Jian Liu

University of North Carolina at Chapel Hill, North Carolina, United States

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Publications (88)478.95 Total impact

  • Jian Liu, Robert J Linhardt
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    ABSTRACT: Covering: up to May 2014Heparan sulfate is a polysaccharide that plays essential physiological functions in the animal kingdom. Heparin, a highly sulfated form of heparan sulfate, is a widely prescribed anticoagulant drug worldwide. The heparan sulfate and heparin isolated from natural sources are highly heterogeneous mixtures differing in their polysaccharide chain lengths and sulfation patterns. The access to structurally defined heparan sulfate and heparin is critical to probe the contribution of specific sulfated saccharide structures to the biological functions as well as for the development of the next generation of heparin-based anticoagulant drugs. The synthesis of heparan sulfate and heparin, using a purely chemical approach, has proven extremely difficult, especially for targets larger than octasaccharides having a high degree of site-specific sulfation. A new chemoenzymatic method has emerged as an effective alternative approach. This method uses recombinant heparan sulfate biosynthetic enzymes combined with unnatural uridine diphosphate-monosaccharide donors. Recent examples demonstrate the successful synthesis of ultra-low molecular weight heparin, low-molecular weight heparin and bioengineered heparin with unprecedented efficiency. The new method provides an opportunity to develop improved heparin-based therapeutics.
    Natural Product Reports 09/2014; 46(4). DOI:10.1039/c4np00076e · 10.72 Impact Factor
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    ABSTRACT: The exquisite control of growth factor function by heparan sulfate (HS) is dictated by tremendous structural heterogeneity of sulfated modifications. How specific HS structures control growth factor-dependent progenitor expansion during organogenesis is unknown. We isolated KIT+ progenitors from fetal salivary glands during a stage of rapid progenitor expansion and profiled HS biosynthetic enzyme expression. Enzymes generating a specific type of 3-O-sulfated-HS (3-O-HS) are enriched, and fibroblast growth factor 10 (FGF10)/FGF receptor 2b (FGFR2b) signaling directly regulates their expression. Bioengineered 3-O-HS binds FGFR2b and stabilizes FGF10/FGFR2b complexes in a receptor- and growth factor-specific manner. Rapid autocrine feedback increases 3-O-HS, KIT, and progenitor expansion. Knockdown of multiple Hs3st isoforms limits fetal progenitor expansion but is rescued with bioengineered 3-O-HS, which also increases adult progenitor expansion. Altering specific 3-O-sulfated epitopes provides a mechanism to rapidly respond to FGFR2b signaling and control progenitor expansion. 3-O-HS may expand KIT+ progenitors in vitro for regenerative therapy.
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    ABSTRACT: Heparan sulfate (HS) is a linear polysaccharide expressed on cell surfaces, in extracellular matrices and cellular granules in metazoan cells. Through non-covalent binding to growth factors, morphogens, chemokines and other protein families, HS is involved in all multicellular physiological activities. Its biological activities depend on the fine structures of its protein-binding domains, the determination of which remains a daunting task. Methods have advanced to the point that information-rich product ion mass spectra may be produced on purified HS saccharides. However, the interpretation of these complex product ion patterns has emerged as the bottleneck to the dissemination of these HS sequencing methods. To solve this problem, we designed HS-SEQ, the first comprehensive algorithm for HS de novo sequencing using high-resolution tandem mass spectra. We tested HS-SEQ using negative electron transfer dissociation (NETD) tandem mass spectra generated from a set of pure synthetic saccharide standards with diverse sulfation patterns. The results showed that HS-SEQ rapidly and accurately determined the correct HS structures from large candidate pools.
    Molecular &amp Cellular Proteomics 06/2014; 13(9). DOI:10.1074/mcp.M114.039560 · 7.25 Impact Factor
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    ABSTRACT: Heparan sulfate and heparin are highly sulfated polysaccharides that consist of a repeating disaccharide unit of glucosamine and glucuronic or iduronic acid. The 2-O-sulfated iduronic acid (IdoA2S) residue is commonly found in heparan sulfate and heparin; however, 2-O-sulfated glucuronic acid (GlcA2S) is a less abundant monosaccharide (∼<5% of total saccharides). Here, we report the synthesis of three GlcA2S-containing hexasaccharides using a chemoenzymatic approach. For comparison purposes, additional IdoA2S-containing hexasaccharides were synthesized. NMR analyses were performed to obtain full chemical shift assignments for the GlcA2S- and IdoA2S-hexasaccharides. These data show that GlcA2S is a more structurally rigid saccharide residue than IdoA2S. The antithrombin binding affinities of a GlcA2S- hexasaccharide and an IdoA2S-hexasaccharide were determined by affinity co-electrophoresis. In contrast to IdoA2S-hexasaccharides, the GlcA2S-hexasaccharide does not bind to antithrombin, confirming that the presence of IdoA2S is critically important for the anticoagulant activity. The availability of pure synthetic GlcA2S-containing oligosaccharides will allow the investigation of the structure and activity relationships of individual sites in heparin or heparan sulfate.
    Glycobiology 04/2014; DOI:10.1093/glycob/cwu032 · 3.75 Impact Factor
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    ABSTRACT: The chemoenzymatic synthesis of heparan sulfate tetrasaccharide (1) and hexasaccharide (2) with a fluorous tag attached at the reducing end is reported. The fluorous tert-butyl dicarbonate ((F)Boc) tag did not interfere with enzymatic recognition for both elongation and specific sulfation, and flash purification was performed by standard fluorous solid-phase extraction (FSPE). Based on an (F)Boc attached disaccharide as acceptor, a series of partial N-sulfated, 6-O-sulfated heparan sulfate oligosaccharides were successfully synthesized employing fluorous techniques.
    Organic Letters 04/2014; 16(8). DOI:10.1021/ol500738g · 6.32 Impact Factor
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    ABSTRACT: Heparan sulfate (HS) is an abundant polysaccharide in the animal kingdom with essential physiological functions. HS is composed of sulfated saccharides that are biosynthesized through a complex pathway involving multiple enzymes. In vivo regulation of this process remains unclear. HS 2-O-sulfotransferase (2OST) is a key enzyme in this pathway. Here, we report the crystal structure of the ternary complex of 2OST, 3-phosphoadenosine 5-phosphate, and a heptasaccharide. Utilizing site-directed mutagenesis and specific oligosaccharide substrate sequences, we probed the molecular basis of specificity and 2OST position in the ordered HS biosynthesis pathway. These studies revealed that Arg80, Lys350, and Arg190 of 2OST interact with the N-sulfo groups near the modification site, consistent with the dependence of 2OST on N-sulfation. In contrast, 6-O-sulfo groups on HS are likely excluded by steric and electrostatic repulsion within the active site supporting the hypothesis that 2-O-sulfation occurs prior to 6-O-sulfation. Our results provide the structural evidence for understanding the sequence of enzymatic events in this pathway.
    Journal of Biological Chemistry 03/2014; 289(19). DOI:10.1074/jbc.M113.530535 · 4.60 Impact Factor
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    ABSTRACT: Heparin, a commonly used anticoagulant drug, is a mixture of highly sulfated polysaccharides with various molecular weights. The unique sulfation pattern dictates the anticoagulant activity of heparin. Commercial heparins are categorized into three forms according to their average molecular weight (MW): unfractionated heparin (UFH, MWavg 14,000), low molecular weight heparin (LMWH, MWavg 3,500-6,500) and the synthetic pentasaccharide (fondaparinux, MW 1508.3). UFH is isolated from porcine intestine while LMWH is derived from UFH by various methods of depolymerization, which generate a wide range of oligosaccharide chain lengths. Different degradation methods result in structurally distinct LMWH products, displaying different pharmacological and pharmacokinetic properties. In this report, we utilized a chemoenzymatic method to synthesize LMWH with the emphasis on controlling the size distribution of the oligosaccharides. A tetrasaccharide primer and a controlled enzyme-based polymerization were employed to build a narrow size oligosaccharide backbone. The oligosaccharide backbones were further modified by a series of sulfation and epimerization steps in order to obtain a full anticoagulation activity. Determination of the anticoagulation activity in vitro and ex vivo indicated that the synthetic LMWH has higher potency than enoxaparin, a commercial LMWH drug in clinical usage.
    Glycobiology 03/2014; DOI:10.1093/glycob/cwu016 · 3.75 Impact Factor
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    ABSTRACT: Low-molecular-weight heparins (LMWHs) are carbohydrate-based anticoagulants clinically used to treat thrombotic disorders, but impurities, structural heterogeneity or functional irreversibility can limit treatment options. We report a series of synthetic LMWHs prepared by cost-effective chemoenzymatic methods. The high activity of one defined synthetic LMWH against human factor Xa (FXa) was reversible in vitro and in vivo using protamine, demonstrating that synthetically accessible constructs can have a critical role in the next generation of LMWHs.
    Nature Chemical Biology 02/2014; DOI:10.1038/nchembio.1459 · 12.95 Impact Factor
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    ABSTRACT: Four well-defined heparan sulfate (HS) block copolymers, containing S-domains (high sulfo group content) placed adjacent to N-domains (low sulfo group content) were chemoenzymatically synthesized and characterized. The domain lengths in these HS block co-polymers were ~40 saccharide units. Microtiter 96-well and 3D cell-based microarray assays utilizing murine immortalized bone marrow (BaF3) cells were developed to evaluate the activity of these HS block co-polymers. Each recombinant BaF3 cell line expresses only a single type of fibroblast growth factor receptor (FGFR), but produces neither HS nor fibroblast growth factors (FGFs). In the presence of different FGFs, BaF3 cell-proliferation showed clear differences for the four HS block co-polymers examined. These data were used to examine the two proposed signaling models, the symmetric FGF2:HS2:FGFR2 ternary complex model and the asymmetric FGF2:HS1:FGFR2 ternary complex model. In the symmetric FGF2:HS2:FGFR2 model, two acidic HS chains bind in a basic canyon located on the top face of the FGF2-FGFR2 protein complex. In this model, the S-domains at the non-reducing ends of the two HS proteoglycan chains are proposed to interact with the FGF2-FGFR2 protein complex. In contrast, in the asymmetric FGF2:HS1:FGFR2 model, a single HS chain interacts with the FGF2-FGFR2 protein complex through a single S-domain that can be located at any position within an HS chain. Our data comparing a series of synthetically prepared HS block copolymers support a preference for the symmetric FGF2:HS2:FGFR2 ternary complex model.
    Journal of Biological Chemistry 02/2014; 289(14). DOI:10.1074/jbc.M113.546937 · 4.60 Impact Factor
  • Nature Chemical Biology 02/2014; · 12.95 Impact Factor
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    ABSTRACT: O-sulfotransferases (OSTs) are critical enzymes in the cellular biosynthesis of the biologically and pharmacologically important heparan sulfate and heparin. Recently, these enzymes have been cloned and expressed in bacteria for application in the chemoenzymatic synthesis of glycosaminoglycan-based drugs. OST activity assays have largely relied on the use of radioisotopic methods using [(35)S] 3'-phosphoadenosine-5'-phosphosulfate and scintillation counting. Herein, we examine alternative assays that are more compatible with a biomanufacturing environment. A high throughput microtiter-based approach is reported that relies on a coupled bienzymic colorimetric assay for heparan sulfate and heparin OSTs acting on polysaccharide substrates using arylsulfotransferase-IV and p-nitrophenylsulfate as a sacrificial sulfogroup donor. A second liquid chromatography-mass spectrometric assay, for heparan sulfate and heparin OSTs acting on structurally defined oligosaccharide substrates, is also reported that provides additional information on the number and positions of the transferred sulfo groups within the product. Together, these assays allow quantitative and mechanistic information to be obtained on OSTs that act on heparan sulfate and heparin precursors.
    Analytical and Bioanalytical Chemistry 11/2013; 406(2). DOI:10.1007/s00216-013-7470-4 · 3.66 Impact Factor
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    ABSTRACT: Heparin, the first biopolymeric drug, possesses a wide range of structural heterogeneity owing to its biosynthesis. Its diverse fine structure is further complicated by an animal tissue-based recovery, leading to considerable structural differences within commercial heparin active pharmaceutical ingredients (APIs). Serious concerns about control of livestock, the primary source of heparin, have been raised since 1990s following a series of incidents involving Bovine spongiform encephalopathy, viral infections and prion contamination. Lack of quality control during initial recovery stages led to adulteration of pharmaceutical heparin with oversulfated chondroitin sulfate (OSCS), resulting in an international crisis in 2008 associated with 100 deaths in US. The inherent problems with the animal tissue-based heparin production have motivated us to develop a commercially feasible chemoenzymatic heparin preparation process. This is based on bacterial fermentation of E. coli K5 to generate a capsular polysaccharide heparin precursor, that is then chemically N-deacetylated and N-sulfonated. A series of six recombinant enzymes, derived from heparin biosynthetic pathway and expressed in E. coli, are then used to epimerize uronic acid residues and sulfonate the C2, C3 and C6 positions. These modifications are carried out in tandem with sensitive analytical techniques and bioassays to result in a series of controlled structural changes. Bioengineered heparin consists of 86% trisulfated disaccharide resembling the highly sulfated structure of heparin. The formation of 9.4% lyase resistant tetrasaccharides upon digestion with heparin lyase 2 is consistent with the presence of antithrombin binding pentasaccharide regions in the range of USP heparins (3.9-6.7%). 1D-1H NMR and HMQC techniques were employed to elucidate presence of 3-O-sulfo and I2S peak in anomeric regions. Peaks corresponding to critical features in the IdoA and GlcN residues, including 2-O-sulfo, N-sulfo, N-acetyl, 3-O-sulfo, and 6-O-sulfo, have been fully assigned by 1H-NMR and HMQC spectra. Investigation of in-vitro anticoagulant activity confirms the biological activity of chemoenzymatically derived heparin with an anti-factor Xa activity of 205.8 U/mg and an anti-factor IIa activity of 225.2 U/mg (anti-factor Xa:anti-factor IIa ratio of 0.91) complying with USP requirements. These analyses showcase generic equivalence of bioengineered heparin to animal derived heparin API. A more robust process based on immobilized enzymes, enhancing enzymatic stability and increased reusability, is under development and will be employed to generate bioengineered heparin for in-vivo studies.
    13 AIChE Annual Meeting; 11/2013
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    ABSTRACT: Testosteronan, an unusual glycosaminoglycan first isolated from the microbe Comamonas testosteroni, was enzymatically synthesized in vitro by transferring uridine diphosphate sugars on β-p-nitrophenyl glucuronide acceptor. After chemically converting testosteronan to N-sulfotestosteronan it was tested as a substrate for sulfotransferases involved in the biosynthesis of the glycosaminoglycan, heparan sulfate. Studies using (35) S-labeled 3'-phosphodenosine-5'-phosphosulfate (PAPS) showed that only 6-O-sulfotransferases acted on N-sulfotestosteronan. An oxidative depolymerization reaction was explored to generate oligosaccharides from (34) S-labeled 6-O-sulfo-N-sulfotestosteroran using (34) S-labeled PAPS because testosteronan was resistant to all of the tested glycosaminoglycan-degrading enzymes. Liquid chromotography-mass spectrometric analysis of the oxidatively depolymerized polysaccharides confirmed the incorporation of (34) S into ~14% of the glucosamine residues. Nuclear magnetic resonance spectroscopy also showed that the sulfo groups were transferred to ~20% of the 6-hydroxyl groups in the glucosamine residue of N-sulfotestosteronan. The bioactivity of 6-O-sulfo-N-sulfotestosteronan was examined by performing protein-binding studies with fibroblast growth factors and antithrombin III using a surface plasmon resonance competition assay. The introduction of 6-O-sulfo groups enhanced N-sulfotestosteronan binding to the fibroblast growth factors, but not to antithrombin III.
    Biopolymers 10/2013; 99(10). DOI:10.1002/bip.22263 · 2.29 Impact Factor
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    ABSTRACT: We have developed an efficient chemoenzymatic synthesis of heparan sulfate oligosaccharides employing the para-nitrophenyl (p-NP) β-glucuronide as an acceptor compatible with enzymatic elongation and one that significantly simplifies oligosaccharide purification on C-18 resin. Employing ceric ammonium nitrate as oxidative reagent to remove the p-NP group unexpectedly also removed the glucuronic acid residue at the reducing-end, affording a smaller oligosaccharide. The application of ceric ammonium sulfate allowed the removal of the p-NP without concomitant loss of the adjacent glucuronic acid offering a route to longer heparin sulfate oligosaccharide products.
    Tetrahedron Letters 08/2013; 54(33):4471-4474. DOI:10.1016/j.tetlet.2013.06.044 · 2.39 Impact Factor
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    ABSTRACT: Abstract From May 2003 to May 2010, a total of 9 patients with severe hemobilia after percutaneous transhepatic cholangial drainage (PTCD) were diagnosed using superselective angiography and cholangiography, and then were treated with interventional procedures. Two patients with hepatic arterio-biliary fistula underwent proximal and distal arterial embolization of the responsible vessel. Six patients with pseudoaneurysm had pseudoaneurysm occlusion with proximal and distal embolization. Another patient with biliary-portal vein fistula received a biliary fully covered stent placement. The effects in these patients were evaluated using superselective angiography immediately after the intervention and at 3- and 6-month follow-up. In all patients, hemobilia was stopped right after the treatment and no sign of recurrence was noted at 3- and 6-month follow-up after the interventional therapy. Our findings demonstrate that interventional therapy is a simple, minimally invasive, and safe approach for treating severe hemobilia in patients receiving PTCD.
    International surgery 08/2013; 98(3):223-8. DOI:10.9738/INTSURG-D-13-CC194 · 0.25 Impact Factor
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    ABSTRACT: Heparan sulfate (HS) is a highly sulfated glycosaminoglycan and exists in all animal tissues. HS and heparin are very similar, except that heparin has higher level of sulfation and higher content of iduronic acid. Despite the fact that it is a century-old drug, heparin remains as a top choice for treating thrombotic disorders. Pharmaceutical heparin is derived from porcine intestine or bovine lung via a long supply chain. This supply chain is vulnerable to the contamination of animal pathogens. Therefore, new methods for manufacturing heparin or heparin-like substances devoid of animal tissues have been explored by many researchers, among which, modifications of heparosan, the capsular polysaccharide of Escherichia coli K5 strain, is one of the promising approaches. Heparosan has a structure similar to unmodified backbone of natural HS and heparin. It is feasible to obtain HS or heparin derivatives by modifying heparosan with chemical or enzymatic methods. These derivatives display different biological activities, such as anticoagulant, anti-inflammatory, anticancer, and antiviral activities. This review focuses on the recent studies of synthesis, activity, and structure-activity relationship of HS/heparin-like derivatives prepared from heparosan. © 2012 Wiley Periodicals, Inc. Med. Res. Rev., 00, No. 00, 1-28, 2012.
    Medicinal Research Reviews 05/2013; 33(3). DOI:10.1002/med.21263 · 8.13 Impact Factor
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    ABSTRACT: The aim of this study was to evaluate the effect of percutaneous one-channel double stent implantation on hilar biliary obstruction involving both hepatic ducts and its clinical value. A total of 8 patients with hilar biliary obstruction involving the left and right hepatic ducts were enrolled. A percutaneous unilateral approach was adopted. Two stents were implanted, one between the left and right hepatic ducts and the other between the hepatic ducts and the common bile duct for biliary drainage. Interventional therapies such as arterial chemoembolization were performed for antitumor treatment. All surgical procedures were successfully accomplished. At 2 weeks after stenting, total bilirubin decreased to 61.2±13.4 μmol/l (the preoperative value was 267.1±154.7 μmol/l). No severe complications or mortalities occurred. Single-channel double stent implantation should be the preferred method of treatment for patients with hilar biliary obstruction involving both hepatic ducts. Drainage and antitumor treatment should also be used when necessary.
    Experimental and therapeutic medicine 04/2013; 5(4):1179-1183. DOI:10.3892/etm.2013.921 · 0.94 Impact Factor
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    Sherket B Peterson, Jian Liu
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    ABSTRACT: Heparan sulfate is a highly sulfated polysaccharide abundantly present in the extracellular matrix. Heparan sulfate consists of a disaccharide repeating unit of glucosamine and glucuronic and iduronic acid residues. The functions of heparan sulfate are largely dictated by its size as well as the sulfation patterns. Heparanase is an enzyme that cleaves heparan sulfate polysaccharide into smaller fragments, regulating the functions of heparan sulfate. Understanding the substrate specificity plays a critical role in dissecting the biological functions of heparanase and heparan sulfate. The prevailing view is that heparanase recognizes specific sulfation patterns in heparan sulfate. However, emerging evidence suggests that heparanase is capable of varying its substrate specificities depending on the saccharide structures around the cleavage site. The plastic substrate specificity suggests a complex role of heparanase in regulating the structures of heparan sulfate in matrix biology.
    Matrix biology: journal of the International Society for Matrix Biology 03/2013; DOI:10.1016/j.matbio.2013.02.006 · 3.56 Impact Factor
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    ABSTRACT: Glycosaminoglycans (GAGs) are complex polysaccharides composed of hexosamine-containing disaccharide repeating units. The three most studied classes of GAGs, heparin/heparan sulfate, hyaluronan, and chondroitin/dermatan sulfate, are essential macromolecules. GAGs isolated from animal and microbial sources have been utilized therapeutically, but naturally occurring GAGs are extremely heterogeneous limiting further development of these agents. These molecules pose difficult targets to construct by classical organic syntheses due to the long chain lengths and complex patterns of modification by sulfation and epimerization. Chemoenzymatic synthesis, a process that employs exquisite enzyme catalysts and various defined precursors (e.g., uridine 5'-diphosphosphate-sugar donors, sulfate donors, acceptors, and oxazoline precursors), promises to deliver homogeneous GAGs. This review covers both theoretical and practical issues of GAG oligosaccharide and polysaccharide preparation as single molecular entities and in library formats. Even at this early stage of technology development, nearly monodisperse GAGs can be made with either natural or artificial structures.
    Glycobiology 03/2013; 23(7). DOI:10.1093/glycob/cwt016 · 3.75 Impact Factor
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    ABSTRACT: A 'clickable' disaccharide was prepared by treating the aldehyde precursor with hydroxylamine, followed by the catalytic hydrogenation and diazotransfer reaction. This disaccharide was successfully applied to the elongation of the backbone construction of ultralow molecular weight (ULMW) heparins using two bacterial glycosyl transferases, N-acetyl glucosaminyl transferase from Escherichia coli K5 (KfiA) and heparosan synthase-2 (pmHS2) from Pasteurella multocida.
    Carbohydrate research 03/2013; 372C:30-34. DOI:10.1016/j.carres.2013.02.010 · 2.03 Impact Factor

Publication Stats

2k Citations
478.95 Total Impact Points

Institutions

  • 2002–2014
    • University of North Carolina at Chapel Hill
      • • Division of Chemical Biology and Medicinal Chemistry
      • • Eshelman School of Pharmacy
      North Carolina, United States
    • Massachusetts Institute of Technology
      • Department of Biology
      Cambridge, Massachusetts, United States
    • Lund University
      • Biomedical Center
      Lund, Skane, Sweden
  • 2012
    • Julphar School of Pharmacy
      Worcester, Massachusetts, United States
    • Broad Institute of MIT and Harvard
      Cambridge, Massachusetts, United States
  • 2007–2012
    • Rensselaer Polytechnic Institute
      • Department of Chemistry and Chemical Biology
      Троя, New York, United States
    • University of North Carolina at Charlotte
      Charlotte, North Carolina, United States
  • 2006–2011
    • University of Illinois at Chicago
      • Department of Ophthalmology and Visual Sciences (Chicago)
      Chicago, IL, United States