Photopharmacologic Vision Restoration Reduces Pathological Rhythmic Field Potentials in Blind Mouse Retina

Photopharmacology has yielded compounds that have potential to restore impaired visual responses resulting from outer retinal degeneration diseases such as retinitis pigmentosa. Here we evaluate two photoswitchable azobenzene ion channel blockers, DAQ and DAA for vision restoration. DAQ exerts its effect primarily on RGCs, whereas DAA induces light-dependent spiking primarily through amacrine cell activation. Degeneration-induced local field potentials remain a major challenge common to all vision restoration approaches. These 5-10 Hz rhythmic potentials increase the background firing rate of retinal ganglion cells (RGCs) and overlay the stimulated response, thereby reducing signal-to-noise ratio. Along with the bipolar cell-selective photoswitch DAD and second-generation RGC-targeting photoswitch PhENAQ, we investigated the effects of DAA and DAQ on rhythmic local field potentials (LFPs) occurring in the degenerating retina. We found that photoswitches targeting neurons upstream of RGCs, DAA (amacrine cells) and DAD (bipolar cells) suppress the frequency of LFPs, while DAQ and PhENAQ (RGCs) had negligible effects on frequency or spectral power of LFPs. Taken together, these results demonstrate remarkable diversity of cell-type specificity of photoswitchable channel blockers in the retina and suggest that specific compounds may counter rhythmic LFPs to produce superior signal-to-noise characteristics in vision restoration

Optical control of NMDA-receptors with a diffusible photoswitch

N-methyl-D-aspartate receptors (NMDARs) play a central role in synaptic plasticity, learning and memory, and are implicated in various neuronal disorders. We synthesized a diffusible photochromic glutamate analogue, azobenzene-triazole-glutamate (ATG), which is specific for NMDARs and functions as a photoswitchable agonist. ATG is inactive in its dark-adapted trans-isoform, but can be converted into its active cis-isoform using one-photon (near UV) or two-photon (740 nm) excitation. Irradiation with violet light photo-inactivates ATG within milliseconds, allowing agonist removal on the timescale of NMDAR deactivation. ATG is compatible with Ca2+ imaging and can be used to optically mimic synaptic coincidence detection protocols. Thus, ATG can be used like traditional caged glutamate compounds, but with the added advantages of NMDAR specificity, low antagonism of GABAR-mediated currents, and precise temporal control of agonist delivery

Domain Model Explains Propagation Dynamics and Stability of Histone H3K27 and H3K36 Methylation Landscapes

Chromatin states must be maintained during cell proliferation to uphold cellular identity and genome integrity. Inheritance of histone modifications is central in this process. However, the histone modification landscape is challenged by incorporation of new unmodified histones during each cell cycle, and the principles governing heritability remain unclear. We take a quantitative computational modeling approach to describe propagation of histone H3K27 and H3K36 methylation states. We measure combinatorial H3K27 and H3K36 methylation patterns by quantitative mass spectrometry on subsequent generations of histones. Using model comparison, we reject active global demethylation and invoke the existence of domains defined by distinct methylation endpoints. We find that H3K27me3 on pre-existing histones stimulates the rate of de novo H3K27me3 establishment, supporting a read-write mechanism in timely chromatin restoration. Finally, we provide a detailed quantitative picture of the mutual antagonism between H3K27 and H3K36 methylation and propose that it stabilizes epigenetic states across cell division

Biomarker characterization in endometrial cancer in Europe: first survey data analysis from 69 pathological academic and hospital labs

Introduction. Endometrial carcinoma (EC) is the commonest gynecological cancer affecting women in Western populations. To predict patient risk, the 2020 edition of the World Health Organization (WHO) Classification of Tumors of the Female Genital Tract stressed the importance of integrated histo-molecular classification of the disease. This survey analysis poses attention on the most frequently used immunohistochemical and molecular markers adopted in daily categorization of ECs in European laboratories. Methods. We analyzed data collected through questionnaires administered to 40 Italian, 20 Spanish, 3 Swiss and 6 United Kingdom (UK) laboratories. We collected information regarding daily practice in EC evaluation, specifically concerning mismatch repair status (MMR) and microsatellite instability (MSI). Summary and descriptive statistical analyses were carried out to evaluate the current practice of each laboratory. Results. The results show that MMR status is mainly evaluated by using immunohistochemistry (IHC) on most EC samples. The most frequent approach for the analysis of MMR status is IHC of four proteins (PMS2, MSH6, MSH2, MLH1). MSI analysis by molecular methods is uncommon but useful as a supplemental tool in specific conditions. MLH1 promoter hypermethylation and BRAF V600 mutations analysis are performed in case of negative expression of MLH1/PMS2. Other markers (mainly p53 followed by POLE and PTEN) are investigated in particular in Spain and Switzerland in a consistent number of cases. Conclusion. Guidelines consultation and standardization of laboratory procedures are efficient means for EC prognostic risk stratification and improving the quality of care

Adenosine A2A receptors facilitate synaptic NMDA currents in CA1 pyramidal neurons

© 2018 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.Background and purpose: NMDA receptors play a key role in both synaptic plasticity and neurodegeneration. Adenosine is an endogenous neuromodulator and through membrane receptors of the A2A subtype can influence both synaptic plasticity and neuronal death. The present work was designed to evaluate the influence of adenosine A2A receptors upon NMDA receptor activity in CA1 hippocampal neurons. We discriminated between modulation of synaptic versus extrasynaptic receptors, since extrasynaptic NMDA receptors are mostly associated with neurodegeneration while synaptic NMDA receptors are linked to plasticity phenomena. Experimental approach: Whole-cell patch-clamp recordings were obtained to evaluate NMDA receptor actions on CA1 pyramidal neurons of young adult (5-10 weeks) male Wistar rat hippocampus. Key results: Activation of A2A receptors with CGS 21680 (30 nM) consistently facilitated chemically-evoked NMDA receptor-currents (NMDA-PSCs) and afferent-evoked NMDA-currents (NMDA-EPSCs), an action prevented by an A2A receptor antagonist (SCH58261, 100 nM) and a PKA inhibitor, H-89 (1 μM). These actions did not reflect facilitation in glutamate release since there was no change in NMDA-EPSCs paired pulse ratio. A2A receptor actions were lost in the presence of an open-channel NMDA receptor blocker, MK-801 (10 μM), but persisted in the presence of memantine, at a concentration (10 μM) known to preferentially block extrasynaptic NMDA receptors. Conclusion and implications: These results show that A2A receptors exert a positive postsynaptic modulatory effect over synaptic, but not extrasynaptic, NMDA receptors in CA1 neurons and, therefore, under non-pathological conditions may contribute to shift the dual role of NMDA receptors towards enhancement of synaptic plasticity.Work supported by LISBOA-01-0145-FEDER-007391, project co-funded by FEDER through POR Lisboa 2020 (Programa Operacional Regional de Lisboa) from PORTUGAL 2020 and Fundação para a Ciência e Tecnologia, (FCT), by an FCT project (PTDC/DTP-FTO/3346/2014), and by a Twinning action (SynaNet) from the EU H2020 programme (project number: 692340). F.M.M. was in receipt of SFRH/BD/89582/2012 FTC fellowship, and D.M.R. is in receipt of a fellowship from Instituto de Medicina Molecular (IMM/BI/38-2017).info:eu-repo/semantics/publishedVersio

Light-induced regulation of ligand-gated channel activity

The control of ligand-gated receptors with light using photochromic compounds has evolved from the first handcrafted examples to accurate, engineered receptors, whose development is supported by rational design, high-resolution protein structures, comparative pharmacology and molecular biology manipulations. Photoswitchable regulators have been designed and characterized for a large number of ligand-gated receptors in the mammalian nervous system, including nicotinic acetylcholine, glutamate and GABA receptors. They provide a well-equipped toolbox to investigate synaptic and neuronal circuits in all-optical experiments. This focused review discusses the design and properties of these photoswitches, their applications and shortcomings and future perspectives in the field

Long term in-vivo studies of a photo-oxidized bovine osteochondral transplant in sheep

BACKGROUND: Articular cartilage has limited capacity to repair. Defects greater than 3 mm heal with formation of inferior fibrous cartilage. Therefore, many attempts have been made to find the ideal graft for larger cartilage lesions. Different grafts, such as untreated or cryopreserved osteochondral transplants, have been used with variable success. METHODS: Photo-oxidized osteochondral grafts were implanted in both femoral condyles of one ovine knee. Untreated xenogeneic and autogeneic grafts served as controls. Three groups of 8 sheep each were formed and they were sacrificed 6, 12 or 18 months after surgery. RESULTS: The macroscopic evaluation of the condyle and graft showed a well-maintained cartilage surface in most grafts at all time points. However, the host cartilage matrix deteriorated considerably in all xenogeneic, most autogeneic and fewer of the photo-oxidized grafts at 12 and 18 months, respectively. The blue colour of the photo-oxidized grafts resulting from the process of photo-oxidation was visible in all grafts at 6 months, had diminished at 12 months and had completely disappeared at 18 months after surgery. Histologically a loss of matrix staining was almost never noticed in untreated xenografts, transiently at 6 months in photo-oxidized grafts and increased at 12 and 18 months. Fusion between graft and host cartilage could be seen in photo-oxidized grafts at 12 and 18 months, but was never seen in autografts and xenografts. CONCLUSIONS: The photo-oxidation of osteochondral grafts and its use as transplant appears to have a beneficial effect on cartilage and bone remodelling. Osteochondral grafts pre-treated with photo-oxidation may be considered for articular cartilage replacement and therefore may delay artificial joint replacements in human patients