Restoration of Visual Function by Expression of a Light-Gated Mammalian Ion Channel in Retinal Ganglion Cells or ON-Bipolar Cells

Most inherited forms of blindness are caused by mutations that lead to photoreceptor cell death but spare second- and third-order retinal neurons. Expression of the light-gated excitatory mammalian ion channel light-gated ionotropic glutamate receptor (LiGluR) in retinal ganglion cells (RGCs) of the retina degeneration (rd1) mouse model of blindness was previously shown to restore some visual functions when stimulated by UV light. Here, we report restored retinal function in visible light in rodent and canine models of blindness through the use of a second-generation photoswitch for LiGluR, maleimide-azobenzene-glutamate 0 with peak efficiency at 460 nm (MAG0460). In the blind rd1 mouse, multielectrode array recordings of retinal explants revealed robust and uniform light-evoked firing when LiGluR-MAG0460 was targeted to RGCs and robust but diverse activity patterns in RGCs when LiGluR-MAG0460 was targeted to ON-bipolar cells (ON-BCs). LiGluR-MAG0460 in either RGCs or ON-BCs of the rd1 mouse reinstated innate light-avoidance behavior and enabled mice to distinguish between different temporal patterns of light in an associative learning task. In the rod-cone dystrophy dog model of blindness, LiGluR-MAG0460 in RGCs restored robust light responses to retinal explants and intravitreal delivery of LiGluR and MAG0460 was well tolerated in vivo. The results in both large and small animal models of photoreceptor degeneration provide a path to clinical translation

Deficiency of caspase recruitment domain family, member 11 (CARD11), causes profound combined immunodeficiency in human subjects.

BACKGROUND: Profound combined immunodeficiency can present with normal numbers of T and B cells, and therefore the functional defect of the cellular and humoral immune response is often not recognized until the first severe clinical manifestation. Here we report a patient of consanguineous descent presenting at 13 months of age with hypogammaglobulinemia, Pneumocystis jirovecii pneumonia, and a suggestive family history. OBJECTIVE: We sought to identify the genetic alteration in a patient with combined immunodeficiency and characterize human caspase recruitment domain family, member 11 (CARD11), deficiency. METHODS: Molecular, immunologic, and functional assays were performed. RESULTS: The immunologic characterization revealed only subtle changes in the T-cell and natural killer cell compartment, whereas B-cell differentiation, although normal in number, was distinctively blocked at the transitional stage. Genetic evaluation revealed a homozygous deletion of exon 21 in CARD11 as the underlying defect. This deletion abrogated protein expression and activation of the canonical nuclear factor κB (NF-κB) pathway in lymphocytes after antigen receptor or phorbol 12-myristate 13-acetate stimulation, whereas CD40 signaling in B cells was preserved. The abrogated activation of the canonical NF-κB pathway was associated with severely impaired upregulation of inducible T-cell costimulator, OX40, cytokine production, proliferation of T cells, and B cell-activating factor receptor expression on B cells. CONCLUSION: Thus in patients with CARD11 deficiency, the combination of impaired activation and especially upregulation of inducible T-cell costimulator on T cells, together with severely disturbed peripheral B-cell differentiation, apparently leads to a defective T-cell/B-cell cooperation and probably germinal center formation and clinically results in severe immunodeficiency. This report discloses the crucial and nonredundant role of canonical NF-κB activation and specifically CARD11 in the antigen-specific immune response in human subjects

Seroprevalence of 34 Human Papillomavirus Types in the German General Population

The natural history of infections with many human papillomavirus (HPV) types is poorly understood. Here, we describe for the first time the age- and sex-dependent antibody prevalence for 29 cutaneous and five mucosal HPV types from 15 species within five phylogenetic genera (alpha, beta, gamma, mu, nu) in a general population. Sera from 1,797 German adults and children (758 males and 1,039 females) between 1 and 82 years (median 37 years) were analysed for antibodies to the major capsid protein L1 by Luminex-based multiplex serology. The first substantial HPV antibody reactions observed already in children and young adults are those to cutaneous types of the genera nu (HPV 41) and mu (HPV 1, 63). The antibody prevalence to mucosal high-risk types, most prominently HPV 16, was elevated after puberty in women but not in men and peaked between 25 and 34 years. Antibodies to beta and gamma papillomaviruses (PV) were rare in children and increased homogeneously with age, with prevalence peaks at 40 and 60 years in women and 50 and 70 years in men. Antibodies to cutaneous alpha PV showed a heterogeneous age distribution. In summary, these data suggest three major seroprevalence patterns for HPV of phylogenetically distinct genera: antibodies to mu and nu skin PV appear early in life, those to mucosal alpha PV in women after puberty, and antibodies to beta as well as to gamma skin PV accumulate later in life

I. Total Synthesis of Smenochromene B and Halenaquinone and II. Studies on the Chemical and Biological Properties of Azobenzene Photoswitches

Part one of this dissertation describes synthetic efforts toward two separate but structurally related groups of marine sesquiterpene quinone natural products, the smenochromene/likonide family and halenaquinone, and culminates in the total synthesis of smenochromene B and halenaquinone. The total synthesis of smenochromene B and efforts towards likonide A centered around the use of smenochromene D as a synthetic intermediate. A macrocyclic ring contraction and double bond isomerization was the key transformation necessary to produce smenochromene B. During the optimization of the synthesis of smenochromene D, two different and remarkable oxidative dimers were isolated, and the mechanism by which they are formed is discussed. Additionally, density functional theory calculations were used to investigate the oxa-6π electrocyclization used to synthesize smenochromene D and are believed to be biologically relevant to the markedly different levels of enantiomeric excess observed within members of the structural family. Halenaquinone, a biologically active pentacyclic quinone isolated from the marine sponge Xestospongia exigua, is biosynthetically related to the smenochromene/likonide family. The total synthesis of halenaquinone reported here features two key steps: a diastereoselective intramolecular Heck reaction, which sets the all-carbon quaternary stereocenter, and an intramolecular inverse-electron-demand Diels-Alder cycloaddition using a vinyl-orthoquinone methide as a diene. This is the first application of such a Diels-Alder cycloaddition to total synthesis. In order to better understand the Diels-Alder reaction, density functional theory calculations were performed to analyze two relevant transition states. Part two describes several projects focused around the synthesis of photoswitchable azobenzene ligands. In order to build on previous work that demonstrated photoregulation of wild-type voltage-gated potassium channels by membrane permeable blockers, a new series of compounds was synthesized with red-shifted absorption maxima. Two new azobenzene containing blockers were identified as being able to regulate voltage-gated potassium channels by toggling between darkness and irradiation with 472 nm light; one compound blocked voltage-gated potassium channels in the trans form, the other in the cis form. Additionally, azobenzene compounds based on the drugs lidocaine and retigabine were synthesized, and preliminary biological data are reported. Finally, the syntheses of azobenzene analogs of the anti-cancer and life-extending (in lower organisms) compound resveratrol and a related protein-tyrosine kinase inhibitor piceatannol are reported

Vinyl Quinones as Diels-Alder Dienes: Concise Synthesis of (-)-Halenaquinone

Eight decades after its discovery, the synthetic appeal of the Diels-Alder reaction has not diminished, and new modes continue to be discovered. The versatility of the reaction and the ease with which its components can be modified is indeed remarkable. Quinones, for instance, are classical electron-deficient dienophiles, yet relatively little is known about their role in neutral or inverseelectron-demand Diels-Alder reactions. With appropriate substitution, however, they can be part of an electron-deficient diene system that can readily engage in cycloadditions. Indeed, vinyl-p-quinones are known to dimerize thermally to yield hydrophenathrenes with excellent regio- and chemoselectivity (1f2; Scheme 1). 1,2 Additionally, Noland and Kedrowski have shown that extremely electron-deficient vinyl nitroquinones undergo facile cycloadditions to electron-rich furans and indoles (e.g., 3f4). 3 Although these modes of reactivity provide rapid access to highl

Restoring Vision to the Blind with Chemical Photoswitches

Degenerative retinal diseases such as retinitis pigmentosa (RP) and age-related macular degeneration (AMD) affect millions of people around the world and lead to irreversible vision loss if left untreated. A number of therapeutic strategies have been developed over the years to treat these diseases or restore vision to already blind patients. In this Review, we describe the development and translational application of light-sensitive chemical photoswitches to restore visual function to the blind retina and compare the translational potential of photoswitches with other vision-restoring therapies. This therapeutic strategy is enabled by an efficient fusion of chemical synthesis, chemical biology, and molecular biology and is broadly applicable to other biological systems. We hope this Review will be of interest to chemists as well as neuroscientists and clinicians

Phospholipase D2 specifically regulates TREK potassium channels via direct interaction and local production of phosphatidic acid

Membrane lipids serve as second messengers and docking sites for proteins and play central roles in cell signaling. A major question about lipid signaling is whether diffusible lipids can selectively target specific proteins. One family of lipid-regulated membrane proteins is the TWIK-related K channel (TREK) subfamily of K2P channels: TREK1, TREK2, and TWIK-related arachdonic acid stimulated K(+) channel (TRAAK). We investigated the regulation of TREK channels by phosphatidic acid (PA), which is generated by phospholipase D (PLD) via hydrolysis of phosphatidylcholine. Even though all three of the channels are sensitive to PA, we found that only TREK1 and TREK2 are potentiated by PLD2 and that none of these channels is modulated by PLD1, indicating surprising selectivity. We found that PLD2, but not PLD1, directly binds to the C terminus of TREK1 and TREK2, but not to TRAAK. The results have led to a model for selective lipid regulation by localization of phospholipid enzymes to specific effector proteins. Finally, we show that regulation of TREK channels by PLD2 occurs natively in hippocampal neurons

A Red-Shifted, Fast-Relaxing Azobenzene Photoswitch for Visible Light Control of an Ionotropic Glutamate Receptor

The use of azobenzene photoswitches has become a dependable method for rapid and exact modulation of biological processes and material science systems. The requirement of ultraviolet light for azobenzene isomerization is not ideal for biological systems due to poor tissue penetration and potentially damaging effects. While modified azobenzene cores with a red-shifted cis-to-trans isomerization have been previously described, they have not yet been incorporated into a powerful method to control protein function: the photoswitchable tethered ligand (PTL) approach. We report the synthesis and characterization of a red-shifted PTL, L-MAG0460, for the light-gated ionotropic glutamate receptor LiGluR. In cultured mammalian cells, the LiGluR+L-MAG0460 system is activated rapidly by illumination with 400-520 nm light to generate a large ionic current. The current rapidly turns off in the dark as the PTL relaxes thermally back to the trans configuration. The visible light excitation and single-wavelength behavior considerably simplify use and should improve utilization in tissue

Phospholipase D2 specifically regulates TREK potassium channels via direct interaction and local production of phosphatidic acid

Membrane lipids serve as second messengers and docking sites for proteins and play central roles in cell signaling. A major question about lipid signaling is whether diffusible lipids can selectively target specific proteins. One family of lipid-regulated membrane proteins is the TWIK-related K channel (TREK) subfamily of K2P channels: TREK1, TREK2, and TWIK-related arachdonic acid stimulated K(+) channel (TRAAK). We investigated the regulation of TREK channels by phosphatidic acid (PA), which is generated by phospholipase D (PLD) via hydrolysis of phosphatidylcholine. Even though all three of the channels are sensitive to PA, we found that only TREK1 and TREK2 are potentiated by PLD2 and that none of these channels is modulated by PLD1, indicating surprising selectivity. We found that PLD2, but not PLD1, directly binds to the C terminus of TREK1 and TREK2, but not to TRAAK. The results have led to a model for selective lipid regulation by localization of phospholipid enzymes to specific effector proteins. Finally, we show that regulation of TREK channels by PLD2 occurs natively in hippocampal neurons