Publications (40)166.53 Total impact
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Article: Integrating high-content imaging and chemical genetics to probe host cellular pathways critical for yersinia pestis infection.
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ABSTRACT: The molecular machinery that regulates the entry and survival of Yersinia pestis in host macrophages is poorly understood. Here, we report the development of automated high-content imaging assays to quantitate the internalization of virulent Y. pestis CO92 by macrophages and the subsequent activation of host NF-κB. Implementation of these assays in a focused chemical screen identified kinase inhibitors that inhibited both of these processes. Rac-2-ethoxy-3 octadecanamido-1-propylphosphocholine (a protein Kinase C inhibitor), wortmannin (a PI3K inhibitor), and parthenolide (an IκB kinase inhibitor), inhibited pathogen-induced NF-κB activation and reduced bacterial entry and survival within macrophages. Parthenolide inhibited NF-κB activation in response to stimulation with Pam3CSK4 (a TLR2 agonist), E. coli LPS (a TLR4 agonist) or Y. pestis infection, while the PI3K and PKC inhibitors were selective only for Y. pestis infection. Together, our results suggest that phagocytosis is the major stimulus for NF-κB activation in response to Y. pestis infection, and that Y. pestis entry into macrophages may involve the participation of protein kinases such as PI3K and PKC. More importantly, the automated image-based screening platform described here can be applied to the study of other bacteria in general and, in combination with chemical genetic screening, can be used to identify host cell functions facilitating the identification of novel antibacterial therapeutics.PLoS ONE 01/2013; 8(1):e55167. · 4.09 Impact Factor -
Article: Bis-imidazolinylindoles are active against methicillin-resistant Staphylococcus aureus and multidrug-resistant Mycobacterium tuberculosis.
The Journal of Antibiotics 11/2012; · 1.65 Impact Factor -
Article: Peptide Conjugated Phosphorodiamidate Morpholino Oligomers Increase Survival of Mice Challenged with Ames Bacillus anthracis.
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ABSTRACT: Targeting bacterial essential genes using antisense phosphorodiamidate morpholino oligomers (PMOs) represents an important strategy in the development of novel antibacterial therapeutics. PMOs are neutral DNA analogues that inhibit gene expression in a sequence-specific manner. In this study, several cationic, membrane-penetrating peptides were conjugated to PMOs (PPMOs) that target 2 bacterial essential genes: acyl carrier protein (acpP) and gyrase A (gyrA). These were tested for their ability to inhibit growth of Bacillus anthracis, a gram-positive spore-forming bacterium and causative agent of anthrax. PPMOs targeted upstream of both target gene start codons and conjugated with the bacterium-permeating peptide (RFF)(3)R were found to be most effective in inhibiting bacterial growth in vitro. Both of the gene-targeted PPMOs protected macrophages from B. anthracis induced cell death. Subsequent, in vivo testing of the PPMOs resulted in increased survival of mice challenged with the virulent Ames strain of B. anthracis. Together, these studies suggest that PPMOs targeting essential genes have the potential of being used as antisense antibiotics to treat B. anthracis infections.Nucleic acid therapeutics. 09/2012; 22(5):316-22. -
Article: Shedding light on filovirus infection with high-content imaging.
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ABSTRACT: Microscopy has been instrumental in the discovery and characterization of microorganisms. Major advances in high-throughput fluorescence microscopy and automated, high-content image analysis tools are paving the way to the systematic and quantitative study of the molecular properties of cellular systems, both at the population and at the single-cell level. High-Content Imaging (HCI) has been used to characterize host-virus interactions in genome-wide reverse genetic screens and to identify novel cellular factors implicated in the binding, entry, replication and egress of several pathogenic viruses. Here we present an overview of the most significant applications of HCI in the context of the cell biology of filovirus infection. HCI assays have been recently implemented to quantitatively study filoviruses in cell culture, employing either infectious viruses in a BSL-4 environment or surrogate genetic systems in a BSL-2 environment. These assays are becoming instrumental for small molecule and siRNA screens aimed at the discovery of both cellular therapeutic targets and of compounds with anti-viral properties. We discuss the current practical constraints limiting the implementation of high-throughput biology in a BSL-4 environment, and propose possible solutions to safely perform high-content, high-throughput filovirus infection assays. Finally, we discuss possible novel applications of HCI in the context of filovirus research with particular emphasis on the identification of possible cellular biomarkers of virus infection.Viruses 08/2012; 4(8):1354-71. · 1.50 Impact Factor -
Article: A limited structural modification results in a significantly more efficacious diazachrysene-based filovirus inhibitor.
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ABSTRACT: Ebola (EBOV) and Marburg (MARV) filoviruses are highly infectious pathogens causing deadly hemorrhagic fever in humans and non-human primates. Promising vaccine candidates providing immunity against filoviruses have been reported. However, the sporadic nature and swift progression of filovirus disease underlines the need for the development of small molecule therapeutics providing immediate antiviral effects. Herein we describe a brief structural exploration of two previously reported diazachrysene (DAAC)-based EBOV inhibitors. Specifically, three analogs were prepared to examine how slight substituent modifications would affect inhibitory efficacy and inhibitor-mediated toxicity during not only EBOV, but also MARV cellular infection. Of the three analogs, one was highly efficacious, providing IC(50) values of 0.696 µM ± 0.13 µM and 2.76 µM ± 0.21 µM against EBOV and MARV infection, respectively, with little or no associated cellular toxicity. Overall, the structure-activity and structure-toxicity results from this study provide a framework for the future development of DAAC-based filovirus inhibitors that will be both active and non-toxic in vivo.Viruses 08/2012; 4(8):1279-88. · 1.50 Impact Factor -
Article: Discovery and Early Development of AVI-7537 and AVI-7288 for the Treatment of Ebola Virus and Marburg Virus Infections.
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ABSTRACT: There are no currently approved treatments for filovirus infections. In this study we report the discovery process which led to the development of antisense Phosphorodiamidate Morpholino Oligomers (PMOs) AVI-6002 (composed of AVI-7357 and AVI-7539) and AVI-6003 (composed of AVI-7287 and AVI-7288) targeting Ebola virus and Marburg virus respectively. The discovery process involved identification of optimal transcript binding sites for PMO based RNA-therapeutics followed by screening for effective viral gene target in mouse and guinea pig models utilizing adapted viral isolates. An evolution of chemical modifications were tested, beginning with simple Phosphorodiamidate Morpholino Oligomers (PMO) transitioning to cell penetrating peptide conjugated PMOs (PPMO) and ending with PMOplus containing a limited number of positively charged linkages in the PMO structure. The initial lead compounds were combinations of two agents targeting separate genes. In the final analysis, a single agent for treatment of each virus was selected, AVI-7537 targeting the VP24 gene of Ebola virus and AVI-7288 targeting NP of Marburg virus, and are now progressing into late stage clinical development as the optimal therapeutic candidates.Viruses 01/2012; 4(11):2806-30. · 1.50 Impact Factor -
Article: High content image based analysis identifies cell cycle inhibitors as regulators of ebola virus infection.
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ABSTRACT: Viruses modulate a number of host biological responses including the cell cycle to favor their replication. In this study, we developed a high-content imaging (HCI) assay to measure DNA content and identify different phases of the cell cycle. We then investigated the potential effects of cell cycle arrest on Ebola virus (EBOV) infection. Cells arrested in G1 phase by serum starvation or G1/S phase using aphidicolin or G2/M phase using nocodazole showed much reduced EBOV infection compared to the untreated control. Release of cells from serum starvation or aphidicolin block resulted in a time-dependent increase in the percentage of EBOV infected cells. The effect of EBOV infection on cell cycle progression was found to be cell-type dependent. Infection of asynchronous MCF-10A cells with EBOV resulted in a reduced number of cells in G2/M phase with concomitant increase of cells in G1 phase. However, these effects were not observed in HeLa or A549 cells. Together, our studies suggest that EBOV requires actively proliferating cells for efficient replication. Furthermore, multiplexing of HCI based assays to detect viral infection, cell cycle status and other phenotypic changes in a single cell population will provide useful information during screening campaigns using siRNA and small molecule therapeutics.Viruses 01/2012; 4(10):1865-77. · 1.50 Impact Factor -
Article: Identification of an antioxidant small-molecule with broad-spectrum antiviral activity.
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ABSTRACT: The highly lethal filoviruses, Ebola and Marburg cause severe hemorrhagic fever in humans and non-human primates. To date there are no licensed vaccines or therapeutics to counter these infections. Identifying novel pathways and host targets that play an essential role during infection will provide potential targets to develop therapeutics. Small molecule chemical screening for Ebola virus inhibitors resulted in identification of a compound NSC 62914. The compound was found to exhibit anti-filovirus activity in cell-based assays and in vivo protected mice following challenge with Ebola or Marburg viruses. Additionally, the compound was found to inhibit Rift Valley fever virus, Lassa virus and Venezuelan equine encephalitis virus in cell-based assays. Investigation of the mechanism of action of the compound revealed that it had antioxidant properties. Specifically, compound NSC 62914 was found to act as a scavenger of reactive oxygen species, and to up-regulate oxidative stress-induced genes. However, four known antioxidant compounds failed to inhibit filovirus infection, thus suggesting that the mechanistic basis of the antiviral function of the antioxidant NSC 62914 may involve modulation of multiple signaling pathways/targets.Antiviral research 01/2012; 93(1):23-9. · 3.61 Impact Factor -
Article: A chemotype that inhibits three unrelated pathogenic targets: the botulinum neurotoxin serotype A light chain, P. falciparum malaria, and the Ebola filovirus.
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ABSTRACT: A 1,7-bis(alkylamino)diazachrysene-based small molecule was previously identified as an inhibitor of the botulinum neurotoxin serotype A light chain metalloprotease. Subsequently, a variety of derivatives of this chemotype were synthesized to develop structure-activity relationships, and all are inhibitors of the BoNT/A LC. Three-dimensional analyses indicated that half of the originally discovered 1,7-DAAC structure superimposed well with 4-amino-7-chloroquinoline-based antimalarial agents. This observation led to the discovery that several of the 1,7-DAAC derivatives are potent in vitro inhibitors of Plasmodium falciparum and, in general, are more efficacious against CQ-resistant strains than against CQ-susceptible strains. In addition, by inhibiting β-hematin formation, the most efficacious 1,7-DAAC-based antimalarials employ a mechanism of action analogous to that of 4,7-ACQ-based antimalarials and are well tolerated by normal cells. One candidate was also effective when administered orally in a rodent-based malaria model. Finally, the 1,7-DAAC-based derivatives were examined for Ebola filovirus inhibition in an assay employing Vero76 cells, and three provided promising antiviral activities and acceptably low toxicities.Journal of Medicinal Chemistry 01/2011; 54(5):1157-69. · 4.80 Impact Factor -
Article: Identification of a small-molecule entry inhibitor for filoviruses.
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ABSTRACT: Ebola virus (EBOV) causes severe hemorrhagic fever, for which therapeutic options are not available. Preventing the entry of EBOV into host cells is an attractive antiviral strategy, which has been validated for HIV by the FDA approval of the anti-HIV drug enfuvirtide. To identify inhibitors of EBOV entry, the EBOV envelope glycoprotein (EBOV-GP) gene was used to generate pseudotype viruses for screening of chemical libraries. A benzodiazepine derivative (compound 7) was identified from a high-throughput screen (HTS) of small-molecule compound libraries utilizing the pseudotype virus. Compound 7 was validated as an inhibitor of infectious EBOV and Marburg virus (MARV) in cell-based assays, with 50% inhibitory concentrations (IC(50)s) of 10 μM and 12 μM, respectively. Time-of-addition and binding studies suggested that compound 7 binds to EBOV-GP at an early stage during EBOV infection. Preliminary Schrödinger SiteMap calculations, using a published EBOV-GP crystal structure in its prefusion conformation, suggested a hydrophobic pocket at or near the GP1 and GP2 interface as a suitable site for compound 7 binding. This prediction was supported by mutational analysis implying that residues Asn69, Leu70, Leu184, Ile185, Leu186, Lys190, and Lys191 are critical for the binding of compound 7 and its analogs with EBOV-GP. We hypothesize that compound 7 binds to this hydrophobic pocket and as a consequence inhibits EBOV infection of cells, but the details of the mechanism remain to be determined. In summary, we have identified a novel series of benzodiazepine compounds that are suitable for optimization as potential inhibitors of filoviral infection.Journal of Virology 01/2011; 85(7):3106-19. · 5.40 Impact Factor -
Article: Comparative in vitro activity profiles of novel bis-indole antibacterials against gram-positive and gram-negative clinical isolates.
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ABSTRACT: Antimicrobial susceptibilities of 233 gram-positive and 180 gram-negative strains to two novel bis-indoles were evaluated. Both compounds were potent inhibitors of gram-positive bacteria, with MIC(90) values of 0.004 to 0.5 microg/ml. One bis-indole, MBX 1162, exhibited potent activity against all gram-negative strains, with MIC(90) values of 0.12 to 4 microg/ml, even against high-level-resistant pathogens, and compared favorably to all comparator antibiotics. The bis-indole compounds show promise for the treatment of multidrug-resistant clinical pathogens.Antimicrobial Agents and Chemotherapy 09/2010; 54(9):3974-7. · 4.84 Impact Factor -
Article: Development of high-content imaging assays for lethal viral pathogens.
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ABSTRACT: Filoviruses such as Ebola (EBOV) and Marburg (MARV) are single-stranded negative sense RNA viruses that cause acute hemorrhagic fever with high mortality rates. Currently, there are no licensed vaccines or therapeutics to counter filovirus infections in humans. The development of higher throughput/high-content primary screening assays followed by validation using the low-throughput traditional plaque or real-time PCR assays will greatly aid efforts toward the discovery of novel antiviral therapeutics. Specifically, high-content imaging technology is increasingly being applied for primary drug screening. In this study, the authors describe the challenges encountered when optimizing bioassays based on image acquisition and analyses for the highly pathogenic filoviruses Ebola and Marburg. A number of biological and imaging-related variables such as plating density, multiplicity of infection, the number of fields scanned per well, fluorescence intensity, and the cell number analyzed were evaluated during the development of these assays. Furthermore, the authors demonstrate the benefits related to the statistical analyses of single-cell data to account for heterogeneity in the subcellular localization and whole-cell integrated intensity of the viral antigen staining pattern. In conclusion, they show that image-based methods represent powerful screening tools for identifying antiviral compounds for highly pathogenic viruses.Journal of Biomolecular Screening 08/2010; 15(7):755-65. · 2.05 Impact Factor -
Article: Efflux-mediated bis-indole resistance in Staphylococcus aureus reveals differential substrate specificities for MepA and MepR.
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ABSTRACT: The bis-indoles are a novel class of compounds with potent antibacterial activity against a broad spectrum of Gram-positive and Gram-negative pathogens. The mechanism of action of these compounds has not been clearly defined. To study the mechanism of action of bis-indoles, selections for mutants of Staphylococcus aureus NCTC 8325 with reduced susceptibility to several chemically related bis-indoles were carried out using serial passages in subinhibitory compound concentrations. Resistant mutants were only obtained for one of the four bis-indoles tested (MBX-1090), and these appeared at concentrations up to 16X MIC within 10-12 passages. MBX-1090 resistance mutations produced a truncated open reading frame of mepR (SAOUHSC_00314), a gene encoding a MarR-like repressor. MepR regulates expression of mepA (SAOUHSC_00315), which encodes a member of the Multidrug and Toxic Compound Extrusion (MATE) family of efflux pumps. MBX-1090 resistance was reverted when mepR (wild type) was provided in trans. Microarray experiments and RT-PCR experiments confirmed that over-expression of mepA is required for resistance. Interestingly, MBX-1090 resistant mutants and strains overexpressing mepA from an expression vector did not exhibit cross-resistance to closely related bis-indole compounds. MBX-1090 did not induce expression of mepA, suggesting that this compound does not directly interact with MepR. Conversely, the bis-indoles that were not substrates of MepA strongly induced mepA expression. The results of this study suggest that MepA and MepR exhibit remarkably distinct substrate specificity for closely related bis-indoles.Bioorganic & medicinal chemistry 02/2010; 18(6):2123-30. · 2.82 Impact Factor -
Article: Reduced levels of protein tyrosine phosphatase CD45 protect mice from the lethal effects of Ebola virus infection.
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ABSTRACT: Ebola virus (EBOV) infection of humans is a lethal but accidental dead-end event. Understanding resistance to EBOV in other species may help establish the basis of susceptibility differences among its hosts. Although rodents are resistant to EBOV, a murine-adapted variant is lethal when injected intraperitoneally into mice. We find that mice expressing reduced levels of the tyrosine phosphatase CD45 are protected against EBOV, whereas wild-type, CD45-deficient, or enzymatically inactive CD45-expressing mice succumbed to infection. Protection was dependent on CD8(+) T cells and interferon gamma. Reduced CD45-expressing mice retained greater control of gene expression and immune cell proliferation following EBOV infection, which contributed to reduced apoptosis, enhanced viral clearance, and increased protection against the virus. Together, these findings suggest that host susceptibility to EBOV is dependent on the delicate balance of immune homeostasis, which, as demonstrated here, can be determined by the levels of a single regulator.Cell host & microbe 09/2009; 6(2):162-73. · 13.02 Impact Factor -
Article: Novel broad-spectrum bis-(imidazolinylindole) derivatives with potent antibacterial activities against antibiotic-resistant strains.
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ABSTRACT: Given the limited number of structural classes of clinically available antimicrobial drugs, the discovery of antibacterials with novel chemical scaffolds is an important strategy in the development of effective therapeutics for both naturally occurring and engineered resistant strains of pathogenic bacteria. In this study, several diarylamidine derivatives were evaluated for their ability to protect macrophages from cell death following infection with Bacillus anthracis, a gram-positive spore-forming bacterium. Four bis-(imidazolinylindole) compounds were identified with potent antibacterial activity as measured by the protection of macrophages and by the inhibition of bacterial growth in vitro. These compounds were effective against a broad range of gram-positive and gram-negative bacterial species, including several antibiotic-resistant strains. Minor structural variations among the four compounds correlated with differences in their effects on bacterial macromolecular synthesis and mechanisms of resistance. In vivo studies revealed protection by two of the compounds of mice lethally infected with B. anthracis, Staphylococcus aureus, or Yersinia pestis. Taken together, these results indicate that the bis-(imidazolinylindole) compounds represent a new chemotype for the development of therapeutics for both gram-positive and gram-negative bacterial species as well as against antibiotic-resistant infections.Antimicrobial Agents and Chemotherapy 08/2009; 53(10):4283-91. · 4.84 Impact Factor -
Article: Reduced expression of CD45 protein-tyrosine phosphatase provides protection against anthrax pathogenesis.
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ABSTRACT: The modulation of cellular processes by small molecule inhibitors, gene inactivation, or targeted knockdown strategies combined with phenotypic screens are powerful approaches to delineate complex cellular pathways and to identify key players involved in disease pathogenesis. Using chemical genetic screening, we tested a library of known phosphatase inhibitors and identified several compounds that protected Bacillus anthracis infected macrophages from cell death. The most potent compound was assayed against a panel of sixteen different phosphatases of which CD45 was found to be most sensitive to inhibition. Testing of a known CD45 inhibitor and antisense phosphorodiamidate morpholino oligomers targeting CD45 also protected B. anthracis-infected macrophages from cell death. However, reduced CD45 expression did not protect anthrax lethal toxin (LT) treated macrophages, suggesting that the pathogen and independently added LT may signal through distinct pathways. Subsequent, in vivo studies with both gene-targeted knockdown of CD45 and genetically engineered mice expressing reduced levels of CD45 resulted in protection of mice after infection with the virulent Ames B. anthracis. Intermediate levels of CD45 expression were critical for the protection, as mice expressing normal levels of CD45 or disrupted CD45 phosphatase activity or no CD45 all succumbed to this pathogen. Mechanism-based studies suggest that the protection provided by reduced CD45 levels results from regulated immune cell homeostasis that may diminish the impact of apoptosis during the infection. To date, this is the first report demonstrating that reduced levels of host phosphatase CD45 modulate anthrax pathogenesis.Journal of Biological Chemistry 04/2009; 284(19):12874-85. · 4.77 Impact Factor -
Article: Burkholderia mallei tssM encodes a putative deubiquitinase that is secreted and expressed inside infected RAW 264.7 murine macrophages.
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ABSTRACT: Burkholderia mallei, a category B biothreat agent, is a facultative intracellular pathogen that causes the zoonotic disease glanders. The B. mallei VirAG two-component regulatory system activates the transcription of approximately 60 genes, including a large virulence gene cluster encoding a type VI secretion system (T6SS). The B. mallei tssM gene encodes a putative ubiquitin-specific protease that is physically linked to, and transcriptionally coregulated with, the T6SS gene cluster. Mass spectrometry and immunoblot analysis demonstrated that TssM was secreted in a virAG-dependent manner in vitro. Surprisingly, the T6SS was found to be dispensable for the secretion of TssM. The C-terminal half of TssM, which contains Cys and His box motifs conserved in eukaryotic deubiquitinases, was purified and biochemically characterized. Recombinant TssM hydrolyzed multiple ubiquitinated substrates and the cysteine at position 102 was critical for enzymatic activity. The tssM gene was expressed within 1 h after uptake of B. mallei into RAW 264.7 murine macrophages, suggesting that the TssM deubiquitinase is produced in this intracellular niche. Although the physiological substrate(s) is currently unknown, the TssM deubiquitinase may provide B. mallei a selective advantage in the intracellular environment during infection.Infection and immunity 02/2009; 77(4):1636-48. · 4.21 Impact Factor -
Article: Sizing the Bacillus anthracis PA63 channel with nonelectrolyte poly(ethylene glycols).
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ABSTRACT: Nonelectrolyte polymers of poly(ethylene glycol) (PEG) were used to estimate the diameter of the ion channel formed by the Bacillus anthracis protective antigen 63 (PA(63)). Based on the ability of different molecular weight PEGs to partition into the pore and reduce channel conductance, the pore appears to be narrower than the one formed by Staphylococcus aureus alpha-hemolysin. Numerical integration of the PEG sample mass spectra and the channel conductance data were used to refine the estimate of the pore's PEG molecular mass cutoff (approximately 1400 g/mol). The results suggest that the limiting diameter of the PA(63) pore is <2 nm, which is consistent with an all-atom model of the PA(63) channel and previous experiments using large ions.Biophysical Journal 08/2008; 95(3):1157-64. · 3.65 Impact Factor -
Chapter: The Detection and Characterization of Ions, DNA, and Proteins Using Nanometer‐Scale Pores
03/2008; , ISBN: 9780470061565 -
Article: Filovirus-like particles produced in insect cells: immunogenicity and protection in rodents.
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ABSTRACT: Virus-like particles (VLPs) of Ebola virus (EBOV) and Marburg virus (MARV) produced in human 293T embryonic kidney cells have been shown to be effective vaccines against filoviral infection. In this study, we explored alternative strategies for production of filovirus-like particle-based vaccines, to accelerate the development process. The goal of this work was to increase the yield of VLPs, while retaining their immunogenic properties. Ebola and Marburg VLPs (eVLPs and mVLPs, respectively) were generated by use of recombinant baculovirus constructs expressing glycoprotein, VP40 matrix protein, and nucleoprotein from coinfected insect cells. The baculovirus-derived eVLPs and mVLPs were characterized biochemically, and then the immune responses produced by the eVLPs in insect cells were studied further. The baculovirus-derived eVLPs elicited maturation of human myeloid dendritic cells (DCs), indicating their immunogenic properties. Mice vaccinated with insect cell-derived eVLPs generated antibody and cellular responses equivalent to those vaccinated with mammalian 293T cell-derived eVLPs and were protected from EBOV challenge in a dose-dependent manner. Together, these data suggest that filovirus-like particles produced by baculovirus expression systems, which are amenable to large-scale production, are highly immunogenic and are suitable as safe and effective vaccines for the prevention of filoviral infection.The Journal of Infectious Diseases 12/2007; 196 Suppl 2:S421-9. · 6.41 Impact Factor
Top co-authors
Top Journals
Institutions
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2011–2012
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University of Belgrade
- Faculty of Chemistry
Belgrade, SE, Serbia
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2007–2012
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United States Army
Washington, WV, USA
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2005–2012
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United States Army Medical Research Institute for Infectious Diseases
Frederick, MD, USA -
SAIC
Frederick, MD, USA -
National Institutes of Health
- Program of Developmental Therapeutics
Bethesda, MD, USA -
United States Naval Research Laboratory (NRL)
- Center for BioMolecular Science and Engineering
Washington, D. C., DC, USA
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2010–2011
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Microbiotix Inc.
Worcester, MA, USA
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2006–2007
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U.S. Army Medical Research Institute of Infectious Diseases
Frederick, MD, USA
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2004–2007
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National Cancer Institute (USA)
- Developmental Therapeutics Program
Bethesda, MD, USA
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2003–2004
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NCI-Frederick
Frederick, MD, USA
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