On the importance of integrating comparative anatomy and One Health perspectives in anatomy education.

As a result of many factors, including climate change, unrestricted population growth, widespread deforestation and intensive agriculture, a new pattern of diseases in humans is emerging. With increasing encroachment by human societies into wild domains, the interfaces between human and animal ecosystems are gradually eroding. Such changes have led to zoonoses, vector-borne diseases, infectious diseases and, most importantly, the emergence of antimicrobial-resistant microbial strains as challenges for human health. Now would seem to be an opportune time to revisit old concepts of health and redefine some of these in the light of emerging challenges. The One Health concept addresses some of the demands of modern medical education by providing a holistic approach to explaining diseases that result from a complex set of interactions between humans, environment and animals, rather than just an amalgamation of isolated signs and symptoms. An added advantage is that the scope of One Health concepts has now expanded to include genetic diseases due to advancements in omics technology. Inspired by such ideas, a symposium was organised as part of the 19th International Federation of Associations of Anatomists (IFAA) Congress (August 2019) to investigate the scope of One Health concepts and comparative anatomy in contemporary medical education. Speakers with expertise in both human and veterinary anatomy participated in the symposium and provided examples where these two disciplines, which have so far evolved largely independent of each other, can collaborate for mutual benefit. Finally, the speakers identified some key concepts of One Health that should be prioritised and discussed the diverse opportunities available to integrate these priorities into a broader perspective that would attempt to explain and manage diseases within the scopes of human and veterinary medicine.

Pharmacological restoration of visual function in a zebrafish model of von-Hippel Lindau disease.

Von Hippel-Lindau (VHL) syndrome is a rare, autosomal dominant disorder, characterised by hypervascularised tumour formation in multiple organ systems. Vision loss associated with retinal capillary hemangioblastomas remains one of the earliest complications of VHL disease. The mortality of Vhl-/- mice in utero restricted modelling of VHL disease in this mammalian model. Zebrafish harbouring a recessive germline mutation in the vhl gene represent a viable, alternative vertebrate model to investigate associated ocular loss-of-function phenotypes. Previous studies reported neovascularisation of the brain, eye and trunk together with oedema in the vhl-/- zebrafish eye. In this study, we demonstrate vhl-/- zebrafish almost entirely lack visual function. Furthermore, hyaloid vasculature networks in the vhl-/- eye are improperly formed and this phenotype is concomitant with development of an ectopic intraretinal vasculature. Sunitinib malate, a multi tyrosine kinase inhibitor, market authorised for cancer, reversed the ocular behavioural and morphological phenotypes observed in vhl-/- zebrafish. We conclude that the zebrafish vhl gene contributes to an endogenous molecular barrier that prevents development of intraretinal vasculature, and that pharmacological intervention with sunitinib can improve visual function and hyaloid vessel patterning while reducing abnormally formed ectopic intraretinal vessels in vhl-/- zebrafish.

A Quininib Analogue and Cysteinyl Leukotriene Receptor Antagonist Inhibits Vascular Endothelial Growth Factor (VEGF)-independent Angiogenesis and Exerts an Additive Antiangiogenic Response with Bevacizumab.

Excess blood vessel growth contributes to the pathology of metastatic cancers and age-related retinopathies. Despite development of improved treatments, these conditions are associated with high economic costs and drug resistance. Bevacizumab (Avastin®), a monoclonal antibody against vascular endothelial growth factor (VEGF), is used clinically to treat certain types of metastatic cancers. Unfortunately, many patients do not respond or inevitably become resistant to bevacizumab, highlighting the need for more effective antiangiogenic drugs with novel mechanisms of action. Previous studies discovered quininib, an antiangiogenic small molecule antagonist of cysteinyl leukotriene receptors 1 and 2 (CysLT1 and CysLT2). Here, we screened a series of quininib analogues and identified a more potent antiangiogenic novel chemical entity (IUPAC name (E)-2-(2-quinolin-2-yl-vinyl)-benzene-1,4-diol HCl) hereafter designated Q8. Q8 inhibits developmental angiogenesis in Tg(fli1:EGFP) zebrafish and inhibits human microvascular endothelial cell (HMEC-1) proliferation, tubule formation, and migration. Q8 elicits antiangiogenic effects in a VEGF-independent in vitro model of angiogenesis and exerts an additive antiangiogenic response with the anti-VEGF biologic bevacizumab. Cell-based receptor binding assays confirm that Q8 is a CysLT1 antagonist and is sufficient to reduce cellular levels of NF-κB and calpain-2 and secreted levels of the proangiogenic proteins intercellular adhesion molecule-1, vascular cell adhesion protein-1, and VEGF. Distinct reductions of VEGF by bevacizumab explain the additive antiangiogenic effects observed in combination with Q8. In summary, Q8 is a more effective antiangiogenic drug compared with quininib. The VEGF-independent activity coupled with the additive antiangiogenic response observed in combination with bevacizumab demonstrates that Q8 offers an alternative therapeutic strategy to combat resistance associated with conventional anti-VEGF therapies.

Brain-Derived Neurotrophic Factor as a Treatment Option for Retinal Degeneration.

This review discusses the therapeutic potential of brain-derived neurotrophic factor (BDNF) for retinal degeneration. BDNF, nerve growth factor (NGF), neurotrophin 3 (NT-3) and NT-4/NT-5 belong to the neurotrophin family. These neuronal modulators activate a common receptor and a specific tropomyosin-related kinase (Trk) receptor. BDNF was identified as a photoreceptor protectant in models of retinal degeneration as early as 1992. However, development of effective therapeutics that exploit this pathway has been difficult due to challenges in sustaining therapeutic levels in the retina.

Phenotype-based Discovery of 2-[(E)-2-(Quinolin-2-yl)vinyl]phenol as a Novel Regulator of Ocular Angiogenesis.

Retinal angiogenesis is tightly regulated to meet oxygenation and nutritional requirements. In diseases such as proliferative diabetic retinopathy and neovascular age-related macular degeneration, uncontrolled angiogenesis can lead to blindness. Our goal is to better understand the molecular processes controlling retinal angiogenesis and discover novel drugs that inhibit retinal neovascularization. Phenotype-based chemical screens were performed using the ChemBridge Diverset(TM)library and inhibition of hyaloid vessel angiogenesis in Tg(fli1:EGFP) zebrafish. 2-[(E)-2-(Quinolin-2-yl)vinyl]phenol, (quininib) robustly inhibits developmental angiogenesis at 4-10 µmin zebrafish and significantly inhibits angiogenic tubule formation in HMEC-1 cells, angiogenic sprouting in aortic ring explants, and retinal revascularization in oxygen-induced retinopathy mice. Quininib is well tolerated in zebrafish, human cell lines, and murine eyes. Profiling screens of 153 angiogenic and inflammatory targets revealed that quininib does not directly target VEGF receptors but antagonizes cysteinyl leukotriene receptors 1 and 2 (CysLT1-2) at micromolar IC50values. In summary, quininib is a novel anti-angiogenic small-molecule CysLT receptor antagonist. Quininib inhibits angiogenesis in a range of cell and tissue systems, revealing novel physiological roles for CysLT signaling. Quininib has potential as a novel therapeutic agent to treat ocular neovascular pathologies and may complement current anti-VEGF biological agents.

Current and emerging therapies for ocular neovascularisation.

Ocular neovascularisation (ONV) is a pathological feature of many human blinding diseases. Here, we review current pharmacological therapies for these disorders and highlight emerging therapies in clinical trial for ONV. Finally, we discuss desirable characteristics of future ONV therapies, including innovative strategies for novel delivery to the back of the eye.

Targeting the PI3K/Akt/mTOR pathway in ocular neovascularization.

Ocular neovascularization, a common pathological feature of wet age-related macular degeneration (AMD), proliferative and diabetic retinopathy (PDR) leads to fluid and blood leakage, scar formation and ultimately blindness. Elucidation of vascular endothelial growth factor (VEGF) as a key mediator of angiogenesis led to clinically approved anti-VEGF agents. However, these drugs are associated with adverse side-effects, high costs and extensive clinical burden. The phosphatidylinositol-3-kinase (PI3K) pathway is an alternative therapeutic target in angiogenic diseases. The PI3K/Akt/mTOR pathway orchestrates an array of normal cellular processes, including growth, survival and angiogenesis. Here, we review the potential of targeting the PI3K pathway, to treat ocular neovascularization.

The Role of Mitochondria in Optic Atrophy With Autosomal Inheritance.

Optic atrophy (OA) with autosomal inheritance is a form of optic neuropathy characterized by the progressive and irreversible loss of vision. In some cases, this is accompanied by additional, typically neurological, extra-ocular symptoms. Underlying the loss of vision is the specific degeneration of the retinal ganglion cells (RGCs) which form the optic nerve. Whilst autosomal OA is genetically heterogenous, all currently identified causative genes appear to be associated with mitochondrial organization and function. However, it is unclear why RGCs are particularly vulnerable to mitochondrial aberration. Despite the relatively high prevalence of this disorder, there are currently no approved treatments. Combined with the lack of knowledge concerning the mechanisms through which aberrant mitochondrial function leads to RGC death, there remains a clear need for further research to identify the underlying mechanisms and develop treatments for this condition. This review summarizes the genes known to be causative of autosomal OA and the mitochondrial dysfunction caused by pathogenic mutations. Furthermore, we discuss the suitability of available in vivo models for autosomal OA with regards to both treatment development and furthering the understanding of autosomal OA pathology.

Protocol for a preclinical systematic review and meta-analysis of pharmacological targeting of peroxisome proliferator-activated receptors in experimental renal injury.

INTRODUCTION: Impaired lipid metabolism in the renal tubule plays a prominent role in the progression of renal fibrosis following acute kidney injury (AKI) and in chronic kidney disease (CKD). Peroxisome proliferator-activated receptors (PPARs) are promising druggable targets to mitigate renal fibrosis by redirecting metabolism, including restoration of fatty acid oxidation (FAO) capacity. We aim to synthesise evidence from preclinical studies of pharmacological PPAR targeting in experimental renal injury, and inform the design of future studies evaluating PPAR-mediated restoration of FAO in AKI and CKD. METHODS AND ANALYSIS: Studies reporting on the impact of pharmacological PPAR modulation in animal models of renal injury will be collected from MEDLINE (Ovid), Embase and Web of Science databases. Predefined eligibility criteria will exclude studies testing medications which are not specific ligands of one or more PPARs and studies involving multimodal pharmacological treatment. The Systematic Review Centre for Laboratory Animal Experimentation risk of bias tool and Collaborative Approach to Meta-Analysis and Review of Animal Experimental Studies checklist will be used to assess quality of the included studies. Data extraction will be followed by a narrative synthesis of the data and meta-analysis where feasible. Analysis will be performed separately for AKI, CKD and renal transplant models. Subgroup analyses will be performed based on study design characteristics, PPAR isotype(s) targeted, and classes of PPAR-targeting medications used. Risk of publication bias will be assessed using funnel plotting, Egger's regression and trim-and-fill analysis. ETHICS AND DISSEMINATION: Ethical approval is not required. Findings will be published in a peer-reviewed journal and presented at scientific meetings. PROSPERO REGISTRATION NUMBER: CRD42021265550.

Curcumin Sensitizes Kidney Cancer Cells to TRAIL-Induced Apoptosis via ROS Mediated Activation of JNK-CHOP Pathway and Upregulation of DR4.

Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL), is a selective anticancer cytokine capable of exerting a targeted therapy approach. Disappointingly, recent research has highlighted the development of TRAIL resistance in cancer cells, thus minimising its usefulness in clinical settings. However, several recent studies have demonstrated that cancer cells can be sensitised to TRAIL through the employment of a combinatorial approach, utilizing TRAIL in conjunction with other natural or synthetic anticancer agents. In the present study, the chemo-sensitising effect of curcumin on TRAIL-induced apoptosis in renal carcinoma cells (RCC) was investigated. The results indicate that exposure of kidney cancer ACHN cells to curcumin sensitised the cells to TRAIL, with the combination treatment of TRAIL and curcumin synergistically targeting the cancer cells without affecting the normal renal proximal tubular epithelial cells (RPTEC/TERT1) cells. Furthermore, this combination treatment was shown to induce caspase-dependent apoptosis, inhibition of the proteasome, induction of ROS, upregulation of death receptor 4 (DR4), alterations in mitogen-activated protein kinase (MAPK) signalling and induction of endoplasmic reticulum stress. An in vivo zebrafish embryo study demonstrated the effectiveness of the combinatorial regime to inhibit tumour formation without affecting zebrafish embryo viability or development. Overall, the results arising from this study demonstrate that curcumin has the ability to sensitise TRAIL-resistant ACHN cells to TRAIL-induced apoptosis.

Pyrazinib (P3), [(E)-2-(2-Pyrazin-2-yl-vinyl)-phenol], a small molecule pyrazine compound enhances radiosensitivity in oesophageal adenocarcinoma.

Oesophageal adenocarcinoma (OAC) is an aggressive disease with 5-year survival rates of <20%. Only 20-30% OAC patients show a beneficial response to neoadjuvant therapy. Altered mitochondrial function is linked with radioresistance in OAC. We identified pyrazinib (P3), a pyrazine phenol small molecule drug with anti-angiogenic and anti-metabolic activity in-vivo in zebrafish and in-vitro isogenic models of OAC radioresistance. Pyrazinib (P3) significantly inhibited blood vessel development in zebrafish (p < 0.001). In-vivo in zebrafish and in-vitro in an isogenic model of OAC radioresistance, pyrazinib (P3) significantly reduced measures of oxidative phosphorylation and glycolysis. Pyrazinib (P3) significantly reduced the surviving fraction in OE33P; radiation-sensitive and OE33R; radiation-resistant cells following irradiation. Under hypoxic conditions pyrazinib (P3) significantly reduced OE33R cell survival following 4 Gy irradiation (p = 0.0216). Multiplex ELISA showed significantly higher secreted levels of 9 of 30 detected inflammatory and angiogenic factors in OE33R radioresistant cells compared to OE33P cells; IL-8, IL-4, IL-6, IL-2, IL-12p70, IL-10, MCP-1, IP-10, ICAM (p < 0.05). Pyrazinib (P3) significantly reduced the secretions of IL-6 (p = 0.0006), IL-8 (p = 0.0488), and IL-4 (p = 0.0111) in OE33R cells. Collectively, these findings support further development of pyrazinib (P3) as a novel therapeutic radiosensitiser in OAC.

The UCD nanosafety workshop (03 December 2018): towards developing a consensus on safe handling of nanomaterials within the Irish university labs and beyond - a report.

Careful handling of the nanomaterials (NMs) in research labs is crucial to ensure a safe working environment. As the largest university in Ireland, University College Dublin (UCD) has invested significant resources to update researchers working with NMs. Due to sizes often <100 nm, the NMs including nanoparticles, harbor unprecedented materialistic properties, for example, enhanced reactivity, conductivity, fluorescence, etc. which albeit conferring the NMs an edge over bulk materials regarding the applied aspects; depending on the dose, also render them to be toxic. Thus, a set of regulatory guidelines have emerged regarding safe handling of the NMs within occupational set-ups. Unfortunately, the current regulations based on the toxic chemicals and carcinogens are often confusing, lack clarity, and difficult to apply for the NMs. As a research-intensive university, a diverse range of research activities occur within the UCD labs, and it is difficult, at times impossible, for the UCD Safety, Insurance, Operational Risk & Compliance (SIRC) office to develop a set of common guidelines and cater throughout all its labs conducting research with the NMs. Hence, a necessity for dialog and exchange of ideas was felt across the UCD which encouraged the researchers including early stage researchers (e.g. PhDs, Postdocs) from multiple schools to participate in a workshop held on the 03 December 2018. The workshop tried to follow a pragmatic approach, where apart from discussing both the in vitro and in vivo scenarios, practical cases simulating situations faced frequently in the labs were discussed. This report summarizes the findings made during the workshop by this emerging critical mass in UCD.

A Brain-Derived Neurotrophic Factor Mimetic Is Sufficient to Restore Cone Photoreceptor Visual Function in an Inherited Blindness Model.

Controversially, histone deacetylase inhibitors (HDACi) are in clinical trial for the treatment of inherited retinal degeneration. Utilizing the zebrafish dye ucd6 model, we determined if treatment with HDACi can rescue cone photoreceptor-mediated visual function. dye exhibit defective visual behaviour and retinal morphology including ciliary marginal zone (CMZ) cell death and decreased photoreceptor outer segment (OS) length, as well as gross morphological defects including hypopigmentation and pericardial oedema. HDACi treatment of dye results in significantly improved optokinetic (OKR) (~43 fold, p < 0.001) and visualmotor (VMR) (~3 fold, p < 0.05) responses. HDACi treatment rescued gross morphological defects and reduced CMZ cell death by 80%. Proteomic analysis of dye eye extracts suggested BDNF-TrkB and Akt signaling as mediators of HDACi rescue in our dataset. Co-treatment with the TrkB antagonist ANA-12 blocked HDACi rescue of visual function and associated Akt phosphorylation. Notably, sole treatment with a BDNF mimetic, 7,8-dihydroxyflavone hydrate, significantly rescued dye visual function (~58 fold increase in OKR, p < 0.001, ~3 fold increase in VMR, p < 0.05). In summary, HDACi and a BDNF mimetic are sufficient to rescue retinal cell death and visual function in a vertebrate model of inherited blindness.

A sustained release formulation of novel quininib-hyaluronan microneedles inhibits angiogenesis and retinal vascular permeability in vivo.

Pathologic neovascularisation and ocular permeability are hallmarks of proliferative diabetic retinopathy and age-related macular degeneration. Current pharmacologic interventions targeting VEGF are effective in only 30-60% of patients and require multiple intraocular injections associated with iatrogenic infection. Thus, our goal is to develop novel small molecule drugs that are VEGF-independent are amenable to sustained ocular-release, and which reduce retinal angiogenesis and retinal vascular permeability. Here, the anti-angiogenic drug quininib was formulated into hyaluronan (HA) microneedles whose safety and efficacy was evaluated in vivo. Quininib-HA microneedles were formulated via desolvation from quininib-HA solution and subsequent cross-linking with 4-arm-PEG-amine prior to freeze-drying. Scanning electron microscopy revealed hollow needle-shaped particle ultrastructure, with a zeta potential of -35.5mV determined by electrophoretic light scattering. The incorporation efficiency and pharmacokinetic profile of quininib released in vitro from the microneedles was quantified by HPLC. Quininib incorporation into these microneedles was 90%. In vitro, 20% quininib was released over 4months; or in the presence of increasing concentrations of hyaluronidase, 60% incorporated quininib was released over 4months. Zebrafish hyaloid vasculature assays demonstrated quininib released from these microneedles significantly (p<0.0001) inhibited ocular developmental angiogenesis compared to control. Sustained amelioration of retinal vascular permeability (RVP) was demonstrated using a bespoke cysteinyl leukotriene induced rodent model. Quininib-HA microparticles significantly inhibited RVP in Brown Norway rats one month after administration compared to neat quininib control (p=0.0071). In summary, quininib-HA microneedles allow for sustained release of quininib; are safe in vivo and quininib released from these microneedles effectively inhibits angiogenesis and RVP in vivo.

Preclinical validation of the small molecule drug quininib as a novel therapeutic for colorectal cancer.

Colorectal cancer (CRC) is a leading cause of cancer deaths. Molecularly targeted therapies (e.g. bevacizumab) have improved survival rates but drug resistance ultimately develops and newer therapies are required. We identified quininib as a small molecule drug with anti-angiogenic activity using in vitro, ex vivo and in vivo screening models. Quininib (2-[(E)-2-(Quinolin-2-yl) vinyl] phenol), is a small molecule drug (molecular weight 283.75 g/mol), which significantly inhibited blood vessel development in zebrafish embryos (p < 0.001). In vitro, quininib reduced endothelial tubule formation (p < 0.001), cell migration was unaffected by quininib and cell survival was reduced by quininib (p < 0.001). Using ex vivo human CRC explants, quininib significantly reduced the secretions of IL-6, IL-8, VEGF, ENA-78, GRO-α, TNF, IL-1ß and MCP-1 ex vivo (all values p < 0.01). Quininib is well tolerated in mice when administered at 50 mg/kg intraperitoneally every 3 days and significantly reduced tumour growth of HT-29-luc2 CRC tumour xenografts compared to vehicle control. In addition, quininib reduced the signal from a αvß3 integrin fluorescence probe in tumours 10 days after treatment initiation, indicative of angiogenic inhibition. Furthermore, quininib reduced the expression of angiogenic genes in xenografted tumours. Collectively, these findings support further development of quininib as a novel therapeutic agent for CRC.

Predominant cone photoreceptor dysfunction in a hyperglycaemic model of non-proliferative diabetic retinopathy.

Approximately 2.5 million people worldwide are clinically blind because of diabetic retinopathy. In the non-proliferative stage, the pathophysiology of this ocular manifestation of diabetes presents as morphological and functional disruption of the retinal vasculature, and dysfunction of retinal neurons. However, it is uncertain whether the vascular and neuronal changes are interdependent or independent events. In addition, the identity of the retinal neurons that are most susceptible to the hyperglycaemia associated with diabetes is unclear. Here, we characterise a novel model of non-proliferative diabetic retinopathy in adult zebrafish, in which the zebrafish were subjected to oscillating hyperglycaemia for 30 days. Visual function is diminished in hyperglycaemic fish. Significantly, hyperglycaemia disrupts cone photoreceptor neurons the most, as evidenced by prominent morphological degeneration and dysfunctional cone-mediated electroretinograms. Disturbances in the morphological integrity of the blood-retinal barrier were also evident. However, we demonstrate that these early vascular changes are not sufficient to induce cone photoreceptor dysfunction, suggesting that the vascular and neuronal complications in diabetic retinopathy can arise independently. Current treatments for diabetic retinopathy target the vascular complications. Our data suggest that cone photoreceptor dysfunction is a clinical hallmark of diabetic retinopathy and that the debilitating blindness associated with diabetic retinopathy may be halted by neuroprotection of cones.

Selective inhibition of retinal angiogenesis by targeting PI3 kinase.

Ocular neovascularisation is a pathological hallmark of some forms of debilitating blindness including diabetic retinopathy, age related macular degeneration and retinopathy of prematurity. Current therapies for delaying unwanted ocular angiogenesis include laser surgery or molecular inhibition of the pro-angiogenic factor VEGF. However, targeting of angiogenic pathways other than, or in combination to VEGF, may lead to more effective and safer inhibitors of intraocular angiogenesis. In a small chemical screen using zebrafish, we identify LY294002 as an effective and selective inhibitor of both developmental and ectopic hyaloid angiogenesis in the eye. LY294002, a PI3 kinase inhibitor, exerts its anti-angiogenic effect in a dose-dependent manner, without perturbing existing vessels. Significantly, LY294002 delivered by intraocular injection, significantly inhibits ocular angiogenesis without systemic side-effects and without diminishing visual function. Thus, targeting of PI3 kinase pathways has the potential to effectively and safely treat neovascularisation in eye disease.

Influence of a quantitative trait locus on mouse chromosome 19 to the light-adapted electroretinogram.

PURPOSE: Both implicit time and amplitude of the cone-mediated electroretinographic (ERG) b-wave differ significantly between the C57BL/6JOlaHsd and 129S2/SvHsd inbred mouse strains. The purpose of this work was to undertake a quantitative genetics study to localize the gene or genes involved. METHODS: Implicit time and amplitude of the a- and b-waves of the single-flash and flicker cone-mediated ERG were recorded as the quantitative traits in reciprocal backcrossed populations. A genome-wide scan was performed with 106 polymorphic markers. Map Manager (release QTXb20) was used to analyze the data and make phenotype-genotype correlations. RESULTS: A quantitative trait locus (QTL) of major effect in controlling variation in both implicit time and amplitude of the cone-mediated ERG localized to the middle of chromosome 19. CONCLUSIONS: Mapping of a QTL influencing both implicit time and b-wave amplitude of the light-adapted ERG represents an initial step toward identifying the gene(s) responsible for this phenotypic variation.