Light-Controlled Cellular Internalization and Cytotoxicity of Nucleic Acid-Binding Agents: Studies in Vitro and in Zebrafish Embryos

This is the peer reviewed version of the following article: C. Penas, M. I. Sánchez, J. Guerra-Varela, L. Sanchez, M. E. Vázquez, J. L. Mascareñas, ChemBioChem 2016, 17, 37-41, which has been published in final form at https://doi.org/10.1002/cbic.201500455. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived VersionsWe synthesized octa‐arginine conjugates of DNA‐binding agents (bisbenzamidine, acridine and Thiazole Orange) and demonstrated that their DNA binding and cell internalization can be inhibited by appending a (negatively charged) oligoglutamic tail through a photolabile linker. UV irradiation released the parent conjugates, thus restoring cell internalization and biological activity. Assays with zebrafish embryos demonstrates the potential of this prodrug strategy for controlling in vivo cytotoxicityWe are thankful for the support given by the Spanish grants SAF2013–41943‐R, CTQ2012–31341 and CTQ2013–49317‐EXP, the Xunta de Galicia GRC2013–041, the ERDF and the European Research Council (Advanced Grant 340055). C.P. and M.I.S. thank the Spanish MCINN for their PhD fellowshipsS

Metallation and mismetallation of iron and manganese proteins in vitro and in vivo: the class I ribonucleotide reductases as a case study

How cells ensure correct metallation of a given protein and whether a degree of promiscuity in metal binding has evolved are largely unanswered questions. In a classic case, iron- and manganese-dependent superoxide dismutases (SODs) catalyze the disproportionation of superoxide using highly similar protein scaffolds and nearly identical active sites. However, most of these enzymes are active with only one metal, although both metals can bind in vitro and in vivo. Iron(II) and manganese(II) bind weakly to most proteins and possess similar coordination preferences. Their distinct redox properties suggest that they are unlikely to be interchangeable in biological systems except when they function in Lewis acid catalytic roles, yet recent work suggests this is not always the case. This review summarizes the diversity of ways in which iron and manganese are substituted in similar or identical protein frameworks. As models, we discuss (1) enzymes, such as epimerases, thought to use Fe[superscript II] as a Lewis acid under normal growth conditions but which switch to Mn[superscript II] under oxidative stress; (2) extradiol dioxygenases, which have been found to use both Fe[superscript II] and Mn[superscript II], the redox role of which in catalysis remains to be elucidated; (3) SODs, which use redox chemistry and are generally metal-specific; and (4) the class I ribonucleotide reductases (RNRs), which have evolved unique biosynthetic pathways to control metallation. The primary focus is the class Ib RNRs, which can catalyze formation of a stable radical on a tyrosine residue in their β2 subunits using either a di-iron or a recently characterized dimanganese cofactor. The physiological roles of enzymes that can switch between iron and manganese cofactors are discussed, as are insights obtained from the studies of many groups regarding iron and manganese homeostasis and the divergent and convergent strategies organisms use for control of protein metallation. We propose that, in many of the systems discussed, “discrimination” between metals is not performed by the protein itself, but it is instead determined by the environment in which the protein is expressed.National Institutes of Health (U.S.) (Grant GM81393

Ein Beitrag zur Kenntnis der Wurmfauna westfälischer Höhlen

von Wiard Griepenburg, Idstein im Taunu

Regulating gene expression with light-activated oligonucleotides

The work in this thesis identifies new photochemical approaches to gain high spatiotemporal control over molecular structure and function, for broad applications in materials and biological science. Caged compounds provide a method for temporarily blocking function until acted upon by an external trigger, typically near-UV light. To enable multiplexing studies, three new biomolecular caging strategies were developed that can be activated with various wavelengths of near-UV or visible light. The first method, an oligonucleotide hairpin structure incorporating one or two nitrobenzyl photolinkers, was applied to a miRNA antagomir and used to turn off let-7 miRNA in zebrafish embryos with 365 nm light. To achieve bidirectional control over miRNA, a circular construct was designed for the ability to turn on the release of exogenous miRNA into zebrafish embryos with 365 nm light. A second oligonucleotide caging method, using a ruthenium-based photolinker (RuBEP), was designed to extend photoactivation to the visible spectrum, with additional potential for two-photon activation. RuBEP was used to cage antisense morpholinos through circularization via a Cu(I)-mediated [3+2] Huisgen cycloaddition reaction. RuBEP-caged morpholinos were photoactivated to turn on antisense activity and successfully knocked down zebrafish chd and ntl genes with 450 nm light, with limited background activity prior to irradiation. A third method of caging was based on encapsulation within photoresponsive nano-polymersomes. Self-assembly of nano-polymersomes was optimized to generate visible-light-responsive vesicles that incorporate a porphyrin dimer in the hydrophobic membrane. These nanovesicles were shown to encapsulate a variety of cargo, including 25mer oligonucleotides, a small molecule fluorescent dye, and two biologically relevant metal ions, Zn 2+ and Ca2+. The photoresponsiveness of the system was modulated with light wavelength, irradiation time, and the presence of dextran in the aqueous core

Activity Center Identification in Medford, OR

53 pagesThe City of Medford seeks to identify activity centers to achieve an array of policy goals, including those that foster nodal development, increase residential density, and encourage alternate forms of transportation. Through the University of Oregon’s Sustainable City Year Program, a class of graduate students from the Community and Regional Planning program identified and analyzed potential activity centers. This report synthesizes information and analyses compiled by four student teams. It contains a description of the class’ methods, analysis of each activity center, limitations, next steps, and supplemental materials

Hyperbranched Poly(phenylene sulfide) and Poly(phenylene sulfone)

Hyperbranched poly(phenylene sulfide) was prepared from 3,4-dichlorobenzenethiol. This monomer was treated with potassium carbonate in an amide solvent, either N,N-dimethylformamide (DMF) or N-methylpyrrolidone (NMP). Polymerization for 24 hours at 100 oC in DMF gave a polymer with a Mw of 17 kD and a polydispersity of 2.0. Polymerization for 8.5 hours at 150 oC in NMP gave a polymer with a Mw of 16 kD and a polydispersity of 1.5. Addition of 1,3,5-trichlorobenzene as a multifunctional core to the polymerizations gave reduced Mw and lower polydispersity. Addition of 1 core for every 50 monomers gave a polymer with a Mw of 8.4 kD and a polydispersity of 1.2 in DMF and a polymer with a Mw of 13 kD and a polydispersity of 1.3 in NMP. The polymers were primarily characterized by size exclusion chromatography with light scattering detection (which provided the molecular weights and distributions) and by thermal methods. Differential scanning calorimetry (DSC) revealed that the hyperbranched PPS was amorphous with a Tg between 60 and 90 oC and no apparent crystallinity. The polymers prepared in DMF had higher Tg’s than those prepared in NMP. Thermogravimetric analysis revealed that the hyperbranched PPS was very thermally stable, with decomposition temperatures between 400 and 450 oC in both air and N2 atmospheres. In air, complete decomposition occurred by about 625 oC, while approximately 25% of the mass remained at 700 oC under N2. The hyperbranched PPS could be oxidized to hyperbranched poly(phenylene sulfone). This material was completely insoluble, but could be analyzed by thermal methods. By DSC, the Tg of the sulfone was approximately 155 oC, while by TGA the decomposition temperature was 325-375 oC in both air and N2. In air, decomposition was complete by 575 oC, while in N2 about 30% of the mass remained at 700 oC. These simple, one-pot approaches to hyperbranched poly(phenylene sulfide) and hyperbranched poly(phenylene sulfone) from commercially available monomers provide an entry to many further studies and applications for these new materials