@article{yao_walker_gamcsik_2023, title={Assessing MTT and sulforhodamine B cell proliferation assays under multiple oxygen environments}, ISSN={["1573-0778"]}, DOI={10.1007/s10616-023-00584-0}, journal={CYTOTECHNOLOGY}, author={Yao, Ming and Walker, Glenn and Gamcsik, Michael P.}, year={2023}, month={Jul} }
 @article{jeffries_gomez_macdonald_gamcsik_2022, title={Direct Detection of Glutathione Biosynthesis, Conjugation, Depletion and Recovery in Intact Hepatoma Cells}, volume={23}, ISSN={["1422-0067"]}, DOI={10.3390/ijms23094733}, abstractNote={Nuclear magnetic resonance (NMR) spectroscopy was used to monitor glutathione metabolism in alginate-encapsulated JM-1 hepatoma cells perfused with growth media containing [3,3′-13C2]-cystine. After 20 h of perfusion with labeled medium, the 13C NMR spectrum is dominated by the signal from the 13C-labeled glutathione. Once 13C-labeled, the high intensity of the glutathione resonance allows the acquisition of subsequent spectra in 1.2 min intervals. At this temporal resolution, the detailed kinetics of glutathione metabolism can be monitored as the thiol alkylating agent monobromobimane (mBBr) is added to the perfusate. The addition of a bolus dose of mBBr results in rapid diminution of the resonance for 13C-labeled glutathione due to a loss of this metabolite through alkylation by mBBr. As the glutathione resonance decreases, a new resonance due to the production of intracellular glutathione-bimane conjugate is detectable. After clearance of the mBBr dose from the cells, intracellular glutathione repletion is then observed by a restoration of the 13C-glutathione signal along with wash-out of the conjugate. These data demonstrate that standard NMR techniques can directly monitor intracellular processes such as glutathione depletion with a time resolution of approximately < 2 min.}, number={9}, journal={INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES}, author={Jeffries, Rex E. and Gomez, Shawn M. and Macdonald, Jeffrey M. and Gamcsik, Michael P.}, year={2022}, month={May} }
 @article{mellinger_muddiman_gamcsik_2022, title={Highlighting Functional Mass Spectrometry Imaging Methods in Bioanalysis}, volume={6}, ISSN={["1535-3907"]}, DOI={10.1021/acs.jproteome.2c00220}, abstractNote={Most mass spectrometry imaging (MSI) methods provide a molecular map of tissue content but little information on tissue function. Mapping tissue function is possible using several well-known examples of "functional imaging" such as positron emission tomography and functional magnetic resonance imaging that can provide the spatial distribution of time-dependent biological processes. These functional imaging methods represent the net output of molecular networks influenced by local tissue environments that are difficult to predict from molecular/cellular content alone. However, for decades, MSI methods have also been demonstrated to provide functional imaging data on a variety of biological processes. In fact, MSI exceeds some of the classic functional imaging methods, demonstrating the ability to provide functional data from the nanoscale (subcellular) to whole tissue or organ level. This Perspective highlights several examples of how different MSI ionization and detection technologies can provide unprecedented detailed spatial maps of time-dependent biological processes, namely, nucleic acid synthesis, lipid metabolism, bioenergetics, and protein metabolism. By classifying various MSI methods under the umbrella of "functional MSI", we hope to draw attention to both the unique capabilities and accessibility with the aim of expanding this underappreciated field to include new approaches and applications.}, journal={JOURNAL OF PROTEOME RESEARCH}, author={Mellinger, Allyson L. and Muddiman, David C. and Gamcsik, Michael P.}, year={2022}, month={Jun} }
 @article{mellinger_kibbe_rabbani_meritet_muddiman_gamcsik_2022, title={Mapping glycine uptake and its metabolic conversion to glutathione in mouse mammary tumors using functional mass spectrometry imaging}, volume={193}, ISSN={["1873-4596"]}, DOI={10.1016/j.freeradbiomed.2022.11.010}, abstractNote={Although glutathione plays a key role in cancer cell viability and therapy response there is no clear trend in relating the level of this antioxidant to clinical stage, histological grade, or therapy response in patient tumors. The likely reason is that static levels of glutathione are not a good indicator of how a tissue deals with oxidative stress. A better indicator is the functional capacity of the tissue to maintain glutathione levels in response to this stress. However, there are few methods to assess glutathione metabolic function in tissue. We have developed a novel functional mass spectrometry imaging (fMSI) method that can map the variations in the conversion of glycine to glutathione metabolic activity across tumor tissue sections by tracking the fate of three glycine isotopologues administered in a timed sequence to tumor-bearing anesthetized mice. This fMSI method generates multiple time point kinetic data for substrate uptake and glutathione production from each spatial location in the tissue. As expected, the fMSI data shows glutathione metabolic activity varies across the murine 4T1 mammary tumor. Although glutathione levels are highest at the tumor periphery there are regions of high content but low metabolic activity. The timed infusion method also detects variations in delivery of the glycine isotopologues thereby providing a measure of tissue perfusion, including evidence of intermittent perfusion, that contributes to the observed differences in metabolic activity. We believe this new approach will be an asset to linking molecular content to tissue function.}, journal={FREE RADICAL BIOLOGY AND MEDICINE}, author={Mellinger, Allyson L. and Kibbe, Russell R. and Rabbani, Zahid N. and Meritet, Danielle and Muddiman, David C. and Gamcsik, Michael P.}, year={2022}, month={Nov}, pages={677–684} }
 @article{mellinger_garrard_khodjaniyazova_rabbani_gamcsik_muddiman_2022, title={Multiple Infusion Start Time Mass Spectrometry Imaging of Dynamic SIL-Glutathione Biosynthesis Using Infrared Matrix-Assisted Laser Desorption Electrospray Ionization}, volume={21}, ISSN={["1535-3907"]}, DOI={10.1021/acs.jproteome.1c00636}, abstractNote={Due to the high association of glutathione metabolism perturbation with a variety of disease states, there is a dire need for analytical techniques to study glutathione kinetics. Additionally, the elucidation of microenvironmental effects on changes in glutathione metabolism would significantly improve our understanding of the role of glutathione in disease. We therefore present a study combining a multiple infusion start time protocol, stable isotope labeling technology, infrared matrix-assisted laser desorption electrospray ionization, and high-resolution accurate mass-mass spectrometry imaging to study spatial changes in glutathione kinetics across in sectioned mouse liver tissues. After injecting a mouse with the isotopologues [2-13C,15N]-glycine, [1,2-13C2]-glycine, and [1,2-13C2,15N]-glycine at three different time points, we were able to fully resolve and spatially map their metabolism into three isotopologues of glutathione and calculate their isotopic enrichment in glutathione. We created a tool in the open-source mass spectrometry imaging software MSiReader to accurately compute the percent isotope enrichment (PIE) of these labels in glutathione and visualize them in heat-maps of the tissue sections. In areas of high flux, we found that each label enriched an approximate median of 1.6%, 1.8%, and 1.5%, respectively, of the glutathione product pool measured in each voxel. This method may be adapted to study the heterogeneity of glutathione flux in diseased versus healthy tissues.}, number={3}, journal={JOURNAL OF PROTEOME RESEARCH}, author={Mellinger, Allyson L. and Garrard, Kenneth P. and Khodjaniyazova, Sitora and Rabbani, Zahid N. and Gamcsik, Michael P. and Muddiman, David C.}, year={2022}, month={Mar}, pages={747–757} }
 @article{yao_walker_gamcsik_2021, title={A multiwell plate-based system for toxicity screening under multiple static or cycling oxygen environments}, volume={11}, ISSN={["2045-2322"]}, DOI={10.1038/s41598-021-83579-1}, abstractNote={Abstract}, number={1}, journal={SCIENTIFIC REPORTS}, author={Yao, Ming and Walker, Glenn and Gamcsik, Michael P.}, year={2021}, month={Feb} }
 @article{yao_rabbani_sattler_nguyen_zaharoff_walker_gamcsik_2019, title={Flow-Encoded Oxygen Control to Track the Time-Dependence of Molecular Changes Induced by Static or Cycling Hypoxia}, volume={91}, ISSN={["1520-6882"]}, DOI={10.1021/acs.analchem.9b03709}, abstractNote={Detecting the effects of low oxygen on cell function is often dependent on monitoring the expression of a number of hypoxia markers. The time dependence of the appearance and stability of these markers varies between cell lines. Assessing cellular marker dynamics is also critical to determining how quickly cells respond to transient changes in oxygen levels that occurs with cycling hypoxia. We fabricated a manifold designed to use flow-encoding to produce sequential changes in gas mixtures delivered to a permeable-bottom 96-well plate. We show how this manifold and plate design can be used to expose cells to either static or cycling hypoxic conditions for eight different time periods thereby facilitating the study of the time-response of cells to altered oxygen environments. Using this device, we monitored the time-dependence of molecular changes in human PANC-1 pancreatic carcinoma and Caco-2 colon adenocarcinoma cells exposed to increasing periods of static or cycling hypoxia. Using immunohistochemistry, both cell lines show detectable levels of the marker protein hypoxia-inducible factor-1α (HIF-1α) after 3 h of exposure to static hypoxia. Cycling hypoxia increased the expression level of HIF-1α compared to static hypoxia. Both static and cycling hypoxia also increased glucose uptake and aldehyde dehydrogenase activity. This new device offers a facile screening approach to determine the kinetics of cellular alterations under varying oxygen conditions.}, number={23}, journal={ANALYTICAL CHEMISTRY}, author={Yao, Ming and Rabbani, Zahid N. and Sattler, Tyler and Nguyen, Khue G. and Zaharoff, David A. and Walker, Glenn and Gamcsik, Michael P.}, year={2019}, month={Dec}, pages={15032–15039} }
 @article{yao_sattler_rabbani_pulliam_walker_gamcsik_2018, title={Mixing and delivery of multiple controlled oxygen environments to a single multiwell culture plate}, volume={315}, ISSN={["1522-1563"]}, DOI={10.1152/ajpcell.00276.2018}, abstractNote={ Precise oxygen control is critical to evaluating cell growth, molecular content, and stress response in cultured cells. We have designed, fabricated, and characterized a 96-well plate-based device that is capable of delivering eight static or dynamically changing oxygen environments to different rows on a single plate. The device incorporates a gas-mixing tree that combines two input gases to generate the eight gas mixtures that supply each row of the plate with a different gas atmosphere via a removable manifold. Using air and nitrogen as feed gases, a single 96-well plate can culture cells in applied gas atmospheres with Po2 levels ranging from 1 to 135 mmHg. Human cancer cell lines MCF-7, PANC-1, and Caco-2 were grown on a single plate under this range of oxygen levels. Only cells grown in wells exposed to Po2 ≤37 mmHg express the endogenous hypoxia markers hypoxia-inducible factor-1α and carbonic anhydrase IX. This design is amenable to multiwell plate-based molecular assays or drug dose-response studies in static or cycling hypoxia conditions. }, number={5}, journal={AMERICAN JOURNAL OF PHYSIOLOGY-CELL PHYSIOLOGY}, author={Yao, Ming and Sattler, Tyler and Rabbani, Zahid N. and Pulliam, Thomas and Walker, Glenn and Gamcsik, Michael P.}, year={2018}, month={Nov}, pages={C766–C775} }
 @article{jiang_jeffries_acosta_tikunov_macdonald_walker_gamcsik_2015, title={Biocompatibility of Tygon® tubing in microfluidic cell culture}, volume={17}, ISSN={1387-2176 1572-8781}, url={http://dx.doi.org/10.1007/S10544-015-9938-9}, DOI={10.1007/S10544-015-9938-9}, abstractNote={Growth of the MDA-MB-231 breast cancer cell line in microfluidic channels was inhibited when culture media was delivered to the channels via microbore Tygon® tubing. Culture media incubated within this tubing also inhibited growth of these cells in conventional 96-well plates. These detrimental effects were not due to depletion of critical nutrients due to adsorption of media components onto the tubing surface. A pH change was also ruled out as a cause. Nuclear magnetic resonance spectroscopy of the cell growth media before and after incubation in the tubing confirmed no detectable loss of media components but did detect the presence of additional unidentified signals in the aliphatic region of the spectrum. These results indicate leaching of a chemical species from microbore Tygon® tubing that can affect cell growth in microfluidic devices.}, number={1}, journal={Biomedical Microdevices}, publisher={Springer Science and Business Media LLC}, author={Jiang, Xiao and Jeffries, Rex E. and Acosta, Miguel A. and Tikunov, Andrey P. and Macdonald, Jeffrey M. and Walker, Glenn M. and Gamcsik, Michael P.}, year={2015}, month={Feb} }
 @article{acosta_jiang_huang_cutler_grant_walker_gamcsik_2014, title={A microfluidic device to study cancer metastasis under chronic and intermittent hypoxia}, volume={8}, ISSN={1932-1058}, url={http://dx.doi.org/10.1063/1.4898788}, DOI={10.1063/1.4898788}, abstractNote={Metastatic cancer cells must traverse a microenvironment ranging from extremely hypoxic, within the tumor, to highly oxygenated, within the host's vasculature. Tumor hypoxia can be further characterized by regions of both chronic and intermittent hypoxia. We present the design and characterization of a microfluidic device that can simultaneously mimic the oxygenation conditions observed within the tumor and model the cell migration and intravasation processes. This device can generate spatial oxygen gradients of chronic hypoxia and produce dynamically changing hypoxic microenvironments in long-term culture of cancer cells.}, number={5}, journal={Biomicrofluidics}, publisher={AIP Publishing}, author={Acosta, Miguel A. and Jiang, Xiao and Huang, Pin-Kang and Cutler, Kyle B. and Grant, Christine S. and Walker, Glenn M. and Gamcsik, Michael P.}, year={2014}, month={Sep}, pages={054117} }
 @article{skamarauskas_oakley_smith_bawn_dunn_vidler_clemence_blain_taylor_gamcsik_et al._2014, title={Noninvasive In Vivo Magnetic Resonance Measures of Glutathione Synthesis in Human and Rat Liver as an Oxidative Stress Biomarker}, volume={59}, ISSN={["1527-3350"]}, DOI={10.1002/hep.26925}, abstractNote={Oxidative stress (OS) plays a central role in the progression of liver disease and in damage to liver by toxic xenobiotics. We have developed methods for noninvasive assessment of hepatic OS defenses by measuring flux through the glutathione (GSH) synthesis pathway. 13C‐labeled GSH is endogenously produced and detected by in vivo magnetic resonance after administration of [2‐13C]‐glycine. We report on a successful first‐ever human demonstration of this approach as well as preclinical studies demonstrating perturbed GSH metabolism in models of acute and chronic OS. Human studies employed oral administration of [2‐13C]‐glycine and 13C spectroscopy on a 3T clinical magnetic resonance (MR) imaging scanner and demonstrated detection and quantification of endogenously produced 13C‐GSH after labeled glycine ingestion. Plasma analysis demonstrated that glycine 13C fractional enrichment achieved steady state during the 6‐hour ingestion period. Mean rate of synthesis of hepatic 13C‐labeled GSH was 0.32 ± 0.18 mmole/kg/hour. Preclinical models of acute OS and nonalcoholic steatohepatitis (NASH) comprised CCl4‐treated and high‐fat, high‐carbohydrate diet‐fed Sprague‐Dawley rats, respectively, using intravenous administration of [2‐13C]‐glycine and observation of 13C‐label metabolism on a 7T preclinical MR system. Preclinical studies demonstrated a 54% elevation of GSH content and a 31% increase in flux through the GSH synthesis pathway at 12 hours after acute insult caused by CCl4 administration, as well as a 23% decrease in GSH content and evidence of early steatohepatitis in the model of NASH. Conclusion: Our data demonstrate in vivo 13C‐labeling and detection of GSH as a biomarker of tissue OS defenses, detecting chronic and acute OS insults. The methods are applicable to clinical research studies of hepatic OS in disease states over time as well as monitoring effects of therapeutic interventions. (Hepatology 2014;59:2321–2330)}, number={6}, journal={HEPATOLOGY}, author={Skamarauskas, John T. and Oakley, Fiona and Smith, Fiona E. and Bawn, Carlo and Dunn, Michael and Vidler, Daniel S. and Clemence, Matthew and Blain, Peter G. and Taylor, Roy and Gamcsik, Michael P. and et al.}, year={2014}, month={Jun}, pages={2321–2330} }
 @article{johnson_tikunov_lee_wolak_pediaditakis_romney_holmuhamedov_gamcsik_macdonald_2012, title={13C magnetic resonance spectroscopy detection of changes in serine isotopomers reflects changes in mitochondrial redox status}, volume={68}, ISSN={["0740-3194"]}, DOI={10.1002/mrm.23296}, abstractNote={Abstract}, number={3}, journal={MAGNETIC RESONANCE IN MEDICINE}, author={Johnson, C. Bryce and Tikunov, Andrey P. and Lee, Haakil and Wolak, Justyna E. and Pediaditakis, Peter and Romney, Doug A. and Holmuhamedov, Ekhson and Gamcsik, Michael P. and Macdonald, Jeffrey M.}, year={2012}, month={Sep}, pages={671–679} }
 @article{thelwall_simpson_rabbani_clark_pourdeyhimi_macdonald_blackband_gamcsik_2012, title={In vivo MR studies of glycine and glutathione metabolism in a rat mammary tumor}, volume={25}, ISSN={["0952-3480"]}, DOI={10.1002/nbm.1745}, abstractNote={The metabolism of glycine into glutathione was monitored noninvasively in vivo in intact rat mammary adenocarcinomas (R3230Ac) by MRI and MRS. Metabolism was tracked by following the isotope label from intravenously infused [2‐13C]‐glycine into the glycinyl residue of glutathione. Signals from [2‐13C]‐glycine and γ‐glutamylcysteinyl‐[2‐13C]‐glycine (13C‐glutathione) were detected by nonlocalized 13C spectroscopy, as these resonances are distinct from background signals. In addition, using spectroscopic imaging methods, heterogeneity in the in vivo tumor distribution of glutathione was observed. In vivo spectroscopy also detected isotope incorporation from [2‐13C]‐glycine into both the 2‐ and 3‐carbons of serine. Analyses of tumor tissue extracts showed single‐ and multiple‐label incorporation from [2‐13C]‐glycine into serine from metabolism through the serine hydroxymethyltransferase and glycine cleavage system pathways. Mass spectrometric analysis of extracts also showed that isotope‐labeled serine is further metabolized via the trans‐sulfuration pathway, as 13C isotope labels appear in both the glycinyl and cysteinyl residues of glutathione. Our studies demonstrate the use of MRI and MRS for the monitoring of tumor metabolic processes central to oxidative stress defense. Copyright © 2011 John Wiley & Sons, Ltd.}, number={2}, journal={NMR IN BIOMEDICINE}, author={Thelwall, Peter E. and Simpson, Nicholas E. and Rabbani, Zahid N. and Clark, M. Daniel and Pourdeyhimi, Roxana and Macdonald, Jeffrey M. and Blackband, Stephen J. and Gamcsik, Michael P.}, year={2012}, month={Feb}, pages={271–278} }
 @article{grahl_puttikamonkul_macdonald_gamcsik_ngo_hohl_cramer_2011, title={In vivo hypoxia and a fungal alcohol dehydrogenase influence the pathogenesis of invasive pulmonary aspergillosis}, volume={7}, number={7}, journal={PLoS Pathogens}, author={Grahl, N. and Puttikamonkul, S. and Macdonald, J. M. and Gamcsik, M. P. and Ngo, L. Y. and Hohl, T. M. and Cramer, R. A.}, year={2011} }
 @article{amoyaw_springer_gamcsik_mutesi_d'alessandro_dempsey_ludeman_2011, title={Synthesis of 13C-labeled derivatives of cysteine for magnetic resonance imaging studies of drug uptake and conversion to glutathione in rat brain}, volume={54}, ISSN={0362-4803}, url={http://dx.doi.org/10.1002/jlcr.1904}, DOI={10.1002/jlcr.1904}, abstractNote={Effects of neurodegeneration have been linked to inefficient detoxification of free radicals due to lowered concentrations of antioxidants, especially glutathione, in the brain. In the biosynthesis of glutathione, cysteine concentration is generally the limiting factor. Glutathione and cysteine administrations are not effective treatments for neurodegeneration because glutathione inefficiently crosses cell membranes and cysteine is neurotoxic at high concentrations. Prodrugs of glutathione and cysteine may have more favorable uptake and/or toxicity profiles. Three such prodrugs were synthesized with a 13C‐label such that in vivo uptake of each and conversion to glutathione in the brain could be monitored by magnetic resonance imaging. L‐[3‐13C]‐Cysteine was treated with sodium acetate trihydrate and acetic anhydride to give 2(R)‐N‐acetyl‐[3‐13C]‐cysteine ([13C]‐NAC; 96%). Addition of triphosgene to L‐[3‐13C]‐cysteine provided 4(R)‐[5‐13C]‐2‐oxothiazolidine‐4‐carboxylic acid ([13C]‐OTZ; 65%). A four‐step pathway was used to synthesize ethyl γ‐L‐glutamyl‐[3‐13C]‐L‐cysteinate ([13C]‐GCEE). L‐[3‐13C]‐Cysteine was esterified (100% yield) and then cyclized with acetaldehyde to give ethyl 2(R,S)‐methyl‐[5‐13C]‐thiazolidine‐4(R)‐carboxylate (73%) as a mixture of two diastereomers (65:35). The thiazolidine was silylated (bis(trimethylsilyl)trifluoroacetamide) and reacted with N‐phthaloyl‐L‐glutamic anhydride. Treatment with hydrazine afforded ethyl N‐[γ‐4′(S)‐glutamyl]‐2(R,S)‐methyl‐[5‐13C]‐thiazolidine‐4(R)‐carboxylate (48%; 73:27 mixture of diastereomers). This was converted to the desired product, [13C]‐GCEE (49%), using mercury (II) acetate and hydrogen sulfide.}, number={9}, journal={Journal of Labelled Compounds and Radiopharmaceuticals}, publisher={Wiley}, author={Amoyaw, Prince N. A. and Springer, James B. and Gamcsik, Michael P. and Mutesi, Rebecca L. and D'Alessandro, Michael A. and Dempsey, Collin R. and Ludeman, Susan M.}, year={2011}, month={Jul}, pages={607–612} }
 @article{gamcsik_daniel clark_ludeman_springer_d’alessandro_simpson_pourdeyhimi_bryce johnson_teeter_blackband_et al._2010, title={Non-invasive Monitoring of L-2-Oxothiazolidine-4-Carboxylate Metabolism in the Rat Brain by In vivo 13C Magnetic Resonance Spectroscopy}, volume={36}, ISSN={0364-3190 1573-6903}, url={http://dx.doi.org/10.1007/S11064-010-0362-5}, DOI={10.1007/S11064-010-0362-5}, abstractNote={The cysteine precursor L-2-oxothiazolidine-4-carboxylate (OTZ, procysteine) can raise cysteine concentration, and thus glutathione levels, in some tissues. OTZ has therefore been proposed as a prodrug for combating oxidative stress. We have synthesized stable isotope labeled OTZ (i.e. L-2-oxo-[5-13C]-thiazolidine-4-carboxylate, 13C-OTZ) and tracked its uptake and metabolism in vivo in rat brain by 13C magnetic resonance spectroscopy. Although uptake and clearance of 13C-OTZ was detectable in rat brain following a bolus dose by in vivo spectroscopy, no incorporation of isotope label into brain glutathione was detectable. Continuous infusion of 13C-OTZ over 20 h, however, resulted in 13C-label incorporation into glutathione, taurine, hypotaurine and lactate at levels sufficient for detection by in vivo magnetic resonance spectroscopy. Examination of brain tissue extracts by mass spectrometry confirmed only low levels of isotope incorporation into glutathione in rats treated with a bolus dose and much higher levels after 20 h of continuous infusion. In contrast to some previous studies, bolus administration of OTZ did not alter brain glutathione levels. Even a continuous infusion of OTZ over 20 h failed to raise brain glutathione levels. These studies demonstrate the utility of in vivo magnetic resonance for non-invasive monitoring of antioxidant uptake and metabolism in intact brain. These types of experiments can be used to evaluate the efficacy of various interventions for maintenance of brain glutathione.}, number={3}, journal={Neurochemical Research}, publisher={Springer Science and Business Media LLC}, author={Gamcsik, Michael P. and Daniel Clark, M. and Ludeman, Susan M. and Springer, James B. and D’Alessandro, Michael A. and Simpson, Nicholas E. and Pourdeyhimi, Roxana and Bryce Johnson, C. and Teeter, Stephanie D. and Blackband, Stephen J. and et al.}, year={2010}, month={Dec}, pages={443–451} }
 @article{da silva_bierbryer_wilds_noronha_colvin_miller_gamcsik_2005, title={Intrastrand base-stacking buttresses widening of major groove in interstrand cross-linked B-DNA}, volume={13}, ISSN={0968-0896}, url={http://dx.doi.org/10.1016/j.bmc.2005.03.032}, DOI={10.1016/j.bmc.2005.03.032}, abstractNote={The introduction of a covalent interstrand cross-link induces changes in the intrinsic structure and deformability of the DNA helix that are recognized by elements of the DNA repair apparatus. In this context, the solution structure of the undecamer d(CGAAAT*TTTCG)2, where T* represents a N3T-butyl-N3T interstrand cross-link, was determined using molecular dynamics calculations restrained by NOE and dihedral angle data obtained from NMR spectroscopy. The structure of this cross-linked undecamer shows dramatic widening of the major groove of the B-DNA stem without disruption of Watson-Crick base pairing. This change in tertiary structure illustrates the cumulative effect of cooperativity in intrastrand base stacking of an A-tract of three adenines. Further, it is the direct result from the imposition of geometric angular constraints by the cross-link chain on an ApT* and T*pT steps in the segment AAAT*T. The widening of the major groove is due to the dominant contribution of base stacking to the stability of the ApT compared to the TpT step suggesting that the latter is more deformable within a DNA stem. Compared to earlier structures of ethyl cross-linked oligonucleotides, this unique perturbation induced by the butyl moiety offers a new probe for systematic studies of DNA repair mechanisms.}, number={14}, journal={Bioorganic & Medicinal Chemistry}, publisher={Elsevier BV}, author={da Silva, Mateus Webba and Bierbryer, Ross G. and Wilds, Christopher J. and Noronha, Anne M. and Colvin, O. Michael and Miller, Paul S. and Gamcsik, Michael P.}, year={2005}, month={Jul}, pages={4580–4587} }
 @article{noll_webba da silva_noronha_wilds_colvin_gamcsik_miller_2005, title={Structure, Flexibility, and Repair of Two Different Orientations of the Same Alkyl Interstrand DNA Cross-Link†}, volume={44}, ISSN={0006-2960 1520-4995}, url={http://dx.doi.org/10.1021/bi050014n}, DOI={10.1021/bi050014n}, abstractNote={Interstrand DNA cross-links are the principal cytotoxic lesions produced by chemotherapeutic bifunctional alkylating agents. Using an N(4)C-ethyl-N(4)C interstrand DNA cross-link to mimic this class of clinically important cancer chemotherapeutic agents, we have characterized the repair, structure, and flexibility of DNA that contains this cross-link in two different orientations. Plasmid DNAs in which the cytosines of single CpG or GpC steps are covalently linked were efficiently processed by repair proficient and homologous recombination deficient strains of Escherichia coli. Repair in a nucleotide excision repair (NER) deficient strain was less efficient overall and displayed a 4-fold difference between the two cross-link orientations. Both the structure and flexibility of DNA containing these cross-links were examined using a combination of (1)H NMR, restrained molecular dynamics simulations, and atomic force microscopy (AFM). The NMR structure of a decamer containing a CpG interstrand cross-link shows the cross-link easily accommodated within the duplex with no disruption of hydrogen bonding and only minor perturbations of helical parameters. In contrast, disruptions caused by the GpC cross-link produced considerable conformational flexibility that precluded structure determination by NMR. AFM imaging of cross-link-containing plasmid DNA showed that the increased flexibility observed in the GpC cross-link persists when it is embedded into much larger DNA fragments. These differences may account for the different repair efficiencies seen in NER deficient cells.}, number={18}, journal={Biochemistry}, publisher={American Chemical Society (ACS)}, author={Noll, David M. and Webba da Silva, Mateus and Noronha, Anne M. and Wilds, Christopher J. and Colvin, O. Michael and Gamcsik, Michael P. and Miller, Paul S.}, year={2005}, month={May}, pages={6764–6775} }
 @article{webba da silva_wilds_noronha_colvin_miller_gamcsik_2004, title={Accommodation of Mispair Aligned N3T-Ethyl-N3T DNA Interstrand Cross Link†}, volume={43}, ISSN={0006-2960 1520-4995}, url={http://dx.doi.org/10.1021/bi0486435}, DOI={10.1021/bi0486435}, abstractNote={The solution structure of the undecamer d(CGAAATTTTCG)(2), where T represents a N(3)T-ethyl-N(3)T interstrand cross link, was elucidated using molecular dynamics calculations restrained by NOE and dihedral data obtained from NMR spectroscopy. The ethyl moiety is particularly well-accommodated between the minor and major grooves. This is an exceptional example of the plasticity along the axis defined by the stem and a unique finding of an interstrand cross link occupying the area associating minor and major grooves. The mismatch-aligned tethered bases preserve good intrastrand stacking with flanking bases. Base-pair steps adjacent to the lesion site are overwound. Accommodation of the lesion also results in an increase in mispair staggering alignment modulated by flexibility because of the tetrahedral geometry of the exocyclic ethyl carbon atoms. This is mechanically coupled with a small measure of concomitant propeller twisting without an increase in intrastrand base-step distance. Both x displacement and sugar puckering are indicative of canonical B DNA throughout the stem. We have thus established that the lesion defined by mismatch-aligned minor groove N(3)T-ethyl-N(3)T cross-linked thymine bases produces very localized distortions in a DNA stem that may be difficult to recognize by repair mechanisms that are not transcription- or replication-coupled. Thus, this synthetic DNA is a valuable structural probe to study mechanisms of repair.}, number={39}, journal={Biochemistry}, publisher={American Chemical Society (ACS)}, author={Webba da Silva, Mateus and Wilds, Christopher J. and Noronha, Anne M. and Colvin, O. Michael and Miller, Paul S. and Gamcsik, Michael P.}, year={2004}, month={Oct}, pages={12549–12554} }
 @article{fishel_gamcsik_delaney_zuhowski_maher_karrison_moschel_egorin_dolan_2004, title={Role of glutathione and nucleotide excision repair in modulation of cisplatin activity with O6-benzylguanine}, volume={55}, ISSN={0344-5704 1432-0843}, url={http://dx.doi.org/10.1007/S00280-004-0901-3}, DOI={10.1007/S00280-004-0901-3}, abstractNote={{"Label"=>"PURPOSE", "NlmCategory"=>"OBJECTIVE"} Modulation of platinating agent cytotoxicity has important clinical implications as a result of their widespread use in the treatment of many different cancers. O6-Benzylguanine (BG) enhances the cytotoxicity of cisplatin against several human tumor lines. The purpose of our work was to elucidate whether BG affects pathways prior to DNA damage (i.e., glutathione, GSH) or following DNA damage (i.e., nucleotide excision repair, NER). {"Label"=>"METHODS", "NlmCategory"=>"METHODS"} In efforts to determine the mechanism of enhancement we: (1) evaluated whether different sequences of BG plus cisplatin treatment differed in their ability to enhance cisplatin-induced cytotoxicity and DNA platination; (2) determined the effect of BG on GSH and glutathione S-transferase (GST) activity and; (3) determined whether BG enhanced cisplatin-induced cytotoxicity in cells lacking specific enzymes in the NER pathway. Colony-forming assay, atomic absorption spectroscopy and HPLC were employed to measure tumor cell growth inhibition, quantitate the amount of platinum on DNA, and determine intracellular GSH concentrations, respectively. {"Label"=>"RESULTS", "NlmCategory"=>"RESULTS"} Increased cytotoxicity and platination of DNA was observed when cells were exposed to BG prior to and/or during cisplatin treatment and not when BG followed cisplatin treatment. BG did not significantly alter GST activity with minimal depletion of GSH. In contrast, buthionine sulfoximine (BSO) caused a much more dramatic decrease in GSH than BG that was not accompanied by a dramatic increase in sensitivity to cisplatin. Furthermore, BG enhanced the cytotoxicity of cisplatin in a series of cell lines deficient in NER. {"Label"=>"CONCLUSIONS", "NlmCategory"=>"CONCLUSIONS"} Overall, our results suggest that the mechanism of enhancement involves neither the GSH nor the NER pathways, but triggers an event prior to DNA platination damage that ultimately results in increased cytotoxicity, apoptosis and increased platination levels.}, number={4}, journal={Cancer Chemotherapy and Pharmacology}, publisher={Springer Science and Business Media LLC}, author={Fishel, Melissa L. and Gamcsik, Michael P. and Delaney, Shannon M. and Zuhowski, Eleanor G. and Maher, Veronica M. and Karrison, Theodore and Moschel, Robert C. and Egorin, Merrill J. and Dolan, M. Eileen}, year={2004}, month={Oct}, pages={333–342} }
 @article{gamcsik_forder_millis_mcgovern_1996, title={A versatile oxygenator and perfusion system for magnetic resonance studies}, volume={49}, DOI={10.1002/bit.260490302}, abstractNote={A compact, reusable membrane oxygenator has been constructed for the perfusion of cultured cells and isolated organs. While the oxygenator was designed to be compatible with nuclear magnetic resonance (NMR) spectroscopy studies, it can also be used for any experiment which requires warming and oxygenation of perfusates. For the NMR studies, the oxygenator can be positioned at the opening of the magnet bore which allows oxygenation and warming of the perfusate immediately prior to delivery to the tissue, therefore eliminating problems with heat or oxygen loss which may occur with the long perfusion lines. (c) 1996 John Wiley & Sons, Inc.}, number={3}, journal={Biotechnology and Bioengineering}, publisher={Wiley}, author={Gamcsik, Michael P. and Forder, John R. and Millis, Kevin K. and McGovern, Kathy Ann}, year={1996}, pages={348–354} }