Ronald M Lynch

Interests

No activities entered.

Courses

Scholarly Contributions

Journals/Publications

• Pendleton, A. L., Antolic, A. T., Kelly, A. C., Davis, M. A., Camacho, L. E., Doubleday, K., Anderson, M. J., Langlais, P. R., Lynch, R. M., & Limesand, S. W. (2020). Lower oxygen consumption and Complex I activity in mitochondria isolated from skeletal muscle of fetal sheep with intrauterine growth restriction. American Journal of Physioly - Endocrinology & Metabolism.
• Frost, T. S., Jiang, L., Lynch, R. M., & Zohar, Y. (2019). Permeability of Epithelial/Endothelial Barriers in Transwells and Microfluidic Bilayer Devices. Micromachines, 10(8).
  More info

  Lung-on-a-chip (LoC) models hold the potential to rapidly change the landscape for pulmonary drug screening and therapy, giving patients more advanced and less invasive treatment options. Understanding the drug absorption in these microphysiological systems, modeling the lung-blood barrier is essential for increasing the role of the organ-on-a-chip technology in drug development. In this work, epithelial/endothelial barrier tissue interfaces were established in microfluidic bilayer devices and transwells, with porous membranes, for permeability characterization. The effect of shear stress on the molecular transport was assessed using known paracellular and transcellular biomarkers. The permeability of porous membranes without cells, in both models, is inversely proportional to the molecular size due to its diffusivity. Paracellular transport, between epithelial/endothelial cell junctions, of large molecules such as transferrin, as well as transcellular transport, through cell lacking required active transporters, of molecules such as dextrans, is negligible. When subjected to shear stress, paracellular transport of intermediate-size molecules such as dextran was enhanced in microfluidic devices when compared to transwells. Similarly, shear stress enhances paracellular transport of small molecules such as Lucifer yellow, but its effect on transcellular transport is not clear. The results highlight the important role that LoC can play in drug absorption studies to accelerate pulmonary drug development.
• Hart, N. J., Weber, C., Papas, K. K., Limesand, S. W., Vagner, J., & Lynch, R. M. (2019). Multivalent activation of GLP-1 and Sulfonylurea receptors modulates β-cell Second Messenger Signaling and Insulin Secretion. American journal of physiology. Cell physiology.
  More info

  Linking two pharmacophores that bind different cell surface receptors into a single molecule can enhance cell-targeting specificity to cells that express the complementary receptor pair. In this report, we developed and tested a synthetic multivalent ligand consisting of glucagon-like peptide-1 (GLP-1) linked to glibenclamide (Glb) for signaling efficacy in β-cells. Expression of receptors for these ligands, as a combination, are relatively specific to the β-cell in the pancreas. The multivalent GLP-1/Glb increased both intracellular cAMP and Ca, although Ca responses were significantly depressed compared to the monomeric Glb. Moreover, GLP-1/Glb increased glucose-stimulated insulin secretion in a dose-dependent manner. However, unlike the combined monomers, GLP-1/Glb did not augment insulin secretion at non-stimulatory glucose concentrations in INS 832/13 β-cells or human islets of Langerhans. These data suggest that linking two binding elements into a single bivalent ligand, such as GLP-1 and Glb, can provide a unique functional agent targeted to β-cells.
• Kelly, A. C., Smith, K. E., Purvis, W. G., Min, C. G., Weber, C. S., Cooksey, A. M., Hasilo, C., Paraskevas, S., Suszynski, T. M., Weegman, B. P., Anderson, M. J., Camacho, L. E., Harland, R. C., Loudovaris, T., Jandova, J., Molano, D. S., Price, N. D., Georgiev, I. G., Scott, W. E., , Manas, D. M., et al. (2019). Oxygen Perfusion (Persufflation) of Human Pancreata Enhances Insulin Secretion and Attenuates Islet Proinflammatory Signaling. Transplantation, 103(1), 160-167.
  More info

  All human islets used in research and for the clinical treatment of diabetes are subject to ischemic damage during pancreas procurement, preservation, and islet isolation. A major factor influencing islet function is exposure of pancreata to cold ischemia during unavoidable windows of preservation by static cold storage (SCS). Improved preservation methods may prevent this functional deterioration. In the present study, we investigated whether pancreas preservation by gaseous oxygen perfusion (persufflation) better preserved islet function versus SCS.
• Kelly, A. C., Camacho, L. E., Pendarvis, K., Davenport, H. M., Steffens, N. R., Smith, K. E., Weber, C. S., Lynch, R. M., Papas, K. K., & Limesand, S. W. (2018). Adrenergic receptor stimulation suppresses oxidative metabolism in isolated rat islets and Min6 cells. Molecular and cellular endocrinology.
  More info

  Insulin secretion is stimulated by glucose metabolism and inhibited by catecholamines through adrenergic receptor stimulation. We determined whether catecholamines suppress oxidative metabolism in β-cells through adrenergic receptors. In Min6 cells and isolated rat islets, epinephrine decreased oxygen consumption rates compared to vehicle control or co-administration of epinephrine with α2-adrenergic receptor antagonist yohimbine. Epinephrine also decreased forskolin-stimulated oxygen consumption rates, indicating cAMP dependent and independent actions. Furthermore, glucose oxidation rates were decreased with epinephrine, independent of the exocytosis of insulin, which was blocked with yohimbine. We evaluated metabolic targets through proteomic analysis after 4 h epinephrine exposure that revealed 466 differentially expressed proteins that were significantly enriched for processes including oxidative metabolism, protein turnover, exocytosis, and cell proliferation. These results demonstrate that acute α2-adrenergic stimulation suppresses glucose oxidation in β-cells independent of nutrient availability and insulin exocytosis, while cAMP concentrations are elevated. Proteomics and immunoblots revealed changes in electron transport chain proteins that were correlated with lower metabolic reducing equivalents, intracellular ATP concentrations, and altered mitochondrial membrane potential implicating a new role for adrenergic control of mitochondrial function and ultimately insulin secretion.
• Smith, K. E., Purvis, W. G., Davis, M. A., Min, C. G., Cooksey, A. M., Weber, C. S., Jandova, J., Price, N. D., Molano, D. S., Stanton, J. B., Kelly, A. C., Steyn, L. V., Lynch, R. M., Limesand, S. W., Alexander, M., Lakey, J. R., Seeberger, K., Korbutt, G. S., Mueller, K. R., , Hering, B. J., et al. (2018). In vitro characterization of neonatal, juvenile, and adult porcine islet oxygen demand, β-cell function, and transcriptomes. Xenotransplantation, 25(6), e12432.
  More info

  There is currently a shortage of human donor pancreata which limits the broad application of islet transplantation as a treatment for type 1 diabetes. Porcine islets have demonstrated potential as an alternative source, but a study evaluating islets from different donor ages under unified protocols has yet to be conducted.
• Chen, X., Kelly, A. C., Yates, D. T., Macko, A. R., Lynch, R. M., & Limesand, S. W. (2017). Islet adaptations in fetal sheep persist following chronic exposure to high norepinephrine. The Journal of endocrinology, 232(2), 285-295.
  More info

  Complications in pregnancy elevate fetal norepinephrine (NE) concentrations. Previous studies in NE-infused sheep fetuses revealed that sustained exposure to high NE resulted in lower expression of α2-adrenergic receptors in islets and increased insulin secretion responsiveness after acutely terminating the NE infusion. In this study, we determined if the compensatory increase in insulin secretion after chronic elevation of NE is independent of hyperglycemia in sheep fetuses and whether it is persistent in conjunction with islet desensitization to NE. After an initial assessment of glucose-stimulated insulin secretion (GSIS) at 129 ± 1 days of gestation, fetuses were continuously infused for seven days with NE and maintained at euglycemia with a maternal insulin infusion. Fetal GSIS studies were performed again on days 8 and 12. Adrenergic sensitivity was determined in pancreatic islets collected at day 12. NE infusion increased (P 
• McKay, B. S., Lynch, R. M., & Stamer, W. D. (2017). Comment on "Identification of Novel G Protein-Coupled Receptor 143 Ligands as Pharmacologic Tools for Investigating X-Linked Ocular Albinism". Investigative ophthalmology & visual science, 58(11), 4733-4734.
• Smith, K. E., Kelly, A. C., Min, C. G., Weber, C. S., McCarthy, F. M., Steyn, L. V., Badarinarayana, V., Stanton, J. B., Kitzmann, J. P., Strop, P., Gruessner, A. C., Lynch, R. M., Limesand, S. W., & Papas, K. K. (2017). Acute Ischemia Induced by High-Density Culture Increases Cytokine Expression and Diminishes the Function and Viability of Highly Purified Human Islets of Langerhans. Transplantation, 101(11), 2705-2712.
  More info

  Encapsulation devices have the potential to enable cell-based insulin replacement therapies (such as human islet or stem cell-derived β cell transplantation) without immunosuppression. However, reasonably sized encapsulation devices promote ischemia due to high β cell densities creating prohibitively large diffusional distances for nutrients. It is hypothesized that even acute ischemic exposure will compromise the therapeutic potential of cell-based insulin replacement. In this study, the acute effects of high-density ischemia were investigated in human islets to develop a detailed profile of early ischemia induced changes and targets for intervention.
• Dehigaspitiya, D. C., Anglin, B. L., Smith, K. R., Weber, C. S., Lynch, R. M., & Mash, E. A. (2015). Linear scaffolds for multivalent targeting of melanocortin receptors. Organic & biomolecular chemistry, 13(47), 11507-17.
  More info

  Molecules bearing one, two, three, or four copies of the tetrapeptide His-dPhe-Arg-Trp were attached to scaffolds based on ethylene glycol, glycerol, and d-mannitol by means of the copper-assisted azide-alkyne cyclization. The abilities of these compounds to block binding of a probe at the melanocortin 4 receptor were evaluated using a competitive binding assay. All of the multivalent molecules studied exhibited 30- to 40-fold higher apparent affinites when compared to a monovalent control. These results are consistent with divalent binding to receptor dimers. No evidence for tri- or tetravalent binding was obtained. Differences in the interligand spacing required for divalent binding, as opposed to tri- or tetravalent binding, may be responsible for these results.
• Dehigaspitiya, D. C., Navath, S., Weber, C. S., Lynch, R. M., & Mash, E. A. (2015). Synthesis and bioactivity of MSH4 oligomers prepared by an A2 + B2 strategy. Tetrahedron letters, 56(23), 3060-3065.
  More info

  Oligomers incorporating the tetrapeptide MSH4, the minimum active sequence of melanocyte stimulating hormone, were synthesized by an A2 + B2 strategy involving microwave-assisted copper-catalyzed azide-alkyne cycloaddition. A2 contained an MSH4 core while B2 contained a (Pro-Gly)3 spacer. Soluble mixtures containing compounds with up to eight MSH4 units were obtained from oligomerizations at high monomer concentrations. The avidities of several oligomeric mixtures were evaluated by means of a competitive binding assay using HEK293 cells engineered to overexpress the melanocortin 4 receptor. When based on total MSH4 concentrations, avidities were only minimally enhanced compared with a monovalent control. The lack of variation in the effect of ligands on probe binding is consistent with high off rates for MSH4 in both monovalent and oligomeric constructs relative to that of the competing probe.
• Elshan, N. G., Jayasundera, T., Anglin, B. L., Weber, C. S., Lynch, R. M., & Mash, E. A. (2015). Trigonal scaffolds for multivalent targeting of melanocortin receptors. Organic & biomolecular chemistry, 13(6), 1778-91.
  More info

  Melanocortin receptors can be used as biomarkers to detect and possibly treat melanoma. To these ends, molecules bearing one, two, or three copies of the weakly binding ligand MSH(4) were attached to scaffolds based on phloroglucinol, tripropargylamine, and 1,4,7-triazacyclononane by means of the copper-assisted azide-alkyne cyclization. This synthetic design allows rapid assembly of multivalent molecules. The bioactivities of these compounds were evaluated using a competitive binding assay that employed human embryonic kidney cells engineered to overexpress the melanocortin 4 receptor. The divalent molecules exhibited 10- to 30-fold higher levels of inhibition when compared to the corresponding monovalent molecules, consistent with divalent binding. The trivalent molecules were only statistically (∼2-fold) better than the divalent molecules, still consistent with divalent binding but inconsistent with trivalent binding. Possible reasons for these behaviors and planned refinements of the multivalent constructs targeting melanocortin receptors based on these scaffolds are discussed.
• Elshan, N. G., Jayasundera, T., Weber, C. S., Lynch, R. M., & Mash, E. A. (2015). Development of a time-resolved fluorescence probe for evaluation of competitive binding to the cholecystokinin 2 receptor. Bioorganic & medicinal chemistry, 23(8), 1841-8.
  More info

  The synthesis, characterization, and use of Eu-DTPA-PEGO-Trp-Nle-Asp-Phe-NH2 (Eu-DTPA-PEGO-CCK4), a luminescent probe targeted to cholecystokinin 2 receptor (CCK2R, aka CCKBR), are described. The probe was prepared by solid phase synthesis. A Kd value of 17±2nM was determined by means of saturation binding assays using HEK-293 cells that overexpress CCK2R. The probe was then used in competitive binding assays against Ac-CCK4 and three new trivalent CCK4 compounds. Repeatable and reproducible binding assay results were obtained. Given its ease of synthesis, purification, receptor binding properties, and utility in competitive binding assays, Eu-DTPA-PEGO-CCK4 could become a standard tool for high-throughput screening of compounds in development targeted to cholecystokinin receptors.
• Marmorstein, A. D., Kinnick, T. R., Stanton, J. B., Johnson, A. A., Lynch, R. M., & Marmorstein, L. Y. (2015). Bestrophin-1 influences transepithelial electrical properties and Ca2+ signaling in human retinal pigment epithelium. Molecular vision, 21, 347-59.
  More info

  Mutations in BEST1, encoding Bestrophin-1 (Best1), cause Best vitelliform macular dystrophy (BVMD) and other inherited retinal degenerative diseases. Best1 is an integral membrane protein localized to the basolateral plasma membrane of the retinal pigment epithelium (RPE). Data from numerous in vitro and in vivo models have demonstrated that Best1 regulates intracellular Ca2+ levels. Although it is known from in vitro and crystal structure data that Best1 is also a calcium-activated anion channel, evidence for Best1 functioning as a channel in human RPE is lacking. To assess Best1-associated channel activity in the RPE, we examined the transepithelial electrical properties of fetal human RPE (fhRPE) cells, which express endogenous Best1.
• Steyn, L. V., Ananthakrishnan, K., Anderson, M. J., Patek, R., Kelly, A., Vagner, J., Lynch, R. M., & Limesand, S. W. (2015). A Synthetic Heterobivalent Ligand Composed of Glucagon-Like Peptide 1 and Yohimbine Specifically Targets β Cells Within the Pancreas. Molecular imaging and biology : MIB : the official publication of the Academy of Molecular Imaging.
  More info

  β Cell specificity for a heterobivalent ligand composed of glucagon-like peptide-1 (GLP-1) linked to yohimbine (GLP-1/Yhb) was evaluated to determine its utility as a noninvasive imaging agent.
• Wojtkowiak, J. W., Cornnell, H. C., Matsumoto, S., Saito, K., Takakusagi, Y., Dutta, P., Kim, M., Zhang, X., Leos, R., Bailey, K. M., Martinez, G., Lloyd, M. C., Weber, C., Mitchell, J. B., Lynch, R. M., Baker, A. F., Gatenby, R. A., Rejniak, K. A., Hart, C., , Krishna, M. C., et al. (2015). Pyruvate sensitizes pancreatic tumors to hypoxia-activated prodrug TH-302. Cancer & metabolism, 3(1), 2.
  More info

  Hypoxic niches in solid tumors harbor therapy-resistant cells. Hypoxia-activated prodrugs (HAPs) have been designed to overcome this resistance and, to date, have begun to show clinical efficacy. However, clinical HAPs activity could be improved. In this study, we sought to identify non-pharmacological methods to acutely exacerbate tumor hypoxia to increase TH-302 activity in pancreatic ductal adenocarcinoma (PDAC) tumor models.
• Hart, N. J., Chung, W. J., Weber, C., Ananthakrishnan, K., Anderson, M., Patek, R., Zhang, Z., Limesand, S. W., Vagner, J., & Lynch, R. M. (2014). Hetero-bivalent GLP-1/glibenclamide for targeting pancreatic β-cells. Chembiochem : a European journal of chemical biology, 15(1), 135-45.
  More info

  G protein-coupled receptor (GPCR) cell signalling cascades are initiated upon binding of a specific agonist ligand to its cell surface receptor. Linking multiple heterologous ligands that simultaneously bind and potentially link different receptors on the cell surface is a unique approach to modulate cell responses. Moreover, if the target receptors are selected based on analysis of cell-specific expression of a receptor combination, then the linked binding elements might provide enhanced specificity of targeting the cell type of interest, that is, only to cells that express the complementary receptors. Two receptors whose expression is relatively specific (in combination) to insulin-secreting pancreatic β-cells are the sulfonylurea-1 (SUR1) and the glucagon-like peptide-1 (GLP-1) receptors. A heterobivalent ligand was assembled from the active fragment of GLP-1 (7-36 GLP-1) and glibenclamide, a small organic ligand for SUR1. The synthetic construct was labelled with Cy5 or europium chelated in DTPA to evaluate binding to β-cells, by using fluorescence microscopy or time-resolved saturation and competition binding assays, respectively. Once the ligand binds to β-cells, it is rapidly capped and presumably removed from the cell surface by endocytosis. The bivalent ligand had an affinity approximately fivefold higher than monomeric europium-labelled GLP-1, likely a result of cooperative binding to the complementary receptors on the βTC3 cells. The high-affinity binding was lost in the presence of either unlabelled monomer, thus demonstrating that interaction with both receptors is required for the enhanced binding at low concentrations. Importantly, bivalent enhancement was accomplished in a cell system with physiological levels of expression of the complementary receptors, thus indicating that this approach might be applicable for β-cell targeting in vivo.
• Kelly, A. C., Steyn, L. V., Kitzmann, J. P., Anderson, M. J., Mueller, K. R., Hart, N. J., Lynch, R. M., Papas, K. K., & Limesand, S. W. (2014). Function and expression of sulfonylurea, adrenergic, and glucagon-like peptide 1 receptors in isolated porcine islets. Xenotransplantation, 21(4), 385-91.
  More info

  The scarcity of human cadaveric pancreata limits large-scale application of islet transplantation for patients with diabetes. Islets isolated from pathogen-free pigs provide an economical and abundant alternative source assuming immunologic barriers are appropriate. Membrane receptors involved in insulin secretion that also have potential as imaging targets were investigated in isolated porcine islets. Quantitative (q)PCR revealed that porcine islets express mRNA transcripts for sulfonylurea receptor 1 (Sur1), inward rectifying potassium channel (Kir6.2, associated with Sur1), glucagon-like peptide 1 receptor (GLP1R), and adrenergic receptor alpha 2A (ADRα2A). Receptor function was assessed in static incubations with stimulatory glucose concentrations, and in the presence of receptor agonists. Glibenclamide, an anti-diabetic sulfonylurea, and exendin-4, a GLP-1 mimetic, potentiated glucose-stimulated insulin secretion >2-fold. Conversely, epinephrine maximally reduced insulin secretion 72 ± 9% (P < 0.05) and had a half maximal inhibitory concentration of 60 nm in porcine islets (95% confidence interval of 45-830 nm). The epinephrine action was inhibited by the ADRα2A antagonist yohimbine. Our findings demonstrate that porcine islets express and are responsive to both stimulatory and inhibitory membrane localized receptors, which can be used as imaging targets after transplantation or to modify insulin secretion.
• Lynch, R. M. (2014). Glucagon-like peptide-1 modulation of calcium homeostasis in human coronary microvascular endothelial cells after ischemia and reperfusion.. J. Endo., Diabetes and Obesity, 2(2), 1095-1102.
• Lynch, R. M. (2014). Synthesis and bioactivity of MSH4 oligomers prepared by an A2 + B2 strategy. Tetrahedron Letters.
  More info

  Dehigaspitiya D, Navath S, Weber CS, Lynch RM, Mash EA.
• Alleti, R., Vagner, J., Dehigaspitiya, D. C., Moberg, V. E., Elshan, N. G., Tafreshi, N. K., Brabez, N., Weber, C. S., Lynch, R. M., Hruby, V. J., Gillies, R. J., Morse, D. L., & Mash, E. A. (2013). Synthesis and characterization of time-resolved fluorescence probes for evaluation of competitive binding to melanocortin receptors. Bioorganic & medicinal chemistry, 21(17), 5029-38.
  More info

  Probes for use in time-resolved fluorescence competitive binding assays at melanocortin receptors based on the parental ligands MSH(4), MSH(7), and NDP-α-MSH were prepared by solid phase synthesis methods, purified, and characterized. The saturation binding of these probes was studied using HEK-293 cells engineered to overexpress the human melanocortin 4 receptor (hMC4R) as well as the human cholecystokinin 2 receptor (hCCK2R). The ratios of non-specific binding to total binding approached unity at high concentrations for each probe. At low probe concentrations, receptor-mediated binding and uptake was discernable, and so probe concentrations were kept as low as possible in determining Kd values. The Eu-DTPA-PEGO-MSH(4) probe exhibited low specific binding relative to non-specific binding, even at low nanomolar concentrations, and was deemed unsuitable for use in competition binding assays. The Eu-DTPA-PEGO probes based on MSH(7) and NDP-α-MSH exhibited Kd values of 27±3.9nM and 4.2±0.48nM, respectively, for binding with hMC4R. These probes were employed in competitive binding assays to characterize the interactions of hMC4R with monovalent and divalent MSH(4), MSH(7), and NDP-α-MSH constructs derived from squalene. Results from assays with both probes reflected only statistical enhancements, suggesting improper ligand spacing on the squalene scaffold for the divalent constructs. The Ki values from competitive binding assays that employed the MSH(7)-based probe were generally lower than the Ki values obtained when the probe based on NDP-α-MSH was employed, which is consistent with the greater potency of the latter probe. The probe based on MSH(7) was also competed with monovalent, divalent, and trivalent MSH(4) constructs that previously demonstrated multivalent binding in competitive binding assays against a variant of the probe based on NDP-α-MSH. Results from these assays confirm multivalent binding, but suggest a more modest increase in avidity for these MSH(4) constructs than was previously reported.
• Brabez, N., Nguyen, K. L., Saunders, K., Lacy, R., Xu, L., Gillies, R. J., Lynch, R. M., Chassaing, G., Lavielle, S., & Hruby, V. J. (2013). Synthesis and evaluation of cholecystokinin trimers: a multivalent approach to pancreatic cancer detection and treatment. Bioorganic & medicinal chemistry letters, 23(8), 2422-5.
  More info

  In the quest for novel tools for early detection and treatment of cancer, we propose the use of multimers targeting overexpressed receptors at the cancer cell surface. Indeed, multimers are prone to create multivalent interactions, more potent and specific than their corresponding monovalent versions, thus enabling the potential for early detection. There is a lack of tools for early detection of pancreatic cancer, one of the deadliest forms of cancer, but CCK2-R overexpression on pancreatic cancer cells makes CCK based multimers potential markers for these cells. In this Letter, we describe the synthesis and evaluation of CCK trimers targeting overexpressed CCK2-R.
• Brabez, N., Saunders, K., Nguyen, K. L., Jayasundera, T. B., Weber, C., Lynch, R. M., Chassaing, G., Lavielle, S., & Hruby, V. J. (2013). Multivalent Interactions: Synthesis and Evaluation of Melanotropin Multimers - Tools for Melanoma Targeting. ACS medicinal chemistry letters, 4(1), 98-102.
  More info

  In order to develop agents for early detection and selective treatment of melanomas, high affinity and high specificity molecular tools are required. Enhanced specificity may be obtained by simultaneously binding to multiple cell surface targets via the use of multimeric analogs of naturally occurring ligands. Trimers targeting overexpressed melanocortin receptors have been found to be potential candidates for this purpose. In the present letter, we describe the synthesis and study of multimers based on a dendrimer-like scaffold. The binding affinity and activity results revealed that dendrimers promote multivalent interactions via statistical and/or cooperative effects on binding. Moreover, viability studies showed no significant toxicity at micromolar concentrations, which will allow these molecular complexes to be used in vivo. Finally, imaging studies showed effective internalization for all the molecules confirming their potential as delivery agents.
• Escalante, A. M., McGrath, R. T., Karolak, M. R., Dorr, R. T., Lynch, R. M., & Landowski, T. H. (2013). Preventing the autophagic survival response by inhibition of calpain enhances the cytotoxic activity of bortezomib in vitro and in vivo. Cancer chemotherapy and pharmacology, 71(6), 1567-76.
  More info

  Bortezomib, a first-generation proteasome inhibitor, induces an endoplasmic reticulum (ER) stress response, which ultimately leads to dysregulation of intracellular Ca(2+) and apoptotic cell death. This study investigated the role of the Ca(2+)-dependent enzyme, calpain, in bortezomib cytotoxicity. A novel therapeutic combination was evaluated in which HIV protease inhibitors were used to block calpain activity and enhance bortezomib cytotoxicity in myeloma cells in vitro and in vivo.
• Martinez-Zaguilan, R., Tompkins, L. S., Gillies, R. J., & Lynch, R. M. (2013). Simultaneous analysis of intracellular pH and Ca²⁺ from cell populations. Methods in molecular biology (Clifton, N.J.), 937, 253-71.
  More info

  Although changes in both pH(in) and [Ca(2+)](i) have been observed in response to a variety of agonists, it is not clear whether these ionic events work independently or are coordinated to lead to a specific physiological response. One of the fundamental problems in studying these ionic events is that changes in pH(in) modify Ca(2+) regulatory mechanisms and changes in Ca(2+) may modify pH regulation. It is desirable to use a technique that allows concomitant monitoring of these two ions in cell populations with high time resolution. Furthermore, like many Ca(2+) binding proteins, all Ca(2+)-sensitive fluoroprobes are inherently sensitive to pH owing to competition of H(+) for the Ca(2+)-binding sites. This chapter describes experimental paradigms that provide optimum conditions for simultaneous measurement of pH from the fluorescence emission of snarf-1, and Ca(2+) using fura-2. The fluorescence spectra of these compounds are sufficiently different to allow simultaneous measurement of pH and Ca(2+) both in vitro and in vivo. Moreover, the ratio of the H(+)-sensitive wavelengths of snarf-1 is unaffected by Ca(2+), or the concomitant presence of fura-2 in cells. Although the fluorescence ratio of fura-2 is insensitive to the presence of snarf-1, it is affected by pH, as indicated above. We describe procedures to correct for this effect and to obtain calibration parameters for fura-2 and snarf-1 required to facilitate analysis of pH and Ca(2+) concentrations within cell populations.
• Sarkar, D. D., Edwards, S. K., Mauser, J. A., Suarez, A. M., Serowoky, M. A., Hudok, N. L., Hudok, P. L., Nuñez, M., Weber, C. S., Lynch, R. M., Miyashita, O., & Tsao, T. (2013). Increased redox-sensitive green fluorescent protein reduction potential in the endoplasmic reticulum following glutathione-mediated dimerization. Biochemistry, 52(19), 3332-45.
  More info

  As the endoplasmic reticulum (ER) is the compartment where disulfide bridges in secreted and cell surface proteins are formed, the disturbance of its redox state has profound consequences, yet regulation of ER redox potential remains poorly understood. To monitor the ER redox state in live cells, several fluorescence-based sensors have been developed. However, these sensors have yielded results that are inconsistent with each other and with earlier non-fluorescence-based studies. One particular green fluorescent protein (GFP)-based redox sensor, roGFP1-iL, could detect oxidizing changes in the ER despite having a reduction potential significantly lower than that previously reported for the ER. We have confirmed these observations and determined the mechanisms by which roGFP1-iL detects oxidizing changes. First, glutathione mediates the formation of disulfide-bonded roGFP1-iL dimers with an intermediate excitation fluorescence spectrum resembling a mixture of oxidized and reduced monomers. Second, glutathione facilitates dimerization of roGFP1-iL, which shifted the equilibrium from oxidized monomers to dimers, thereby increasing the molecule's reduction potential compared with that of a dithiol redox buffer. We conclude that the glutathione redox couple in the ER significantly increased the reduction potential of roGFP1-iL in vivo by facilitating its dimerization while preserving its ratiometric nature, which makes it suitable for monitoring oxidizing and reducing changes in the ER with a high degree of reliability in real time. The ability of roGFP1-iL to detect both oxidizing and reducing changes in ER and its dynamic response in glutathione redox buffer between approximately -190 and -130 mV in vitro suggests a range of ER redox potentials consistent with those determined by earlier approaches that did not involve fluorescent sensors.
• Green, A. S., Chen, X., Macko, A. R., Anderson, M. J., Kelly, A. C., Hart, N. J., Lynch, R. M., & Limesand, S. W. (2012). Chronic pulsatile hyperglycemia reduces insulin secretion and increases accumulation of reactive oxygen species in fetal sheep islets. The Journal of endocrinology, 212(3), 327-42.
  More info

  Children from diabetic pregnancies have a greater incidence of type 2 diabetes. Our objective was to determine if exposure to mild-moderate hyperglycemia, by modeling managed diabetic pregnancies, affects fetal β-cell function. In sheep fetuses, β-cell responsiveness was examined after 2 weeks of sustained hyperglycemia with 3 pulses/day, mimicking postprandial excursions, and compared to saline-infused controls (n = 10). Two pulsatile hyperglycemia (PHG) treatments were studied: mild (mPHG, n = 5) with +15% sustained and +55% pulse; and moderate (PHG, n = 10) with +20% sustained and +100% pulse. Fetal glucose-stimulated insulin secretion and glucose-potentiated arginine insulin secretion were lower (P < 0.05) in PHG (0.86 ± 0.13 and 2.91 ± 0.39  ng/ml plasma insulin) but not in mPHG fetuses (1.21 ± 0.08 and 4.25 ± 0.56  ng/ml) compared to controls (1.58 ± 0.25 and 4.51 ± 0.56  ng/ml). Islet insulin content was 35% lower in PHG and 35% higher in mPHG vs controls (P < 0.01). Insulin secretion and maximally stimulated insulin release were also reduced (P < 0.05) in PHG islets due to lower islet insulin content. Isolated PHG islets also had 63% greater (P < 0.01) reactive oxygen species (ROS) accumulation at 11.1  mmol/l glucose than controls (P < 0.01), but oxidative damage was not detected in islet proteins. PHG fetuses showed evidence of oxidative damage to skeletal muscle proteins (P < 0.05) but not insulin resistance. Our findings show that PHG induced dysregulation of islet ROS handling and decreased islet insulin content, but these outcomes are independent. The β-cell outcomes were dependent on the severity of hyperglycemia because mPHG fetuses had no distinguishable impairments in ROS handling or insulin secretion but greater insulin content.
• Konhilas, J. P., Behunin, S. M., & Lynch, R. M. (2012). Keeping the beat. Focus on "enrichment of neonatal rat cardiomyocytes in primary culture facilitates long-term maintenance of contractility in vitro". American journal of physiology. Cell physiology, 303(12), C1218-9.
• Xu, L., Josan, J. S., Vagner, J., Caplan, M. R., Hruby, V. J., Mash, E. A., Lynch, R. M., Morse, D. L., & Gillies, R. J. (2012). Heterobivalent ligands target cell-surface receptor combinations in vivo. Proceedings of the National Academy of Sciences of the United States of America, 109(52), 21295-300.
  More info

  A challenge in tumor targeting is to deliver payloads to cancers while sparing normal tissues. A limited number of antibodies appear to meet this challenge as therapeutics themselves or as drug-antibody conjugates. However, antibodies suffer from their large size, which can lead to unfavorable pharmacokinetics for some therapeutic payloads, and that they are targeted against only a single epitope, which can reduce their selectivity and specificity. Here, we propose an alternative targeting approach based on patterns of cell surface proteins to rationally develop small, synthetic heteromultivalent ligands (htMVLs) that target multiple receptors simultaneously. To gain insight into the multivalent ligand strategy in vivo, we have generated synthetic htMVLs that contain melanocortin (MSH) and cholecystokinin (CCK) pharmacophores that are connected via a fluorescent labeled, rationally designed synthetic linker. These ligands were tested in an experimental animal model containing tumors that expressed only one (control) or both (target) MSH and CCK receptors. After systemic injection of the htMVL in tumor-bearing mice, label was highly retained in tumors that expressed both, compared with one, target receptors. Selectivity was quantified by using ex vivo measurement of Europium-labeled htMVL, which had up to 12-fold higher specificity for dual compared with single receptor expressing cells. This proof-of-principle study provides in vivo evidence that small, rationally designed bivalent htMVLs can be used to selectively target cells that express both, compared with single complimentary cell surface targets. These data open the possibility that specific combinations of targets on tumors can be identified and selectively targeted using htMVLs.
• Brabez, N., Lynch, R. M., Xu, L., Gillies, R. J., Chassaing, G., Lavielle, S., & Hruby, V. J. (2011). Design, synthesis, and biological studies of efficient multivalent melanotropin ligands: tools toward melanoma diagnosis and treatment. Journal of medicinal chemistry, 54(20), 7375-84.
  More info

  To achieve early detection and specific cancer treatment, we propose the use of multivalent interactions in which a series of binding events leads to increased affinity and consequently to selectivity. Using melanotropin (MSH) ligands, our aim is to target melanoma cells which overexpress melanocortin receptors. In this study, we report the design and efficient synthesis of new trivalent ligands bearing MSH ligands. Evaluation of these multimers on a cell model engineered to overexpress melanocortin 4 receptors (MC4R) showed up to a 350-fold increase in binding compared to the monomer, resulting in a trivalent construct with nanomolar affinity starting from a micromolar affinity ligand. Cyclic adenosine monophosphate (cAMP) production was also investigated, leading to more insights into the effects of multivalent compounds on transduction mechanisms.
• Josan, J. S., De Silva, C. R., Yoo, B., Lynch, R. M., Pagel, M. D., Vagner, J., & Hruby, V. J. (2011). Fluorescent and lanthanide labeling for ligand screens, assays, and imaging. Methods in molecular biology (Clifton, N.J.), 716, 89-126.
  More info

  The use of fluorescent (or luminescent) and metal contrast agents in high-throughput screens, in vitro assays, and molecular imaging procedures has rapidly expanded in recent years. Here we describe the development and utility of high-affinity ligands for cancer theranostics and other in vitro screening -studies. In this context, we also illustrate the syntheses and use of heteromultivalent ligands as targeted imaging agents.
• Josan, J. S., Handl, H. L., Sankaranarayanan, R., Xu, L., Lynch, R. M., Vagner, J., Mash, E. A., Hruby, V. J., & Gillies, R. J. (2011). Cell-specific targeting by heterobivalent ligands. Bioconjugate chemistry, 22(7), 1270-8.
  More info

  Current cancer therapies exploit either differential metabolism or targeting to specific individual gene products that are overexpressed in aberrant cells. The work described herein proposes an alternative approach--to specifically target combinations of cell-surface receptors using heteromultivalent ligands ("receptor combination approach"). As a proof-of-concept that functionally unrelated receptors can be noncovalently cross-linked with high avidity and specificity, a series of heterobivalent ligands (htBVLs) were constructed from analogues of the melanocortin peptide ligand ([Nle(4), dPhe(7)]-α-MSH) and the cholecystokinin peptide ligand (CCK-8). Binding of these ligands to cells expressing the human Melanocortin-4 receptor and the Cholecystokinin-2 receptor was analyzed. The MSH(7) and CCK(6) were tethered with linkers of varying rigidity and length, constructed from natural and/or synthetic building blocks. Modeling data suggest that a linker length of 20-50 Å is needed to simultaneously bind these two different G-protein coupled receptors (GPCRs). These ligands exhibited up to 24-fold enhancement in binding affinity to cells that expressed both (bivalent binding), compared to cells with only one (monovalent binding) of the cognate receptors. The htBVLs had up to 50-fold higher affinity than that of a monomeric CCK ligand, i.e., Ac-CCK(6)-NH(2). Cell-surface targeting of these two cell types with labeled heteromultivalent ligand demonstrated high avidity and specificity, thereby validating the receptor combination approach. This ability to noncovalently cross-link heterologous receptors and target individual cells using a receptor combination approach opens up new possibilities for specific cell targeting in vivo for therapy or imaging.
• Josan, J. S., R, C., Yoo, B., Lynch, R. M., Pagel, M. D., Vagner, J., Hruby, V. J., Josan, J. S., R, C., Yoo, B., Lynch, R. M., Pagel, M. D., Vagner, J., & Hruby, V. J. (2011). Fluorescent and lanthanide labeling for ligand screens, assays, and imaging.. Methods in molecular biology (Clifton, N.J.), 716, 89-126.
  More info

  PMID: 21318902;PMCID: PMC3365840;Abstract: The use of fluorescent (or luminescent) and metal contrast agents in high-throughput screens, in vitro assays, and molecular imaging procedures has rapidly expanded in recent years. Here we describe the development and utility of high-affinity ligands for cancer theranostics and other in vitro screening -studies. In this context, we also illustrate the syntheses and use of heteromultivalent ligands as targeted imaging agents.
• Carmines, P., & Lynch, R. M. (2010). Changes in APS sectional programming for EB2011. Expanded programming and meeting-within-a-meeting structure. The Physiologist, 53(5), 137, 140.
• Pritchard, T. J., Bowman, P. S., Jefferson, A., Tosun, M., Lynch, R. M., & Paul, R. J. (2010). Na(+)-K(+)-ATPase and Ca(2+) clearance proteins in smooth muscle: a functional unit. American journal of physiology. Heart and circulatory physiology, 299(2), H548-56.
  More info

  The Na(+)-K(+)-ATPase (NKA) can affect intracellular Ca(2+) concentration regulation via coupling to the Na(+)-Ca(2+) exchanger and may be important in myogenic tone. We previously reported that in mice carrying a transgene for the NKA alpha(2)-isoform in smooth muscle (alpha(2sm+)), the alpha(2)-isoform protein as well as the alpha(1)-isoform (not contained in the transgene) increased to similar degrees (2-7-fold). Aortas from alpha(2sm+) mice relaxed faster from a KCl-induced contraction, hypothesized to be related to more rapid Ca(2+) clearance. To elucidate the mechanisms underlying this faster relaxation, we therefore measured the expression and distribution of proteins involved in Ca(2+) clearance. Na(+)-Ca(2+) exchanger, sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA), and plasma membrane Ca(2+)-ATPase (PMCA) proteins were all elevated up to approximately fivefold, whereas actin, myosin light chain, and calponin proteins were not changed in smooth muscle from alpha(2sm+) mice. Interestingly, the corresponding Ca(2+) clearance mRNA levels were unchanged. Immunocytochemical data indicate that the Ca(2+) clearance proteins are distributed similarly in wild-type and alpha(2sm+) aorta cells. In studies measuring relaxation half-times from a KCl-induced contraction in the presence of pharmacological inhibitors of SERCA and PMCA, we estimated that together these proteins were responsible for approximately 60-70% of relaxation in aorta. Moreover, the percent contribution of SERCA and PMCA to relaxation rates in alpha(2sm+) aorta was not significantly different from that in wild-type aorta. The coordinate expressions of NKA and Ca(2+) clearance proteins without change in the relative contributions of each individual protein to smooth muscle function suggest that NKA may be but one component of a larger functional Ca(2+) clearance system.

Presentations

• Lynch, R. M. (2016, August). Molecular Theranostics: Targeting Receptor Combinations with Multivalent Agents. PhysSoc Mtg.. Dublin, IRE: The Physiological Society.
• Lynch, R. M. (2016, March). Multivalent Agents for Targeted Therapy and Diagnostics of Metabolic Diseases. Physiology Seminar. North Chicago, IL: Rosiland Franklin University Collge of Medcine.
• Lynch, R. M. (2015, Apr 2., 2015). Accessing the molecular barcode: multivalent ligands for beta-cell specific targeting and therapeutics. American Physiological Society Symposium: Rejuvenating the beta-cell. Boston, MA: Experimental Biology.
• Lynch, R. M. (2015, Sept 13, 2015). Title: Multivalent approaches for beta-cell specific targeting. Symposium: Beta cell Imaging. Stockholm Sweden: BetaTrain, European Diabetes Association.
• Lynch, R. M. (2014, July). Invited Speaker. Seminar: St. Vincent's Research Institute. Melbourne, AUS: St. Vincent's Research Institute;.
  More info

  Invited Speaker, Tom Loudovaris, Targeting Diabetes: Enhancing Therapeutic Efficacy with Multivalancy
• Lynch, R. M. (2014, June). Invited Speaker. Seminar: IMB Division of Chemistry and Human Therapeutics. Brisbane, AUS: Univ. Queensland,.
  More info

  Invited Speaker, Univ. Queensland, IMB Division of Chemistry and Human Therapeutics, David Fairlie
• Lynch, R. M. (2014, June). Invited Speaker. Seminar: Translational Research Institute, University of Queensland, AUS.
  More info

  Invited Speaker, Univ. Queensland, Translational Research Institute; Invited by: Glenda Ph.D.
• Lynch, R. M. (2014, May). Invited Speaker. Symposium: Imaging and Energetics. National institutes of Health , Bethesda, MD: National institutes of Health.
  More info

  Invited Speaker, Symposium: Imaging and Energetics, May 30, 2014 at the National institutes of Health
• Lynch, R. M. (2014, October). Invited Speaker. Cayman Peptide Meeting. St. Croix VI: Cayman Peptide Meeting.
  More info

  Invited Speaker, Cayman Peptide Meeting, St. Croix VI Oct. 15-19, 2014