Liwen Niu

Chinese Academy of Sciences, Peping, Beijing, China

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Publications (107)525.98 Total impact

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    ABSTRACT: Staphylococcus aureus is a highly versatile pathogen that can infect human tissue by producing a large arsenal of virulence factors that are tightly regulated by a complex regulatory network. Rot, which shares sequence similarity with SarA homologues, is a global regulator that regulates numerous virulence genes. However, the recognition model of Rot for the promoter region of target genes and the putative regulation mechanism remain elusive. In this study, the 1.77 Å resolution X-ray crystal structure of Rot is reported. The structure reveals that two Rot molecules form a compact homodimer, each of which contains a typical helix–turn–helix module and a β-hairpin motif connected by a flexible loop. Fluorescence polarization results indicate that Rot preferentially recognizes AT-rich dsDNA with ∼30-base-pair nucleotides and that the conserved positively charged residues on the winged-helix motif are vital for binding to the AT-rich dsDNA. It is proposed that the DNA-recognition model of Rot may be similar to that of SarA, SarR and SarS, in which the helix–turn–helix motifs of each monomer interact with the major grooves of target dsDNA and the winged motifs contact the minor grooves. Interestingly, the structure shows that Rot adopts a novel dimerization model that differs from that of other SarA homologues. As expected, perturbation of the dimer interface abolishes the dsDNA-binding ability of Rot, suggesting that Rot functions as a dimer. In addition, the results have been further confirmed in vivo by measuring the transcriptional regulation of α-toxin, a major virulence factor produced by most S. aureus strains.
    Acta Crystallographica Section D Biological Crystallography 09/2014; 70(9). · 12.67 Impact Factor
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    ABSTRACT: DnaT is a primosomal protein that is required for the stalled replication fork restart in Escherichia coli. As an adapter, DnaT mediates the PriA-PriB-ssDNA ternary complex and the DnaB/C complex. However, the fundamental function of DnaT during PriA-dependent primosome assembly is still a black box. Here, we report the 2.83 Å DnaT(84-153)-dT10 ssDNA complex structure, which reveals a novel three-helix bundle single-stranded DNA binding mode. Based on binding assays and negative-staining electron microscopy results, we found that DnaT can bind to phiX 174 ssDNA to form nucleoprotein filaments for the first time, which indicates that DnaT might function as a scaffold protein during the PriA-dependent primosome assembly. In combination with biochemical analysis, we propose a cooperative mechanism for the binding of DnaT to ssDNA and a possible model for the assembly of PriA-PriB-ssDNA-DnaT complex that sheds light on the function of DnaT during the primosome assembly and stalled replication fork restart. This report presents the first structure of the DnaT C-terminal complex with ssDNA and a novel model that explains the interactions between the three-helix bundle and ssDNA.
    Nucleic Acids Research 07/2014; · 8.81 Impact Factor
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    ABSTRACT: Yeast Hif1, a homologue of human nuclear autoantigenic sperm protein (NASP), is a histone chaperone that involved in various protein complexes modifying histones during telomeric silencing and chromatin reassembly. For elucidating the structural basis of Hif1, here, we present crystal structure of Hif1 that consists of a superhelixed TPR domain and an extended acid loop covering the rear of TPR domain, which represents typical characters of SHNi-TPR (Sim3-Hif1-NASP interrupted TPR) proteins. Our binding assay indicates that Hif1 could bind to histone octamer via histone H3 and H4. However, the acid loop is crucial for the binding of histones while it may also change the conformation of TPR groove. By binding to core histone complex Hif1 may recruit functional protein complexes to modify histones during chromatin reassembly.
    The Biochemical journal. 06/2014;
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    ABSTRACT: Arginine methylation plays vital roles in the cellular functions of the protozoan Trypanosoma brucei. The T. brucei arginine methyltransferase 6 (TbPRMT6) is a type I arginine methyltransferase homologous to human PRMT6. In this study, we report the crystal structures of apo-TbPRMT6 and its complex with the reaction product S-adenosyl-homocysteine (SAH). The structure of apo-TbPRMT6 displays several features that are different from those of type I PRMTs that were structurally characterized previously, including four stretches of insertion, the absence of strand β15, and a distinct dimerization arm. The comparison of the apo-TbPRMT6 and SAH-TbPRMT6 structures revealed the fine rearrangements in the active site upon SAH binding. The isothermal titration calorimetry results demonstrated that SAH binding greatly increases the affinity of TbPRMT6 to a substrate peptide derived from bovine histone H4. The western blotting and mass spectrometry results revealed that TbPRMT6 methylates bovine histone H4 tail at arginine 3 but cannot methylate several T. brucei histone tails. In summary, our results highlight the structural differences between TbPRMT6 and other type I PRMTs and reveal that the active site rearrangement upon SAH binding is important for the substrate binding of TbPRMT6.
    PLoS ONE 01/2014; 9(2):e87267. · 3.53 Impact Factor
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    ABSTRACT: Adenylate kinase plays a very important role in regulating adenylate species in the cell. Methanocaldococcus jannaschii is a rich resource of unique enzymes. Here, MJ0458, an adenylate kinase from M. jannaschii, was crystallized. A set of X-ray diffraction data to 2.70 Å resolution was collected on beamline BL-17U of the Shanghai Synchrotron Radiation Facility (SSRF). The crystal belonged to space group P41212 or P43212. The unit-cell parameters were a = b = 76.18, c = 238.70 Å, α = β = γ = 90°.
    Acta Crystallographica Section F Structural Biology and Crystallization Communications 11/2013; 69(Pt 11):1272-4. · 0.55 Impact Factor
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    ABSTRACT: Heterogeneous nuclear ribonucleoprotein L (hnRNP L) is an abundant RNA-binding protein implicated in many bioprocesses including pre-mRNA processing, mRNA export of intronless genes, internal ribosomal entry site (IRES)-mediated translation, and chromatin modification. It contains four RNA recognition motifs (RRMs) that bind with CA repeats or CA-rich elements. In this study, surface plasmon resonance (SPR) spectroscopy assays revealed that all four RRM domains contribute to RNA-binding. Furthermore, we elucidated the crystal structures of hnRNP L RRM1 and RRM34 at 2.0 Å and 1.8 Å, respectively. These RRMs all adopt the typical β1α1β2β3α2β4 topology, except for an unusual fifth β-strand in RRM3. RRM3 and RRM4 interact intimately with each other mainly through helical surfaces, leading the two β-sheets to face opposite directions. Structure-based mutations and SPR assay results suggested that the β-sheets of RRM1 and RRM34 are accessible for RNA binding. FRET-based gel shift assays (FRET-EMSA) and steady-state FRET assays (ss-FRET), together with cross-linking and dynamic light scattering (DLS) assays, demonstrated that hnRNP L RRM34 facilitates RNA looping when binding to two appropriately separated binding sites within the same target pre-mRNA. EMSA and ITC binding studies with in vivo target RNA suggested that hnRNP L-mediated RNA looping may occur in vivo. Our study provides a mechanistic explanation for the dual functions of hnRNP L in alternative-splicing regulation as an activator or repressor.
    Journal of Biological Chemistry 08/2013; 288(31):22636-22649. · 4.65 Impact Factor
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    ABSTRACT: The Nit (nitrilase-like) protein subfamily constitutes branch 10 of the nitrilase superfamily. Nit proteins are widely distributed in nature. Mammals possess two members of the Nit subfamily, namely Nit1 and Nit2. Based on sequence similarity, yeast Nit2 (yNit2) is a homologue of mouse Nit1, a tumour-suppressor protein whose substrate specificity is not yet known. Previous studies have shown that mammalian Nit2 (also a putative tumour suppressor) is identical to ω-amidase, an enzyme that catalyzes the hydrolysis of α-ketoglutaramate (α-KGM) and α-ketosuccinamate (α-KSM) to α-ketoglutarate (α-KG) and oxaloacetate (OA), respectively. In the present study, crystal structures of wild-type (WT) yNit2 and of WT yNit2 in complex with α-KG and with OA were determined. In addition, the crystal structure of the C169S mutant of yNit2 (yNit2-C169S) in complex with an endogenous molecule of unknown structure was also solved. Analysis of the structures revealed that α-KG and OA are covalently bound to Cys169 by the formation of a thioester bond between the sulfhydryl group of the cysteine residue and the γ-carboxyl group of α-KG or the β-carboxyl group of OA, reflecting the presumed reaction intermediates. However, an enzymatic assay suggests that α-KGM is a relatively poor substrate of yNit2. Finally, a ligand was found in the active site of yNit2-C169S that may be a natural substrate of yNit2 or an endogenous regulator of enzyme activity. These crystallographic analyses provide information on the mode of substrate/ligand binding at the active site of yNit2 and insights into the catalytic mechanism. These findings suggest that yNit2 may have broad biological roles in yeast, especially in regard to nitrogen homeostasis, and provide a framework for the elucidation of the substrate specificity and biological role of mammalian Nit1.
    Acta Crystallographica Section D Biological Crystallography 08/2013; 69(Pt 8):1470-81. · 12.67 Impact Factor
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    ABSTRACT: Ricin belongs to the type II ribosome-inactivating proteins (RIPs) which depurinate the universally conserved α-sarcin loop of rRNA. Ricin's RNA-N-glycosidase activity also largely depends on the ribosomal proteins which play an important role during the process of rRNA depurination. Therefore, the study of the interaction between ricin and the ribosomal elements will be better to understand the catalysis mechanism of ricin. The antibody 6C2 is a mouse monoclonal antibody exhibiting unusually potent neutralizing ability against ricin, but the neutralization mechanism remains unknown. Here, we report the 2.8 Å crystal structure of 6C2 Fab in complex with the A-chain of ricin (RTA), which reveals an extensive antigen-antibody interface containing both hydrogen bonds and van der Waals contacts. The CDR loops H1, H2, H3, and L3 form a pocket to accommodate the epitope on the RTA (residues D96-T116). ELISA results show that Gln98, Glu99, Glu102 and Thr105 (RTA) are the key residues that play an important role in recognizing 6C2. With the perturbation of the 6C2 Fab-RTA interface, 6C2 loses its neutralization ability, measured based on the inhibition of protein synthesis in a cell-free system. Finally, we propose that the neutralization mechanism of 6C2 against ricin is that the binding of 6C2 hinders the interaction between RTA and the ribosome, and the SPR and pull-down results confirm our hypothesis. In short, our data explain the neutralization mechanism of mAb 6C2 against ricin and provide a structural basis for the development of improved antibody drugs with better specificity and higher affinity.
    Journal of Biological Chemistry 07/2013; · 4.65 Impact Factor
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    ABSTRACT: AhV_aPA, the acidic PLA2 purified from Agkistrodon halys pallas venom, was previously reported to possess a strong enzymatic activity and can remarkably induce a further contractile response on the 60 mM K(+)-induced contraction with an EC50 in 369 nM on mouse thoracic aorta rings. In the present study, we found that the p-bromo-phenacyl-bromide (pBPB), which can completely inhibit the enzymatic activity of AhV_aPA, did not significantly reduce the contractile response on vessel rings induced by AhV_aPA, indicating that the vasoconstrictor effects of AhV_aPA are independent of the enzymatic activity. The inhibitor experiments showed that the contractile response induced by AhV_aPA is mainly attributed to the Ca(2+) releasing from Ca(2+) store, especially sarcoplasmic reticulum (SR). Detailed studies showed that the Ca(2+) release from SR is related to the activation of inositol trisphosphate receptors (IP3Rs) rather than ryanodine receptors (RyRs). Furthermore, the vasoconstrictor effect could be strongly reduced by pre-incubation with hepain, indicating that the basic amino acid residues on the surface of AhV_aPA may be involved in the interaction between AhV_aPA and the molecular receptors. These findings offer new insights into the functions of snake PLA2 and provide a novel pathogenesis of A. halys pallas venom.
    Toxicon 05/2013; · 2.92 Impact Factor
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    ABSTRACT: Human RNA-binding protein (HuR), a ubiquitously expressed member of the Hu protein family, plays an important role in mRNA degradation and has been implicated as a key post-transcriptional regulator. HuR contains three RNA-recognition motif (RRM) domains. The two N-terminal tandem RRM domains can selectively bind AU-rich elements (AREs), while the third RRM domain (RRM3) contributes to interactions with the poly-A tail of target mRNA and other ligands. Here, the X-ray structure of two methylated tandem RRM domains (RRM1/2) of HuR in their RNA-free form was solved at 2.9 Å resolution. The crystal structure of RRM1/2 complexed with target mRNA was also solved at 2.0 Å resolution; comparisons of the two structures show that HuR RRM1/2 undergoes conformational changes upon RNA binding. Fluorescence polarization assays (FPA) were used to study the protein-RNA interactions. Both the structure and the FPA analysis indicated that RRM1 is the primary ARE-binding domain in HuR and that the conformational changes induce subsequent contacts of the RNA substrate with the inter-domain linker and RRM2 which greatly improve the RNA-binding affinity of HuR.
    Acta Crystallographica Section D Biological Crystallography 03/2013; 69(Pt 3):373-80. · 12.67 Impact Factor
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    ABSTRACT: Phosphorylated derivatives of phosphatidylinositol (PtdIns), also called phosphoinositides (PIPs), are basic components of membrane-associated signalling systems. A family of PtdIns-transfer proteins (PITPs) called the Sec14 family have been predicted to form a set of functional modules that can sense different types of lipid metabolism and transmit the information to the PIP signalling system. In eukaryotic cells, the Sec14 family exhibits a wide diversity of activity, but the structural basis of this diversity remains unclear. In the present study, the dimeric structure of Sfh3 (Sec14 family homologue 3 in yeast) is reported for the first time and differs from the Sec14 proteins reported to date, all of which are monomeric. Some variations in the binding pocket of Sfh3 were observed and the dimer interface was identified and proposed to provide a link between dimer-monomer state changes and PtdIns binding. Together, these structural changes and the oligomeric state transformation of Sfh3 support ideas of diversity within the Sec14 family and provide some new clues to function.
    Acta Crystallographica Section D Biological Crystallography 03/2013; 69(Pt 3):313-23. · 12.67 Impact Factor
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    ABSTRACT: Ap(4)A hydrolase (asymmetrical diadenosine tetraphosphate hydrolase, EC 3.6.1.17), an enzyme involved in a number of biological processes, is characterized as cleaving the polyphosphate chain at the fourth phosphate from the bound adenosine moiety. This paper presents the crystal structure of wild-type and E58A mutant human Ap(4)A hydrolase. Similar to the canonical Nudix fold, human Ap(4)A hydrolase shows the common αβα-sandwich architecture. Interestingly, two sulfate ions and one diphosphate coordinated with some conserved residues were observed in the active cleft, which affords a better understanding of a possible mode of substrate binding.
    Biochemical and Biophysical Research Communications 02/2013; · 2.41 Impact Factor
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    ABSTRACT: Phospholipases A(2) (PLA(2)s) are the major component of snake venoms and exert a variety of relevant toxic actions such as neurotoxicity and myotoxicity, amongst others. An acidic PLA(2), here named AhV_aPA, was purified from Agkistrodon halys pallas venom by means of a three-step chromatographic procedure. AhV_aPA migrated as a single band on SDS-PAGE gels, with a molecular weight of about 14 kDa. Like other acidic aPLA(2)s, AhV_aPA has high enzymatic activity. Tension measurements of mouse thoracic aortic rings remarkably indicated that AhV_aPA could induce a further contractile response on the 60 mM K(+)-induced contraction, with an EC(50) of 369 nmol l(-1). Rod-shaped crystals were obtained by the hanging-drop vapour-diffusion method and diffracted to a resolution limit of 2.30 Å. The crystals belonged to space group P222, with unit-cell parameters a = 44.27, b = 68.39, c = 81.54 Å.
    Acta Crystallographica Section F Structural Biology and Crystallization Communications 11/2012; 68(Pt 11):1329-1332. · 0.55 Impact Factor
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    ABSTRACT: A snake venom thrombin-like enzyme (SVTLE) from Agkistrodon halys pallas venom was isolated by means of a two-step chromatographic procedure. The purified enzyme, named AhV_TL-I, showed fibrinogenolytic activity against both the Aα and Bβ chains of bovine fibrinogen. Unlike the other SVTLEs, AhV_TL-I has poor esterolytic activity upon BAEE substrate. The N-terminal sequence of AhV_TL-I was determined to be IIGGDEXNINEHRFLVALYT, and the molecular mass was confirmed to 29389.533 Da by MALDI-TOF mass spectrometry. Its complete cDNA and derived amino acid sequence were obtained by RT-PCR. The crystal structure of AhV_TL-I was determined at a resolution of 1.75 Å. A disaccharide was clearly mapped in the structure, which involved in regulating the esterolytic activity of AhV_TL-I. The presence of the N-glycan deformed the 99-loop, and the resulting steric hindrances hindered the substrates to access the active site. Furthermore, with the carbohydrate moiety, AhV_TL-I could induce mouse thoracic aortic ring contraction with the EC(50) of 147 nmol/L. Besides, the vasoconstrictor effects of AhV_TL-I were also independent of the enzymatic activity. The results of [Ca(2+)](i) measurement showed that the vasoconstrictor effects of AhV_TL-I were attributed to Ca(2+) releasing from Ca(2+) store. Further studies showed that it was related to the activation of ryanodine receptors (RyRs). These offer new insights into the snake SVTLEs functions and provide a novel pathogenesis of A. halys pallas venom.
    Archives of Toxicology 10/2012; · 5.22 Impact Factor
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    ABSTRACT: The yeast Paf1 complex (Paf1C), which is composed of the proteins Paf1, Cdc73, Ctr9, Leo1 and Rtf1, accompanies RNA polymerase II from the promoter to the 3'-end formation site of mRNA- and snoRNA-encoding genes. As one of the first identified subunits of Paf1C, yeast Cdc73 (yCdc73) takes part in many transcription-related processes, including binding to RNA polymerase II, recruitment and activation of histone-modification factors and communication with other transcriptional activators. The human homologue of yCdc73, parafibromin, has been identified as a tumour suppressor linked to breast, renal and gastric cancers. However, the functional mechanism of yCdc73 has until recently been unclear. Here, a 2.2 Å resolution crystal structure of the highly conserved C-terminal region of yCdc73 is reported. It revealed that yCdc73 appears to have a GTPase-like fold. However, no GTPase activity was observed. The crystal structure of yCdc73 will shed new light on the modes of function of Cdc73 and Paf1C.
    Acta Crystallographica Section D Biological Crystallography 08/2012; 68(Pt 8):953-9. · 12.67 Impact Factor
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    ABSTRACT: Mannonate dehydratase (ManD; EC4.2.1.8) catalyzes the dehydration of d-mannonate to 2-keto-3-deoxygluconate. It is the third enzyme in the pathway for dissimilation of d-glucuronate to 2-keto-3-deoxygluconate involving in the Entner-Doudoroff pathway in certain bacterial and archaeal species. ManD from Gram negative bacteria has an insert sequence as compared to those from Gram positives revealed by sequence analysis. To evaluate the impact of this insert sequence on the catalytic efficiency, we solved the crystal structures of ManD from Escherichia coli strain K12 and its complex with d-mannonate, which reveal that this insert sequence forms two α helices locating above the active site. The two insert α helices introduce a loop that forms a cap covering the substrate binding pocket, which restricts the tunnels of substrate entering and product releasing from the active site. Site-directed mutations and enzymatic activity assays confirm that the catalytic rate is decreased by this loop. These features are conserved among Gram negative bacteria. Thus, the insert sequence of ManD from Gram negative bacteria acts as a common inducer to decrease the catalytic rate and consequently the glucuronate metabolic rate as compared to those from Gram positives. Moreover, residues essential for substrate to enter the active site were characterized via structural analysis and enzymatic activity assays.
    Journal of Structural Biology 07/2012; · 3.36 Impact Factor
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    ABSTRACT: Palmitoylation/depalmitoylation plays an important role in protein modification. yApt1 is the only enzyme in Saccharomyces cerevisiae that catalyses depalmitoylation. In the present study, recombinant full-length yApt1 was cloned, expressed, purified and crystallized. The crystals diffracted to 2.40 Å resolution and belonged to space group P4(2)2(1)2, with unit-cell parameters a = b = 146.43, c = 93.29 Å. A preliminary model of the three-dimensional structure has been built and further refinement is ongoing.
    Acta Crystallographica Section F Structural Biology and Crystallization Communications 07/2012; 68(Pt 7):775-7. · 0.55 Impact Factor
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    ABSTRACT: The yeast Shu complex, consisting of the proteins Shu1, Shu2, Psy3, and Csm2, maintains genomic stability by coupling post-replication repair to homologous recombination. However, a lack of biochemical and structural information on the Shu proteins precludes revealing their precise roles within the pathway. Here, we report on the 1.9-Å crystal structure of the Psy3-Csm2 complex. The crystal structure shows that Psy3 forms a heterodimer with Csm2 mainly through a hydrophobic core. Unexpectedly, Psy3 and Csm2 share a similar architecture that closely resembles the ATPase core domain of Rad51. The L2 loop present in Psy3 and Csm2 is similar to that of Rad51 and confers the DNA binding activity of the Shu complex. As with Rad51, the Shu complex appears to form a nucleoprotein filament by binding nonspecifically to DNA. Structure-based mutagenesis studies have demonstrated that the DNA binding activity of the Shu complex is essential for repair of the methyl methanesulfonate-induced DNA damage. Our findings provide good foundations for the understanding of the Srs2 regulation by the Shu complex.
    Journal of Biological Chemistry 03/2012; 287(24):20231-9. · 4.65 Impact Factor
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    ABSTRACT: Agkisacucetin is a snake C-type lectin isolated from the venom of Agkistrodon acutus (A. acutus). It binds specifically to the platelet glycoprotein (GP) Ib and prevents the von Willebrand factor (VWF) accessing it. We determined the crystal structure of agkisacucetin to 1.9Å resolution. The structure of agkisacucetin has an (αβ) fold similar to another GPIb-binding protein, flavocetin-A, but lacks the C-terminal cysteine in the β-subunit, does not form (βα)(4) tetramers, and does not cluster GPIbs, like flavocetin-A.
    Proteins Structure Function and Bioinformatics 02/2012; 80(6):1707-11. · 3.34 Impact Factor

Publication Stats

747 Citations
525.98 Total Impact Points

Institutions

  • 2003–2014
    • Chinese Academy of Sciences
      • Institute of High Energy Physics
      Peping, Beijing, China
  • 2002–2013
    • Anhui University
      • School of Life Sciences
      Luchow, Anhui Sheng, China
  • 1997–2013
    • University of Science and Technology of China
      • School of Life Sciences
      Hefei, Anhui Sheng, China
    • State Key Laboratory of Medical Genetics of China
      Ch’ang-sha-shih, Hunan, China
  • 2004–2012
    • Hefei Institute of Physical Sciences, Chinese Academy of Sciences
      Luchow, Anhui Sheng, China
  • 2002–2011
    • Northeast Institute of Geography and Agroecology
      • • Department of Science
      • • Molecular Biology and Cell Biology Laboratory
      Beijing, Beijing Shi, China
  • 2007
    • Cornell University
      Ithaca, New York, United States