Mark O Lively

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

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Publications (57)312.55 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.
    No preview · Article · Feb 2015 · Tetrahedron Letters
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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.
    No preview · Article · Jul 2014 · Neuro-Oncology
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    ABSTRACT: Purpose: WHO grade 2 low-grade gliomas (LGG) with high risk factors for recurrence are mostly lethal despite current treatments. 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 HLA-A2(+) adults with high-risk LGGs in the following three cohorts: (i) patients without prior progression, chemotherapy, or radiotherapy (RT); (ii) patients without prior progression or chemotherapy but with prior RT; and (iii) recurrent patients. Experimental design: GAAs were IL13Rα2, EphA2, WT1, and Survivin. Synthetic peptides were emulsified in Montanide-ISA-51 and given every 3 weeks for eight courses with intramuscular injections of poly-ICLC, followed by q12 week booster vaccines. Results: Cohorts 1, 2, and 3 enrolled 12, 1, and 10 patients, respectively. No regimen-limiting toxicity was encountered except for one case with grade 3 fever, fatigue, and mood disturbance (cohort 1). ELISPOT assays demonstrated robust IFNγ responses against at least three of the four GAA epitopes in 10 and 4 cases of cohorts 1 and 3, respectively. Cohort 1 patients demonstrated significantly higher IFNγ responses than cohort 3 patients. Median progression-free survival (PFS) periods since the first vaccine are 17 months in cohort 1 (range, 10-47+) and 12 months in cohort 3 (range, 3-41+). The only patient with large astrocytoma in cohort 2 has been progression-free for more than 67 months since diagnosis. Conclusion: The current regimen is well tolerated and induces robust GAA-specific responses in WHO grade 2 glioma patients. These results warrant further evaluations of this approach. Clin Cancer Res; 21(2); 286-94. ©2014 AACR.
    No preview · Article · Jul 2014 · Neuro-Oncology
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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.
    Full-text · Article · Jan 2014 · Bioconjugate Chemistry
  • Stephen J. Walker · Mark O. Lively

    No preview · Article · Dec 2013
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    ABSTRACT: BACKGROUND: Patients with high grade gliomas (HGGs) routinely receive radiation (RT), temozolomide (TMZ), and glucocorticoids and 40% develop severe lymphopenia (lymphocyte counts <500 cells/mm3) 2-months after initiation of concurrent RT/TMZ (Grossman, 2011). The lymphopenia resulting from these three lymphotoxic therapies is associated with shorter survival from tumor progression and appears primarily related to the radiation of circulating lymphocytes (Yovino, 2013). This study was designed to evaluate the feasibility of removing circulating lymphocytes prior to beginning radiation and reinfusing them once radiation is complete in patients with newly diagnosed HGGs. METHODS: Ten patients with newly diagnosed HGGs and baseline total lymphocyte counts (TLC) >1000 cells/mm3 were enrolled. Lymphocytes were harvested using peripheral access and a single 120 minute apheresis prior to initiating concurrent RT/TMZ therapy, cryopresevered for 6 weeks, and reinfused immediately after the completion of RT. Weekly blood counts and bimonthly lymphocyte subtypes were monitored. RESULTS: Baseline TLC ranged from 1060-2550 cells/mm3 (median 1570) and a median of 8.44 × 107 lymphocytes/Kg were collected (range 3.44-11.1x107 lymphocytes/Kg in the first 9 patients). In the five patients who have completed six weeks of concurrent RT/TMZ, the TLC fell 68% to a median of 460 cells/mm3 (range 390-1050). Four of these patients had lymphocyte reinfusion via a peripheral IV and one patient required a PICC line. A median of 6.99 × 107 lymphocytes/Kg (range 2.69-9.47 × 107 lymphocytes/Kg) were infused. No adverse events occurred with collection or reinfusion. Three weeks after reinfusion the TLC had increased to a median of 640 cells/mm3 (range 543-1486). CONCLUSIONS: Preliminary data from this study suggests that lymphocyte harvesting and reinfusion are feasible in this patient population which develops severe lymphopenia that is associated with early tumor progression and death. Additional data on serial lymphocyte subtypes and cytokine levels will be available once all patients have been reinfused and followed.
    Full-text · Article · Nov 2013 · Neuro-Oncology
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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.
    Full-text · Article · Sep 2013 · Nucleic Acids Research
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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.
    Full-text · Article · Aug 2013 · ACS Chemical Biology
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    Henry R Bourne · Mark O Lively

    Preview · Article · Jul 2012 · Science
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    ABSTRACT: Osteogenic differentiation of mesenchymal stem cells (MSC) is important in the field of bone tissue engineering. The identification of biological factors that influence osteogenesis is vital for developing a broader understanding of how complex microenvironments play a role in differentiation. The aim of this study was to demonstrate that adipose-derived stem cell (ADSC) osteogenesis is enhanced through interaction with extracellular matrices (ECM) secreted by ADSC undergoing osteogenesis. ADSC were obtained from human patients following elective abdominoplasty. Cells were selected for plastic adherence, characterized, and induced to differentiate using osteogenic supplements (OS; dexamethasone, ascorbic acid, and beta-glycerol phosphate). Cells were removed at several time points during osteogenesis and the secreted ECM was isolated. Undifferentiated cells were re-seeded onto the cell secreted ECMs and induced to differentiate with OS. At several time points, cells cultured on ECMs or tissue culture plastic controls (i.e. uncoated surface) were collected and RNA isolated. QPCR and gene array analysis revealed enrichment of osteogenic markers and more rapid progression through osteogenic maturational phases in cells seeded onto ECM secreted at the midpoint in differentiation (ca. 15 days). Our results demonstrate that the cumulative deposition of ECM reaches a critical point at approximately 15 days, before which there appear to be no definitive osteogenic cues from the matrix, and after which, strong drivers of osteogenesis are present. The creation of microenvironments that contain essential morphogenic matrix signals is an important step towards methods of growing and differentiating MSC in a rapid effective manner, particularly for bone-related clinical applications.
    No preview · Article · Jul 2012 · Journal of Biomimetics Biomaterials and Tissue Engineering
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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.
    Preview · Article · Jun 2012 · Molecular and Cellular Biology
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    ABSTRACT: Background: Coenzyme Q10 (CoQ10) 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 CoQ10 in female breast cancer patients with the primary objective of determining CoQ10'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 CoQ10 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 CoQ10 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 1, 8%; stage II, 1%), ethnicity (white, 87%; black, 11%; Hispanic, 2%), and planned therapy. Baseline CoQ10 levels in the CoQ10 and placebo arms were 0.70 and 0.73 microg/mL, respectively; the 24-week CoQ10 levels were 1.83 and 0.79 microg/mL, respectively. There were no significant differences between the CoQ10 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 CoQ10 led to sustained increases in plasma CoQ10 levels but did not result in improved self-reported fatigue or QOL after 24 weeks of treatment.
    No preview · Article · Jun 2012 · The journal of supportive oncology
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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.
    Full-text · Article · Apr 2012 · ACS Chemical Biology
  • Henry R. Bourne · Mark O. Lively

    No preview · Article · Jan 2012 · Science
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    Jeane M Govan · Mark O Lively · Alexander Deiters
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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.
    Full-text · Article · Aug 2011 · Journal of the American Chemical Society
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    ABSTRACT: We provide evidence that a factor other than the previously identified lipid mobilizing factor, zinc alpha-2 glycoprotein, promotes lipolysis in the MCA-induced sarcoma-bearing cachexia model. Cachexia is characterized by progressive loss of adipose tissue and skeletal muscle without a concurrent increase in food intake to restore lost tissue stores. We compared tumor-bearing ad lib fed (TB) animals to nontumor bearing ad lib fed (NTB) animals or nontumor-bearing pair-fed (PF) animals at various time points throughout development of tumor derived cachexia. Prior to cachexia, the TB animals lost more than 10 +/- 0.7% of their body fat before losing protein mass and decreasing their food intake. Fat loss occurred because adipocyte size, not number, was reduced. Increased turnover of palmitate and significantly higher serum triglyceride levels prior to cachexia were further indicators of an early loss of lipid from the adipocytes. Yet, circulating levels of norepinephrine, epinephrine, TNF-alpha, and zinc alpha-2 glycoprotein were not increased prior to the loss of fat mass. We provide evidence for a serum factor(s), other than zinc alpha-2 glycoprotein, that stimulates release of glycerol from 3T3-L1 adipocytes and promotes the loss of stored adipose lipid prior to the loss of lean body mass in this model.
    No preview · Article · Apr 2010 · Nutrition and Cancer
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    Douglas D Young · Mark O Lively · Alexander Deiters
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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.
    Full-text · Article · Apr 2010 · Journal of the American Chemical Society
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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.
    Full-text · Article · Jul 2009 · ChemBioChem
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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.
    Full-text · Article · May 2009 · Biochemistry
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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.
    Full-text · Article · Apr 2009 · Nucleic Acids Research

Publication Stats

1k Citations
312.55 Total Impact Points

Institutions

  • 2007-2015
    • Wake Forest School of Medicine
      • • Center for Structural Biology
      • • Department of Biochemistry
      • • Section on Hematology and Oncology
      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
  • 2009
    • North Carolina State University
      • Department of Chemistry
      Raleigh, NC, United States