From 2,3-Diazabicyclo[2.2.2]oct-2-ene to Fluorazophore-L, a membrane-bound fluorescent probe for antioxidants

The aim of this work was to synthesize and to establish a new fluorescent membrane probe for antioxidants by exploiting the exceptional properties of the long-lived fluorophore 2,3-diazabicyclo[2.2.2]oct-2-ene (DBO) alias Fluorazophore-P. The first step was to find an appropriate synthetic route towards a lipophilic derivative of Fluorazophore-P, namely Fluorazophore-L, that should enable an efficient and facile incorporation into model membrane systems. The water-soluble hydroxy-substituted Fluorazophore-H was chosen as a key compound and served as a versatile precursor for various members of the Fluorazophore-family, including Fluorazophore-L. For example, substantial contributions in the synthesis of fluorazophore-labeled peptides to monitor the length-dependence of end-to-end collision rates of polypeptides were done within this work: "A Fluorescence Based Method for Direct Measurement of Submicrosecond Intramolecular Contact Formation in Biopolymers: An Exploratory Study with Polypeptides", R. R. Hudgins, F. Huang, G. Gramlich, W. M. Nau, J. Am. Chem. Soc. 2002, 124, 556-564. (Appendix) In this context, the search for a mild and selective method to substitute a harsh hydrolysis step, led to a study about a photo-cleavable Fluorazophore: "A Photoactivable Fluorophore Based on Thiadiazolidinedione as Caging Group", G. Gramlich, W. M. Nau, Org. Let. 1999, 1, 603-605. (Appendix) Fluorazophore-L (Fluoazophore-L) was designed as a head-labeled palmitic acid derivative. Experiments in homogeneous solution confirmed that Fluoazophore-L preserves its photophysical properties, namely the long-lived fluorescence and the essentially diffusion-controlled reactivity towards α-tocopherol (α-Toc). Its capability to serve as a membrane probe was assessed by air/water monolayer experiments (surface pressure-area isotherms) and preliminary spectroscopic measurements. It could be shown that Fluoazophore-L partitions into monolayers of 1-palmitoyl-2-oleoyl-sn-glycero-3- phosphocholine (POPC) and that even pure Fluoazophore-L forms stable monolayers at the air-water interface thus presents a highly amphiphilic molecule: "A Long-Lived Amphiphilic Fluorescent Probe studied in POPC Air-Water Monolayer and Solution Bilayer Systems", G. Gramlich, J. Zhang, M. Winterhalter, W. M. Nau, Chem. Phys. Lipids 2001, 113, 1-9 (Appendix). The first assignment of Fluoazophore-L in model membranes was a study of its interaction with the water-soluble antioxidant vitamin C, thus examining interfacial reactivity. Singlet-excited Fluoazophore-L was used as a mimic for highly reactive lipid alkoxyl and peroxyl radicals. This work revealed an unexpected inversion of the pHdependent activity pattern, which could be ascribed to an interesting surface effect: "Increased Antioxidant Reactivity of Vitamin C at low pH in Model Membranes", G. Gramlich, J. Zhang, W. M. Nau, J. Am. Chem. Soc. 2002, 124, 11252-11253 (Appendix). Finally, the intrafacial reactivity of α-Toc in liposomes and micelles could be probed by means of Fluoazophore-L. In micelles and in membrane structures a more demanding quenching kinetics than in usual organic solvents arises. In the case of small micelles Poissonian statistics has to be applied while in liposomes a two dimensional diffusion rate limits the maximum reactivity. In this study, the "immobile" probe/quencher pair Fluoazophore-L/α-Toc was used for the first time and the validity of different quenching models was discussed. The resulting diffusion rate constants for α-Toc provide important benchmark values for antioxidant research: "Diffusion of α-Tocopherol in Membrane Models: Probing the Kinetics of Vitamin E Antioxidant Action by Fluorescence in Real Time", G. Gramlich, J. Zhang, W. M. Nau, J. Am. Chem. Soc. 2004, 126, 5482-5492 (Appendix). A global fitting routine was developed to allow appropriate data processing of fluorescence quenching in membrane models. This fitting procedure was also successfully employed in the simultaneous fitting of steady-state and time-resolved fluorescence quenching by host-guest complexation with cyclodextrins. "A Joint Structural, Kinetic, and Thermodynamic Investigation of Substituent Effects on Host- Guest Complexation of Bicyclic Azoalkanes by β-Cyclodextrin", X. Zhang, G. Gramlich, X. Wang, W. M. Nau, J. Am. Chem. Soc. 2002, 124, 254-263 (Appendix). For the quenching models used, it is essential to ensure that reaction between singletexcited fluorazophores and hydrogen donors as antioxidants occurs only by hydrogen transfer and upon contact of probe and quencher. To clarify this process experiments using spectroscopic methods were contributed to a detailed theoretical study of reaction pathways: "Fluorescence Quenching by Sequential Hydrogen, Electron, and Proton Transfer in the Proximity of a Conical Intersection", A. Sinicropi, R. Pogni, R. Basosi, M. A. Robb, G. Gramlich, W. M. Nau, M. Olivucci, Angew. Chem., Int. Ed. 2001, 40, 4185-4189 (Appendix). In summary, the result of this study was the design and synthesis of the new fluorescent membrane probe Fluorazophore-L that combines the unusual properties of DBO with a complete incorporation into model membranes. The properties of the new probe were assessed in monolayer and by fluorescence lifetime experiments. Its potency was proven by the interaction with natural antioxidants located in the proximity of membrane mimetic systems. These quenching experiments allowed a new insight into the processes involving antioxidants in microheterogeneous environments, especially an unusual inversion of the well-known reactivity pattern of ascorbic acid and the observation of the lateral diffusion of α-tocopherol along the surface of supramolecular assemblies

Antisense-mediated exon skipping: a therapeutic strategy for titin-based dilated cardiomyopathy

Frameshift mutations in the TTN gene encoding titin are a major cause for inherited forms of dilated cardiomyopathy (DCM), a heart disease characterized by ventricular dilatation, systolic dysfunction, and progressive heart failure. To date, there are no specific treatment options for DCM patients but heart transplantation. Here, we show the beneficial potential of reframing titin transcripts by antisense oligonucleotide (AON)-mediated exon skipping in human and murine models of DCM carrying a previously identified autosomal-dominant frameshift mutation in titin exon 326. Correction of TTN reading frame in patient-specific cardiomyocytes derived from induced pluripotent stem cells rescued defective myofibril assembly and stability and normalized the sarcomeric protein expression. AON treatment in Ttn knock-in mice improved sarcomere formation and contractile performance in homozygous embryos and prevented the development of the DCM phenotype in heterozygous animals. These results demonstrate that disruption of the titin reading frame due to a truncating DCM mutation canbe restored by exon skipping in both patient cardiomyocytes invitro and mouse heart invivo, indicating RNA-based strategies as a potential treatment option for DCM

Activity-dependence of synaptic vesicle dynamics

The proper function of synapses relies on efficient recycling of synaptic vesicles. The small size of synaptic boutons has hampered efforts to define the dynamical states of vesicles during recycling. Moreover, whether vesicle motion during recycling is regulated by neural activity remains largely unknown. We combined nanoscale-resolution tracking of individual synaptic vesicles in cultured hippocampal neurons from rats of both sexes with advanced motion analyses to demonstrate that the majority of recently endocytosed vesicles undergo sequences of transient dynamical states including epochs of directed, diffusional, and stalled motion. We observed that vesicle motion is modulated in an activity-dependent manner, with dynamical changes apparent in ∼20% of observed boutons. Within this subpopulation of boutons, 35% of observed vesicles exhibited acceleration and 65% exhibited deceleration, accompanied by corresponding changes in directed motion. Individual vesicles observed in the remaining ∼80% of boutons did not exhibit apparent dynamical changes in response to stimulation. More quantitative transient motion analyses revealed that the overall reduction of vesicle mobility, and specifically of the directed motion component, is the predominant activity-evoked change across the entire bouton population. Activity-dependent modulation of vesicle mobility may represent an important mechanism controlling vesicle availability and neurotransmitter release.SIGNIFICANCE STATEMENTMechanisms governing synaptic vesicle dynamics during recycling remain poorly understood. Using nanoscale resolution tracking of individual synaptic vesicles in hippocampal synapses and advanced motion analysis tools we demonstrate that synaptic vesicles undergo complex sets of dynamical states that include epochs of directed, diffusive, and stalled motion. Most importantly, our analyses revealed that vesicle motion is modulated in an activity-dependent manner apparent as the reduction in overall vesicle mobility in response to stimulation. These results define the vesicle dynamical states during recycling and reveal their activity-dependent modulation. Our study thus provides fundamental new insights into the principles governing synaptic function

Extending Context-Sensitivity in Term Rewriting

We propose a generalized version of context-sensitivity in term rewriting based on the notion of "forbidden patterns". The basic idea is that a rewrite step should be forbidden if the redex to be contracted has a certain shape and appears in a certain context. This shape and context is expressed through forbidden patterns. In particular we analyze the relationships among this novel approach and the commonly used notion of context-sensitivity in term rewriting, as well as the feasibility of rewriting with forbidden patterns from a computational point of view. The latter feasibility is characterized by demanding that restricting a rewrite relation yields an improved termination behaviour while still being powerful enough to compute meaningful results. Sufficient criteria for both kinds of properties in certain classes of rewrite systems with forbidden patterns are presented

The 1991 State and Local Fiscal Crisis

macroeconomics, fiscal crisis, 1991

An Econometric Examination of the New Federalism

macroeconomics, econometrics, Federalism

Social Security Reform Proposals: USAs, Clawbacks, and Other Add-Ons

Faced with Social Security's impending deficits, some lawmakers have proposed supplementing the program's benefits with personal, market-based retirement accounts for all workers. Those proposals, dubbed "add-ons" because they would be added to the existing Social Security system, do not address Social Security's financial crisis. They would merely create another centralized retirement plan requiring a new funding stream. Proposed funding sources include voluntary individual contributions, general tax revenue, and mandatory payroll tax increases. Depending on which funding mechanism is selected, the market-based retirement accounts threaten to become tax shelters for higher-wage earners, become new entitlements, or increase the payroll tax burden. Although some add-ons are designed to "shore up" Social Security by cutting its benefits by the amounts accumulated in the accounts, such plans rely on a vast infusion of government money and offer no greater retirement income for workers. Studies show that if workers could invest what is currently taken from them in the form of Social Security payroll taxes, they would retire comfortably. Since workers already save enough to secure a comfortable retirement, it would be more sensible to let them get a better deal on their current payroll taxes by putting that money in personal accounts. Those accounts can be integrated with Social Security and therefore have the potential to eliminate Social Security's financial crisis. In addition, the accounts can ensure that all workers, not just the wealthy, can retire with financial security

Abstract involutions of algebraic groups and of Kac-Moody groups

Based on the second author's thesis in this article we provide a uniform treatment of abstract involutions of algebraic groups and of Kac-Moody groups using twin buildings, RGD systems, and twisted involutions of Coxeter groups. Notably we simultaneously generalize the double coset decompositions established by Springer and by Helminck-Wang for algebraic groups and by Kac-Wang for certain Kac-Moody groups, we analyze the filtration studied by Devillers-Muhlherr in the context of arbitrary involutions, and we answer a structural question on the combinatorics of involutions of twin buildings raised by Bennett-Gramlich-Hoffman-Shpectorov