The vascular geometry of the choriocapillaris is associated with spatially heterogeneous molecular exchange with the outer retina.

Vision relies on the continuous exchange of material between the photoreceptors, retinal pigment epithelium and choriocapillaris, a dense microvascular bed located underneath the outer retina. The anatomy and physiology of the choriocapillaris and their association with retinal homeostasis have proven difficult to characterize, mainly because of the unusual geometry of this vascular bed. By analysing tissue dissected from 81 human eyes, we show that the thickness of the choriocapillaris does not vary significantly over large portions of the macula or with age. Assessments of spatial variations in the anatomy of the choriocapillaris in three additional human eyes indicate that the location of arteriolar and venular vessels connected to the plane of the choriocapillaris is non-random, and that venular insertions cluster around arteriolar ones. Mathematical models built upon these anatomical analyses reveal that the choriocapillaris contains regions where the transport of passive elements is dominated by diffusion, and that these diffusion-limited regions represent areas of reduced exchange with the outer retina. The width of diffusion-limited regions is determined by arterial flow rate and the relative arrangement of arteriolar and venular insertions. These analyses demonstrate that the apparent complexity of the choriocapillaris conceals a fine balance between several anatomical and functional parameters to effectively support homeostasis of the outer retina. KEY POINTS: The choriocapillaris is the capillary bed supporting the metabolism of photoreceptors and retinal pigment epithelium, two critical components of the visual system located in the outer part of the retina. The choriocapillaris has evolved a planar multipolar vascular geometry that differs markedly from the branched topology of most vasculatures in the human body. Here, we report that this planar multipolar vascular geometry is associated with spatially heterogenous molecular exchange between choriocapillaris and outer retina. Our data and analyses highlight a necessary balance between choriocapillaris anatomical and functional parameters to effectively support homeostasis of the outer retina.

Chronic Granulomatous Dacryoadenitis Associated With Pleomorphic Adenoma.

Chronic granulomatous inflammation occurs rarely alongside pleomorphic adenomas of the major salivary glands but would not appear to have been reported with lacrimal gland adenomas. We describe the clinical features, imaging and histopathology for 4 patients (3 female) who had granulomatous inflammation alongside lacrimal gland adenomas-the patients being with age 39, 44, 48, and 53 years at time of surgery. One patient had an asymptomatic lesion found on imaging, and the other 3 had symptoms for between 3 years and several decades. Conjecturally, this rare phenomenon might arise from an inflammatory response to leakage of secretions from the ductular components of the glands.

Extensive Sclerosis ("mummification") in Lacrimal Gland Pleomorphic Adenoma May Indicate Neighboring Malignant Transformation.

Salivary gland pleomorphic adenomas not uncommonly show extensive sclerosis ("dense hyalinization," "coagulative necrosis," or "mummification"), which arise spontaneously or after prior surgery, and this change is considered a high-risk factor for malignant transformation of benign salivary adenomas. While minor hyalinization is common in lacrimal gland adenomas, massive sclerosis-where almost all (90% or more) of the tumor is replaced by an amorphous hyaline material-is extremely rare. Four patients (2 males) are described in whom, despite not having an acute inflammatory episode, their lacrimal gland tumor showed marked sclerotic necrosis within the majority of either the benign or malignant parts of the tumor. Three tumors had evidence of malignant change, 2 to adenocarcinoma, and 1 to mucoepidermoid carcinoma, but none of the malignant areas showed perineural or endovascular invasion. Extensive sclerosis in association with a lacrimal gland pleomorphic adenoma (LGPA) is rare (4/110 of the cases) and-as with salivary adenomas-appears to be associated with a high chance of associated malignant change. Both the surgeon and the histopathologist should regard extensive sclerotic necrosis as a harbinger for malignant change in association with benign pleomorphic adenomas of the lacrimal gland.

Atypical, Extremely Rapid Growth of Pediatric Orbital Neurilemmoma.

Solitary orbital neurilemmoma-a benign tumor of Schwann cells in a peripheral nerve-sheath-are typically very slow growing and present in middle age; in the absence of neurofibromatosis, they are almost never seen in childhood. We describe the clinical presentation, imaging, pathology, and management of this tumor in a 12 years old-the tumor growing extremely rapidly over 5 months and without any evidence of cystic degeneration, hemorrhage, or sarcomatous features. The possibility of tumor growth having been accelerated by prior biopsy is discussed.

Features of ectopic lymphoid-like structures in human uveitis.

Persistent non-infectious uveitis has a significant morbidity, but the extent to which this is accompanied by inflammation driven remodelling of the tissue is unclear. To address this question, we studied a series of samples selected from two ocular tissue repositories and identified 15 samples with focal infiltration. Eleven of fifteen contained lymphocytes, both B cells (CD20 positive) and T cells (CD3 positive). In 20% of the samples there was evidence of ectopic lymphoid like structures with focal aggregations of B cells and T cells, segregated into anatomically different adjacent zones. To investigate inflammation in the tissue, an analysis of 520 immune relevant transcripts was carried out and 24 genes were differentially upregulated, compared with control tissue. Two of these (CD14 and fibronectin) were increased in ocular inflammation compared to control immune tissue (tonsil). We demonstrate that in a significant minority of patients, chronic persistent uveitis leads to dysregulation of ocular immune surveillance, characterized by the development of areas of local ectopic lymphoid like structures, which may be a target for therapeutic intervention directed at antibody producing cells.

Lipoprotein-associated phospholipase A2 (Lp-PLA2) as a therapeutic target to prevent retinal vasopermeability during diabetes.

Lipoprotein-associated phospholipase A2 (Lp-PLA2) hydrolyses oxidized low-density lipoproteins into proinflammatory products, which can have detrimental effects on vascular function. As a specific inhibitor of Lp-PLA2, darapladib has been shown to be protective against atherogenesis and vascular leakage in diabetic and hypercholesterolemic animal models. This study has investigated whether Lp-PLA2 and its major enzymatic product, lysophosphatidylcholine (LPC), are involved in blood-retinal barrier (BRB) damage during diabetic retinopathy. We assessed BRB protection in diabetic rats through use of species-specific analogs of darapladib. Systemic Lp-PLA2 inhibition using SB-435495 at 10 mg/kg (i.p.) effectively suppressed BRB breakdown in streptozotocin-diabetic Brown Norway rats. This inhibitory effect was comparable to intravitreal VEGF neutralization, and the protection against BRB dysfunction was additive when both targets were inhibited simultaneously. Mechanistic studies in primary brain and retinal microvascular endothelial cells, as well as occluded rat pial microvessels, showed that luminal but not abluminal LPC potently induced permeability, and that this required signaling by the VEGF receptor 2 (VEGFR2). Taken together, this study demonstrates that Lp-PLA2 inhibition can effectively prevent diabetes-mediated BRB dysfunction and that LPC impacts on the retinal vascular endothelium to induce vasopermeability via VEGFR2. Thus, Lp-PLA2 may be a useful therapeutic target for patients with diabetic macular edema (DME), perhaps in combination with currently administered anti-VEGF agents.

Nasal and Lacrimal Sac Histopathology in Patients With Systemic Sarcoidosis Undergoing External Lacrimal Drainage Surgery.

PURPOSE: To review the histological findings in the lacrimal sac and nasal mucosa from patients with sarcoidosis undergoing external lacrimal drainage surgery. METHODS: All patients undergoing external dacryocystorhinostomy at Moorfields Eye Hospital with a known history of sarcoidosis had biopsies taken from the lacrimal sac and/or nasal mucosa during surgery. These patients were identified from databases at Moorfields Eye Hospital and the Institute of Ophthalmology, and their clinical notes were reviewed retrospectively for intraoperative findings with a view to identifying common trends. The histological findings of each biopsy were reviewed and classified as showing granulomas, nongranulomatous inflammation, or nonspecific fibrosis. RESULTS: Forty patients (29 females; 72%) were known to have systemic sarcoidosis prior to surgery, and they underwent 60 external dacryocystorhinostomies. Paired histological samples were available from 49/60 (82%) procedures, nasal biopsies alone in 3 dacryocystorhinostomies (5%), and solely lacrimal sac biopsies in 8 (13%). The main site of systemic sarcoidosis was pulmonary involvement (19 patients; 48%). Recorded operative findings included 9 large lacrimal sac mucoceles (29%), a "thick" (26%) or "inflamed" (9.7%) lacrimal sac mucosa, and "thick" (36%) or "friable" (32%) nasal mucosa. Noncaseating granulomas were identified in 34/57 (60%) sacs, and 45/52 (87%) nasal tissues-this being in 31/49 (63%) of paired tissues. Chronic inflammation, without granulomas, was present in 20/57 (35%) lacrimal sacs but only in 5/52 (9.6%) of nasal biopsies. CONCLUSIONS: In patients with sarcoidosis undergoing external dacryocystorhinostomy, the characteristic histological feature-noncaseating granulomas-is present in most patients' lacrimal sac mucosa and in almost all of their nasal mucosae. The lacrimal sac and nasal mucosa often appears abnormal-thickened or friable-during surgery.

Detection of mutations in SF3B1, EIF1AX and GNAQ in primary orbital melanoma by candidate gene analysis.

BACKGROUND: Ocular melanoma is a rare but often deadly malignancy that arises in the uvea (commonest primary site), conjunctiva or the orbit. Primary orbital melanoma (POM) is exceedingly rare, with approximately 60 cases reported to date. Despite recent advances in our understanding of the genetics of primary uveal and conjunctival melanomas, this information is lacking for POM. METHODS: DNA was extracted from 12 POM tissues, with matched germline DNA (where available). MLPA was conducted to detect chromosomal alterations and Sanger sequencing used to identify point mutations in candidate melanoma driver genes (BRAF, NRAS, KRAS, GNA11, GNAQ), and other genes implicated in melanoma prognosis (EIF1AX, SF3B1). Immunohistochemistry was performed to analyse BAP1 nuclear expression. RESULTS: MLPA detected copy number alterations in chromosomes 1p, 3, 6 and 8. Sequencing of melanoma driver genes revealed GNAQ (p.Q209L) mutations in two samples; although it is possible that these samples represent extraocular spread of an occult uveal melanoma. A recurrent mutation in SF3B1 (p.R625H) was observed in indolent, but not aggressive, tumours; a mutation in EIF1AX (p.N4S) was detected in one patient with non-aggressive disease. CONCLUSIONS: EIF1AX and SF3B1 mutations appear have a role in determining the clinical course of POM and detection of these changes could have clinical significance. Further in depth analysis of this rare group using differing 'omic technologies will provide novel insights into tumour pathogenesis.

Mathematical models of retinitis pigmentosa: The oxygen toxicity hypothesis.

The group of genetically mediated diseases, known collectively as retinitis pigmentosa (RP), cause retinal degeneration and, hence, loss of vision. The most common inherited retinal degeneration, RP is currently untreatable. The retina detects light using cells known as photoreceptors, of which there are two types: rods and cones. In RP, genetic mutations cause patches of photoreceptors to degenerate and typically directly affect either rods or cones, but not both. During disease progression, degenerate patches spread and the unaffected photoreceptor type also begins to degenerate. The cause underlying these phenomena is currently unknown. The oxygen toxicity hypothesis proposes that secondary photoreceptor loss is due to hyperoxia (toxically high oxygen levels), which results from the decrease in oxygen uptake following the initial loss of photoreceptors. In this paper, we construct mathematical models, formulated as 1D systems of partial differential equations, to investigate this hypothesis. Using a combination of numerical simulations, asymptotic analysis and travelling wave analysis, we find that degeneration may spread due to hyperoxia, and generate spatio-temporal patterns of degeneration similar to those seen in vivo. We determine the conditions under which a degenerate patch will spread and show that the wave speed of degeneration is a monotone decreasing function of the local photoreceptor density. Lastly, the effects of treatment with antioxidants and trophic factors, and of capillary loss, upon the dynamics of photoreceptor loss and recovery are considered.

P2X7R modulation of visually evoked synaptic responses in the retina.

P2X7Rs are distributed throughout all layers of the retina, and thus, their localisation on various cell types puts into question their specific site(s) of action. Using a dark-adapted, ex vivo mouse retinal whole mount preparation, the present study aimed to characterise the effect of P2X7R activation on light-evoked, excitatory RGC ON-field excitatory post-synaptic potentials (fEPSPs) and on outer retinal electroretinogram (ERG) responses under comparable conditions. The pharmacologically isolated NMDA receptor-mediated RGC ON-fEPSP was reduced in the presence of BzATP, an effect which was significantly attenuated by A438079 and other selective P2X7R antagonists A804598 or AF27139. In physiological Krebs medium, BzATP induced a significant potentiation of the ERG a-wave, with a concomitant reduction in the b-wave and the power of the oscillatory potentials. Conversely, in the pharmacologically modified Mg2+-free perfusate, BzATP reduced both the a-wave and b-wave. The effects of BzATP on the ERG components were suppressed by A438079. A role for P2X7R function in visual processing in both the inner and outer retina under physiological conditions remains controversial. The ON-fEPSP was significantly reduced in the presence of A804598 but not by A438079 or AF27139. Furthermore, A438079 did not have any effect on the ERG components in physiological Krebs but potentiated and reduced the a-wave and b-wave, respectively, when applied to the pharmacologically modified medium. Therefore, activation of P2X7Rs affects the function in the retinal ON pathway. The presence of a high concentration of extracellular ATP would most likely contribute to the modulation of visual transmission in the retina in the pathophysiological microenvironment.

Retinal oxygen distribution and the role of neuroglobin.

The retina is the tissue layer at the back of the eye that is responsible for light detection. Whilst equipped with a rich supply of oxygen, it has one of the highest oxygen demands of any tissue in the body and, as such, supply and demand are finely balanced. It has been suggested that the protein neuroglobin (Ngb), which is found in high concentrations within the retina, may help to maintain an adequate supply of oxygen via the processes of transport and storage. We construct mathematical models, formulated as systems of reaction-diffusion equations in one-dimension, to test this hypothesis. Numerical simulations show that Ngb may play an important role in oxygen transport, but not in storage. Our models predict that the retina is most susceptible to hypoxia in the regions of the photoreceptor inner segment and inner plexiform layers, where Ngb has the potential to prevent hypoxia and increase oxygen uptake by 30-40 %. Analysis of a simplified model confirms the utility of Ngb in transport and shows that its oxygen affinity ([Formula: see text] value) is near optimal for this process. Lastly, asymptotic analysis enables us to identify conditions under which the piecewise linear and quadratic approximations to the retinal oxygen profile, used in the literature, are valid.

Resistance to the most common optic neuropathy is associated with systemic mitochondrial efficiency.

Glaucomatous optic neuropathy, an important neurodegenerative condition and the commonest optic neuropathy in humans, is the leading cause of irreversible blindness worldwide. Its prevalence and incidence increase exponentially with ageing and raised intraocular pressure (IOP). Using glaucomatous optic neuropathy as an exemplar for neurodegeneration, this study investigates putative factors imparting resistance to neurodegeneration. Systemic mitochondrial function, oxidative stress and vascular parameters were compared from isolated lymphocytes, whole blood and urine samples between 30 patients who have not developed the neuropathy despite being exposed for many years to very high IOP ('resistant'), 30 fast deteriorating glaucoma patients despite having low IOP ('susceptible'), and 30 age-similar controls. We found that 'resistant' individuals showed significantly higher rates of ADP phosphorylation by mitochondrial respiratory complexes I, II and IV, hyperpolarised mitochondrial membrane potential, higher levels of mitochondrial DNA, and enhanced capacity to deal with cytosolic calcium overload and exogenous oxidative stress, as compared to both controls and glaucoma patients. While it has been known for some years that mitochondrial dysfunction is implicated in neurodegeneration, this study provides a fresh perspective to the field of neurodegeneration by providing, for the first time, evidence that systemic mitochondrial efficiency above normal healthy levels is associated with an enhanced ability to withstand optic nerve injury. These results demonstrate the importance of cellular bioenergetics in glaucomatous disease progression, with potential relevance for other neurodegenerative disorders, and raise the possibility for new therapeutic targets in the field of neurodegeneration.

Congenital respiratory epithelial cysts of the orbit: a rare cause of major orbital impairment.

Congenital respiratory epithelial cysts of the orbit are rare lesions with few reported cases. Extensive disease may cause an orbital apex syndrome, resulting in significant visual loss. Two such cases were described, the risk factors associated with surgical morbidity were identified, and the embryological origins of these cysts were reviewed.

Daily Light Onset and Plasma Membrane Tethers Regulate Mitochondria Redistribution within the Retinal Pigment Epithelium.

The retinal pigment epithelium (RPE) is an essential component of the retina that plays multiple roles required to support visual function. These include light onset- and circadian rhythm-dependent tasks, such as daily phagocytosis of photoreceptor outer segments. Mitochondria provide energy to the highly specialized and energy-dependent RPE. In this study, we examined the positioning of mitochondria and how this is influenced by the onset of light. We identified a population of mitochondria that are tethered to the basal plasma membrane pre- and post-light onset. Following light onset, mitochondria redistributed apically and interacted with melanosomes and phagosomes. In a choroideremia mouse model that has regions of the RPE with disrupted or lost infolding of the plasma membrane, the positionings of only the non-tethered mitochondria were affected. This provides evidence that the tethering of mitochondria to the plasma membrane plays an important role that is maintained under these disease conditions. Our work shows that there are subpopulations of RPE mitochondria based on their positioning after light onset. It is likely they play distinct roles in the RPE that are needed to fulfil the changing cellular demands throughout the day.

Papilloma development and long-term ciclosporin use in chronic ocular allergy with associated keratoconus.

PURPOSE: Conjunctival papillomata are squamous epithelial tumors with a strong association with human papilloma virus (HPV) types 6 and 11. They are benign conjunctival tumors that can be treated by surgical excision. We report a case where topical immunosuppressive therapy modified the local T-cell immunity in the conjunctiva resulting in papilloma development in a patient with keratoconus and a strong atopic history. METHODS: A case report of a 44-year-old man with a history of severe ocular and generalized atopy is presented. We present the problems encountered in management of his severe ocular allergy and how these impeded the management of his keratoconus. RESULTS: Conventional antiallergy topical medication was not producing symptom relief in this patient, and so topical immunosuppression was commenced using ciclosporin ointment 0.2%. This therapy modified the local T-cell immunity in the conjunctiva resulting in the development of papillomata which contributed to the intolerance of contact lens wear for visual rehabilitation of the keratoconus in the patient. These lesions were surgically removed but typically recurred and required further surgical excision. Adjunct cryotherapy was also performed at the time of the surgery to try to stem the recurrence of the papillomas. CONCLUSIONS: To the best of our knowledge and following a review of the published literature using key databases that include Medline and PubMed, this is the first report confirming the development of conjunctival papillomas secondary to HPV type 6 in a ciclosporin-treated patient.

Whole-cell energy modeling reveals quantitative changes of predicted energy flows in RAS mutant cancer cell lines.

Cellular utilization of available energy flows to drive a multitude of forms of cellular "work" is a major biological constraint. Cells steer metabolism to address changing phenotypic states but little is known as to how bioenergetics couples to the richness of processes in a cell as a whole. Here, we outline a whole-cell energy framework that is informed by proteomic analysis and an energetics-based gene ontology. We separate analysis of metabolic supply and the capacity to generate high-energy phosphates from a representation of demand that is built on the relative abundance of ATPases and GTPases that deliver cellular work. We employed mouse embryonic fibroblast cell lines that express wild-type KRAS or oncogenic mutations and with distinct phenotypes. We observe shifts between energy-requiring processes. Calibrating against Seahorse analysis, we have created a whole-cell energy budget with apparent predictive power, for instance in relation to protein synthesis.

RETINAL NEURONAL ECTOPIA: A NEW ENTITY IN THE DIFFERENTIAL DIAGNOSIS OF RETINOBLASTOMA.

BACKGROUND: To present a rare retinal disorder that should be considered in the differential diagnosis of retinoblastoma. METHODS: A 2-year-old boy presented with left ocular discomfort, leukocoria, and a left divergent squint. Examination of the left eye revealed abnormalities in the anterior segment, and fundoscopy showed an irregular white calcified mass with fibrosis and traction toward the lens. As the ocular discomfort worsened, enucleation of the left eye was performed. RESULTS: Histopathological and immunohistochemical assessment of the enucleated eye established the diagnosis of retinal neuronal ectopia. CONCLUSION: We believe that this case is unique in the human retina and highlights the need for specialist differential diagnosis. Although rare, retinal neuronal ectopia should be considered in the differential diagnosis of retinoblastoma.

Analysis of Ras-effector interaction competition in large intestine and colorectal cancer context.

Cancer is the second leading cause of death globally, and colorectal cancer (CRC) is among the five most common cancers. The small GTPase KRAS is an oncogene that is mutated in ~30% of all CRCs. Pharmacological treatments of CRC are currently unsatisfactory, but much hope rests on network-centric approaches to drug development and cancer treatment. These approaches, however, require a better understanding of how networks downstream of Ras oncoproteins are connected in a particular tissue context - here colon and CRC. Previously we have shown that competition for binding to a 'hub' protein, such as Ras, can induce a rewiring of signal transduction networks. In this study, we analysed 56 established and predicted effectors that contain a structural domain with the potential ability to bind to Ras oncoproteins and their link to pathways coordinating intestinal homoeostasis and barrier function. Using protein concentrations in colon tissue and Ras-effector binding affinities, a computational network model was generated that predicted how effectors differentially and competitively bind to Ras in colon context. The model also predicted both qualitative and quantitative changes in Ras-effector complex formations with increased levels of active Ras - to simulate its upregulation in cancer - simply as an emergent property of competition for the same binding interface on the surface of Ras. We also considered how the number of Ras-effector complexes at the membrane can be increased by additional domains present in some effectors that are recruited to the membrane in response to specific conditions (inputs/stimuli/growth factors) in colon context and CRC.

Combining Gene-Disease Associations with Single-Cell Gene Expression Data Provides Anatomy-Specific Subnetworks in Age-Related Macular Degeneration.

Background: Age-related macular degeneration (AMD) is the most common cause of visual impairment in the developed world. Despite some treatment options for late AMD, there is no intervention that blocks early AMD proceeding to the late and blinding forms. This is partly due to the lack of precise drug targets, despite great advances in genetics, epidemiology, and protein-protein interaction (PPI) networks proposed to be driving the disease pathology. A systems approach to narrow down PPI networks to specific protein drug targets would provide new therapeutic options. Materials and Methods: In this study we analyzed single cell RNAseq (RNA sequencing) datasets of 17 cell types present in choroidal, retinal pigment epithelium (RPE), and neural retina (NR) tissues to explore if a more granular analysis incorporating different cell types exposes more specific pathways and relationships. Furthermore, we developed a novel and systematic gene ontology database (SysGO) to explore if a subcellular classification of processes will further enhance the understanding of the pathogenesis of this complex disorder and its comorbidities with other age-related diseases. Results: We found that 57% of the AMD (risk) genes are among the top 25% expressed genes in ∼1 of the 17 choroidal/RPE/NR cell types, and 9% were among the top 1% of expressed genes. Using SysGO, we identified an enrichment of AMD genes in cell membrane and extracellular anatomical locations, and we found both functional enrichments (e.g., cell adhesion) and cell types (e.g., fibroblasts, microglia) not previously associated with AMD pathogenesis. We reconstructed PPI networks among the top expressed AMD genes for all 17 choroidal/RPE/NR cell types, which provides molecular and anatomical definitions of AMD phenotypes that can guide therapeutic approaches to target this complex disease. Conclusion: We provide mechanism-based AMD endophenotypes that can be exploited in vitro, using computational models and for drug discovery/repurposing.

Repository of proposed pathways and protein-protein interaction networks in age-related macular degeneration.

Age-related macular degeneration (AMD) is one of the commonest causes of sight loss in the elderly population and to date there is no intervention that slows or prevents early AMD disease progressing to blinding neovascularization or geographic atrophy. AMD is a complex disease and factors proposed to contribute to the development and progression of disease include aging, genetics, epigenetics, oxidative stress, pro-inflammatory state, and life-style factors such as smoking, alcohol, and high fat diet. Here, we generate a knowledge repository of pathways and protein-protein interaction (PPI) networks likely to be implicated in AMD pathogenesis, such as complement activation, lipid trafficking and metabolism, vitamin A cycle, oxidative stress, proteostasis, bioenergetics, autophagy/mitophagy, extracellular matrix (ECM) turnover, and choroidal vascular dropout. Two disctinct clusters ermerged from the networks for parainflamation and ECM homeostasis, which may represent two different disease modules underlying AMD pathology. Our analyses also suggest that the disease manifests primarily in RPE/choroid and less in neural retina. The use of standardized syntax when generating maps of these biological processes (SBGN standard) and networks (PSI standard) enables visualization of complex information in graphical programs such as CellDesigner and Cytoscape and enhances reusability and extension of data. The ability to focus onto subnetworks, multiple visualizations and simulation options will enable the AMD research community to computationally model subnetworks or to test experimentally new hypotheses arising from connectivities in the AMD pathway map.