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1.  A New Oligonucleotide Microarray for Detection of Pathogenic and Non-Pathogenic Legionella spp. 
PLoS ONE  2014;9(12):e113863.
Legionella pneumophila has been recognized as the major cause of legionellosis since the discovery of the deadly disease. Legionella spp. other than L. pneumophila were later found to be responsible to many non-pneumophila infections. The non-L. pneumophila infections are likely under-detected because of a lack of effective diagnosis. In this report, we have sequenced the 16S-23S rRNA gene internal transcribed spacer (ITS) of 10 Legionella species and subspecies, including L. anisa, L. bozemanii, L. dumoffii, L. fairfieldensis, L. gormanii, L. jordanis, L. maceachernii, L. micdadei, L. pneumophila subspp. fraseri and L. pneumophila subspp. pasculleii, and developed a rapid oligonucleotide microarray detection technique accordingly to identify 12 most common Legionella spp., which consist of 11 pathogenic species of L. anisa, L. bozemanii, L. dumoffii, L. gormanii, L. jordanis, L. longbeachae, L. maceachernii, L. micdadei, and L. pneumophila (including subspp. pneumophila, subspp. fraseri, and subspp. pasculleii) and one non-pathogenic species, L. fairfieldensis. Twenty-nine probes that reproducibly detected multiple Legionella species with high specificity were included in the array. A total of 52 strains, including 30 target pathogens and 22 non-target bacteria, were used to verify the oligonucleotide microarray assay. The sensitivity of the detection was at 1.0 ng with genomic DNA or 13 CFU/100 mL with Legionella cultures. The microarray detected seven samples of air conditioner-condensed water with 100% accuracy, validating the technique as a promising method for applications in basic microbiology, clinical diagnosis, food safety, and epidemiological surveillance. The phylogenetic study based on the ITS has also revealed that the non-pathogenic L. fairfieldensis is the closest to L. pneumophila than the nine other pathogenic Legionella spp.
PMCID: PMC4254607  PMID: 25469776
2.  Light Spatial Distribution in the Canopy and Crop Development in Cotton 
PLoS ONE  2014;9(11):e113409.
The partitioning of light is very difficult to assess, especially in discontinuous or irregular canopies. The aim of the present study was to analyze the spatial distribution of photosynthetically active radiation (PAR) in a heterogeneous cotton canopy based on a geo-statistical sampling method. Field experiments were conducted in 2011 and 2012 in Anyang, Henan, China. Field plots were arranged in a randomized block design with the main plot factor representing the plant density. There were 3 replications and 6 densities used in every replicate. The six plant density treatments were 15,000, 33,000, 51,000, 69,000, 87,000 and 105,000 plants ha−1. The following results were observed: 1) transmission within the canopy decreased with increasing density and significantly decreased from the top to the bottom of the canopy, but the greatest decreases were observed in the middle layers of the canopy on the vertical axis and closing to the rows along the horizontal axis; 2) the transmitted PAR (TPAR) of 6 different cotton populations decreased slowly and then increased slightly as the leaves matured, the TPAR values were approximately 52.6–84.9% (2011) and 42.7–78.8% (2012) during the early cotton developmental stage, and were 33.9–60.0% (2011) and 34.5–61.8% (2012) during the flowering stage; 3) the Leaf area index (LAI) was highly significant exponentially correlated (R2 = 0.90 in 2011, R2 = 0.91 in 2012) with the intercepted PAR (IPAR) within the canopy; 4) and a highly significant linear correlation (R2 = 0.92 in 2011, R2 = 0.96 in 2012) was observed between the accumulated IPAR and the biomass. Our findings will aid researchers to improve radiation-use efficiency by optimizing the ideotype for cotton canopy architecture based on light spatial distribution characteristics.
PMCID: PMC4237451  PMID: 25409026
3.  Development of a DNA Microarray Method for Detection and Identification of All 15 Distinct O-Antigen Forms of Legionella pneumophila 
Applied and Environmental Microbiology  2013;79(21):6647-6654.
Legionella is ubiquitous in many environments. At least 50 species and 70 serogroups of the Gram-negative bacterium have been identified. Of the 50 species, 20 are pathogenic, and Legionella pneumophila is responsible for the great majority (approximately 90%) of the Legionnaires' disease cases that occur. Furthermore, of the 15 L. pneumophila serogroups identified, O1 alone causes more than 84% of the Legionnaires' disease cases that occur worldwide. Rapid and reliable assays for the detection and identification of L. pneumophila in water, environmental, and clinical samples are in great demand. L. pneumophila bacteria are traditionally identified by their O antigens by immunological methods. We have recently developed an O serogroup-specific DNA microarray for the detection of all 15 distinct O-antigen forms of L. pneumophila, including serogroups O1 to O15. A total of 35 strains were used to verify the specificity of the microarray, including 15 L. pneumophila O-antigen standard reference strains and seven L. pneumophila clinical isolates as target strains, seven reference strains of other non-pneumophila Legionella species as closely related strains, and six non-Legionella bacterial species as nonrelated strains. The detection sensitivity was 1 ng of genomic DNA or 0.4 CFU/ml in water samples with filter enrichment and plate culturing. This study demonstrated that the microarray allows specific, sensitive, and reproducible detection of L. pneumophila serogroups. To the best of our knowledge, this is the first report of a microarray serotyping method for all 15 distinct O-antigen forms of L. pneumophila.
PMCID: PMC3811488  PMID: 23974134
4.  Cost Template for Meaningful Activity Intervention for Mild Cognitive Impairment 
Clinical nurse specialist CNS  2013;27(2):88-95.
To describe and compare cost estimates for a pilot study of the Daily Enhancement of Meaningful Activity (DEMA) intervention for persons with mild cognitive impairment (PwMCI)-caregiver dyads.
The increasing complexity of the health care system and rising health care costs, have forced nurse scientists to find ways to effectively improve health care quality and control cost, but no studies have examined costs for new programs that target PwMCI-caregiver dyads.
Description of the project
Pilot study data were used to develop a cost template and calculate the cost of implementing the DEMA.
Mean cost per dyad was estimated to be $1,327.97 in the clinical setting, compared with $1,069.06 if a telephone delivery mode had been used for four of the six face-to-face sessions. This difference was largely due to transportation-related expenses and staff cost.
DEMA should be evaluated further with larger and more diverse samples as a technology-delivered health promotion program that could reduce costs.
PMCID: PMC3636762  PMID: 23392066
Cost; intervention studies; cognitive impairment
5.  Clinical characteristics and prognostic analysis of triple-negative breast cancer patients 
Molecular and Clinical Oncology  2013;2(2):245-251.
It is well-established that triple-negative breast cancer (TNBC) is a subtype of breast cancer, characterized by a poor prognosis and aggressive biological behavior. However, the available relevant data on TNBC in non-Western populations are limited. In order to analyze the clinicopathological and molecular biological characteristics and observe survival and prognostic factors, 972 breast cancer patients (156 of whom had TNBC) who received treatment at the First Affiliated Hospital of Medical School of Xi'an Jiaotong University and the First Hospital of China Medical University, between January, 2004 and January, 2007 were retrospectively evaluated. In the univariate analysis, tumor size, TNM stage, axillary lymph node status and recurrence or metastasis were identified as prognostic factors for 7-year disease-free survival (DFS) and overall survival (OS). Our multivariate Cox's regression analysis demonstrated that tumor size and axillary lymph node status were significant prognostic factors for 7-year DFS and OS. Notably, tumor subgroup (TNBC vs. non-TNBC) was a significant prognostic factor associated with 7-year DFS and OS in breast cancer. It was suggested that TNBC exhibited a worse 7-year survival compared with that in non-TNBC patients, most likely due to its more aggressive behavior and insensitivity to specific therapy.
PMCID: PMC3917786  PMID: 24649341
clinicopathological characteristics; prognostic analysis; survival analysis; triple-negative breast cancer
6.  Development of a DNA Microarray for Molecular Identification of All 46 Salmonella O Serogroups 
Applied and Environmental Microbiology  2013;79(11):3392-3399.
Salmonella is a major cause of food-borne disease in many countries. Serotype determination of Salmonella is important for disease assessment, infection control, and epidemiological surveillance. In this study, a microarray system that targets the O antigen-specific genes was developed for simultaneously detecting and identifying all 46 Salmonella O serogroups. Of these, 40 serogroups can be confidently identified, and the remaining 6, in three pairs (serogroups O67 and B, E1 and E4, and A and D1), need to be further distinguished from each other using PCR methods or conventional serotyping methods. The microarray was shown to be highly specific when evaluated against 293 Salmonella strains, 186 Shigella strains, representative Escherichia coli strains, and 10 strains of other bacterial species. The assay correctly identified 288 (98%) of the Salmonella strains. The detection sensitivity was determined to be 50 ng genomic DNA per sample. By testing simulated samples in a tomato background, 2 to 8 CFU per gram inoculated could be detected after enrichment. This newly developed microarray assay is the first molecular protocol that can be used for the comprehensive detection and identification of all 46 Salmonella O serogroups. Compared to the traditional serogrouping method, the microarray provides a reliable, high-throughput, and sensitive approach that can be used for rapid identification of multiple Salmonella O serogroups simultaneously.
PMCID: PMC3648052  PMID: 23524674
7.  Characterization of a Novel Rieske-Type Alkane Monooxygenase System in Pusillimonas sp. Strain T7-7 
Journal of Bacteriology  2013;195(9):1892-1901.
The cold-tolerant bacterium Pusillimonas sp. strain T7-7 is able to utilize diesel oils (C5 to C30 alkanes) as a sole carbon and energy source. In the present study, bioinformatics, proteomics, and real-time reverse transcriptase PCR approaches were used to identify the alkane hydroxylation system present in this bacterium. This system is composed of a Rieske-type monooxygenase, a ferredoxin, and an NADH-dependent reductase. The function of the monooxygenase, which consists of one large (46.711 kDa) and one small (15.355 kDa) subunit, was further studied using in vitro biochemical analysis and in vivo heterologous functional complementation tests. The purified large subunit of the monooxygenase was able to oxidize alkanes ranging from pentane (C5) to tetracosane (C24) using NADH as a cofactor, with greatest activity on the C15 substrate. The large subunit also showed activity on several alkane derivatives, including nitromethane and methane sulfonic acid, but it did not act on any aromatic hydrocarbons. The optimal reaction condition of the large subunit is pH 7.5 at 30°C. Fe2+ can enhance the activity of the enzyme evidently. This is the first time that an alkane monooxygenase system belonging to the Rieske non-heme iron oxygenase family has been identified in a bacterium.
PMCID: PMC3624578  PMID: 23417490
8.  Role of the Phenylalanine-Hydroxylating System in Aromatic Substance Degradation and Lipid Metabolism in the Oleaginous Fungus Mortierella alpina 
Applied and Environmental Microbiology  2013;79(10):3225-3233.
Mortierella alpina is a filamentous fungus commonly found in soil that is able to produce lipids in the form of triacylglycerols that account for up to 50% of its dry weight. Analysis of the M. alpina genome suggests that there is a phenylalanine-hydroxylating system for the catabolism of phenylalanine, which has never been found in fungi before. We characterized the phenylalanine-hydroxylating system in M. alpina to explore its role in phenylalanine metabolism and its relationship to lipid biosynthesis. Significant changes were found in the profile of fatty acids in M. alpina grown on medium containing an inhibitor of the phenylalanine-hydroxylating system compared to M. alpina grown on medium without inhibitor. Genes encoding enzymes involved in the phenylalanine-hydroxylating system (phenylalanine hydroxylase [PAH], pterin-4α-carbinolamine dehydratase, and dihydropteridine reductase) were expressed heterologously in Escherichia coli, and the resulting proteins were purified to homogeneity. Their enzymatic activity was investigated by high-performance liquid chromatography (HPLC) or visible (Vis)-UV spectroscopy. Two functional PAH enzymes were observed, encoded by distinct gene copies. A novel role for tetrahydrobiopterin in fungi as a cofactor for PAH, which is similar to its function in higher life forms, is suggested. This study establishes a novel scheme for the fungal degradation of an aromatic substance (phenylalanine) and suggests that the phenylalanine-hydroxylating system is functionally significant in lipid metabolism.
PMCID: PMC3685260  PMID: 23503309
9.  Genomic diversity and adaptation of Salmonella enterica serovar Typhimurium from analysis of six genomes of different phage types 
BMC Genomics  2013;14:718.
Salmonella enterica serovar Typhimurium (or simply Typhimurium) is the most common serovar in both human infections and farm animals in Australia and many other countries. Typhimurium is a broad host range serovar but has also evolved into host-adapted variants (i.e. isolated from a particular host such as pigeons). Six Typhimurium strains of different phage types (defined by patterns of susceptibility to lysis by a set of bacteriophages) were analysed using Illumina high-throughput genome sequencing.
Variations between strains were mainly due to single nucleotide polymorphisms (SNPs) with an average of 611 SNPs per strain, ranging from 391 SNPs to 922 SNPs. There were seven insertions/deletions (indels) involving whole or partial gene deletions, four inactivation events due to IS200 insertion and 15 pseudogenes due to early termination. Four of these inactivated or deleted genes may be virulence related. Nine prophage or prophage remnants were identified in the six strains. Gifsy-1, Gifsy-2 and the sopE2 and sspH2 phage remnants were present in all six genomes while Fels-1, Fels-2, ST64B, ST104 and CP4-57 were variably present. Four strains carried the 90-kb plasmid pSLT which contains several known virulence genes. However, two strains were found to lack the plasmid. In addition, one strain had a novel plasmid similar to Typhi strain CT18 plasmid pHCM2.
The genome data suggest that variations between strains were mainly due to accumulation of SNPs, some of which resulted in gene inactivation. Unique genetic elements that were common between host-adapted phage types were not found. This study advanced our understanding on the evolution and adaptation of Typhimurium at genomic level.
PMCID: PMC3853940  PMID: 24138507
Typhimurium; Genome; Next generation sequencing; Phage type; Evolution; Single nucleotide polymorphism
10.  Absence of Mutations in Exon 6 of the TARDBP Gene in 207 Chinese Patients with Sporadic Amyotrohic Lateral Sclerosis 
PLoS ONE  2013;8(7):e68106.
Mutations in the TARDBP gene, which encodes the Tar DNA binding protein, have been shown to causes of both familial amyotrophic lateral sclerosis (FALS) and sporadic ALS (SALS). Recently, several novel TARDBP exon 6 mutants have been reported in patients with ALS in Europe and America but not in Asia. To further examine the spectrum and frequency of TARDBP exon 6 mutations, we investigated their frequency in ethnic Chinese patients with sporadic ALS. TARDBP exon 6 was screened by direct sequencing in 207 non-SOD1 SALS patients and 230 unrelated healthy controls but no mutations were identified. Our data indicate that exon 6 mutations in TARDBP are not a common cause of SALS in Han Chinese population from Southern Mainland China.
PMCID: PMC3706600  PMID: 23874513
11.  A case of adaptation through a mutation in a tandem duplication during experimental evolution in Escherichia coli 
BMC Genomics  2013;14:441.
DNA duplications constitute important precursors for genome variation. Here we analyzed an unequal duplication harboring a beneficial mutation that may provide alternative evolutionary outcomes.
We characterized this evolutionary event during experimental evolution for only 100 generations of an Escherichia coli strain under glucose limitation within chemostats. By combining Insertion Sequence based Restriction Length Polymorphism experiments, pulsed field gel electrophoresis and two independent genome re-sequencing experiments, we identified an evolved lineage carrying a 180 kb duplication of the 46’ region of the E. coli chromosome. This evolved duplication revealed a heterozygous state, with one copy harboring a 2668 bp deletion that included part of the ogrK gene and both the yegR and yegS genes. By genetically manipulating ancestral and evolved strains, we showed that the single yegS inactivation was sufficient to confer a frequency dependent fitness increase under the chemostat selective conditions in both the ancestor and evolved genetic contexts, implying that the duplication itself was not a direct fitness contributor. Nonetheless, the heterozygous duplicated state was relatively stable in the conditions prevailing during evolution in chemostats, in striking contrast to non selective conditions in which the duplication resolved at high frequency into either its ancestral or deleted copy.
Our results suggest that the duplication state may constitute a second order selection process providing higher evolutionary potential. Moreover, its heterozygous nature may provide differential evolutionary opportunities in alternating environments. Our results also highlighted how careful analyses of whole genome data are needed to identify such complex rearrangements.
PMCID: PMC3708739  PMID: 23822838
Experimental evolution; Chemostats; Escherichia coli; Duplication; Heterozygous duplication; Insertion sequence elements; Frequency-dependent selection; Indirect selection
12.  Genetic Analysis of the Cronobacter sakazakii O4 to O7 O-Antigen Gene Clusters and Development of a PCR Assay for Identification of All C. sakazakii O Serotypes 
Applied and Environmental Microbiology  2012;78(11):3966-3974.
The Gram-negative bacterium Cronobacter sakazakii is an emerging food-borne pathogen that causes severe invasive infections in neonates. Variation in the O-antigen lipopolysaccharide in the outer membrane provides the basis for Gram-negative bacteria serotyping. The O-antigen serotyping scheme for C. sakazakii, which includes seven serotypes (O1 to O7), has been recently established, and the O-antigen gene clusters and specific primers for three C. sakazakii serotypes (O1, O2, and O3) have been characterized. In this study, the C. sakazakii O4, O5, O6, and O7 O-antigen gene clusters were sequenced, and gene functions were predicted on the basis of homology. C. sakazakii O4 shared a similar O-antigen gene cluster with Escherichia coli O103. The general features and anomalies of all seven C. sakazakii O-antigen gene clusters were evaluated and the relationship between O-antigen structures and their gene clusters were investigated. Serotype-specific genes for O4 to O7 were identified, and a molecular serotyping method for all C. sakazakii O serotypes, a multiplex PCR assay, was developed by screening against 136 strains of C. sakazakii and closely related species. The sensitivity of PCR-based serotyping method was determined to be 0.01 ng of genomic DNA and 103 CFU of each strain/ml. This study completes the elucidation of C. sakazakii O-antigen genetics and provides a molecular method suitable for the identification of C. sakazakii O1 to O7 strains.
PMCID: PMC3346386  PMID: 22447597
13.  The other face of the other-race effect: An fMRI investigation of the other-race face categorization advantage 
Neuropsychologia  2011;49(13):3739-3749.
The present study was the first to use the functional magnetic resonance imaging (fMRI) methodology to investigate the neural correlates of race categorization of own- and other-race faces. We found that Chinese participants categorized the race of Caucasian faces more accurately and faster than that of Chinese faces, replicating the robust effect of the other-race categorization advantage. Regions of interest (ROI) analyses revealed greater neural activations when participants were categorizing own-race faces than other-race faces in the bilateral ventral occipito-temporal cortex (VOT) such as the fusiform face areas (FFA) and the occipital face areas (OFA). Within the left FFA, there was also a significant negative correlation between the behavioral difference of own- and other-race face categorization accuracy and the activation difference between categorizing own- and other-race faces. Whole brain analyses showed that categorizing own-race faces induced greater activations in the right medial frontal cortex (MFC) and right inferior frontal gyrus (IFG) than categorizing other-race faces. Psychophysiological interaction (PPI) analyses revealed that the frontal cortical regions interacted more strongly with the posterior VOT during the categorization of own-race faces than that of other-race faces. Overall, our findings suggest that relative to the categorization of other-race faces, more cortical resources are engaged during the categorization of own-race faces with which we have a higher level of processing expertise. This increased involvement of cortical neural sources perhaps serves to provide more in-depth processing of own-race faces (such as individuation), which in turn paradoxically results in the behavioral other-race categorization advantage.
PMCID: PMC3221326  PMID: 21971308
face processing; other-race effect; face categorization; face recognition; fusiform face area; occipital face area; fMRI; cortical neural resources
14.  Biochemical Characterization of the CDP-d-Arabinitol Biosynthetic Pathway in Streptococcus pneumoniae 17F 
Journal of Bacteriology  2012;194(8):1868-1874.
Streptococcus pneumoniae is a major human pathogen associated with many diseases worldwide. Capsular polysaccharides (CPSs) are the major virulence factor. The biosynthetic pathway of d-arabinitol, which is present in the CPSs of several S. pneumoniae serotypes, has never been identified. In this study, the genes abpA (previously known as abp1) and abpB (previously known as abp2), which have previously been reported to be responsible for nucleoside diphosphate (NDP)-d-arabinitol (the nucleotide-activated form of d-arabinitol) synthesis, were cloned. The enzyme products were overexpressed, purified, and analyzed for their respective activities. Novel products produced by AbpA- and AbpB-catalyzing reactions were detected by capillary electrophoresis, and the structures of the products were elucidated using electrospray ionization mass spectrometry and nuclear magnetic resonance spectroscopy. As a result, abpA was identified to be a d-xylulose-5-phosphate cytidylyltransferase-encoding gene, responsible for the transfer of CTP to d-xylulose-5-phosphate (d-Xlu-5-P) to form CDP-d-xylulose, and abpB was characterized to be a CDP-d-xylulose reductase-encoding gene, responsible for the conversion of CDP-d-xylulose to CDP-d-arabinitol as the final product. The kinetic parameters of AbpA for the substrates d-Xlu-5-P and CTP and those of AbpB for the substrate CDP-d-xylulose and the cofactors NADH or NADPH were measured, and the effects of temperature, pH, and cations on the two enzymes were analyzed. This study confirmed the involvement of the genes abpA and abpB and their products in the biosynthetic pathway of CDP-d-arabinitol.
PMCID: PMC3318453  PMID: 22328666
15.  Intra- and postoperative complications in 137 cases of giant thyroid gland tumor 
Oncology Letters  2012;4(5):965-969.
The intra- and postoperative complications resulting from surgery for giant thyroid gland tumors (diameter greater than 10 cm) present serious challenges to patient recovery. Although there are a number of methods, all have limitations. In this study, we present our experience with several complications of surgical treatment of giant thyroid gland tumors to increase the awareness and aid the prevention of these complications. A total of 137 consecutive patients who underwent surgical treatment in Henan Tumor Hospital were retrospectively analyzed. Statistics pertaining to the patients’ clinical factors were gathered. We found that the most common surgical complications were recurrent laryngeal nerve (RLN) injury and symptomatic hypoparathyroidism. Other complications included incision site infections, bleeding, infection and chyle fistula, the incidence of which increased significantly with increasing extent of surgery from group I (near-total thyroidectomy) to group V (total thyroidectomy plus lateral neck dissection). Low complication rates may be achieved with more accurate knowledge of the surgical anatomy, skilled surgical treatment and experience. More extensive surgery results in a greater number of complications.
PMCID: PMC3499596  PMID: 23162632
complications; giant thyroid gland; recurrent laryngeal nerve; hypoparathyroidism
16.  Use of a DNA Microarray for Detection and Identification of Bacterial Pathogens Associated with Fishery Products▿ 
Applied and Environmental Microbiology  2011;77(23):8219-8225.
We established a microarray for the simultaneous detection and identification of diverse putative pathogens often associated with fishery products by targeting specific genes of Listeria monocytogenes, Salmonella, Shigella, Staphylococcus aureus, Streptococcus pyogenes, Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus, and Yersinia enterocolitica and the 16S-23S rRNA gene internal transcribed spacer (ITS) region of Proteus mirabilis and Proteus vulgaris. The microarray contained 26 specific probes and was tested against a total of 123 target bacterial strains that included 55 representative strains, 68 clinical isolates, and 45 strains of other bacterial species that belonged to 8 genera and 34 species, and it was shown to be specific and reproducible. A detection sensitivity of 10 ng DNA or 10 CFU/ml for pure cultures of each target organism demonstrated that the assay was highly sensitive and reproducible. Mock and real fishery product samples were tested by the microarray, and the accuracy was 100%. The DNA microarray method described in this communication is specific, sensitive, and reliable and has several advantages over traditional methods of bacterial culture and antiserum agglutination assays.
PMCID: PMC3233069  PMID: 21965411
17.  Complete Genome Sequence of Pusillimonas sp. T7-7, a Cold-Tolerant Diesel Oil-Degrading Bacterium Isolated from the Bohai Sea in China▿ 
Journal of Bacteriology  2011;193(15):4021-4022.
Pusillimonas sp. T7-7 is a diesel oil-degrading cold-tolerant bacterium isolated from the benthal mud of a petroleum-contaminated site in Bohai Sea, China. We present here the complete genome sequence of T7-7. Genome analysis revealed many features of typical marine bacteria, including the absence of intact sugar metabolic pathways, the presence of glyoxylate and gluconeogenesis pathways, and the abilities for nitrate assimilation and denitrification, as well as sulfate reduction and sulfite oxidation. The presence of novel genes for the degradation of diesel oils was suggested.
PMCID: PMC3147516  PMID: 21622753
18.  A novel non-homologous recombination-mediated mechanism for Escherichia coli unilateral flagellar phase variation 
Nucleic Acids Research  2012;40(10):4530-4538.
Flagella contribute to the virulence of bacteria through chemotaxis, adhesion to and invasion of host surfaces. Flagellar phase variation is believed to facilitate bacterial evasion of the host immune response. In this study, the flnA gene that encodes Escherichia coli H17 flagellin was examined by whole genome sequencing and genetic deletion analysis. Unilateral flagellar phase variation has been reported in E. coli H3, H47 and H17 strains, although the mechanism for phase variation in the H17 strain has not been previously understood. Analysis of phase variants indicated that the flagellar phase variation in the H17 strain was caused by the deletion of an ∼35 kb DNA region containing the flnA gene from diverse excision sites. The presence of covalently closed extrachromosomal circular forms of this excised 35 kb region was confirmed by the two-step polymerase chain reaction. The deletion and complementation test revealed that the Int1157 integrase, a tyrosine recombinase, mediates the excision of this region. Unlike most tyrosine recombinases, Int1157 is suggested to recognize diverse sites and mediate recombination between non-homologous DNA sequences. This is the first report of non-homologous recombination mediating flagellar phase variation.
PMCID: PMC3378880  PMID: 22287625
19.  Genome Characterization of the Oleaginous Fungus Mortierella alpina 
PLoS ONE  2011;6(12):e28319.
Mortierella alpina is an oleaginous fungus which can produce lipids accounting for up to 50% of its dry weight in the form of triacylglycerols. It is used commercially for the production of arachidonic acid. Using a combination of high throughput sequencing and lipid profiling, we have assembled the M. alpina genome, mapped its lipogenesis pathway and determined its major lipid species. The 38.38 Mb M. alpina genome shows a high degree of gene duplications. Approximately 50% of its 12,796 gene models, and 60% of genes in the predicted lipogenesis pathway, belong to multigene families. Notably, M. alpina has 18 lipase genes, of which 11 contain the class 2 lipase domain and may share a similar function. M. alpina's fatty acid synthase is a single polypeptide containing all of the catalytic domains required for fatty acid synthesis from acetyl-CoA and malonyl-CoA, whereas in many fungi this enzyme is comprised of two polypeptides. Major lipids were profiled to confirm the products predicted in the lipogenesis pathway. M. alpina produces a complex mixture of glycerolipids, glycerophospholipids and sphingolipids. In contrast, only two major sterol lipids, desmosterol and 24(28)-methylene-cholesterol, were detected. Phylogenetic analysis based on genes involved in lipid metabolism suggests that oleaginous fungi may have acquired their lipogenic capacity during evolution after the divergence of Ascomycota, Basidiomycota, Chytridiomycota and Mucoromycota. Our study provides the first draft genome and comprehensive lipid profile for M. alpina, and lays the foundation for possible genetic engineering of M. alpina to produce higher levels and diverse contents of dietary lipids.
PMCID: PMC3234268  PMID: 22174787
20.  Genomic and Proteomic Analyses of the Fungus Arthrobotrys oligospora Provide Insights into Nematode-Trap Formation 
PLoS Pathogens  2011;7(9):e1002179.
Nematode-trapping fungi are “carnivorous” and attack their hosts using specialized trapping devices. The morphological development of these traps is the key indicator of their switch from saprophytic to predacious lifestyles. Here, the genome of the nematode-trapping fungus Arthrobotrys oligospora Fres. (ATCC24927) was reported. The genome contains 40.07 Mb assembled sequence with 11,479 predicted genes. Comparative analysis showed that A. oligospora shared many more genes with pathogenic fungi than with non-pathogenic fungi. Specifically, compared to several sequenced ascomycete fungi, the A. oligospora genome has a larger number of pathogenicity-related genes in the subtilisin, cellulase, cellobiohydrolase, and pectinesterase gene families. Searching against the pathogen-host interaction gene database identified 398 homologous genes involved in pathogenicity in other fungi. The analysis of repetitive sequences provided evidence for repeat-induced point mutations in A. oligospora. Proteomic and quantitative PCR (qPCR) analyses revealed that 90 genes were significantly up-regulated at the early stage of trap-formation by nematode extracts and most of these genes were involved in translation, amino acid metabolism, carbohydrate metabolism, cell wall and membrane biogenesis. Based on the combined genomic, proteomic and qPCR data, a model for the formation of nematode trapping device in this fungus was proposed. In this model, multiple fungal signal transduction pathways are activated by its nematode prey to further regulate downstream genes associated with diverse cellular processes such as energy metabolism, biosynthesis of the cell wall and adhesive proteins, cell division, glycerol accumulation and peroxisome biogenesis. This study will facilitate the identification of pathogenicity-related genes and provide a broad foundation for understanding the molecular and evolutionary mechanisms underlying fungi-nematodes interactions.
Author Summary
The fungus Arthrobotrys oligospora has multiple lifestyles. It's not only a nematode pathogen, but also a saprophyte, a pathogen of other fungi, and a colonizer of plant roots. As a nematode pathogen, A. oligospora forms adhesive networks to capture nematodes and is a model organism for understanding the interaction between these fungi and their host nematodes. In this study, the whole genome sequence of A. oligospora was reported. Our analyses of the proteome profiles of intracellular proteins from cells treated with nematode extracts for 10 h and 48 h revealed a key set of genes involved in trap formation. The changes in protein levels for some trap formation related genes were further confirmed by qPCR. The combined genome and proteome analysis identified the major genetic and metabolic pathways involved in trap formation in A. oligospora. Our results provide the first glimpse into the genome and proteome of this fascinating group of carnivorous fungi. The data should serve as a roadmap for further investigations into the interaction between nematode-trapping fungi and their host nematodes, providing broad foundations for research on the biocontrol of pathogenic nematodes.
PMCID: PMC3164635  PMID: 21909256
21.  Biochemical Characterization of UDP-Gal:GlcNAc-Pyrophosphate-Lipid β-1,4-Galactosyltransferase WfeD, a New Enzyme from Shigella boydii Type 14 That Catalyzes the Second Step in O-Antigen Repeating-Unit Synthesis▿ †  
Journal of Bacteriology  2010;193(2):449-459.
The O antigen is the outer part of the lipopolysaccharide (LPS) in the outer membrane of Gram-negative bacteria and contains many repeats of an oligosaccharide unit. It contributes to antigenic variability and is essential to the full function and virulence of bacteria. Shigella is a Gram-negative human pathogen that causes diarrhea in humans. The O antigen of Shigella boydii type 14 consists of repeating oligosaccharide units with the structure [→6-d-Galpα1→4-d-GlcpAβ1→6-d-Galpβ1→4-d-Galpβ1→4-d-GlcpNAcβ1→]n. The wfeD gene in the O-antigen gene cluster of Shigella boydii type 14 was proposed to encode a galactosyltransferase (GalT) involved in O-antigen synthesis. We confirmed here that the wfeD gene product is a β4-GalT that synthesizes the Galβ1-4GlcNAcα-R linkage. WfeD was expressed in Escherichia coli, and the activity was characterized by using UDP-[3H]Gal as the donor substrate as well as the synthetic acceptor substrate GlcNAcα-pyrophosphate-(CH2)11-O-phenyl. The enzyme product was analyzed by liquid chromatography-mass spectrometry (LC-MS), high-performance liquid chromatography (HPLC), nuclear magnetic resonance (NMR), and galactosidase digestion. The enzyme was shown to be specific for the UDP-Gal donor substrate and required pyrophosphate in the acceptor substrate. Divalent metal ions such as Mn2+, Ni2+, and, surprisingly, also Pb2+ enhanced the enzyme activity. Mutational analysis showed that the Glu101 residue within a DxD motif is essential for activity, possibly by forming the catalytic nucleophile. The Lys211 residue was also shown to be required for activity and may be involved in the binding of the negatively charged acceptor substrate. Our study revealed that the β4-GalT WfeD is a novel enzyme that has virtually no sequence similarity to mammalian β4-GalT, although it catalyzes a similar reaction.
PMCID: PMC3019819  PMID: 21057010
22.  Characterization of the CDP-2-Glycerol Biosynthetic Pathway in Streptococcus pneumoniae▿ †  
Journal of Bacteriology  2010;192(20):5506-5514.
Capsule polysaccharide (CPS) plays an important role in the virulence of Streptococcus pneumoniae and is usually used as the pneumococcal vaccine target. Glycerol-2-phosphate is found in the CPS of S. pneumoniae types 15A and 23F and is rarely found in the polysaccharides of other bacteria. The biosynthetic pathway of the nucleotide-activated form of glycerol-2-phosphate (NDP-2-glycerol) has never been identified. In this study, three genes (gtp1, gtp2, and gtp3) from S. pneumoniae 23F that have been proposed to be involved in the synthesis of NDP-2-glycerol were cloned and the enzyme products were expressed, purified, and assayed for their respective activities. Capillary electrophoresis was used to detect novel products from the enzyme-substrate reactions, and the structure of the product was elucidated using electrospray ionization mass spectrometry and nuclear magnetic resonance spectroscopy. Gtp1 was identified as a reductase that catalyzes the conversion of 1,3-dihydroxyacetone to glycerol, Gtp3 was identified as a glycerol-2-phosphotransferase that catalyzes the conversion of glycerol to glycerol-2-phosphate, and Gtp2 was identified as a cytidylyltransferase that transfers CTP to glycerol-2-phosphate to form CDP-2-glycerol as the final product. The kinetic parameters of Gtp1 and Gtp2 were characterized in depth, and the effects of temperature, pH, and cations on these two enzymes were analyzed. This is the first time that the biosynthetic pathway of CDP-2-glycerol has been identified biochemically; this pathway provides a method to enzymatically synthesize this compound.
PMCID: PMC2950487  PMID: 20729354
23.  Molecular and Genetic Analyses of the Putative Proteus O Antigen Gene Locus▿ † 
Applied and Environmental Microbiology  2010;76(16):5471-5478.
Proteus species are well-characterized opportunistic pathogens primarily associated with urinary tract infections (UTI) of humans. The Proteus O antigen is one of the most variable constituents of the cell surface, and O antigen heterogeneity is used for serological classification of Proteus isolates. Even though most Proteus O antigen structures have been identified, the O antigen locus has not been well characterized. In this study, we identified the putative Proteus O antigen locus and demonstrated this region's high degree of heterogeneity by comparing sequences of 40 Proteus isolates using PCR-restriction fragment length polymorphism (RFLP). This analysis identified five putative Proteus O antigen gene clusters, and the probable functions of these O antigen-related genes were proposed, based on their similarity to genes in the available databases. Finally, Proteus-specific genes from these five serogroups were identified by screening 79 strains belonging to the 68 Proteus O antigen serogroups. To our knowledge, this is the first molecular characterization of the putative Proteus O antigen locus, and we describe a novel molecular classification method for the identification of different Proteus serogroups.
PMCID: PMC2918944  PMID: 20581173
24.  Development of a DNA Microarray for Detection and Serotyping of Enterotoxigenic Escherichia coli ▿  
Journal of Clinical Microbiology  2010;48(6):2066-2074.
Enterotoxigenic Escherichia coli (ETEC) is a common pathogen worldwide causing infectious diarrhea, especially traveler's diarrhea. Traditional physiological assays, immunoassays, and PCR-based methods for the detection of ETEC target the heat-labile enterotoxin and/or the heat-stable enterotoxin. Separate serotyping methods using antisera are required to determine the ETEC serogroup. In this study, we developed a DNA microarray that can simultaneously detect enterotoxin genes and the 19 most common O serogroup genes in ETEC strains. The specificity and reproducibility of this approach were verified by hybridization to 223 strains: 50 target reference or clinical strains and 173 other strains, including those belonging to other E. coli O serogroups and closely related species. The sensitivity of detection was determined to be 50 ng of genomic DNA or 108 CFU per ml of organisms in pure culture. The random PCR strategy used in this study with minimal bias provides an effective alternative to multiplex PCR for the detection of pathogens using DNA microarrays. The assay holds promise for applications in the clinical diagnosis and epidemiological surveillance of pathogenic microorganisms.
PMCID: PMC2884529  PMID: 20351209
25.  Utility of BRAF protein overexpression in predicting the metastasis potential of papillary thyroid carcinoma 
Oncology Letters  2010;2(1):59-63.
V-raf murine sarcoma viral oncogene homolog B1 (BRAF) is a significant member of the MAPK pathway, the point mutation (V600E) of which is a common genetic event in papillary thyroid carcinoma (PTC). Investigators showed that the variations in BRAF expression levels were independent of the V600E mutation. These variations were involved in the pathogenesis of thyroid carcinomas. This study evaluated the feasibility of BRAF, proliferating cell nuclear antigen (PCNA) and hMSH2 as markers for the prediction of the metastatic potential of PTC. Using immunohistochemistry, the expression of BRAF, PCNA and hMSH2 proteins was studied in 70 PTC and 29 nodular goiter (NG) tissues. The results indicated that i) the positive rate of BRAF, PCNA and hMSH2 expression in PTCs was significantly higher than that in NGs (P=0.000, P=0.000 and P=0.003, respectively), ii) the positive rate of BRAF expression in the lymph node metastasis (LNM) group was significantly higher than that in the non-LNM group (P=0.019), iii) the age at diagnosis of PTC patients with LNM was significantly older compared to that without LNM (P=0.021) and iv) the positive rate of BRAF expression significantly correlated with that of PCNA and hMSH2 expression (P=0.000 and P=0.019, respectively). In conclusion, BRAF, PCNA and hMSH2 overexpression appeared to be molecular events of PTC carcinogenesis. Older patients with BRAF overexpression appear to be a high-risk group for PTC metastasis. Detection of BRAF expression is likely to aid in the prediction of the metastatic potential of the carcinoma.
PMCID: PMC3412503  PMID: 22870129
papillary thyroid carcinoma; V-raf murine sarcoma viral oncogene homolog B1; proliferating cell nuclear antigen; hMSH2; immunohistochemistry

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