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author:("Xu, huiyang")
1.  Therapeutic Effects of TianDiJingWan on the Aβ25–35-Induced Alzheimer's Disease Model Rats 
The main purpose of this study was to demonstrate the therapeutic effects and mechanism of TDJW, a modern Chinese medicine prescription developed based on the basic traditional Chinese medicine theory of “tonifying the kidney essence,” on the Aβ25–35-induced AD rats. The AD model was established by the intracerebroventricular administrations of Aβ25–35 into the hippocampus CA1 tissue of SD male rats. 72 rats were randomly divided into six groups: sham operation, AD model, donepezil, high TDJW group, medium TDJW group, and low TDJW group. After oral administration of TDJW, the results of Morris water maze and step-down test showed that the learning and memory abilities of AD rats were significantly improved. And biochemical measurement demonstrated that Ach and Glu in hippocampus tissues of AD rats were increased as well. Moreover, the Aβ deposits and p-Tau aggregations in hippocampus CA1 tissues of AD rats were attenuated as observed in the micrographs of immunohistochemistry study, and the results of ELISA indicated that the expressions of TNF-α, IL-1β, and IL-6 in hippocampus tissues were significantly decreased. In conclusion, the present study demonstrated that TDJW could be used as a promising therapeutic agent for the clinical applications of AD treatment in patients.
PMCID: PMC4357045  PMID: 25815030
2.  Conceptual fluency increases recollection: behavioral and electrophysiological evidence 
It is widely established that fluency can contribute to recognition memory. Previous studies have found that enhanced fluency increases familiarity, but not recollection. The present study was motivated by a previous finding that conceptual priming affected recollection. We used event-related potentials to investigate the electrophysiological correlates of these effects with conceptually related two-character Chinese words. We found that previous conceptual priming effects on conceptual fluency only increased the incidence of recollection responses. We also found that enhanced conceptual fluency was associated with N400 attenuation, which was also correlated with the behavioral indicator of recollection. These results suggest that the N400 effect might be related to the impact of conceptual fluency on recollection recognition. These study findings provide further evidence for the relationship between fluency and recollection.
PMCID: PMC4485059  PMID: 26175678
conceptual fluency; recollection; recognition; event-related potential; N400
3.  Consistently High Unprotected Anal Intercourse (UAI) and factors correlated with UAI among men who have sex with men: implication of a serial cross-sectional study in Guangzhou, China 
BMC Infectious Diseases  2014;14:696.
China experiencing an increasing HIV epidemic among men who have sex with men (MSM), and unprotected anal intercourse (UAI) has played a key role in this process. The aims of this study were to examine the trend of UAI and to explore the factors correlated with UAI among MSM in Guangzhou, China.
Data from 2008 to 2013 were retrieved from the annual serological and behavioral surveys system. We collected information on demographic, HIV related sexual behavior with men and women, access to HIV prevention services, and symptoms of sexually transmitted infections. Chi-square test was used to examine the similarity of the participants during the study period. Univariate and multivariate logistic regression were conducted to test the factors associated with UAI. Trend test was used to check the change of UAI in different characteristic stratums during the study period.
In total, 58.4% (range from 54.5% to 62.0%) of the participants reported that they engaged in UAI in the past six months. Participants who aged less than 20 [Adjusted Odds Ratio (AOR): 2.22, 95% Confidential Interval (CI): 1.07-4.61], only attended elementary school (or less) (AOR: 1.41, 95% CI: 1.04-1.90), cohabiting with male partner (AOR: 2.15, 95% CI: 1.66-2.79), divorced or widowed (AOR: 2.80, 95% CI: 1.54-5.07), did not test for HIV in the past year (AOR: 1.36, 95% CI: 1.12-1.65), and had 10 or more partners in the past six months (AOR: 1.85, 95% CI: 1.18-2.91) had higher odds of UAI. However, the proportions of UAI were stable in different stratums during the study period.
The proportion of MSM engaged in UAI was consistently high during the study period. Effective intervention strategies, which include but not limit to risk reduction counseling and testing services, are urgently needed to bring down the risk behaviors of the MSM in Guangzhou, in order to control HIV/STIs epidemic in this specific population.
PMCID: PMC4279965  PMID: 25519034
Unprotected anal intercourse (UAI); Men who have sex with men (MSM); Human immunodeficiency virus (HIV); Trend; Serial cross-sectional study; China
4.  Effect of extractions from Ephedra sinica Stapf on hyperlipidemia in mice 
The aim of the present study was to investigate the hypolipidemic and antioxidant potential of ephedra extractions in diet-induced hyperlipidemic mice. Mice were fed a diet high in fat to establish the hyperlipidemic model. A total of 48 mice were randomly divided into six groups, which included the normal control, model control, positive control, ephedra alkaloid, ephedra polysaccharide and ephedra non-alkaloid groups. Intragastric administration of the respective treatments was provided continuously for four weeks and the body weight was recorded weekly. The total levels of cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDL-C) and malondialdehyde (MDA), and the activity levels of superoxide dismutase (SOD), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the serum were recorded. In addition, changes in liver morphology and organ coefficients (ratio of organ to body weight) were evaluated, while the acute toxicity reactions of ephedra extractions were investigated using the modified Spearman-Karber method. Compared with the mice in the model control group, the weight, liver coefficient, serum levels of TC, TG and MDA, and activities of ALT and AST were significantly lower (P<0.05) in the mice in the ephedra non-alkaloid group. However, the level of HDL-C and the activity of SOD were markedly higher (P<0.05). Fatty degeneration of the liver in the ephedra alkaloid and non-alkaloid groups was notably improved compared with the model control group. The mean lethal dose (LD50) of ephedra alkaloids was 610 mg/kg, and the maximum tolerated dose of oral ephedra non-alkaloids in the mice was 367.5-fold larger than the clinical dosage in humans. In conclusion, ephedra non-alkaloids have therapeutic potential for the treatment of hyperlipidemia, since they are able to improve lipid metabolism and are relatively safe for use under the maximum tolerated dose.
PMCID: PMC4280985  PMID: 25574244
ephedra alkaloids; ephedra polysaccharides; ephedra non-alkaloids; hyperlipidemia; acute toxicity
5.  Increasing HIV and Decreasing Syphilis Prevalence in a Context of Persistently High Unprotected Anal Intercourse, Six Consecutive Annual Surveys among Men Who Have Sex with Men in Guangzhou, China, 2008 to 2013 
PLoS ONE  2014;9(7):e103136.
Previous studies have reported a possibly increasing HIV prevalence among men who have sex with men (MSM) in China. However there have been limited systematic analyses of existing surveillance data to learn the trend of HIV prevalence and factors driving the trend. The aims of this study were to examine the trend of HIV prevalence among MSM in Guangzhou and to explore the role of unprotected anal intercourse (UAI) in the trend.
Snow-ball sampling was applied in the subject recruitment for the annual serological and behavioral surveys among MSM from 2008 to 2013. Data collected in the behavioral survey include demographic information, HIV related sexual behavior with men and women, access to HIV prevention services, and symptoms of sexually transmitted infections. Chi-square test was used to analyze the trend of HIV prevalence. Multivariate logistic regression was conducted to test the factors associated with HIV infection.
HIV prevalence increased significantly from 5.0% in 2008 to 11.4% in 2013 while syphilis prevalence decreased from 17.4% to 3.3% in the same period. UAI rates were high and stable in every single year, ranging from 54.5% to 62.0%. Those who were having UAI (OR = 1.80, 95% confidence interval (CI): 1.26–2.58), being migrants, having more than 10 partners, and infected with syphilis had higher risk for HIV infection.
HIV epidemic is expanding in Guangzhou. The persistently high UAI may have played a major role in the increasing trend of HIV prevalence. Targeted prevention program should be conducted among MSM who are migrants, low educational level, syphilis infected, or having multiple partners to encourage HIV test and change UAI behavior. The general high UAI calls for tailored intervention program to promote healthy culture and form a safe sex social norm in the MSM community.
PMCID: PMC4111575  PMID: 25061936
6.  A novel mutation in DDR2 causing spondylo-meta-epiphyseal dysplasia with short limbs and abnormal calcifications (SMED-SL) results in defective intra-cellular trafficking 
BMC Medical Genetics  2014;15:42.
The rare autosomal genetic disorder, Spondylo-meta-epiphyseal dysplasia with short limbs and abnormal calcifications (SMED-SL), is reported to be caused by missense or splice site mutations in the human discoidin domain receptor 2 (DDR2) gene. Previously our group has established that trafficking defects and loss of ligand binding are the underlying cellular mechanisms of several SMED-SL causing mutations. Here we report the clinical characteristics of two siblings of consanguineous marriage with suspected SMED-SL and identification of a novel disease-causing mutation in the DDR2 gene.
Clinical evaluation and radiography were performed to evaluate the patients. All the coding exons and splice sites of the DDR2 gene were sequenced by Sanger sequencing. Subcellular localization of the mutated DDR2 protein was determined by confocal microscopy, deglycosylation assay and Western blotting. DDR2 activity was measured by collagen activation and Western analysis.
In addition to the typical features of SMED-SL, one of the patients has an eye phenotype including visual impairment due to optic atrophy. DNA sequencing revealed a novel homozygous dinucleotide deletion mutation (c.2468_2469delCT) on exon 18 of the DDR2 gene in both patients. The mutation resulted in a frameshift leading to an amino acid change at position S823 and a predicted premature termination of translation (p.S823Cfs*2). Subcellular localization of the mutant protein was analyzed in mammalian cell lines, and it was found to be largely retained in the endoplasmic reticulum (ER), which was further supported by its N-glycosylation profile. In keeping with its cellular mis-localization, the mutant protein was found to be deficient in collagen-induced receptor activation, suggesting protein trafficking defects as the major cellular mechanism underlying the loss of DDR2 function in our patients.
Our results indicate that the novel mutation results in defective trafficking of the DDR2 protein leading to loss of function and disease. This confirms our previous findings that DDR2 missense mutations occurring at the kinase domain result in retention of the mutant protein in the ER.
PMCID: PMC4001364  PMID: 24725993
DDR2; Spondylo-meta-epiphyseal dysplasia; Trafficking defect; SMED-SL; ERAD; Optic atrophy
7.  Normal Activation of Discoidin Domain Receptor 1 Mutants with Disulfide Cross-links, Insertions, or Deletions in the Extracellular Juxtamembrane Region 
The Journal of Biological Chemistry  2014;289(19):13565-13574.
Background: DDR1 is a constitutively dimeric receptor tyrosine kinase that is activated by collagen. The mechanism of transmembrane signaling is unknown.
Results: DDR1 activation is unaffected by disulfide cross-links, insertions, or deletions in the extracellular juxtamembrane region.
Conclusion: The extracellular juxtamembrane region of DDR1 does not transmit a conformational change across the cell membrane.
Significance: The activation mechanism of DDR1 appears to be unique among receptor tyrosine kinases.
The discoidin domain receptors, DDR1 and DDR2, are receptor tyrosine kinases that are activated by collagen. DDR activation does not appear to occur by the common mechanism of ligand-induced receptor dimerization: the DDRs form stable noncovalent dimers in the absence of ligand, and ligand-induced autophosphorylation of cytoplasmic tyrosines is unusually slow and sustained. Here we sought to identify functionally important dimer contacts within the extracellular region of DDR1 by using cysteine-scanning mutagenesis. Cysteine substitutions close to the transmembrane domain resulted in receptors that formed covalent dimers with high efficiency, both in the absence and presence of collagen. Enforced covalent dimerization did not result in constitutive activation and did not affect the ability of collagen to induce receptor autophosphorylation. Cysteines farther away from the transmembrane domain were also cross-linked with high efficiency, but some of these mutants could no longer be activated. Furthermore, the extracellular juxtamembrane region of DDR1 tolerated large deletions as well as insertions of flexible segments, with no adverse effect on activation. These findings indicate that the extracellular juxtamembrane region of DDR1 is exceptionally flexible and does not constrain the basal or ligand-activated state of the receptor. DDR1 transmembrane signaling thus appears to occur without conformational coupling through the juxtamembrane region, but requires specific receptor interactions farther away from the cell membrane. A plausible mechanism to explain these findings is signaling by DDR1 clusters.
PMCID: PMC4036362  PMID: 24671415
Collagen; Mutagenesis Site-specific; Receptor Regulation; Receptor Structure-Function; Receptor Tyrosine Kinase; Signal Transduction; Activation Mechanism; Cysteine Scanning
8.  Phosphoproteomics of collagen receptor networks reveals SHP-2 phosphorylation downstream of wild-type DDR2 and its lung cancer mutants 
Biochemical Journal  2013;454(Pt 3):501-513.
Collagen is an important extracellular matrix component that directs many fundamental cellular processes including differentiation, proliferation and motility. The signalling networks driving these processes are propagated by collagen receptors such as the β1 integrins and the DDRs (discoidin domain receptors). To gain an insight into the molecular mechanisms of collagen receptor signalling, we have performed a quantitative analysis of the phosphorylation networks downstream of collagen activation of integrins and DDR2. Temporal analysis over seven time points identified 424 phosphorylated proteins. Distinct DDR2 tyrosine phosphorylation sites displayed unique temporal activation profiles in agreement with in vitro kinase data. Multiple clustering analysis of the phosphoproteomic data revealed several DDR2 candidate downstream signalling nodes, including SHP-2 (Src homology 2 domain-containing protein tyrosine phosphatase 2), NCK1 (non-catalytic region of tyrosine kinase adaptor protein 1), LYN, SHIP-2 [SH2 (Src homology 2)-domain-containing inositol phosphatase 2], PIK3C2A (phosphatidylinositol-4-phosphate 3-kinase, catalytic subunit type 2α) and PLCL2 (phospholipase C-like 2). Biochemical validation showed that SHP-2 tyrosine phosphorylation is dependent on DDR2 kinase activity. Targeted proteomic profiling of a panel of lung SCC (squamous cell carcinoma) DDR2 mutants demonstrated that SHP-2 is tyrosine-phosphorylated by the L63V and G505S mutants. In contrast, the I638F kinase domain mutant exhibited diminished DDR2 and SHP-2 tyrosine phosphorylation levels which have an inverse relationship with clonogenic potential. Taken together, the results of the present study indicate that SHP-2 is a key signalling node downstream of the DDR2 receptor which may have therapeutic implications in a subset of DDR2 mutations recently uncovered in genome-wide lung SCC sequencing screens.
The present study characterizes integrin and DDR2 signalling networks activated by collagen. Using clustering approaches, DDR2-specific signalling components such as SHP-2 were identified. We further demonstrate that SHP-2 is phosphorylated by a subset of DDR2 lung cancer mutants.
PMCID: PMC3893797  PMID: 23822953
cell signalling; collagen; discoidin domain receptor; lung cancer; mass spectrometry; phosphoproteomics; CDK1, cyclin-dependent kinase 1; DDR, discoidin domain receptor; DMEM, Dulbecco’s modified Eagle’s medium; DYRK1A, dual-specificity tyrosine-phosphorylation-regulated kinase 1A; EGFR, epidermal growth factor receptor; ERK, extracellular-signal-regulated kinase; EV, empty vector; GO, Gene Ontology; HEK, human embryonic kidney; HRP, horseradish peroxidase; IL, interleukin; IMAC, immobilized metal-ion-affinity chromatography; KD, kinase domain; mAb, monoclonal antibody; MCAM, multiple clustering analysis methodology; NCK1, non-catalytic region of tyrosine kinase adaptor protein 1; PIK3C2A, phosphatidylinositol-4-phosphate 3-kinase, catalytic subunit type 2α; PLCL2, phospholipase C-like 2; RFB, radiometric filter binding; RTK, receptor tyrosine kinase; SCC, squamous cell carcinoma; SHIP-2, SH2 (Src homology 2)-domain-containing inositol phosphatase 2; SHP-2, Src homology 2 domain-containing protein tyrosine phosphatase 2; SRM, selective reaction monitoring; TDA, template-directed assembly; TEAB, triethylammonium bicarbonate; TFA, trifluoroacetic acid
9.  Microbial Lithotrophic Oxidation of Structural Fe(II) in Biotite 
Applied and Environmental Microbiology  2012;78(16):5746-5752.
Microorganisms are known to participate in the weathering of primary phyllosilicate minerals through the production of organic ligands and acids and through the uptake of products of weathering. Here we show that the lithotrophic Fe(II)-oxidizing, nitrate-reducing enrichment culture described by Straub et al. (K. L. Straub, M. Benz, B. Schink, and F. Widdel, Appl. Environ. Microbiol. 62:1458–1460, 1996) can grow via oxidation of structural Fe(II) in biotite, a Fe(II)-rich trioctahedral mica found in granitic rocks. Oxidation of silt/clay-sized biotite particles was detected by a decrease in extractable Fe(II) content and simultaneous nitrate reduction. Mössbauer spectroscopy confirmed structural Fe(II) oxidation. Approximately 1.5 × 107 cells were produced per μmol of Fe(II) oxidized, in agreement with previous estimates of the growth yield of lithoautotrophic circumneutral-pH Fe(II)-oxidizing bacteria. Microbial oxidation of structural Fe(II) resulted in biotite alterations similar to those found in nature, including a decrease in the unit cell b dimension toward dioctahedral levels and Fe and K release. Structural Fe(II) oxidation may involve either direct enzymatic oxidation, followed by solid-state mineral transformation, or indirect oxidation as a result of the formation of aqueous Fe, followed by electron transfer from Fe(II) in the mineral to Fe(III) in solution. Although it is not possible to distinguish between these two mechanisms with available data, the complete absence of aqueous Fe in oxidation experiments favors the former alternative. The demonstration of microbial oxidation of structural Fe(II) suggests that microorganisms are directly responsible for the initial step in the weathering of biotite in granitic aquifers and the plant rhizosphere.
PMCID: PMC3406116  PMID: 22685132
10.  Discoidin Domain Receptors Promote α1β1- and α2β1-Integrin Mediated Cell Adhesion to Collagen by Enhancing Integrin Activation 
PLoS ONE  2012;7(12):e52209.
The discoidin domain receptors, DDR1 and DDR2, are receptor tyrosine kinases that bind to and are activated by collagens. Similar to collagen-binding β1 integrins, the DDRs bind to specific motifs within the collagen triple helix. However, these two types of collagen receptors recognize distinct collagen sequences. While GVMGFO (O is hydroxyproline) functions as a major DDR binding motif in fibrillar collagens, integrins bind to sequences containing Gxx’GEx”. The DDRs are thought to regulate cell adhesion, but their roles have hitherto only been studied indirectly. In this study we used synthetic triple-helical collagen-derived peptides that incorporate either the DDR-selective GVMGFO motif or integrin-selective motifs, such as GxOGER and GLOGEN, in order to selectively target either type of receptor and resolve their contributions to cell adhesion. Our data using HEK293 cells show that while cell adhesion to collagen I was completely inhibited by anti-integrin blocking antibodies, the DDRs could mediate cell attachment to the GVMGFO motif in an integrin-independent manner. Cell binding to GVMGFO was independent of DDR receptor signalling and occurred with limited cell spreading, indicating that the DDRs do not mediate firm adhesion. However, blocking the interaction of DDR-expressing cells with collagen I via the GVMGFO site diminished cell adhesion, suggesting that the DDRs positively modulate integrin-mediated cell adhesion. Indeed, overexpression of the DDRs or activation of the DDRs by the GVMGFO ligand promoted α1β1 and α2β1 integrin-mediated cell adhesion to medium- and low-affinity integrin ligands without regulating the cell surface expression levels of α1β1 or α2β1. Our data thus demonstrate an adhesion-promoting role of the DDRs, whereby overexpression and/or activation of the DDRs leads to enhanced integrin-mediated cell adhesion as a result of higher integrin activation state.
PMCID: PMC3527415  PMID: 23284937
11.  Isolation of Phyllosilicate–Iron Redox Cycling Microorganisms from an Illite–Smectite Rich Hydromorphic Soil 
The biogeochemistry of phyllosilicate–Fe redox cycling was studied in a Phalaris arundinacea (reed canary grass) dominated redoximorphic soil from Shovelers Sink, a small glacial depression near Madison, WI. The clay size fraction of Shovelers Sink soil accounts for 16% of the dry weight of the soil, yet contributes 74% of total Fe. The dominant mineral in the clay size fraction is mixed layer illite–smectite, and in contrast to many other soils and sediments, Fe(III) oxides are present in low abundance. We examined the Fe biogeochemistry of Shovelers Sink soils, estimated the abundance of Fe redox cycling microorganisms, and isolated in pure culture representative phyllosilicate–Fe oxidizing and reducing organisms. The abundance of phyllosilicate–Fe reducing and oxidizing organisms was low compared to culturable aerobic heterotrophs. Both direct isolation and dilution-to-extinction approaches using structural Fe(II) in Bancroft biotite as a Fe(II) source, and O2 as the electron acceptor, resulted in recovery of common rhizosphere organisms including Bradyrhizobium spp. and strains of Cupriavidus necator and Ralstonia solanacearum. In addition to oxidizing biotite and soluble Fe(II) with O2, each of these isolates was able to oxidize Fe(II) in reduced NAu-2 smectite with NO3- as the electron acceptor. Oxidized NAu-2 smectite or amorphous Fe(III) oxide served as electron acceptors for enrichment and isolation of Fe(III)-reducing microorganisms, resulting in recovery of a strain related to Geobacter toluenoxydans. The ability of the recovered microorganisms to cycle phyllosilicate–Fe was verified in an experiment with native Shovelers Sink clay. This study confirms that Fe in the native Shovelers Sink clay is readily available for microbial redox transformation and can be cycled by the Fe(III)-reducing and Fe(II)-oxidizing microorganisms recovered from the soil.
PMCID: PMC3318192  PMID: 22493596
smectite; phyllosilicate; Fe(III) reducing microorganisms; Fe(II) oxidizing microorganisms; neutrophilic; soil; hydromorphic; Shovelers Sink
12.  Repeated Anaerobic Microbial Redox Cycling of Iron▿† 
Applied and Environmental Microbiology  2011;77(17):6036-6042.
Some nitrate- and Fe(III)-reducing microorganisms are capable of oxidizing Fe(II) with nitrate as the electron acceptor. This enzymatic pathway may facilitate the development of anaerobic microbial communities that take advantage of the energy available during Fe-N redox oscillations. We examined this phenomenon in synthetic Fe(III) oxide (nanocrystalline goethite) suspensions inoculated with microflora from freshwater river floodplain sediments. Nitrate and acetate were added at alternate intervals in order to induce repeated cycles of microbial Fe(III) reduction and nitrate-dependent Fe(II) oxidation. Addition of nitrate to reduced, acetate-depleted suspensions resulted in rapid Fe(II) oxidation and accumulation of ammonium. High-resolution transmission electron microscopic analysis of material from Fe redox cycling reactors showed amorphous coatings on the goethite nanocrystals that were not observed in reactors operated under strictly nitrate- or Fe(III)-reducing conditions. Microbial communities associated with N and Fe redox metabolism were assessed using a combination of most-probable-number enumerations and 16S rRNA gene analysis. The nitrate-reducing and Fe(III)-reducing cultures were dominated by denitrifying Betaproteobacteria (e.g., Dechloromonas) and Fe(III)-reducing Deltaproteobacteria (Geobacter), respectively; these same taxa were dominant in the Fe cycling cultures. The combined chemical and microbiological data suggest that both Geobacter and various Betaproteobacteria participated in nitrate-dependent Fe(II) oxidation in the cycling cultures. Microbially driven Fe-N redox cycling may have important consequences for both the fate of N and the abundance and reactivity of Fe(III) oxides in sediments.
PMCID: PMC3165426  PMID: 21742920
13.  Coexistence of Different Genotypes in the Same Bat and Serological Characterization of Rousettus Bat Coronavirus HKU9 Belonging to a Novel Betacoronavirus Subgroup▿  
Journal of Virology  2010;84(21):11385-11394.
Rousettus bat coronavirus HKU9 (Ro-BatCoV HKU9), a recently identified coronavirus of novel Betacoronavirus subgroup D, from Leschenault's rousette, was previously found to display marked sequence polymorphism among genomes of four strains. Among 10 bats with complete RNA-dependent RNA polymerase (RdRp), spike (S), and nucleocapsid (N) genes sequenced, three and two sequence clades for all three genes were codetected in two and five bats, respectively, suggesting the coexistence of two or three distinct genotypes of Ro-BatCoV HKU9 in the same bat. Complete genome sequencing of the distinct genotypes from two bats, using degenerate/genome-specific primers with overlapping sequences confirmed by specific PCR, supported the coexistence of at least two distinct genomes in each bat. Recombination analysis using eight Ro-BatCoV HKU9 genomes showed possible recombination events between strains from different bat individuals, which may have allowed for the generation of different genotypes. Western blot assays using recombinant N proteins of Ro-BatCoV HKU9, Betacoronavirus subgroup A (HCoV-HKU1), subgroup B (SARSr-Rh-BatCoV), and subgroup C (Ty-BatCoV HKU4 and Pi-BatCoV HKU5) coronaviruses were subgroup specific, supporting their classification as separate subgroups under Betacoronavirus. Antibodies were detected in 75 (43%) of 175 and 224 (64%) of 350 tested serum samples from Leschenault's rousette bats by Ro-BatCoV HKU9 N-protein-based Western blot and enzyme immunoassays, respectively. This is the first report describing coinfection of different coronavirus genotypes in bats and coronavirus genotypes of diverse nucleotide variation in the same host. Such unique phenomena, and the unusual instability of ORF7a, are likely due to recombination which may have been facilitated by the dense roosting behavior and long foraging range of Leschenault's rousette.
PMCID: PMC2953156  PMID: 20702646
14.  Collagen binding specificity of the discoidin domain receptors: Binding sites on collagens II and III and molecular determinants for collagen IV recognition by DDR1 
Matrix Biology  2011;30(1):16-26.
The discoidin domain receptors, DDR1 and DDR2 are cell surface receptor tyrosine kinases that are activated by triple-helical collagen. While normal DDR signalling regulates fundamental cellular processes, aberrant DDR signalling is associated with several human diseases. We previously identified GVMGFO (O is hydroxyproline) as a major DDR2 binding site in collagens I–III, and located two additional DDR2 binding sites in collagen II. Here we extend these studies to the homologous DDR1 and the identification of DDR binding sites on collagen III. Using sets of overlapping triple-helical peptides, the Collagen II and Collagen III Toolkits, we located several DDR2 binding sites on both collagens. The interaction of DDR1 with Toolkit peptides was more restricted, with DDR1 mainly binding to peptides containing the GVMGFO motif. Triple-helical peptides containing the GVMGFO motif induced DDR1 transmembrane signalling, and DDR1 binding and receptor activation occurred with the same amino acid requirements as previously defined for DDR2. While both DDRs exhibit the same specificity for binding the GVMGFO motif, which is present only in fibrillar collagens, the two receptors display distinct preferences for certain non-fibrillar collagens, with the basement membrane collagen IV being exclusively recognised by DDR1. Based on our recent crystal structure of a DDR2-collagen complex, we designed mutations to identify the molecular determinants for DDR1 binding to collagen IV. By replacing five amino acids in DDR2 with the corresponding DDR1 residues we were able to create a DDR2 construct that could function as a collagen IV receptor.
PMCID: PMC3034869  PMID: 21044884
DDR, discoidin domain receptor; DS, discoidin homology; HEK, human embryonic kidney; RTK, receptor tyrosine kinase; VWF, von Willebrand factor; Collagen receptor; Discoidin domain receptor; Receptor tyrosine kinase; Cell–extracellular matrix interaction; Collagen binding specificity
15.  Ecoepidemiology and Complete Genome Comparison of Different Strains of Severe Acute Respiratory Syndrome-Related Rhinolophus Bat Coronavirus in China Reveal Bats as a Reservoir for Acute, Self-Limiting Infection That Allows Recombination Events▿ †  
Journal of Virology  2010;84(6):2808-2819.
Despite the identification of severe acute respiratory syndrome-related coronavirus (SARSr-CoV) in Rhinolophus Chinese horseshoe bats (SARSr-Rh-BatCoV) in China, the evolutionary and possible recombination origin of SARSr-CoV remains undetermined. We carried out the first study to investigate the migration pattern and SARSr-Rh-BatCoV genome epidemiology in Chinese horseshoe bats during a 4-year period. Of 1,401 Chinese horseshoe bats from Hong Kong and Guangdong, China, that were sampled, SARSr-Rh-BatCoV was detected in alimentary specimens from 130 (9.3%) bats, with peak activity during spring. A tagging exercise of 511 bats showed migration distances from 1.86 to 17 km. Bats carrying SARSr-Rh-BatCoV appeared healthy, with viral clearance occurring between 2 weeks and 4 months. However, lower body weights were observed in bats positive for SARSr-Rh-BatCoV, but not Rh-BatCoV HKU2. Complete genome sequencing of 10 SARSr-Rh-BatCoV strains showed frequent recombination between different strains. Moreover, recombination was detected between SARSr-Rh-BatCoV Rp3 from Guangxi, China, and Rf1 from Hubei, China, in the possible generation of civet SARSr-CoV SZ3, with a breakpoint at the nsp16/spike region. Molecular clock analysis showed that SARSr-CoVs were newly emerged viruses with the time of the most recent common ancestor (tMRCA) at 1972, which diverged between civet and bat strains in 1995. The present data suggest that SARSr-Rh-BatCoV causes acute, self-limiting infection in horseshoe bats, which serve as a reservoir for recombination between strains from different geographical locations within reachable foraging range. Civet SARSr-CoV is likely a recombinant virus arising from SARSr-CoV strains closely related to SARSr-Rh-BatCoV Rp3 and Rf1. Such frequent recombination, coupled with rapid evolution especially in ORF7b/ORF8 region, in these animals may have accounted for the cross-species transmission and emergence of SARS.
PMCID: PMC2826035  PMID: 20071579
16.  Trafficking defects and loss of ligand binding are the underlying causes of all reported DDR2 missense mutations found in SMED-SL patients 
Human Molecular Genetics  2010;19(11):2239-2250.
Spondylo-meta-epiphyseal dysplasia (SMED) with short limbs and abnormal calcifications (SMED-SL) is a rare, autosomal recessive human growth disorder, characterized by disproportionate short stature, short limbs, short broad fingers, abnormal metaphyses and epiphyses, platyspondyly and premature calcifications. Recently, three missense mutations and one splice-site mutation in the DDR2 gene were identified as causative genetic defects for SMED-SL, but the underlying cellular and biochemical mechanisms were not explored. Here we report a novel DDR2 missense mutation, c.337G>A (p.E113K), that causes SMED-SL in two siblings in the United Arab Emirates. Another DDR2 missense mutation, c.2254C>T (p.R752C), matching one of the previously reported SMED-SL mutations, was found in a second affected family. DDR2 is a plasma membrane receptor tyrosine kinase that functions as a collagen receptor. We expressed DDR2 constructs with the identified point mutations in human cell lines and evaluated their localization and functional properties. We found that all SMED-SL missense mutants were defective in collagen-induced receptor activation and that the three previously reported mutants (p.T713I, p.I726R and p.R752C) were retained in the endoplasmic reticulum. The novel mutant (p.E113K), in contrast, trafficked normally, like wild-type DDR2, but failed to bind collagen. This finding is in agreement with our recent structural data identifying Glu113 as an important amino acid in the DDR2 ligand-binding site. Our data thus demonstrate that SMED-SL can result from at least two different loss-of-function mechanisms: namely defects in DDR2 targeting to the plasma membrane or the loss of its ligand-binding activity.
PMCID: PMC2865377  PMID: 20223752
17.  U(VI) Sequestration in Hydroxyapatite Produced by Microbial Glycerol 3-Phosphate Metabolism▿ †  
Applied and Environmental Microbiology  2009;75(18):5773-5778.
Previous studies have demonstrated the potential for removal of U(VI) from solution via precipitation of U(VI)-bearing calcium-phosphate (Ca-P) minerals coupled to microbial hydrolysis of glycerol phosphate compounds. We evaluated this process in circumneutral-pH groundwater from Area 2 of the U.S. Department of Energy Field Research Center at Oak Ridge National Laboratory. Area 2 groundwater contains high concentrations of dissolved calcium (ca. 4 mM), and thus, release of phosphate during glycerol phosphate metabolism has the potential to create conditions favorable for U(VI) sequestration in Ca-P minerals. Microbial enumeration and isolation studies verified the presence of aerobic and nitrate-reducing glycerol 3-phosphate (G3P)-metabolizing microorganisms in Area 2 sediments. Coprecipitation of U(VI) with Ca-P minerals coupled to microbial G3P hydrolysis was demonstrated in artificial groundwater under aerobic and nitrate-reducing conditions. Transmission electron microscopy analysis and mineral-washing experiments demonstrated that U(VI) was incorporated into the structure of the insoluble Ca-P mineral hydroxyapatite [Ca5(PO4)3OH]. Our results support the idea that U(VI) can be effectively removed from solution in contaminated aquifers through stimulation of microbial organophosphate metabolism.
PMCID: PMC2747859  PMID: 19633115
18.  A HIV-1 heterosexual transmission chain in Guangzhou, China: a molecular epidemiological study 
Virology Journal  2009;6:148.
We conducted molecular analyses to confirm four clustering HIV-1 infections (Patient A, B, C & D) in Guangzhou, China. These cases were identified by epidemiological investigation and suspected to acquire the infection through a common heterosexual transmission chain.
Env C2V3V4 region, gag p17/p24 junction and partial pol gene of HIV-1 genome from serum specimens of these infected cases were amplified by reverse transcription polymerase chain reaction (RT-PCR) and nucleotide sequenced.
Phylogenetic analyses indicated that their viral nucleotide sequences were significantly clustered together (bootstrap value is 99%, 98% and 100% in env, gag and pol tree respectively). Evolutionary distance analysis indicated that their genetic diversities of env, gag and pol genes were significantly lower than non-clustered controls, as measured by unpaired t-test (env gene comparison: p < 0.005; gag gene comparison: p < 0.005; pol gene comparison: p < 0.005).
Epidemiological results and molecular analyses consistently illustrated these four cases represented a transmission chain which dispersed in the locality through heterosexual contact involving commercial sex worker.
PMCID: PMC2761389  PMID: 19778458
19.  Comparative Analysis of Twelve Genomes of Three Novel Group 2c and Group 2d Coronaviruses Reveals Unique Group and Subgroup Features▿  
Journal of Virology  2006;81(4):1574-1585.
Twelve complete genomes of three novel coronaviruses—bat coronavirus HKU4 (bat-CoV HKU4), bat-CoV HKU5 (putative group 2c), and bat-CoV HKU9 (putative group 2d)—were sequenced. Comparative genome analysis showed that the various open reading frames (ORFs) of the genomes of the three coronaviruses had significantly higher amino acid identities to those of other group 2 coronaviruses than group 1 and 3 coronaviruses. Phylogenetic trees constructed using chymotrypsin-like protease, RNA-dependent RNA polymerase, helicase, spike, and nucleocapsid all showed that the group 2a and 2b and putative group 2c and 2d coronaviruses are more closely related to each other than to group 1 and 3 coronaviruses. Unique genomic features distinguishing between these four subgroups, including the number of papain-like proteases, the presence or absence of hemagglutinin esterase, small ORFs between the membrane and nucleocapsid genes and ORFs (NS7a and NS7b), bulged stem-loop and pseudoknot structures downstream of the nucleocapsid gene, transcription regulatory sequence, and ribosomal recognition signal for the envelope gene, were also observed. This is the first time that NS7a and NS7b downstream of the nucleocapsid gene has been found in a group 2 coronavirus. The high Ka/Ks ratio of NS7a and NS7b in bat-CoV HKU9 implies that these two group 2d-specific genes are under high selective pressure and hence are rapidly evolving. The four subgroups of group 2 coronaviruses probably originated from a common ancestor. Further molecular epidemiological studies on coronaviruses in the bats of other countries, as well as in other animals, and complete genome sequencing will shed more light on coronavirus diversity and their evolutionary histories.
PMCID: PMC1797546  PMID: 17121802
20.  Importance of c-Type cytochromes for U(VI) reduction by Geobacter sulfurreducens 
BMC Microbiology  2007;7:16.
In order to study the mechanism of U(VI) reduction, the effect of deleting c-type cytochrome genes on the capacity of Geobacter sulfurreducens to reduce U(VI) with acetate serving as the electron donor was investigated.
The ability of several c-type cytochrome deficient mutants to reduce U(VI) was lower than that of the wild type strain. Elimination of two confirmed outer membrane cytochromes and two putative outer membrane cytochromes significantly decreased (ca. 50–60%) the ability of G. sulfurreducens to reduce U(VI). Involvement in U(VI) reduction did not appear to be a general property of outer membrane cytochromes, as elimination of two other confirmed outer membrane cytochromes, OmcB and OmcC, had very little impact on U(VI) reduction. Among the periplasmic cytochromes, only MacA, proposed to transfer electrons from the inner membrane to the periplasm, appeared to play a significant role in U(VI) reduction. A subpopulation of both wild type and U(VI) reduction-impaired cells, 24–30%, accumulated amorphous uranium in the periplasm. Comparison of uranium-accumulating cells demonstrated a similar amount of periplasmic uranium accumulation in U(VI) reduction-impaired and wild type G. sulfurreducens. Assessment of the ability of the various suspensions to reduce Fe(III) revealed no correlation between the impact of cytochrome deletion on U(VI) reduction and reduction of Fe(III) hydroxide and chelated Fe(III).
This study indicates that c-type cytochromes are involved in U(VI) reduction by Geobacter sulfurreducens. The data provide new evidence for extracellular uranium reduction by G. sulfurreducens but do not rule out the possibility of periplasmic uranium reduction. Occurrence of U(VI) reduction at the cell surface is supported by the significant impact of elimination of outer membrane cytochromes on U(VI) reduction and the lack of correlation between periplasmic uranium accumulation and the capacity for uranium reduction. Periplasmic uranium accumulation may reflect the ability of uranium to penetrate the outer membrane rather than the occurrence of enzymatic U(VI) reduction. Elimination of cytochromes rarely had a similar impact on both Fe(III) and U(VI) reduction, suggesting that there are differences in the routes of electron transfer to U(VI) and Fe(III). Further studies are required to clarify the pathways leading to U(VI) reduction in G. sulfurreducens.
PMCID: PMC1829397  PMID: 17346345
22.  SARS-CoV Infection in a Restaurant from Palm Civet 
Emerging Infectious Diseases  2005;11(12):1860-1865.
Contact with food animals was associated with SARS-CoV infection in the People’s Republic of China.
Epidemiologic investigations showed that 2 of 4 patients with severe acute respiratory syndrome (SARS) identified in the winter of 2003–2004 were a waitress at a restaurant in Guangzhou, China, that served palm civets as food and a customer who ate in the restaurant a short distance from animal cages. All 6 palm civets at the restaurant were positive for SARS-associated coronavirus (SARS-CoV). Partial spike (S) gene sequences of SARS-CoV from the 2 patients were identical to 4 of 5 S gene viral sequences from palm civets. Phylogenetic analysis showed that SARS-CoV from palm civets in the restaurant was most closely related to animal isolates. SARS cases at the restaurant were the result of recent interspecies transfer from the putative palm civet reservoir, and not the result of continued circulation of SARS-CoV in the human population.
PMCID: PMC3367621  PMID: 16485471
Severe acute respiratory syndrome; coronavirus; palm civet; research
23.  Molecular Evolution Analysis and Geographic Investigation of Severe Acute Respiratory Syndrome Coronavirus-Like Virus in Palm Civets at an Animal Market and on Farms‡  
Journal of Virology  2005;79(18):11892-11900.
Massive numbers of palm civets were culled to remove sources for the reemergence of severe acute respiratory syndrome (SARS) in Guangdong Province, China, in January 2004, following SARS coronavirus detection in market animals. The virus was identified in all 91 palm civets and 15 raccoon dogs of animal market origin sampled prior to culling, but not in 1,107 palm civets later sampled at 25 farms, spread over 12 provinces, which were claimed to be the source of traded animals. Twenty-seven novel signature variation residues (SNVs) were identified on the spike gene and were analyzed for their phylogenetic relationships, based on 17 sequences obtained from animals in our study and from other published studies. Analysis indicated that the virus in palm civets at the live-animal market had evolved to infect humans. The evolutionary starting point was a prototype group consisting of three viral sequences of animal origin. Initially, seven SNV sites caused six amino acid changes, at positions 147, 228, 240, 479, 821, and 1080 of the spike protein, to generate low-pathogenicity viruses. One of these was linked to the first SARS patient in the 2003-2004 period. A further 14 SNVs caused 11 amino acid residue changes, at positions 360, 462, 472, 480, 487, 609, 613, 665, 743, 765, and 1163. The resulting high-pathogenicity groups were responsible for infections during the so-called early-phase epidemic of 2003. Finally, the remaining six SNVs caused four amino acid changes, at positions 227, 244, 344, and 778, which resulted in the group of viruses responsible for the global epidemic.
PMCID: PMC1212604  PMID: 16140765

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