Single particle cryo-EM of the complex between interphotoreceptor retinoid-binding protein and a monoclonal antibody

Interphotoreceptor retinoid-binding protein (IRBP) is a highly expressed protein secreted by rod and cone photoreceptors that has major roles in photoreceptor homeostasis as well as retinoid and polyunsaturated fatty acid transport between the neural retina and retinal pigment epithelium. Despite two crystal structures reported on fragments of IRBP and decades of research, the overall structure of IRBP and function within the visual cycle remain unsolved. Here, we studied the structure of native bovine IRBP in complex with a monoclonal antibody (mAb5) by cryo-electron microscopy, revealing the tertiary and quaternary structure at sufficient resolution to clearly identify the complex components. Complementary mass spectrometry experiments revealed the structure and locations of N-linked carbohydrate post-translational modifications. This work provides insight into the structure of IRBP, displaying an elongated, flexible three-dimensional architecture not seen among other retinoid-binding proteins. This work is the first step in elucidation of the function of this enigmatic protein

Improvement of the Heisenberg and Fisher-information-based uncertainty relations for D-dimensional central potentials

The Heisenberg and Fisher-information-based uncertainty relations are improved for stationary states of single-particle systems in a D-dimensional central potential. The improvement increases with the squared orbital hyperangular quantum number. The new uncertainty relations saturate for the isotropic harmonic oscillator wavefunction.We are very grateful for partial support to Junta de Andalucía (under the grants FQM- 0207 and FQM-481), Ministerio de Educaci´on y Ciencia (under the project FIS2005-00973), and the European Research Network NeCCA (under the project INTAS-03-51-6637). RGF acknowledges the support of Junta de Andalucía under the program of Retorno de Investigadores a Centros de Investigación Andaluces

Rapid Chromosome Evolution in Recently Formed Polyploids in Tragopogon (Asteraceae)

Polyploidy, frequently termed "whole genome duplication", is a major force in the evolution of many eukaryotes. Indeed, most angiosperm species have undergone at least one round of polyploidy in their evolutionary history. Despite enormous progress in our understanding of many aspects of polyploidy, we essentially have no information about the role of chromosome divergence in the establishment of young polyploid populations. Here we investigate synthetic lines and natural populations of two recently and recurrently formed allotetraploids Tragopogon mirus and T. miscellus (formed within the past 80 years) to assess the role of aberrant meiosis in generating chromosomal/genomic diversity. That diversity is likely important in the formation, establishment and survival of polyploid populations and species.Applications of fluorescence in situ hybridisation (FISH) to natural populations of T. mirus and T. miscellus suggest that chromosomal rearrangements and other chromosomal changes are common in both allotetraploids. We detected extensive chromosomal polymorphism between individuals and populations, including (i) plants monosomic and trisomic for particular chromosomes (perhaps indicating compensatory trisomy), (ii) intergenomic translocations and (iii) variable sizes and expression patterns of individual ribosomal DNA (rDNA) loci. We even observed karyotypic variation among sibling plants. Significantly, translocations, chromosome loss, and meiotic irregularities, including quadrivalent formation, were observed in synthetic (S(0) and S(1) generations) polyploid lines. Our results not only provide a mechanism for chromosomal variation in natural populations, but also indicate that chromosomal changes occur rapidly following polyploidisation.These data shed new light on previous analyses of genome and transcriptome structures in de novo and establishing polyploid species. Crucially our results highlight the necessity of studying karyotypes in young (<150 years old) polyploid species and synthetic polyploids that resemble natural species. The data also provide insight into the mechanisms that perturb inheritance patterns of genetic markers in synthetic polyploids and populations of young natural polyploid species

Overexposure to apoptosis via disrupted glial specification perturbs Drosophila macrophage function and reveals roles of the CNS during injury

Apoptotic cell clearance by phagocytes is a fundamental process during development, homeostasis and the resolution of inflammation. However, the demands placed on phagocytic cells such as macrophages by this process, and the limitations these interactions impose on subsequent cellular behaviours are not yet clear. Here, we seek to understand how apoptotic cells affect macrophage function in the context of a genetically tractable Drosophila model in which macrophages encounter excessive amounts of apoptotic cells. Loss of the glial-specific transcription factor Repo prevents glia from contributing to apoptotic cell clearance in the developing embryo. We show that this leads to the challenge of macrophages with large numbers of apoptotic cells in vivo. As a consequence, macrophages become highly vacuolated with cleared apoptotic cells, and their developmental dispersal and migration is perturbed. We also show that the requirement to deal with excess apoptosis caused by a loss of repo function leads to impaired inflammatory responses to injury. However, in contrast to migratory phenotypes, defects in wound responses cannot be rescued by preventing apoptosis from occurring within a repo mutant background. In investigating the underlying cause of these impaired inflammatory responses, we demonstrate that wound-induced calcium waves propagate into surrounding tissues, including neurons and glia of the ventral nerve cord, which exhibit striking calcium waves on wounding, revealing a previously unanticipated contribution of these cells during responses to injury. Taken together, these results demonstrate important insights into macrophage biology and how repo mutants can be used to study macrophage–apoptotic cell interactions in the fly embryo. Furthermore, this work shows how these multipurpose cells can be ‘overtasked’ to the detriment of their other functions, alongside providing new insights into which cells govern macrophage responses to injury in vivo

COVID-19: Rapid antigen detection for SARS-CoV-2 by lateral flow assay: A national systematic evaluation of sensitivity and specificity for mass-testing

Background Lateral flow device (LFD) viral antigen immunoassays have been developed around the world as diagnostic tests for SARS-CoV-2 infection. They have been proposed to deliver an infrastructure-light, cost-economical solution giving results within half an hour. Methods LFDs were initially reviewed by a Department of Health and Social Care team, part of the UK government, from which 64 were selected for further evaluation from 1st August to 15th December 2020. Standardised laboratory evaluations, and for those that met the published criteria, field testing in the Falcon-C19 research study and UK pilots were performed (UK COVID-19 testing centres, hospital, schools, armed forces). Findings 4/64 LFDs so far have desirable performance characteristics (orient Gene, Deepblue, Abbott and Innova SARS-CoV-2 Antigen Rapid Qualitative Test). All these LFDs have a viral antigen detection of >90% at 100,000 RNA copies/ml. 8951 Innova LFD tests were performed with a kit failure rate of 5.6% (502/8951, 95% CI: 5.1–6.1), false positive rate of 0.32% (22/6954, 95% CI: 0.20–0.48). Viral antigen detection/sensitivity across the sampling cohort when performed by laboratory scientists was 78.8% (156/198, 95% CI 72.4–84.3). Interpretation Our results suggest LFDs have promising performance characteristics for mass population testing and can be used to identify infectious positive individuals. The Innova LFD shows good viral antigen detection/sensitivity with excellent specificity, although kit failure rates and the impact of training are potential issues. These results support the expanded evaluation of LFDs, and assessment of greater access to testing on COVID-19 transmission. Funding Department of Health and Social Care. University of Oxford. Public Health England Porton Down, Manchester University NHS Foundation Trust, National Institute of Health Research

Lecithin:Retinol Acyltransferase: A Key Enzyme Involved in the Retinoid (visual) Cycle

Lecithin:retinol acyltransferase (LRAT) catalyzes the acyl transfer from the sn-1 position of phosphatidylcholine (PC) to all-trans-retinol, creating fatty acid retinyl esters (palmitoyl, stearoyl, and some unsaturated derivatives). In the eye, these retinyl esters are substrates for the 65 kDa retinoid isomerase (RPE65). LRAT is well characterized biochemically, and recent structural data from closely related family members of the NlpC/P60 superfamily and a chimeric protein have established its catalytic mechanism. Mutations in the LRAT gene are responsible for approximately 1% of reported cases of Leber congenital amaurosis (LCA). Lack of functional LRAT, expressed in the retinal pigmented epithelium (RPE), results in loss of the visual chromophore and photoreceptor degeneration. LCA is a rare hereditary retinal dystrophy with an early onset associated with mutations in one of 21 known genes. Protocols have been devised to identify therapeutics that compensate for mutations in RPE65, also associated with LCA. The same protocols can be adapted to combat dystrophies associated with LRAT. Improvement in the visual function of clinical recipients of therapy with recombinant adeno-associated virus (rAAV) vectors incorporating the RPE65 gene provides a proof of concept for LRAT, which functions in the same cell type and metabolic pathway as RPE65. In parallel, a clinical trial that employs oral 9-cis-retinyl acetate to replace the missing chromophore in RPE65 and LRAT causative disease has proven to be effective and free of adverse effects. This article summarizes the biochemistry of LRAT and examines chromophore replacement as a treatment for LCA caused by LRAT&nbsp;mutations

Stable Retinoid Analogue Targeted Dual pH-Sensitive Smart Lipid ECO/pDNA Nanoparticles for Specific Gene Delivery in the Retinal Pigment Epithelium

Dysfunctions caused by gene mutations in the retinal pigment epithelium (RPE) lead to retinal degeneration, visual function loss, and even blindness. RPE-specific gene replacement therapy holds great promise for treating monogenic ocular disorders in the RPE. Although the adeno-associated virus (AAV) has been approved for gene therapy to treat monogenic visual disorders, broad clinical applications of AAV-based gene therapy are limited by its gene loading capacity. In this work, we intended to design and develop a stable retinylamine analogue ACU4429-modified dual pH-sensitive ECO/pDNA nanoparticles for specific delivery of large therapeutic genes to the RPE. ACU4429 was first conjugated to a PEG 3.4 kD spacer with a pH-sensitive hydrazone linker at the distal end (ACU-PEG-HZ-MAL). The targeted dual pH-sensitive ECO/pDNA nanoparticles were then prepared by self-assembly of ACU-PEG-HZ-MAL, pH-sensitive lipid carrier ECO, and a plasmid DNA expressing the large ABCA4 gene. The formation of targeted ACU-PEG-HZ-ECO/pDNA nanoparticles was characterized by dynamic light scattering and gel electrophoresis. The incorporation of a hydrazone linker enhanced the cytosolic gene delivery, which translated to high ABCA4 expression in ARPE-19 cells for ACU-PEG-HZ-ECO/pABCA4 nanoparticles. The targeted nanoparticles also demonstrated excellent targeting efficiency in the interphotoreceptor matrix of Abca4-/- mice, resulting in enhanced expression of the ABCA4 gene in the RPE. The ACU4429 PEG hydrazone-modified ECO/pDNA nanoparticles provide a promising nonviral platform to safely and effectively deliver therapeutic genes with unlimited sizes for the treatment of monogenic visual disorders

Non-viral Gene Therapy for Stargardt Disease with ECO/pRHO-ABCA4 Self-Assembled Nanoparticles

Stargardt disease (STGD) is an autosomal recessive retinal disorder caused by a monogenic ABCA4 mutation. Currently, there is no effective therapy to cure Stargardt disease. The replacement of mutated ABCA4 with a functional gene remains an attractive strategy. In this study, we have developed a non-viral gene therapy using nanoparticles self-assembled by a multifunctional pH-sensitive amino lipid ECO and a therapeutic ABCA4 plasmid. The nanoparticles mediated efficient intracellular gene transduction in wild-type (WT) and Abca4-/- mice. Specific ABCA4 expression in the outer segment of photoreceptors was achieved by incorporating a rhodopsin promoter into the plasmids. The ECO/pRHO-ABCA4 nanoparticles induced substantial and specific ABCA4 expression for at least 8&nbsp;months, 35% reduction in A2E accumulation on average, and a delayed Stargardt disease progression for at least 6&nbsp;months in Abca4-/- mice. ECO/plasmid nanoparticles constitute a promising non-viral gene therapy platform for Stargardt disease and other visual dystrophies

Two-photon imaging of the mammalian retina with ultrafast pulsing laser

Noninvasive imaging of visual system components in vivo is critical for understanding the causal mechanisms of retinal diseases and for developing therapies for their treatment. However, ultraviolet light needed to excite endogenous fluorophores that participate in metabolic processes of the retina is highly attenuated by the anterior segment of the human eye. In contrast, 2-photon excitation fluorescence imaging with pulsed infrared light overcomes this obstacle. Reducing retinal exposure to laser radiation remains a major barrier in advancing this technology to studies in humans. To increase fluorescence intensity and reduce the requisite laser power, we modulated ultrashort laser pulses with high-order dispersion compensation and applied sensorless adaptive optics and custom image recovery software and observed an over 300% increase in fluorescence of endogenous retinal fluorophores when laser pulses were shortened from 75 fs to 20 fs. No functional or structural changes to the retina were detected after exposure to 2-photon excitation imaging light with 20-fs pulses. Moreover, wide bandwidth associated with short pulses enables excitation of multiple fluorophores with different absorption spectra and thus can provide information about their relative changes and intracellular distribution. These data constitute a substantial advancement for safe 2-photon fluorescence imaging of the human eye