The isolation, characterisation and investigation into the In vitro behaviour of human ocular vascular endothelial cells

Intraocular angiogenesis is associated with a number of common, blinding conditions including wet age-related macular degeneration, proliferative diabetic retinopathy, retinopathy of prematurity and rubeotic glaucoma. The pathogenesis of these disorders is centered on choroidal, retinal and iris microvascular endothelial cells (ECs) respectively. When studying these conditions, workers have applied conclusions from in vitro studies of human umbilical vein EC (HUVEC) and microvascular endothelial cells, derived from different species or organs. However it is now widely accepted that endothelial cells are very heterogeneous and .extrapolation of results from these cells may not provide reliable data applicable to human eye disease. I have successfully isolated and cultured matched human retinal, choroidal and iris endothelial cells (and sourced HUVECs) and have examined their gene expression profiles. I found wide differences in gene expression between HUVEC and ocular ECs and in addition, between matched human retinal and choroidal endothelial cells. Taken together, these results suggest that HUVECs are not suitable surrogates for studying human ocular disease and secondly. It has implications for our understanding and treatment of retinal and choroidal vascular diseases. To further define the heterogeneity of endothelial cells within a vascular bed, I developed a technique for the isolation of human inner choroidal ECs. After the cells were characterized by surface marker expression, the response of these unique cells to various growth factors was defined and gene expression microarray analysis was performed and compared with matching outer choroidal ECs. This demonstrated differences in genes involved in fenestration formation and growth factor expression. These findings may have implications for our understanding of macular diseases. Overall, this body of work provides an insight into endothelial cell heterogeneity within the eye and may provide clues as to the reasons for ocular vascular bed disease susceptibility and helps identify potential future selective anti-angiogenic treatments

The isolation, characterisation and investigation into the In vitro behaviour of human ocular vascular endothelial cells

Intraocular angiogenesis is associated with a number of common, blinding conditions including wet age-related macular degeneration, proliferative diabetic retinopathy, retinopathy of prematurity and rubeotic glaucoma. The pathogenesis of these disorders is centered on choroidal, retinal and iris microvascular endothelial cells (ECs) respectively. When studying these conditions, workers have applied conclusions from in vitro studies of human umbilical vein EC (HUVEC) and microvascular endothelial cells, derived from different species or organs. However it is now widely accepted that endothelial cells are very heterogeneous and .extrapolation of results from these cells may not provide reliable data applicable to human eye disease. I have successfully isolated and cultured matched human retinal, choroidal and iris endothelial cells (and sourced HUVECs) and have examined their gene expression profiles. I found wide differences in gene expression between HUVEC and ocular ECs and in addition, between matched human retinal and choroidal endothelial cells. Taken together, these results suggest that HUVECs are not suitable surrogates for studying human ocular disease and secondly. It has implications for our understanding and treatment of retinal and choroidal vascular diseases. To further define the heterogeneity of endothelial cells within a vascular bed, I developed a technique for the isolation of human inner choroidal ECs. After the cells were characterized by surface marker expression, the response of these unique cells to various growth factors was defined and gene expression microarray analysis was performed and compared with matching outer choroidal ECs. This demonstrated differences in genes involved in fenestration formation and growth factor expression. These findings may have implications for our understanding of macular diseases. Overall, this body of work provides an insight into endothelial cell heterogeneity within the eye and may provide clues as to the reasons for ocular vascular bed disease susceptibility and helps identify potential future selective anti-angiogenic treatments

Reticular pseudodrusen in late-onset retinal degeneration

PURPOSE: To characterize the association of reticular pseudodrusen (RPD) with late-onset retinal degeneration (L-ORD) using multimodal imaging. DESIGN: Prospective, two-center, longitudinal case series. SUBJECTS: Twenty-nine cases with L-ORD. METHODS: All subjects were evaluated within a three-year interval with near-infrared reflectance, fundus autofluorescence, and spectral-domain optical coherence tomography. In addition, a subset of patients also underwent indocyanine green angiography, fundus fluorescein angiography, mesopic microperimetry, and multifocal electroretinography. Main outcome measures: Prevalence, topographic distribution, and temporal phenotypic changes of RPD in L-ORD. RESULTS: A total of 29 molecularly confirmed L-ORD cases were included in this prospective study. RPD was detected in 18 cases (62%) at baseline, of which 10 were male. The prevalence of RPD varied with age. The mean age of RPD patients was 57.3±7.2 years. RPD was not seen in cases below the fifth decade (n=3 patients) or in the eighth decade (n=5 patients). RPD were found commonly in the macula with relative sparing of the fovea and were also identified in the peripheral retina. The morphology of RPD changed with follow-up. Two cases (3 eyes) demonstrated RPD regression. CONCLUSIONS: RPD is found frequently in cases with L-ORD and at a younger age than in individuals with AMD. RPD exhibits quick formation and collapse, change in type and morphology with time, relative foveal-sparing, and also has a peripheral retinal location in L-ORD

Avalanches on a conical bead pile: scaling with tuning parameters

Uniform spherical beads were used to explore the behavior of a granular system near its critical angle of repose on a conical bead pile. We found two tuning parameters that could take the system to a critical point where a simple power-law described the avalanche size distribution as predicted by self-organized criticality, which proposed that complex dynamical systems self-organize to a critical point without need for tuning. Our distributions were well described by a simple power-law with the power {\tau} = 1.5 when dropping beads slowly onto the apex of a bead pile from a small height. However, we could also move the system from the critical point using either of two tuning parameters: the height from which the beads fell onto the top of the pile or the region over which the beads struck the pile. As the drop height increased, the system did not reach the critical point yet the resulting distributions were independent of the bead mass, coefficient of friction, or coefficient of restitution. All our apex-dropping distributions for any type of bead (glass, stainless steel, zirconium) showed universality by scaling onto a common curve with {\tau} = 1.5 and {\sigma} = 1.0, where 1/{\sigma} is the power of the tuning parameter. From independent calculations using the moments of the distribution, we find values for {\tau} = 1.6 \pm 0.1 and {\sigma} = 0.91 \pm 0.15. When beads were dropped across the surface of the pile instead of solely on the apex, then the system also moved from the critical point and again the avalanche size distributions fell on a common curve when scaled similarly using the same values of {\tau} and {\sigma}. We also observed that an hcp structure on the base of the pile caused an emergent structure in the pile that had six faces with some fcc or hcp structure.Comment: 8 pages, 6 figures; submitted to Granular Matter; Reformatted into LaTeX from Word; Fixed typo in uncertainty of tau; Rearranged two paragraphs to improve flo

Performance of Genotype Imputation for Rare Variants Identified in Exons and Flanking Regions of Genes

Genotype imputation has the potential to assess human genetic variation at a lower cost than assaying the variants using laboratory techniques. The performance of imputation for rare variants has not been comprehensively studied. We utilized 8865 human samples with high depth resequencing data for the exons and flanking regions of 202 genes and Genome-Wide Association Study (GWAS) data to characterize the performance of genotype imputation for rare variants. We evaluated reference sets ranging from 100 to 3713 subjects for imputing into samples typed for the Affymetrix (500K and 6.0) and Illumina 550K GWAS panels. The proportion of variants that could be well imputed (true r2>0.7) with a reference panel of 3713 individuals was: 31% (Illumina 550K) or 25% (Affymetrix 500K) with MAF (Minor Allele Frequency) less than or equal 0.001, 48% or 35% with 0.001<MAF< = 0.005, 54% or 38% with 0.005<MAF< = 0.01, 78% or 57% with 0.01<MAF< = 0.05, and 97% or 86% with MAF>0.05. The performance for common SNPs (MAF>0.05) within exons and flanking regions is comparable to imputation of more uniformly distributed SNPs. The performance for rare SNPs (0.01<MAF< = 0.05) was much more dependent on the GWAS panel and the number of reference samples. These results suggest routine use of genotype imputation for extending the assessment of common variants identified in humans via targeted exon resequencing into additional samples with GWAS data, but imputation of very rare variants (MAF< = 0.005) will require reference panels with thousands of subjects

Existence of families of spacetimes with a Newtonian limit

J\"urgen Ehlers developed \emph{frame theory} to better understand the relationship between general relativity and Newtonian gravity. Frame theory contains a parameter $\lambda$, which can be thought of as $1/c^2$, where $c$ is the speed of light. By construction, frame theory is equivalent to general relativity for $\lambda >0$, and reduces to Newtonian gravity for $\lambda =0$. Moreover, by setting \ep=\sqrt{\lambda}, frame theory provides a framework to study the Newtonian limit \ep \searrow 0 (i.e. $c\to \infty$). A number of ideas relating to frame theory that were introduced by J\"urgen have subsequently found important applications to the rigorous study of both the Newtonian limit and post-Newtonian expansions. In this article, we review frame theory and discuss, in a non-technical fashion, some of the rigorous results on the Newtonian limit and post-Newtonian expansions that have followed from J\"urgen's work

Treatment Strategy with Gene Editing for Late-onset Retinal Degeneration Caused by a Founder Variant in C1QTNF5

AbstractPurpose: Genome editing is an emerging group of technologies with the potential to ameliorate dominant, monogenic human diseases such as late-onset retinal degeneration (L-ORD). The goal of this study was to identify disease stages and retinal locations optimal for evaluating the efficacy of a future genome editing trial.Methods: Twenty five L-ORD patients (age range, 33-77 years; median age, 59 years) harboring the founder variant S163R in C1QTNF5 were enrolled from three centers in the United Kingdom and United States. Patients were examined with widefield optical coherence tomography (OCT) and chromatic perimetry under dark-adapted and light-adapted conditions to derive phenomaps of retinal disease. Results were analyzed with a model of a shared natural history of a single delayed exponential across all subjects and all retinal locations.Results: Critical age for the initiation of photoreceptor loss ranged from 48 years at the temporal paramacular retina to 74 years at the inferior midperipheral retina. Subretinal deposits (sRET-Ds) became more prevalent as critical age was approached. Subretinal pigment epithelial deposits (sRPE-Ds) were detectable in the youngest patients showing no other structural or functional abnormalities at the retina. The sRPE-D thickness continuously increased, reaching 25 µm in the extrafoveal retina and 19 µm in the fovea at critical age. Loss of light sensitivity preceded shortening of outer segments and loss of photoreceptors by more than a decade.Conclusions: Retinal regions providing an ideal treatment window exist across all severity stages of L-ORD

Control of resistance against bacteriophage killing by a metabolic regulator in meningitis-associated Escherichia coli

Ecologically beneficial traits in bacteria are encoded by intrinsic and horizontally acquired genes. However, such traits are not universal, and the highly mosaic nature of bacterial genomes requires control at the transcriptional level to drive these processes. It has emerged that regulatory flexibility is widespread in the Escherichia coli species, whereby preexisting transcription factors can acquire new and unrelated roles in regulating beneficial traits. DsdC is the regulator of D-serine tolerance in E. coli, is essential for D-serine catabolism, and is often encoded by two copies in neonatal meningitis-associated E. coli (NMEC). Here, we reveal that DsdC is a global regulator of transcription in NMEC and does not require D-serine for the control of novel beneficial traits. We show that DsdC binds the chromosome in an unusual manner, with many binding sites arranged in clusters spanning entire operons and within gene coding sequences, such as neuO. Importantly, we identify neuO as the most significantly down-regulated gene in a strain deleted for both dsdC copies, in both the presence and absence of D-serine. NeuO is prophage encoded in several NMEC K1 isolates and mediates capsule O-acetylation but has no effect on attachment to or invasion of human brain endothelial cells. Instead, we demonstrate that NeuO provides resistance against K1 bacteriophage attack and that this critical function is regulated by DsdC. This work highlights how a horizontally acquired enzyme that functions in cell-surface modulation can be controlled by an intrinsic regulator to provide a key ecological benefit to an E. coli pathotype

The Generation of Successive Unmarked Mutations and Chromosomal Insertion of Heterologous Genes in Actinobacillus pleuropneumoniae Using Natural Transformation

We have developed a simple method of generating scarless, unmarked mutations in Actinobacillus pleuropneumoniae by exploiting the ability of this bacterium to undergo natural transformation, and with no need to introduce plasmids encoding recombinases or resolvases. This method involves two successive rounds of natural transformation using linear DNA: the first introduces a cassette carrying cat (which allows selection by chloramphenicol) and sacB (which allows counter-selection using sucrose) flanked by sequences to either side of the target gene; the second transformation utilises the flanking sequences ligated directly to each other in order to remove the cat-sacB cassette. In order to ensure efficient uptake of the target DNA during transformation, A. pleuropneumoniae uptake sequences are added into the constructs used in both rounds of transformation. This method can be used to generate multiple successive deletions and can also be used to introduce targeted point mutations or insertions of heterologous genes into the A. pleuropneumoniae chromosome for development of live attenuated vaccine strains. So far, we have applied this method to highly transformable isolates of serovars 8 (MIDG2331), which is the most prevalent in the UK, and 15 (HS143). By screening clinical isolates of other serovars, it should be possible to identify other amenable strains