Mark O Lively

Wake Forest School of Medicine, Winston-Salem, North Carolina, United States

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Publications (52)297.43 Total impact

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    ABSTRACT: Bacterial and viral CpG oligonculeotides are unmethylated cytosine-phosphate-guanosine dinucleotide sequences and trigger an innate immune response through activation of the toll-like receptor 9 (TLR9). We have developed synthetic photocaged CpGs via site-specific incorporation of nitropiperonyloxymethyl (NPOM)-caged thymidine residues. These oligonucleotides enable the optical control of TLR9 function and thereby provide light-activation of an immune response. We provide a proof-of-concept model by applying a reporter assay in live cells and by quantification of endogenous production of interleukin 6.
    Tetrahedron Letters 02/2015; 56(23). DOI:10.1016/j.tetlet.2015.01.165 · 2.39 Impact Factor
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    ABSTRACT: Tumor associated macrophages (TAMS) have been suggested to be key players in the tumor microenvironment. The M2 activation phenotype is believed to be the predominant TAM phenotype in solid tumors. To further define the role of M2 TAMs and analyze the cross-talk between tumor cells and TAMs, we examined the presence of macrophage markers in glioblastoma (GBM) tumors, their responses to various treatments, the genetic profile of M2 macrophages and also potential mediators of tumor cells communication with TAMs.
    Neuro-Oncology 07/2014; 16 Suppl 3:iii40-iii41. DOI:10.1093/neuonc/nou208.66 · 5.29 Impact Factor
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    ABSTRACT: WHO grade II low-grade gliomas (LGGs) with high risk factors for recurrence are mostly lethal despite current treatments, and novel approaches are needed. We conducted a phase I study to evaluate the safety and immunogenicity of subcutaneous vaccinations with synthetic peptides for glioma-associated antigen (GAA) epitopes in human leukocyte antigen (HLA)-A2+ adults with high-risk LGGs in the following three cohorts: 1) newly diagnosed patients without prior radiation therapy (RT); 2) newly diagnosed patients with prior RT, and 3) recurrent patients.
    Neuro-Oncology 07/2014; 16 Suppl 3(2):iii39. DOI:10.1093/neuonc/nou208.62 · 5.29 Impact Factor
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    ABSTRACT: Doxorubicin (Dox) is widely used for breast cancer treatment but causes serious side-effects including cardiotoxicity that may adversely impact patient lifespan even if treatment is successful. Herein, we describe selective conjugation of Dox to a single site in a DNA hairpin resulting in a highly stable complex that enables Dox to be used more effectively. Selective conjugation of Dox to G15 in the hairpin loop was verified using site-specific labeling with [2-15N]-2'-deoxyguanosine in conjunction with [1H-15N] 2D NMR while 1:1 stoichiometry for the conjugate was validated by ESI-QTOF mass spectrometry and UV spectroscopy. Molecular modeling indicated covalently bound Dox also intercalated into the stem of the hairpin and stability studies demonstrated the resulting Dox-conjugated hairpin (DCH) complex had a half-life > 30 h, considerably longer than alternative covalent and non-covalent complexes. Secondary conjugation of DCH with folic acid (FA) resulted in increased internalization into breast cancer cells. The dual conjugate, DCH-FA, can be used for safer and more effective chemotherapy with Dox and this conjugation strategy can be expanded to include additional anti-cancer drugs.
    Bioconjugate Chemistry 01/2014; 25(2). DOI:10.1021/bc4005427 · 4.82 Impact Factor
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    ABSTRACT: Short interfering RNAs (siRNAs) and microRNAs (miRNAs) have been widely used in mammalian tissue culture and model organisms to selectively silence genes of interest. One limitation of this technology is the lack of precise external control over the gene-silencing event. The use of photocleavable protecting groups installed on nucleobases is a promising strategy to circumvent this limitation, providing high spatial and temporal control over siRNA or miRNA activation. Here, we have designed, synthesized and site-specifically incorporated new photocaged guanosine and uridine RNA phosphoramidites into short RNA duplexes. We demonstrated the applicability of these photocaged siRNAs in the light-regulation of the expression of an exogenous green fluorescent protein reporter gene and an endogenous target gene, the mitosis motor protein, Eg5. Two different approaches were investigated with the caged RNA molecules: the light-regulation of catalytic RNA cleavage by RISC and the light-regulation of seed region recognition. The ability to regulate both functions with light enables the application of this optochemical methodology to a wide range of small regulatory RNA molecules.
    Nucleic Acids Research 09/2013; 41(22). DOI:10.1093/nar/gkt806 · 8.81 Impact Factor
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    ABSTRACT: Antisense oligonucleotides are powerful tools to regulate gene expression in cells and model organisms. However, a transfection or microinjection is needed for efficient delivery of the antisense agent. We report the conjugation of multiple HIV TAT peptides to a hairpin-protected antisense agent through a light-cleavable nucleobase caging group. This conjugation allows for the facile delivery of the antisense agent without a transfection reagent and photochemical activation offers precise control over gene expression. The developed approach is highly modular, as demonstrated by the conjugation of folic acid to the caged antisense agent. This enabled targeted cell delivery through cell-surface folate receptors followed by photochemical triggering of antisense activity. Importantly, the presented strategy delivers native oligonucleotides after light-activation, devoid of any delivery functionalities or modifications that could otherwise impair their antisense activity.
    ACS Chemical Biology 08/2013; 8(10). DOI:10.1021/cb400293e · 5.36 Impact Factor
  • Henry R Bourne, Mark O Lively
    Science 07/2012; 337(6093):390. DOI:10.1126/science.1226460 · 31.48 Impact Factor
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    ABSTRACT: EphrinA1 is a glycosylphosphatidylinositol (GPI)-linked ligand for the EphA2 receptor, which is overexpressed in glioblastoma (GBM), among other cancers. Activation of the receptor by ephrinA1 leads to a suppression of oncogenic properties of GBM cells. We documented that a monomeric functional form of ephrinA1 is released from cancer cells and thus explored the mechanism of ephrinA1 release and the primary protein sequence. We demonstrate here that multiple metalloproteases (MMPs) are able to cleave ephrinA1, most notably MMP-1, -2, -9, and -13. The proteolytic cleavage that releases ephrinA1 occurs at three positions near the C terminus, producing three forms ending in valine-175, histidine-177, or serine-178. Moreover, deletion of amino acids 174 to 181 or 175 to 181 yields ephrinA1 that is still GPI linked but not released by proteolysis, underlining the necessity of amino acids 175 to 181 for release from the membrane. Furthermore, recombinant ephrinA1 ending at residue 175 retains activity toward the EphA2 receptor. These findings suggest a mechanism of release and provide evidence for the existence of several forms of monomeric ephrinA1. Moreover, ephrinA1 should be truncated at a minimum at amino acid 175 in fusions or conjugates with other molecules in order to prevent likely proteolysis within physiological and pathobiological environments.
    Molecular and Cellular Biology 06/2012; 32(16):3253-64. DOI:10.1128/MCB.06791-11 · 5.04 Impact Factor
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    ABSTRACT: BACKGROUND: Coenzyme Q(10) (CoQ(10)) is a common antioxidant supplement with known cardioprotective effects and potential anticancer benefits. OBJECTIVES: We performed a randomized, double-blind, placebo-controlled study of oral CoQ(10) in female breast cancer patients with the primary objective of determining CoQ(10)'s effects on self-reported fatigue, depression, and quality of life (QOL). METHODS: Eligible women with newly diagnosed breast cancer and planned adjuvant chemotherapy were randomized to oral supplements of 300 mg CoQ(10) or placebo, each combined with 300 IU vitamin E, divided into 3 daily doses. Treatment was continued for 24 weeks. Blood tests, QOL measures, and levels of plasma CoQ(10) and vitamin E were obtained at baseline and at 8, 16, and 24 weeks. Mixed-effects models were used to assess treatment differences in outcomes over time. RESULTS: Between September 2004 and March 2009, 236 women were enrolled. Treatment arms were well balanced with respect to age (range, 28-85 years), pathologic stage (stage 0, 91%; stage I, 8%; stage II, 1%), ethnicity (white, 87%; black, 11%; Hispanic, 2%), and planned therapy. Baseline CoQ(10) levels in the CoQ(10) and placebo arms were 0.70 and 0.73 μg/mL, respectively; the 24-week CoQ(10) levels were 1.83 and 0.79 μg/mL, respectively. There were no significant differences between the CoQ(10) and placebo arms at 24 weeks for scores on the Profile of Mood States-Fatigue questionnaire (least squares means, 7.08 vs 8.24, P = .257), the Functional Assessment of Chronic Illness Therapy-Fatigue tool (37.6 vs 37.6, P = .965), the Functional Assessment of Cancer Therapy-Breast Cancer instrument (111.9 vs 110.4, P = .577), or the Center for Epidemiologic Studies-Depression scale (11.6 vs 12.3, P = .632). CONCLUSIONS: Supplementation with conventional doses of CoQ(10) led to sustained increases in plasma CoQ(10) levels but did not result in improved self-reported fatigue or QOL after 24 weeks of treatment.
    The journal of supportive oncology 06/2012; DOI:10.1016/j.suponc.2012.03.003
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    ABSTRACT: Triplex-forming oligonucleotides (TFOs) are efficient tools to regulate gene expression through the inhibition of transcription. Here, nucleobase-caging technology was applied to the temporal regulation of transcription through light-activated TFOs. Through site-specific incorporation of caged thymidine nucleotides, the TFO:DNA triplex formation is blocked, rendering the TFO inactive. However, after a brief UV irradiation, the caging groups are removed, activating the TFO and leading to the inhibition of transcription. Furthermore, the synthesis and site-specific incorporation of caged deoxycytidine nucleotides within TFO inhibitor sequences was developed, allowing for the light-deactivation of TFO function and thus photochemical activation of gene expression. After UV-induced removal of the caging groups, the TFO forms a DNA dumbbell structure, rendering it inactive, releasing it from the DNA, and activating transcription. These are the first examples of light-regulated TFOs and their application in the photochemical activation and deactivation of gene expression. In addition, hairpin loop structures were found to significantly increase the efficacy of phosphodiester DNA-based TFOs in tissue culture.
    ACS Chemical Biology 04/2012; 7(7):1247-56. DOI:10.1021/cb300161r · 5.36 Impact Factor
  • Henry R. Bourne, Mark O. Lively
    Science 01/2012; 337(6093). DOI:10.2307/23267885 · 31.48 Impact Factor
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    ABSTRACT: DNA decoys have been developed for the inhibition of transcriptional regulation of gene expression. However, the present methodology lacks the spatial and temporal control of gene expression that is commonly found in nature. Here, we report the application of photoremovable protecting groups on nucleobases of nuclear factor κB (NF-κB) DNA decoys to regulate NF-κB-driven transcription of secreted alkaline phosphatase using light as an external control element. The NF-κB family of proteins is comprised of important eukaryotic transcription factors that regulate a wide range of cellular processes and are involved in immune response, development, cellular growth, and cell death. Several diseases, including cancer, arthritis, chronic inflammation, asthma, neurodegenerative diseases, and heart disease, have been linked to constitutively active NF-κB. Through the direct incorporation of caging groups into an NF-κB decoy, we were able to disrupt DNA:DNA hybridization and inhibit the binding of the transcription factor to the DNA decoy until UV irradiation removed the caging groups and restored the activity of the oligonucleotide. Excellent light-switching behavior of transcriptional regulation was observed. This is the first example of a caged DNA decoy for the photochemical regulation of gene expression in mammalian cells and represents an important addition to the toolbox of light-controlled gene regulatory agents.
    Journal of the American Chemical Society 08/2011; 133(33):13176-82. DOI:10.1021/ja204980v · 11.44 Impact Factor
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    ABSTRACT: The photochemical regulation of biological systems represents a very precise means of achieving high-resolution control over gene expression in both a spatial and a temporal fashion. DNAzymes are enzymatically active deoxyoligonucleotides that enable the site-specific cleavage of RNA and have been used in a variety of in vitro applications. We have previously reported the photochemical activation of DNAzymes and antisense agents through the preparation of a caged DNA phosphoramidite and its site-specific incorporation into oligonucleotides. The presence of the caging group disrupts either DNA:RNA hybridization or catalytic activity until removed via a brief irradiation with UV light. Here, we are expanding this concept by investigating the photochemical deactivation of DNAzymes and antisense agents. Moreover, we report the application of light-activated and light-deactivated antisense agents to the regulation of gene function in mammalian cells. This represents the first example of gene silencing antisense agents that can be turned on and turned off in mammalian tissue culture.
    Journal of the American Chemical Society 04/2010; 132(17):6183-93. DOI:10.1021/ja100710j · 11.44 Impact Factor
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    ABSTRACT: Removal by the light: The photochemical regulation of restriction endonucleases, which are important enzymes in molecular biology, has been investigated. Photolabile protecting groups have been installed on DNA substrates and have been demonstrated to inhibit restriction endonuclease activity until removed by UV light irradiation. Interestingly, these groups do not appear to dramatically affect initial binding of the enzyme to the DNA substrate, but rather prevent recognition of the specific cleavage site.
    ChemBioChem 07/2009; 10(10):1612-6. DOI:10.1002/cbic.200900090 · 3.06 Impact Factor
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    ABSTRACT: The naturally occurring nitroalkenes, nitrolinoleic (NO(2)-LA) and nitrooleic (NO(2)-OA) acids, are among the most potent endogenous ligand activators of PPARgamma-dependent transcription. In order to understand mechanisms that regulate cellular response to these nitroalkenes, we previously demonstrated that glutathione conjugation of NO(2)-LA and MRP1-mediated efflux of the conjugates were associated with significant attenuation of PPARgamma activation by this nitroalkene [(2006) Biochemistry 45, 7889-7896]. Here we show that NO(2)-OA activation of PPARgamma is similarly affected by nonenzymatic conjugation and MRP1-mediated efflux. Moreover, the roles of glutathione S-transferases (GSTs) in the glutathione conjugation and bioactivities of NO(2)-LA and NO(2)-OA were investigated. While none of the GST isozymes tested (GSTA1-1, A4-4, M1a-1a, and P1a-1a) enhanced the rate of glutathione conjugation, expression of GSTA1-1, M1a-1a, or P1a-1a in MCF7 cells significantly reduced the magnitude of PPARgamma-dependent reporter gene transcription in response to NO(2)-LA and NO(2)-OA treatment, with GSTP1a-1a expression mediating the most potent inhibition of PPARgamma. Although these GSTs failed to catalyze nitroalkene conjugation with glutathione, the nitroalkenes were found to associate avidly with all four GST isozymes as indicated by their ability to inhibit GST activity with K(i)'s in the nanomolar range. Treatment of purified GSTP1a-1a with excess NO(2)-LA and NO(2)-OA resulted in the formation of covalent adducts between GSTP1a monomers and nitroalkenes, although separate experiments indicated that such covalent bond formation was not necessary for avid GST-nitroalkene interactions. These results suggest that GSTs can inhibit the activation of transcription by nitroalkenes via noncatalytic sequestration of these ligands, and their glutathione conjugates, away from their nuclear target, PPARgamma.
    Biochemistry 05/2009; 48(19):4159-69. DOI:10.1021/bi900224c · 3.19 Impact Factor
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    ABSTRACT: The effects of photocaged nucleosides on the DNA polymerization reaction was investigated, finding that most polymerases are unable to recognize and read through the presence of a single caging group on the DNA template. Based on this discovery, a new method of introducing mutations into plasmid DNA via a light-mediated mutagenesis protocol was developed. This methodology is advantageous over several common approaches in that it requires the use of only two polymerase chain reaction primers, and does not require any restriction sites or use of restriction enzymes. Additionally, this approach enables not only site-directed mutations, but also the insertion of DNA strands of any length into plasmids and the deletion of entire genes from plasmids.
    Nucleic Acids Research 04/2009; 37(8):e58. DOI:10.1093/nar/gkp150 · 8.81 Impact Factor
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    ChemBioChem 12/2008; 9(18):2937-40. DOI:10.1002/cbic.200800627 · 3.06 Impact Factor
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    ABSTRACT: A number of studies have clearly demonstrated that flagellin is a potent adjuvant that promotes robust immune responses when it is given with a protein antigen. In view of the potential biological and practical benefits of a recombinant protein vaccine composed of a single fusion protein containing flagellin and antigen, we have evaluated the efficacy of a fusion protein composed of flagellin and two protective antigens of Yersinia pestis (F1 and V) in eliciting protection against respiratory challenge with Y. pestis. Flagellin-F1-V was produced and purified in high yield under good manufacturing practices conditions. The fusion protein retains full Toll-like receptor 5-stimulating activity in vitro. Using a prime-boost immunization protocol, we found that flagellin-F1-V elicits robust antigen-specific humoral immunity in mice and two species of nonhuman primates. Immune mice were fully protected against intranasal challenge with 150 mean tolerated doses of Y. pestis CO92. In immune mice, the bacteria were completely cleared within 3 days after challenge. Flagellin-F1-V exhibited full stability for at least 297 days at 4 degrees C and at least 168 days at 25 degrees C. At between 29 and 84 days at 37 degrees C, the protein exhibited a loss of biological activity that appeared to be associated with a substantial change in protein diameter, possibly due to oligomerization. On the basis of our results, we believe that flagellin-F1-V is an outstanding candidate for evaluation in studies with humans.
    Clinical and vaccine Immunology: CVI 12/2008; 16(1):21-8. DOI:10.1128/CVI.00333-08 · 2.37 Impact Factor
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    ABSTRACT: Three complete cDNAs encoding different forms of vitellogenin (Vtg) were isolated from a white perch (Morone americana) liver cDNA library and characterized with respect to immunobiochemical and functional features of the three Vtgs and their product yolk proteins (YPs) in this species and in the congeneric striped bass (Morone saxatilis). The two longest cDNAs encoded Vtgs with a complete suite of yolk protein domains that, based on comparisons with vtg sequences from other species, were categorized as VtgAa and VtgAb using the current nomenclature for multiple teleost Vtgs. The shorter cDNA encoded a Vtg that lacked a phosvitin domain, had a shortened C-terminus, and was categorized as VtgC. Mapping of peptide sequences from the purified Vtgs and their derived YPs to Vtg sequences deduced from the cDNAs definitively identified the white perch VtgAa, VtgAb, and VtgC proteins. Detailed comparisons of the primary structures of each Vtg with partial or complete sequences of Morone yolk proteins or of Vtgs from other fishes revealed conserved and variant structural elements of teleost Vtgs with functional significance, including, as examples, signal peptide cleavage sites, dimerization sites, cathepsin D protease recognition sites, and receptor-binding domains. These comparisons also yielded an interim revision of the classification scheme for multiple teleost Vtgs.
    Marine Biotechnology 10/2008; 11(2):169-87. DOI:10.1007/s10126-008-9133-6 · 3.15 Impact Factor
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    ABSTRACT: Photochemical activation of a deoxyribozyme with peroxidase activity was achieved by the synthesis and incorporation of a caged deoxyguanosine.
    Molecular BioSystems 07/2008; 4(6):508-11. DOI:10.1039/b800166a · 3.18 Impact Factor

Publication Stats

1k Citations
297.43 Total Impact Points

Institutions

  • 2007–2015
    • Wake Forest School of Medicine
      • • Center for Structural Biology
      • • Department of Biochemistry
      Winston-Salem, North Carolina, United States
  • 2014
    • University of Pittsburgh
      Pittsburgh, Pennsylvania, United States
  • 2013
    • College of William and Mary
      • Department of Chemistry
      Williamsburg, Virginia, United States
  • 1986–2013
    • Wake Forest University
      • • Department of Biochemistry
      • • Department of Otolaryngology
      Winston-Salem, North Carolina, United States
  • 2007–2009
    • North Carolina State University
      • Department of Chemistry
      Raleigh, NC, United States
  • 1981
    • University of Washington Seattle
      • Department of Biochemistry
      Seattle, Washington, United States