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1.  Peripheral Blood Mononuclear Cell Traffic Plays a Crucial Role in Mother-to-Infant Transmission of Hepatitis B Virus 
The role of peripheral blood mononuclear cells (PBMCs) in HBV intrauterine infection is not fully defined. Particularly the origin of PBMCs in HBV-infected neonates remains to be addressed. We carried out a population-based nested case-control study by enrolling 312 HBsAg-positive mothers and their babies. PBMC HBV DNA as well as serum HBsAg and HBV DNA was tested in cohort entry samples. Totally, 45.5% (142/312) of the newborns were found to be infected with HBV in perinatal transmission. 119 mother-infant pairs were identified to be different in the genetic profile of maternal and fetal PBMCs by AS-PCR and hemi-nested PCR. Among them, 57.1% (68/119) of the maternal PBMCs in index cases were positive for HBV DNA while 83.8% (57/68) of the HBV DNA positive maternal PBMCs passed the placental barrier and entered the fetus. Furthermore, maternal PBMC HBV infection was significantly associated with newborn infants HBV infection. PBMC traffic from mother to fetus resulted in a 9.5-fold increased risk of HBV infection in PBMC HBV DNA positive newborn infants. These data indicate that maternal PBMCs infected with HBV contribute to HBV intrauterine infection of newborn infants via PBMC traffic from mother to fetus.
doi:10.7150/ijbs.10813
PMCID: PMC4323366
Hepatitis B virus; mother-to-infant transmission; peripheral blood mononuclear cell; fetomaternal cellular traffic.
2.  The Widely Used ATB FUNGUS 3 Automated Readings in China and Its Misleading High MICs of Candida spp. to Azoles: Challenges for Developing Countries' Clinical Microbiology Labs 
PLoS ONE  2014;9(12):e114004.
The rapid development in the clinical microbiology diagnostic assays presents more challenges for developing countries than for the developed world, especially in the area of test validation before the introduction of new tests. Here we report on the misleading high MICs of Candida spp. to azoles using the ATB FUNGUS 3 (bioMérieux, La Balme-les Grottes, France) with automated readings in China to highlight the dangers of introducing a diagnostic assay without validation. ATB FUNGUS 3 is the most commonly used commercial antifungal susceptibility testing method in China. An in-depth analysis of data showed higher levels of resistance to azoles when ATB FUNGUS 3 strips were read automatically than when read visually. Based on this finding, the performance of ATB FUNGUS 3, read both visually and automatically, was evaluated by testing 218 isolates of five clinically important Candida species, using broth microdilution (BMD) following CLSI M27-A3 as the gold-standard. The overall essential agreement (EA) between ATB visual readings and BMD was 99.1%. In contrast, the ATB automated readings showed higher discrepancies with BMD, with overall EA of 86.2%, and specifically lower EA was observed for fluconazole (80.7%), voriconazole (77.5%), and itraconazole (73.4%), which was most likely due to the trailing effect of azoles. The major errors in azole drug susceptibilities by ATB automated readings is a concern in China that can result in misleading clinical antifungal drug selection and pseudo high rates of antifungal resistance. Therefore, the ATB visual reading is generally recommended. In the meantime, we propose a practical algorithm to be followed for ATB FUNGUS 3 antifungal susceptibility for Candida spp. before the improvement in the automated reading system.
doi:10.1371/journal.pone.0114004
PMCID: PMC4252076  PMID: 25460351
3.  A Highly Selective and Sensitive Turn-On Fluorescent Chemosensor Based on Rhodamine 6G for Iron(III)** 
ChemistryOpen  2014;3(6):264-268.
Recently, more and more rhodamine derivatives have been used as fluorophores to construct sensors due to their excellent spectroscopic properties. A rhodamine-based fluorescent and colorimetric Fe3+ chemosensor 3’,6’-bis(ethylamino)-2-acetoxyl-2’,7’-dimethyl-spiro[1H-isoindole-1,9’-[9H]xanthen]-3(2H)-one (RAE) was designed and synthesized. Upon the addition of Fe3+, the dramatic enhancement of both fluorescence and absorbance intensity, as well as the color change of the solution, could be observed. The detection limit of RAE for Fe3+ was around 7.98 ppb. Common coexistent metal ions showed little or no interference in the detection of Fe3+. Moreover, the addition of CN− could quench the fluorescence of the acetonitrile solution of RAE and Fe3+, indicating the regeneration of the chemosensor RAE. The robust nature of the sensor was shown by the detection of Fe3+ even after repeated rounds of quenching. As iron is a ubiquitous metal in cells and plays vital roles in many biological processes, this chemosensor could be developed to have applications in biological studies.
doi:10.1002/open.201402065
PMCID: PMC4280826  PMID: 25558445
chemosensors; fluorescence; iron(III); rhodamine; selective response
4.  Risk factor analysis of perioperative mortality after ruptured bleeding in hepatocellular carcinoma 
World Journal of Gastroenterology : WJG  2014;20(40):14921-14926.
AIM: To discuss strategies and prognosis for the emergency treatment of ruptured bleeding in primary hepatocellular carcinoma.
METHODS: The retrospective analysis was performed by examining the emergency treatment experiences of 60 cases of ruptured bleeding in primary hepatocellular carcinoma. The treatment methods included surgical tumour resection, transcatheter arterial embolization (TAE) and non-surgical treatment. Univariate and multivariate analyses were performed to identify the risk factors that impacted 30-d mortality in the research groups.
RESULTS: The 30-d mortality of all patients was 28.3% (n = 17). The univariate analysis showed that Child-Pugh C level liver function, shock, massive blood transfusion and large tumour volume were risk factors that influenced 30-d mortality. The multivariate analysis showed that shock and massive blood transfusion were independent risk factors that impacted the 30-d mortality of surgical resection. As for the TAE patients, larger tumour volume was a risk factor towards prognosis.
CONCLUSION: Radical resection and TAE therapy would achieve better results in carefully selected ruptured hepatocellular tumours.
doi:10.3748/wjg.v20.i40.14921
PMCID: PMC4209555  PMID: 25356052
Hepatocellular carcinoma; Spontaneous rupture; Liver resection
5.  PI3K/AKT Pathway Involvement in the Osteogenic Effects of Osteoclast Culture Supernatants on Preosteoblast Cells 
Tissue Engineering. Part A  2013;19(19-20):2226-2232.
This study aimed to investigate the ability of osteoclasts during bone resorption activities to regulate the differentiation and calcification of osteoblast precursor cells. The bone resorption model was established using in vitro cortical bone slices and mouse RAW264.7 cells, which were differentiated into osteoclasts by stimulation with the receptor activator of nuclear factor-κB ligand and macrophage colony-stimulating factor. Tartrate-resistant acid phosphatase (TRAP) staining, reverse transcriptase–polymerase chain reaction (RT-PCR), and scanning electron microscopy (SEM) were used to detect osteoclast differentiation. The osteoblast precursor cell line MC3T3-E1 was cultured with the bone resorption supernatant (BRS). Involvement of the phosphatidylinositol 3-kinase (PI3K)/AKT pathway in osteogenesis was evaluated by Western blotting, RT-PCR, and ELISA analysis of markers of the early (runt-related transcription factor-2 and alkaline phosphatase) and late (osteocalcin [OCN]) stages of osteogenesis, and Alizarin Red S staining of matrix mineralization. TRAP staining, RT-PCR, and SEM analysis demonstrated the successful establishment of the bone resorption model. Osteoclast BRS effectively increased the differentiation and calcification of MC3T3-E1 cells. Western blot analysis indicated that the BRS enhanced AKT and p-AKT expression levels in MC3T3-E1 cells. Following AKT2 knockdown and treatment with the PI3K/AKT pathway inhibitor LY294002, the expression of OCN in MC3T3-E1 cells was decreased (p<0.05), as was the calcification area (p<0.05). The data obtained in this study indicated that the osteoclast bone resorption medium promoted the differentiation and calcification of MC3T3-E1 cells and that the PI3K/AKT pathway played a role in this process.
doi:10.1089/ten.tea.2012.0469
PMCID: PMC3761438  PMID: 23617625
6.  National Surveillance of Methicillin-Resistant Staphylococcus aureus in China Highlights a Still-Evolving Epidemiology with 15 Novel Emerging Multilocus Sequence Types 
Journal of Clinical Microbiology  2013;51(11):3638-3644.
The global spread of methicillin-resistant Staphylococcus aureus (MRSA) is a serious problem, particularly in mainland China. In order to better understand the national molecular epidemiology and resistance profiles of hospital-associated MRSA (HA-MRSA) in China, a laboratory-based multicenter surveillance study was conducted. Sixty-nine hospitals in 45 large cities in 27 provinces were involved, and a total of 1,141 HA-MRSA isolates were collected during the 6-month study period in 2011. All MRSA isolates were characterized by multilocus sequence typing (MLST), staphylococcal cassette chromosome mec (SCCmec) typing, spa typing, detection of the Panton-Valentine leukocidin (PVL) locus (lukS-PV and lukF-PV), and antibiogram analysis. ST239-III-t030, ST239-III-t037, and ST5-II-t002 were the predominant HA-MRSA clones (overall prevalence rates, 57.1%, 12.9%, and 8.1%, respectively), although the prevalence rates of these major clones varied markedly in different administrative regions. Of note, 6.6% of the HA-MRSA isolates were found to belong to ST59, which had typical community-associated MRSA (CA-MRSA) features, including carriage of SCCmec type IV or V and PVL and less antimicrobial resistance than other major HA-MRSA clones. Moreover, among 36 MLST sequence types (STs) identified, 15 STs, accounting for 3.5% of total isolates, were novel. A novel ST designated ST2590, which is a single-locus variant of ST5-II-t002, was identified in three hospitals in two large cities, with a total of 17 isolates. To further monitor trends in HA-MRSA prevalence, epidemic clonal shifts, clone emergence, and transmission between community and health care settings, longitudinal national MRSA surveillance is required.
doi:10.1128/JCM.01375-13
PMCID: PMC3889745  PMID: 23985906
7.  Unusual Compression Behavior of Nanocrystalline CeO2 
Scientific Reports  2014;4:4441.
The x-ray diffraction study of 12 nm CeO2 was carried out up to ~40 GPa using an angle dispersive synchrotron-radiation in a diamond-anvil cell with different pressure transmitting medium (PTM) (4:1 methanol: ethanol mixture, silicone oil and none) at room temperature. While the cubic fluorite-type structure CeO2 was retained to the highest pressure, there is progressive broadening and intensity reduction of the reflections with increasing pressure. At pressures above 12 GPa, an unusual change in the compression curve was detected in all experiments. Significantly, apparent negative volume compressibility was observed at P = 18–27 GPa with silicone oil as PTM, however it was not detected in other circumstances. The expansion of the unit cell volume of cubic CeO2 was about 1% at pressures of 15–27 GPa. To explain this abnormal phenomenon, a dual structure model (hard amorphous shell and relatively soft crystalline core) has been proposed.
doi:10.1038/srep04441
PMCID: PMC3963033  PMID: 24658049
8.  Cytochrome P450 107U1 is required for sporulation and antibiotic production in Streptomyces coelicolor 
The filamentous bacterium Streptomyces coelicolor has a complex life cycle involving the formation of hair-like aerial mycelia on the colony surface, which differentiate into chains of spores. Genes required for the initiation of aerial mycelium formation have been termed ‘bld’ (bald), describing the smooth, undifferentiated colonies of mutant strains. We report the identification of a new bld gene designated as sco3099 and biochemical analysis of its encoded enzyme, cytochrome P450 (P450, or CYP) 107U1. Deletion of sco3099 resulted in a mutant defective in aerial hyphae sporulation and sensitive to heat shock, indicating that P450 107U1 plays a key role in growth and development of S. coelicolor. This is the first P450 reported to participate in a sporulation process in Streptomycetes. The substrate and catalytic properties of P450 107U1 were further investigated in mass spectrometry-based metabolomic studies. Glycocholic acid (from the medium) was identified as a substrate of P450 107U1 and was oxidized to glyco-7-oxo-deoxycholic acid. Although this reaction is apparently not relevant to the observed sporulation deficiency, it suggests that P450 107U1 might exert its physiological function by oxidizing other steroid-like molecules.
doi:10.1016/j.abb.2013.01.001
PMCID: PMC3600146  PMID: 23357279
Cytochrome P450; Streptomyces; antibiotic synthesis; sporulation; steroid oxidation; metabolomics
9.  Hormonal Regulation of Ovarian Bursa Fluid in Mice and Involvement of Aquaporins 
PLoS ONE  2013;8(5):e63823.
In rodent species, the ovary and the end of oviduct are encapsulated by a thin membrane called ovarian bursa. The biological functions of ovarian bursa remain unexplored despite its structural arrangement in facilitating oocytes transport into oviduct. In the present study, we observed a rapid fluid accumulation and reabsorption within the ovarian bursa after ovarian stimulation (PMSG-primed hCG injection), suggesting that the ovarian bursa might play an active role in regulating local fluid homeostasis around the timing of ovulation. We hypothesized that the aquaporin proteins, which are specialized channels for water transport, might be involved in this process. By screening the expression of aquaporin family members (Aqp1-9) in the ovarian tissue and isolated ovarian bursa (0, 1, 2 and 5 h after hCG injection), we found that AQP2 and AQP5 mRNA showed dynamic changes after hCG treatment, showing upregulation at 1–2 h followed by gradually decrease at 5 h, which is closely related with the intra-bursa fluid dynamics. Further immunofluorescence examinations of AQP2 and AQP5 in the ovarian bursa revealed that AQP2 is specifically localized in the outer layer (peritoneal side) while AQP5 localized in the inner layer (ovarian side) of the bursa, such cell type specific and spatial-temporal expressions of AQP2 and 5 support our hypothesis that they might be involved in efficient water transport through ovarian bursa under ovulation related hormonal regulation. The physiological significance of aquaporin-mediated water transport in the context of ovarian bursa still awaits further clarification.
doi:10.1371/journal.pone.0063823
PMCID: PMC3661669  PMID: 23717491
10.  The Role of Ile87 of CYP158A2 in Oxidative Coupling Reaction 
Both CYP158A1 and CYP158A2 are able to catalyze an oxidative C-C coupling reaction producing biflaviolin or triflaviolin in Streptomyces coelicolor A3(2). The substrate-bound crystal structures of CYP158A2 and CYP158A1 reveal that the side chain of Ile87 in CYP158A2 points to the active site contacting the distal flaviolin molecule, however, the bulkier side chain of Lys90 in CYP158A1 (corresponding to Ile87 in CYP158A2) is toward the distal surface of the protein. These results suggest that these residues could be important in determining product regiospecificity. In order to explore the role of the two residues in catalysis, the reciprocal mutants, Ile87Lys and Lys90Ile, of CYP158A2 and CYP158A1, respectively, were generated and characterized. The mutant Ile87Lys enzyme forms two isomers of biflaviolin instead of three isomers of biflaviolin in wild-type CYP158A2. CYP158A1 containing the substitution of lysine with isoleucine has the same catalytic activity compared with the wild-type CYP158A1. The crystal structure of Ile87Lys showed that the BC loop in the mutant is in a very different orientation compared with the BC loop in both CYP158A1/A2 structures. These results shed light on the mechanism of the oxidative coupling reaction catalyzed by cytochrome P450.
doi:10.1016/j.abb.2011.12.007
PMCID: PMC3274592  PMID: 22203090
Cytochrome P450; Oxidative coupling reaction; CYP158A1; CYP158A2; Regiospecificity
11.  SUMO-1 Modification on K166 of PolyQ-Expanded aTaxin-3 Strengthens Its Stability and Increases Its Cytotoxicity 
PLoS ONE  2013;8(1):e54214.
Post-translational modification by SUMO was proposed to modulate the pathogenesis of several neurodegenerative diseases. Spinocerebellar ataxia type 3/Machado-Joseph disease (SCA3/MJD) is an autosomal dominant neurodegenerative disease caused by polyQ-expanded ataxin-3. We have previously shown that ataxin-3 was a new target of SUMOylation in vitro and in vivo. Here we identified that the major SUMO-1 binding site was located on lysine 166. SUMOylation did not influence the subcellular localization, ubiquitination or aggregates formation of mutant-type ataxin-3, but partially increased its stability and the cell apoptosis. Our findings revealed the role of ataxin-3 SUMOylation in SCA3/MJD pathogenesis.
doi:10.1371/journal.pone.0054214
PMCID: PMC3561348  PMID: 23382880
13.  Aquaporin3 is a sperm water channel essential for postcopulatory sperm osmoadaptation and migration 
Cell Research  2010;21(6):922-933.
In the journey from the male to female reproductive tract, mammalian sperm experience a natural osmotic decrease (e.g., in mouse, from ∼415 mOsm in the cauda epididymis to ∼310 mOsm in the uterine cavity). Sperm have evolved to utilize this hypotonic exposure for motility activation, meanwhile efficiently silence the negative impact of hypotonic cell swelling. Previous physiological and pharmacological studies have shown that ion channel-controlled water influx/efflux is actively involved in the process of sperm volume regulation; however, no specific sperm proteins have been found responsible for this rapid osmoadaptation. Here, we report that aquaporin3 (AQP3) is a sperm water channel in mice and humans. Aqp3-deficient sperm show normal motility activation in response to hypotonicity but display increased vulnerability to hypotonic cell swelling, characterized by increased tail bending after entering uterus. The sperm defect is a result of impaired sperm volume regulation and progressive cell swelling in response to physiological hypotonic stress during male-female reproductive tract transition. Time-lapse imaging revealed that the cell volume expansion begins at cytoplasmic droplet, forcing the tail to angulate and form a hairpin-like structure due to mechanical membrane stretch. The tail deformation hampered sperm migration into oviduct, resulting in impaired fertilization and reduced male fertility. These data suggest AQP3 as an essential membrane pathway for sperm regulatory volume decrease (RVD) that balances the “trade-off” between sperm motility and cell swelling upon physiological hypotonicity, thereby optimizing postcopulatory sperm behavior.
doi:10.1038/cr.2010.169
PMCID: PMC3343308  PMID: 21135872
aquaporin3; sperm; osmoregulation; postcopulation
14.  Expansion and diversification of the SET domain gene family following whole-genome duplications in Populus trichocarpa 
Background
Histone lysine methylation modifies chromatin structure and regulates eukaryotic gene transcription and a variety of developmental and physiological processes. SET domain proteins are lysine methyltransferases containing the evolutionarily-conserved SET domain, which is known to be the catalytic domain.
Results
We identified 59 SET genes in the Populus genome. Phylogenetic analyses of 106 SET genes from Populus and Arabidopsis supported the clustering of SET genes into six distinct subfamilies and identified 19 duplicated gene pairs in Populus. The chromosome locations of these gene pairs and the distribution of synonymous substitution rates showed that the expansion of the SET gene family might be caused by large-scale duplications in Populus. Comparison of gene structures and domain architectures of each duplicate pair indicated that divergence took place at the 3'- and 5'-terminal transcribed regions and at the N- and C-termini of the predicted proteins, respectively. Expression profile analysis of Populus SET genes suggested that most Populus SET genes were expressed widely, many with the highest expression in young leaves. In particular, the expression profiles of 12 of the 19 duplicated gene pairs fell into two types of expression patterns.
Conclusions
The 19 duplicated SET genes could have originated from whole genome duplication events. The differences in SET gene structure, domain architecture, and expression profiles in various tissues of Populus suggest that members of the SET gene family have a variety of developmental and physiological functions. Our study provides clues about the evolution of epigenetic regulation of chromatin structure and gene expression.
doi:10.1186/1471-2148-12-51
PMCID: PMC3402991  PMID: 22497662
15.  Structural Analysis of Cytochrome P450 105N1 Involved in the Biosynthesis of the Zincophore, Coelibactin 
Coelibactin is a putative non-ribosomally synthesized peptide with predicted zincophore activity and which has been implicated in antibiotic regulation in Streptomyces coelicolor A3(2). The coelibactin biosynthetic pathway contains a stereo- and regio-specific monooxygenation step catalyzed by a cytochrome P450 enzyme (CYP105N1). We have determined the X-ray crystal structure of CYP105N1 at 2.9 Å and analyzed it in the context of the bacterial CYP105 family as a whole. The crystal structure reveals a channel between the α-helical domain and the β-sheet domain exposing the heme pocket and the long helix I to the solvent. This wide-open conformation of CYP105N1 may be related to the bulky substrate coelibactin. The ligand-free CYP105N1 structure has enough room in the substrate access channel to allow the coelibactin to enter into the active site. Analysis of typical siderophore ligands suggests that CYP105N1 may produce derivatives of coelibactin, which would then be able to chelate the zinc divalent cation.
doi:10.3390/ijms13078500
PMCID: PMC3430247  PMID: 22942716
cytochrome P450; CYP105N1; siderophore; Streptomyces coelicolor A3(2); zinc chelation
16.  Cyclization of a Cellular Dipentaenone by Streptomyces coelicolor Cytochrome P450 154A1 without Oxidation/Reduction 
Journal of the American Chemical Society  2010;132(43):15173-15175.
We report a comprehensive genetic, metabolomic, and biochemical study on the catalytic properties of Streptomyces coelicolor cytochrome P450 (P450) 154A1, known to have a unique heme orientation in its crystal structure. Deletion of the P450 154A1 gene compromised the long-term stability of the bacterial spores. A novel dipentaenone (1) with a high degree of conjugation was identified as an endogenous substrate of P450 154A1 using a metabolomics approach. The biotransformation of 1 by P450 154A1 was shown to be an unexpected intramolecular cyclization to a Paternò–Büchi-like product, without oxidation/reduction.
doi:10.1021/ja107801v
PMCID: PMC3118511  PMID: 20979426
17.  Seroprevalence of Toxoplasma gondii infection in dairy goats in Shaanxi Province, Northwestern China 
Parasites & Vectors  2011;4:47.
Background
Toxoplasma gondii is an important zoonotic pathogen causing significant human and animal health problems. Infection in dairy goats not only results in significant reproductive losses, but also represents an important source of human infection due to consumption of infected meat and milk. In the present study we report for the first time seroprevalence of T. gondii infection in Guanzhong and Saanen dairy goats in Shaanxi province, Northwestern China.
Results
Sera from 751 dairy goats from 9 farms in 6 counties were examined for T. gondii antibodies with an indirect haemagglutination (IHA) test. Antibodies to T. gondii were detected in 106 (14.1%) serum samples, with antibody titres ranging from 1:64 to 1:1024. Seropositive goats were found in all 9 farms and seroprevalences in Guanzhong (16.3%, 75/461) and Saanen (10.7%, 31/290) dairy goats were not statistically significantly different. All the factors (sex, age and location) reported in the present study affected prevalence of infection, and seroprevalence increased with age, suggesting postnatal acquisition of T. gondii infection.
Conclusions
The results of the present survey indicate that infection by T. gondii is widely prevalent in dairy goats in Shaanxi province, Northwestern China, and this has implications for prevention and control of toxoplasmosis in this province.
doi:10.1186/1756-3305-4-47
PMCID: PMC3079685  PMID: 21457538
18.  Transient β2-Adrenoceptor Activation Confers Pregnancy Loss by Disrupting Embryo Spacing at Implantation* 
The Journal of Biological Chemistry  2010;286(6):4349-4356.
Pregnancy loss is a serious social and medical issue, with one important cause associated with aberrant embryo implantation during early pregnancy. However, whether and how the process of embryo implantation is affected by environmental factors such as stress-induced sympathetic activation remained elusive. Here we report an unexpected, transient effect of β2-adrenoreceptor (β2-AR) activation (day 4 postcoitus) in disrupting embryo spacing at implantation, leading to substantially increased midterm pregnancy loss. The abnormal embryo spacing could be prevented by pretreatment of β2-AR antagonist or genetic ablation of β-AR. Similar β2-AR activation at day 5 postcoitus, when implantation sites have been established, did not affect embryo spacing or pregnancy outcome, indicating that the adverse effect of β2-AR activation is limited to the preimplantation period before embryo attachment. In vitro and in vivo studies demonstrated that the transient β2-AR activation abolished normal preimplantation uterine contractility without adversely affecting blastocyst quality. The contractility inhibition is mediated by activation of the cAMP-PKA pathway and accompanied by specific down-regulation of lpa3, a gene previously found to be critical for uterine contraction and embryo spacing. These results indicated that normal uterine contraction-mediated correct intrauterine embryo distribution is crucial for successful ongoing pregnancy. Abnormal β2-AR activation at early pregnancy provided a molecular clue in explaining how maternal stress at early stages could adversely affect the pregnancy outcome.
doi:10.1074/jbc.M110.197202
PMCID: PMC3039384  PMID: 21148315
Adrenergic Receptor; Development; Embryo; Gene Knockout; Reproduction; cAMP; Embryo Implantation; Embryo Spacing; Pregnancy Loss; Uterine Contraction
19.  Streptomyces coelicolor A3(2) CYP102 Protein, a Novel Fatty Acid Hydroxylase Encoded as a Heme Domain without an N-Terminal Redox Partner▿  
The gene from Streptomyces coelicolor A3(2) encoding CYP102B1, a recently discovered CYP102 subfamily which exists solely as a single P450 heme domain, has been cloned, expressed in Escherichia coli, purified, characterized, and compared to its fusion protein family members. Purified reconstitution metabolism experiments with spinach ferredoxin, ferredoxin reductase, and NADPH revealed differences in the regio- and stereoselective metabolism of arachidonic acid compared to that of CYP102A1, exclusively producing 11,12-epoxyeicosa-5,8,14-trienoic acid in addition to the shared metabolites 18-hydroxy arachidonic acid and 14,15-epoxyeicosa-5,8,11-trienoic acid. Consequently, in order to elucidate the physiological function of CYP102B1, transposon mutagenesis was used to generate an S. coelicolor A3(2) strain lacking CYP102B1 activity and the phenotype was assessed.
doi:10.1128/AEM.03000-09
PMCID: PMC2838009  PMID: 20097805
20.  Deposition of silver nanoparticles on titanium surface for antibacterial effect 
Microbial colonization on implanted devices and biofilm formation is a recurrent complication in implant surgery and may result in loss of implants. The aim of this study was to deposit silver nanoparticles on a titanium surface to obtain antibacterial properties. In the present study, we prepared a silver nanoparticle-modified titanium (Ti-nAg) surface using silanization method. The morphology and chemical components of the Ti-nAg surface were characterized by scanning electron microscopy (SEM) equipped with energy-dispersive spectroscopy (EDS). Two species of bacteria, Staphylococcus aureus and Escherichia coli, were utilized to test the antibacterial effect of the Ti-nAg treated surface. The SEM examination revealed that a small quantity of silver nanoparticles was sparsely deposited on the titanium surface. The diameter of these nanoparticles ranged from ten to several hundred nm. EDS analyses revealed that there was 4.26% of Ag present on the surface. After a 24-hour incubation, 94% of Staphylococcus aureus and over 95% of Escherichia coli had been killed on the Ti-nAg surface, and the SEM examination of anti-adhesive efficacy test showed that there were less bacteria attached to Ti-nAg surface than to a control surface of untreated Titanium. These data suggest that silver nanoparticle-modified titanium is a promising material with an antibacterial property that may be used as an implantable biomaterial.
PMCID: PMC2865021  PMID: 20463942
nano-silver; titanium; antibacterial activity; silanization method
21.  The First Virally Encoded Cytochrome P450▿  
Journal of Virology  2009;83(16):8266-8269.
The genome sequence of the giant virus Acanthamoeba polyphaga mimivirus revealed the presence of two putative cytochrome P450 (CYP) genes. The product of one of the two predicted CYP genes (YP_143162) showed low-level homology to sterol 14-demethylase (CYP51) and contained a C-terminal polypeptide domain of unknown function. YP_143162 expression (without an N-terminal membrane binding domain) in Escherichia coli yields a CYP protein which gives a reduced CO difference maximum at 448 nm and was formally demonstrated as the first viral cytochrome P450. Analysis of binding of lipid and sterol substrates indicated no perturbation in CYP heme environment, and an absence of activity was seen when 14-methyl sterols were used as a substrate. The function of the CYP protein and its C-terminal domain remain unknown.
doi:10.1128/JVI.00289-09
PMCID: PMC2715754  PMID: 19515774
22.  Hypoxia-Inducible Factor-Dependent Degeneration, Failure, and Malignant Transformation of the Heart in the Absence of the von Hippel-Lindau Protein▿  
Molecular and Cellular Biology  2008;28(11):3790-3803.
Hypoxia-inducible transcription factor 1 (HIF-1) and HIF-2α regulate the expression of an expansive array of genes associated with cellular responses to hypoxia. Although HIF-regulated genes mediate crucial beneficial short-term biological adaptations, we hypothesized that chronic activation of the HIF pathway in cardiac muscle, as occurs in advanced ischemic heart disease, is detrimental. We generated mice with cardiac myocyte-specific deletion of the von Hippel-Lindau protein (VHL), an essential component of an E3 ubiquitin ligase responsible for suppressing HIF levels during normoxia. These mice were born at expected frequency and thrived until after 3 months postbirth, when they developed severe progressive heart failure and premature death. VHL-null hearts developed lipid accumulation, myofibril rarefaction, altered nuclear morphology, myocyte loss, and fibrosis, features seen for various forms of human heart failure. Further, nearly 50% of VHL−/− hearts developed malignant cardiac tumors with features of rhabdomyosarcoma and the capacity to metastasize. As compelling evidence for the mechanistic contribution of HIF-1α, the concomitant deletion of VHL and HIF-1α in the heart prevented this phenotype and restored normal longevity. These findings strongly suggest that chronic activation of the HIF pathway in ischemic hearts is maladaptive and contributes to cardiac degeneration and progression to heart failure.
doi:10.1128/MCB.01580-07
PMCID: PMC2423296  PMID: 18285456
23.  Endothelial Expression of β1 Integrin Is Required for Embryonic Vascular Patterning and Postnatal Vascular Remodeling▿  
Molecular and Cellular Biology  2007;28(2):794-802.
The largest subgroup of integrins is that containing the β1 subunit. β1 integrins have been implicated in a wide array of biological processes ranging from adhesion to cell growth, organogenesis, and mechanotransduction. Global deletion of β1 integrin expression results in embryonic death at ca. embryonic day 5 (E5), a developmental time point too early to determine the effects of this integrin on vascular development. To elucidate the specific role of β1 integrin in the vasculature, we conditionally deleted the β1 gene in the endothelium. Homozygous deletion of β1 integrins in the endothelium resulted in failure of normal vascular patterning, severe fetal growth retardation, and embryonic death at E9.5 to 10, although there were no overt effects on vasculogenesis. Heterozygous endothelial β1 gene deletion did not diminish fetal or postnatal survival, but it reduced β1 subunit expression in endothelial cells from adult mice by approximately 40%. These mice demonstrated abnormal vascular remodeling in response to experimentally altered in vivo blood flow and diminished vascularization in healing wounds. These data demonstrate that endothelial expression of β1 integrin is required for developmental vascular patterning and that endothelial β1 gene dosing has significant functional effects on vascular remodeling in the adult. Understanding how β1 integrin expression is modulated may have significant clinical importance.
doi:10.1128/MCB.00443-07
PMCID: PMC2223431  PMID: 17984225
24.  Association between the frequency of class II HLA antigens and the susceptibility to intrauterine infection of hepatitis B virus 
Multiple factors determine the susceptibility to intrauterine hepatitis B virus (HBV) infection. These factors include the HBV structure, HBV mutation, HBV DNA level, placental barrier, the immune status of the mother, and the genetic make-ups of the newborn infants. Since HLA system is an integral component of the immune response, we hypothesized that the highly polymorphic HLA genes are the key determinants of intrauterine HBV infection. In this study, we selected newborn infants of HBsAg-positive mothers, and divided the infants into 2 groups: intrauterine infection group and non-intrauterine infection group according to the status whether or not they were infected at birth. Each infected infant was compared with 2 controls from the same birth cohort. HLA-DR allele typing was performed using a PCR-sequence specific primer (PCR-SSP) for 24 subjects with intrauterine infection and 48 controls without infection. We found that, among the fifteen (15) HLA-DR alleles assessed, HLA-DRB1*07 was the one, and the only one, significantly in excess (OR = 6.66, P = 0.004) in the intrauterine infection group compared to the non-intrauterine infection group. Our findings thus suggest that high frequency of HLA class II molecules, e.g. HLA-DRB1*07, is associated with the susceptibility of the infants to intrauterine HBV infection.
PMCID: PMC2359901  PMID: 18463715
Hepatitis B virus (HBV); Human leukocyte antigen (HLA); Genetic susceptibility; Intrauterine infection; Nested case-control study
25.  Insight into gene fusion from molecular dynamics simulation of fused and un-fused IGPS (Imidazole Glycerol Phosphate Synthetase)  
Bioinformation  2006;1(3):99-104.
Gene fusion produces proteins with novel structural architectures during evolution. Recent comparative genome analysis shows several cases of fusion÷fission across distant phylogeny. However, the selection forces driving gene fusion are not fully understood due to the lack of structural, dynamics and kinetics data. Available structural data at PDB (protein databank) contains limited cases of structural pairs describing fused and un-fused structures. Nonetheless, we identified a pair of IGPS (imidazole glycerol phosphate synthetase) structures (comprising of HisF ­ glutaminase unit and HisH ­ cyclase unit) from S. cerevisiae (SC) and T. thermophilus (TT). The HisF­HisH structural units are domains in SC and subunits in TT. Hence, they are fused in SC and un-fused in TT. Subsequently, a domain-domain interface is formed in SC and a subunit-subunit interface in TT between HisF and HisH. Our interest is to document the structure and dynamics differences between fused and un-fused IGPS. Therefore, we probed into the structures of fused IGPS in SC and un-fused IGPS in TT using molecular dynamics simulation for 5ns. Simulation shows that fused IGPS in SC has larger interface area between HisF­HisH and greater radius of gyration compared to un-fused IGPS in TT. These structural features for the first time demonstrate the evolutionary advantage in generating proteins with novel structural architecture through gene fusion.
PMCID: PMC1904513  PMID: 17611615
gene fusion; fused proteins; evolution; molecular dynamics; interface; domains; subunits

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