598839
14.  Information theory applied to the sparse gene ontology annotation network to predict novel gene function 
Bioinformatics (Oxford, England)  2007;23(13):i529-i538.
Despite advances in the gene annotation process, the functions of a large portion of the gene products remain insufficiently characterized. In addition, the “in silico” prediction of novel Gene Ontology (GO) annotations for partially characterized gene functions or processes is highly dependent on reverse genetic or function genomics approaches.
We propose a novel approach, Information Theory-based Semantic Similarity (ITSS), to automatically predict molecular functions of genes based on Gene Ontology annotations. We have demonstrated using a 10-fold cross-validation that the ITSS algorithm obtains prediction accuracies (Precision 97%, Recall 77%) comparable to other machine learning algorithms when applied to similarly dense annotated portions of the GO datasets. In addition, such method can generate highly accurate predictions in sparsely annotated portions of GO, in which previous algorithm failed to do so. As a result, our technique generates an order of magnitude more gene function predictions than previous methods. Further, this paper presents the first historical rollback validation for the predicted GO annotations, which may represent more realistic conditions for an evaluation than generally used cross-validations type of evaluations. By manually assessing a random sample of 100 predictions conducted in a historical roll-back evaluation, we estimate that a minimum precision of 51% (95% confidence interval: 43%–58%) can be achieved for the human GO Annotation file dated 2003.
The program is available on request. The 97,732 positive predictions of novel gene annotations from the 2005 GO Annotation dataset are available at
PMCID: PMC2882681  PMID: 17646340
15.  A Tool for Abstracting Relevant Classes of Concepts: the Common Ancestry Summarizer 
Controlled Medical Terminologies (CMTs) are indispensable tools for present medical information systems and medical informatics researches. Concept-oriented architecture and multiple-hierarchy have been accepted as two of the desiderata of contemporary CMTs. A common problem for informaticians working with class-based methods for terminologies is to find the Most Relevant Common Ancestor (MRCA) for a group of concepts, the common ancestor which has the closest semantic distance to all the given concepts. Finding an ancestor concept to summarize a group of concepts is required to map concepts from the knowledgebase to those of appropriate granularity in CMTs. However, manually exploring the hierarchical relationships and determining the MRCA are daunting tasks due to the massive size, the multiple hierarchies and in some case the cycles of the hierarchical networks of terminologies. In our study, we developed a web-based visualization tool, the Common Ancestry Summarizer (CAS), that graphically displays the hierarchical relations of multiple concepts within CMTs, and also assist users to identify the MRCA of these concepts by identifying the Lowest Common Ancestors (LCAs). The CAS has been shown useful in organizing classes for the controlled terms used in clinical guidelines (e.g. “the bottom-up method”).
PMCID: PMC2879046  PMID: 15360853
Controlled medical terminologies; Semantic network; Multiple hierarchies; Common ancestors
16.  High-Performance Signal Detection for Adverse Drug Events using MapReduce Paradigm 
Post-marketing pharmacovigilance is important for public health, as many Adverse Drug Events (ADEs) are unknown when those drugs were approved for marketing. However, due to the large number of reported drugs and drug combinations, detecting ADE signals by mining these reports is becoming a challenging task in terms of computational complexity. Recently, a parallel programming model, MapReduce has been introduced by Google to support large-scale data intensive applications. In this study, we proposed a MapReduce-based algorithm, for common ADE detection approach, Proportional Reporting Ratio (PRR), and tested it in mining spontaneous ADE reports from FDA. The purpose is to investigate the possibility of using MapReduce principle to speed up biomedical data mining tasks using this pharmacovigilance case as one specific example. The results demonstrated that MapReduce programming model could improve the performance of common signal detection algorithm for pharmacovigilance in a distributed computation environment at approximately liner speedup rates.
PMCID: PMC3041463  PMID: 21347109
17.  Detection of Novel SARS-like and Other Coronaviruses in Bats from Kenya 
Emerging Infectious Diseases  2009;15(3):482-485.
Diverse coronaviruses have been identified in bats from several continents but not from Africa. We identified group 1 and 2 coronaviruses in bats in Kenya, including SARS-related coronaviruses. The sequence diversity suggests that bats are well-established reservoirs for and likely sources of coronaviruses for many species, including humans.
PMCID: PMC2681120  PMID: 19239771
Coronavirus; SARS; bat viruses; pathogen discovery; emerging viral infections; dispatch
18.  Information-Theoretic Classification of SNOMED Improves the Organization of Context-Sensitive Excerpts from Cochrane Reviews 
The emphasis on evidence based medicine (EBM) has placed increased focus on finding timely answers to clinical questions in presence of patients. Using a combination of natural language processing for the generation of clinical excerpts and information theoretic distance based clustering, we evaluated multiple approaches for the efficient presentation of context-sensitive EBM excerpts.
PMCID: PMC2655812  PMID: 18694197
19.  Natural Language Processing in the Molecular Imaging Domain 
Molecular imaging represents the intersection between imaging and genomic sciences. There has been a surge in research literature and information in both sciences. Information contained within molecular imaging literature could be used to 1) link to genomic and imaging information resources and 2) to organize and index images. This research focuses on the adaptation, evaluation, and application of BioMedLEE, a natural language processing system (NLP), in the automated extraction of information from molecular imaging abstracts.
PMCID: PMC1560602  PMID: 16779429
20.  Improving Patient Compliance with Best Practices Guidelines: A Web based Automated and Personalized Reminders System 
Health Reminders is a prototype application that delivers personalized guidelines and recommended testing to the patient at the appropriate time and through multiple communication channels such as email, web, cellular text messaging (Simplified Message Service), automatic insertion into Personal Information Managers (schedulers like MS Outlook) as appointments and tasks.
PMCID: PMC1560457  PMID: 16779226
21.  Oxidation of Benzene to Phenol, Catechol, and 1,2,3-Trihydroxybenzene by Toluene 4-Monooxygenase of Pseudomonas mendocina KR1 and Toluene 3-Monooxygenase of Ralstonia pickettii PKO1 
Aromatic hydroxylations are important bacterial metabolic processes but are difficult to perform using traditional chemical synthesis, so to use a biological catalyst to convert the priority pollutant benzene into industrially relevant intermediates, benzene oxidation was investigated. It was discovered that toluene 4-monooxygenase (T4MO) of Pseudomonas mendocina KR1, toluene 3-monooxygenase (T3MO) of Ralstonia pickettii PKO1, and toluene ortho-monooxygenase (TOM) of Burkholderia cepacia G4 convert benzene to phenol, catechol, and 1,2,3-trihydroxybenzene by successive hydroxylations. At a concentration of 165 μM and under the control of a constitutive lac promoter, Escherichia coli TG1/pBS(Kan)T4MO expressing T4MO formed phenol from benzene at 19 ± 1.6 nmol/min/mg of protein, catechol from phenol at 13.6 ± 0.3 nmol/min/mg of protein, and 1,2,3-trihydroxybenzene from catechol at 2.5 ± 0.5nmol/min/mg of protein. The catechol and 1,2,3-trihydroxybenzene products were identified by both high-pressure liquid chromatography and mass spectrometry. When analogous plasmid constructs were used, E. coli TG1/pBS(Kan)T3MO expressing T3MO formed phenol, catechol, and 1,2,3-trihydroxybenzene at rates of 3 ± 1, 3.1 ± 0.3, and 0.26 ± 0.09 nmol/min/mg of protein, respectively, and E. coli TG1/pBS(Kan)TOM expressing TOM formed 1,2,3-trihydroxybenzene at a rate of 1.7 ± 0.3 nmol/min/mg of protein (phenol and catechol formation rates were 0.89 ± 0.07 and 1.5 ± 0.3 nmol/min/mg of protein, respectively). Hence, the rates of synthesis of catechol by both T3MO and T4MO and the 1,2,3-trihydroxybenzene formation rate by TOM were found to be comparable to the rates of oxidation of the natural substrate toluene for these enzymes (10.0 ± 0.8, 4.0 ± 0.6, and 2.4 ± 0.3 nmol/min/mg of protein for T4MO, T3MO, and TOM, respectively, at a toluene concentration of 165 μM).
PMCID: PMC444830  PMID: 15240250
22.  Altering Toluene 4-Monooxygenase by Active-Site Engineering for the Synthesis of 3-Methoxycatechol, Methoxyhydroquinone, and Methylhydroquinone 
Journal of Bacteriology  2004;186(14):4705-4713.
Wild-type toluene 4-monooxygenase (T4MO) of Pseudomonas mendocina KR1 oxidizes toluene to p-cresol (96%) and oxidizes benzene sequentially to phenol, to catechol, and to 1,2,3-trihydroxybenzene. In this study T4MO was found to oxidize o-cresol to 3-methylcatechol (91%) and methylhydroquinone (9%), to oxidize m-cresol and p-cresol to 4-methylcatechol (100%), and to oxidize o-methoxyphenol to 4-methoxyresorcinol (87%), 3-methoxycatechol (11%), and methoxyhydroquinone (2%). Apparent Vmax values of 6.6 ± 0.9 to 10.7 ± 0.1 nmol/min/ mg of protein were obtained for o-, m-, and p-cresol oxidation by wild-type T4MO, which are comparable to the toluene oxidation rate (15.1 ± 0.8 nmol/min/mg of protein). After these new reactions were discovered, saturation mutagenesis was performed near the diiron catalytic center at positions I100, G103, and A107 of the alpha subunit of the hydroxylase (TmoA) based on directed evolution of the related toluene o-monooxygenase of Burkholderia cepacia G4 (K. A. Canada, S. Iwashita, H. Shim, and T. K. Wood, J. Bacteriol. 184:344-349, 2002) and a previously reported T4MO G103L regiospecific mutant (K. H. Mitchell, J. M. Studts, and B. G. Fox, Biochemistry 41:3176-3188, 2002). By using o-cresol and o-methoxyphenol as model substrates, regiospecific mutants of T4MO were created; for example, TmoA variant G103A/A107S produced 3-methylcatechol (98%) from o-cresol twofold faster and produced 3-methoxycatechol (82%) from 1 mM o-methoxyphenol seven times faster than the wild-type T4MO (1.5 ± 0.2 versus 0.21 ± 0.01 nmol/min/mg of protein). Variant I100L produced 3-methoxycatechol from o-methoxyphenol four times faster than wild-type T4MO, and G103S/A107T produced methylhydroquinone (92%) from o-cresol fourfold faster than wild-type T4MO and there was 10 times more in terms of the percentage of the product. Variant G103S produced 40-fold more methoxyhydroquinone from o-methoxyphenol than the wild-type enzyme produced (80 versus 2%) and produced methylhydroquinone (80%) from o-cresol. Hence, the regiospecific oxidation of o-methoxyphenol and o-cresol was changed for significant synthesis of 3-methoxycatechol, methoxyhydroquinone, 3-methylcatechol, and methylhydroquinone. The enzyme variants also demonstrated altered monohydroxylation regiospecificity for toluene; for example, G103S/A107G formed 82% o-cresol, so saturation mutagenesis converted T4MO into an ortho-hydroxylating enzyme. Furthermore, G103S/A107T formed 100% p-cresol from toluene; hence, a better para-hydroxylating enzyme than wild-type T4MO was formed. Structure homology modeling suggested that hydrogen bonding interactions of the hydroxyl groups of altered residues S103, S107, and T107 influence the regiospecificity of the oxygenase reaction.
PMCID: PMC438599  PMID: 15231803
23.  Toluene 3-Monooxygenase of Ralstonia pickettii PKO1 Is a para-Hydroxylating Enzyme 
Journal of Bacteriology  2004;186(10):3117-3123.
Oxygenases are promising biocatalysts for performing selective hydroxylations not accessible by chemical methods. Whereas toluene 4-monooxygenase (T4MO) of Pseudomonas mendocina KR1 hydroxylates monosubstituted benzenes at the para position and toluene ortho-monooxygenase (TOM) of Burkholderia cepacia G4 hydroxylates at the ortho position, toluene 3-monooxygenase (T3MO) of Ralstonia pickettii PKO1 was reported previously to hydroxylate toluene at the meta position, producing primarily m-cresol (R. H. Olsen, J. J. Kukor, and B. Kaphammer, J. Bacteriol. 176:3749-3756, 1994). Using gas chromatography, we have discovered that T3MO hydroxylates monosubstituted benzenes predominantly at the para position. TG1/pBS(Kan)T3MO cells expressing T3MO oxidized toluene at a maximal rate of 11.5 ± 0.33 nmol/min/mg of protein with an apparent Km value of 250 μM and produced 90% p-cresol and 10% m-cresol. This product mixture was successively transformed to 4-methylcatechol. T4MO, in comparison, produces 97% p-cresol and 3% m-cresol. Pseudomonas aeruginosa PAO1 harboring pRO1966 (the original T3MO-bearing plasmid) also exhibited the same product distribution as that of TG1/pBS(Kan)T3MO. TG1/pBS(Kan)T3MO produced 66% p-nitrophenol and 34% m-nitrophenol from nitrobenzene and 100% p-methoxyphenol from methoxybenzene, as well as 62% 1-naphthol and 38% 2-naphthol from naphthalene; similar results were found with TG1/pBS(Kan)T4MO. Sequencing of the tbu locus from pBS(Kan)T3MO and pRO1966 revealed complete identity between the two, thus eliminating any possible cloning errors. 1H nuclear magnetic resonance analysis confirmed the structural identity of p-cresol in samples containing the product of hydroxylation of toluene by pBS(Kan)T3MO.
PMCID: PMC400597  PMID: 15126473
25.  Extended attributes of event monitor systems for criteria-based notification modalities. 
The efficacy of event monitors (EMs) at reducing morbidity and mortality of certain clinical conditions (CCs) is well established. In addition, studies have shown that user inverted exclamation mark s preferences on the modality of notification are correlated to the type of reminder or alert. Nonetheless, few institutions have implemented large scale automated monitoring of a considerable number of distinct CCs, and to our knowledge, none of these sizable projects also offer user-customizable communication modalities (CMs) over all monitored conditions. As both the numbers of CMs and CCs increase, the complexity of customizing user preferences amplifies following a geometric progression. This paper demonstrates an automated approach, based on generic notification attributes (NAs) and notification criteria (NC), which significantly simplifies the management and personalization of the CMs for institutions where the manual assignment of a CM for every alert is forbidding. The methods by which these NAs were developed, their significance for existing CCs and their implementation using the Arden Syntax and Guideline interchange format (GLIF) are described. The proposed Criteria-Based Notification is shown to improve two facets of the management of event monitors: 1) the assignment of CMs becomes independent from clinical conditions, de-facto removing institution-specific CMs from the knowledge bases of the event monitors and inserting CC-specific and institution-independent NAs, thus increasing their reusability and sharability; 2) knowledge-based independent NAs facilitate both institution-level management and user-level preference configuration.
PMCID: PMC2244499  PMID: 12463927

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