RESUMO
Alu RNA accumulation due to DICER1 deficiency in the retinal pigmented epithelium (RPE) is implicated in geographic atrophy (GA), an advanced form of age-related macular degeneration that causes blindness in millions of individuals. The mechanism of Alu RNA-induced cytotoxicity is unknown. Here we show that DICER1 deficit or Alu RNA exposure activates the NLRP3 inflammasome and triggers TLR-independent MyD88 signaling via IL18 in the RPE. Genetic or pharmacological inhibition of inflammasome components (NLRP3, Pycard, Caspase-1), MyD88, or IL18 prevents RPE degeneration induced by DICER1 loss or Alu RNA exposure. These findings, coupled with our observation that human GA RPE contains elevated amounts of NLRP3, PYCARD, and IL18 and evidence of increased Caspase-1 and MyD88 activation, provide a rationale for targeting this pathway in GA. Our findings also reveal a function of the inflammasome outside the immune system and an immunomodulatory action of mobile elements.
Assuntos
Elementos Alu , RNA Helicases DEAD-box/metabolismo , Atrofia Geográfica/imunologia , Atrofia Geográfica/patologia , Inflamassomos/imunologia , Fator 88 de Diferenciação Mieloide/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Ribonuclease III/metabolismo , Animais , Proteínas de Transporte/metabolismo , Atrofia Geográfica/metabolismo , Humanos , Inflamassomos/metabolismo , Camundongos , Proteína 3 que Contém Domínio de Pirina da Família NLR , Epitélio Pigmentado da Retina/patologia , Receptores Toll-Like/metabolismoRESUMO
Alu retroelements propagate via retrotransposition by hijacking long interspersed nuclear element-1 (L1) reverse transcriptase (RT) and endonuclease activities. Reverse transcription of Alu RNA into complementary DNA (cDNA) is presumed to occur exclusively in the nucleus at the genomic integration site. Whether Alu cDNA is synthesized independently of genomic integration is unknown. Alu RNA promotes retinal pigmented epithelium (RPE) death in geographic atrophy, an untreatable type of age-related macular degeneration. We report that Alu RNA-induced RPE degeneration is mediated via cytoplasmic L1-reverse-transcribed Alu cDNA independently of retrotransposition. Alu RNA did not induce cDNA production or RPE degeneration in L1-inhibited animals or human cells. Alu reverse transcription can be initiated in the cytoplasm via self-priming of Alu RNA. In four health insurance databases, use of nucleoside RT inhibitors was associated with reduced risk of developing atrophic macular degeneration (pooled adjusted hazard ratio, 0.616; 95% confidence interval, 0.493-0.770), thus identifying inhibitors of this Alu replication cycle shunt as potential therapies for a major cause of blindness.
Assuntos
Elementos Alu/genética , Elementos Nucleotídeos Longos e Dispersos/genética , Degeneração Macular/genética , Pigmentos da Retina/metabolismo , Animais , Citoplasma/genética , DNA Complementar/genética , Epitélio/metabolismo , Epitélio/patologia , Humanos , Degeneração Macular/patologia , Pigmentos da Retina/biossíntese , Retroelementos/genética , Transcrição Reversa/genéticaRESUMO
Toxicity observed in aquatic ecosystems often cannot be explained by the action of a single pollutant. Likewise, evaluation standards formulated by a single effect cannot truly reflect the environmental quality requirements. The study of mixtures is needed to provide environmental relevance and knowledge of combined toxicity. In this study, the embryos of Japanese medaka (Oryzias latipes) were treated with individual and binary mixture of copper (Cu) and cadmium (Cd) until 12 days post-fertilization (dpf). Hatching, mortality, development, histology and gene expression were assessed. Our results showed that the highest concentration mixture of Cd (10 mg/L) and Cu (1 mg/L) affected survival, hatching time and hatching success. Occurrence of uninflated swim bladder was the highest (value) with exposure to 10 mg/L Cd. Swim bladder was commonly over-inflated in a mixture (0.1 mg/L Cd + 1.0 mg/L Cu) exposure. Individuals exposed to the mixture (0.1 Cd + 1.0 Cu mg/L) showed up to a 7.69% increase in swim bladder area compared to the control group. The mixtures containing 0.1 or 10 mg/L Cd, each with 1.0 mg/L Cu resulted in significantly increased of Pbx1b expression, higher than any Cd or Cu alone (p < 0.01). In the co-exposure group (0.1/10 Cd + 1.0 Cu mg/L), Pbx1b expression was found at 12 dpf but not 7 dpf in controls. Higher concentrations of Cd may progressively reduce Pbx1b expression, potentially explaining why 75% of individuals in the 10 mg/L Cd group failed to inflate their swim bladders. Additionally, the swim bladder proved to be a valuable bio-indicator for biological evaluation.
Assuntos
Oryzias , Poluentes Químicos da Água , Humanos , Animais , Cobre/toxicidade , Cádmio/toxicidade , Ecossistema , Bexiga Urinária , Poluentes Químicos da Água/toxicidade , Embrião não MamíferoRESUMO
Alzheimer's disease (AD) pathologies were discovered in the accessible neurosensory retina. However, their exact nature and topographical distribution, particularly in the early stages of functional impairment, and how they relate to disease progression in the brain remain largely unknown. To better understand the pathological features of AD in the retina, we conducted an extensive histopathological and biochemical investigation of postmortem retina and brain tissues from 86 human donors. Quantitative examination of superior and inferior temporal retinas from mild cognitive impairment (MCI) and AD patients compared to those with normal cognition (NC) revealed significant increases in amyloid ß-protein (Aß42) forms and novel intraneuronal Aß oligomers (AßOi), which were closely associated with exacerbated retinal macrogliosis, microgliosis, and tissue atrophy. These pathologies were unevenly distributed across retinal layers and geometrical areas, with the inner layers and peripheral subregions exhibiting most pronounced accumulations in the MCI and AD versus NC retinas. While microgliosis was increased in the retina of these patients, the proportion of microglial cells engaging in Aß uptake was reduced. Female AD patients exhibited higher levels of retinal microgliosis than males. Notably, retinal Aß42, S100 calcium-binding protein B+ macrogliosis, and atrophy correlated with severity of brain Aß pathology, tauopathy, and atrophy, and most retinal pathologies reflected Braak staging. All retinal biomarkers correlated with the cognitive scores, with retinal Aß42, far-peripheral AßOi and microgliosis displaying the strongest correlations. Proteomic analysis of AD retinas revealed activation of specific inflammatory and neurodegenerative processes and inhibition of oxidative phosphorylation/mitochondrial, and photoreceptor-related pathways. This study identifies and maps retinopathy in MCI and AD patients, demonstrating the quantitative relationship with brain pathology and cognition, and may lead to reliable retinal biomarkers for noninvasive retinal screening and monitoring of AD.
Assuntos
Doença de Alzheimer , Masculino , Humanos , Feminino , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/metabolismo , Proteoma/metabolismo , Proteômica , Retina/patologia , Atrofia/patologia , Biomarcadores/metabolismoRESUMO
Oxidative stress in the retinal pigment epithelium (RPE) can cause mitochondrial dysfunction and is likely a causative factor in the pathogenesis of age-related macular degeneration (AMD). Under oxidative stress conditions, some of the RPE cells become senescent and a contributory role for RPE senescence in AMD pathology has been proposed. The purpose of this study is to 1) characterize senescence in human RPE; 2) investigate the effect of an αB Crystallin chaperone peptide (mini Cry) in controlling senescence, in particular by regulating mitochondrial function and senescence-associated secretory phenotype (SASP) production and 3) develop mouse models for studying the role of RPE senescence in dry and nAMD. Senescence was induced in human RPE cells in two ways. First, subconfluent cells were treated with 0.2 µg/ml doxorubicin (DOX); second, subconfluent cells were treated with 500 µM H2O2. Senescence biomarkers (senescence-associated beta-galactosidase (SA-ßgal), p21, p16) and mitochondrial proteins (Fis1, DRP1, MFN2, PGC1-α, mtTFA) were analyzed in control and experimental groups. The effect of mini Cry on mitochondrial bioenergetics, glycolysis and SASP was determined. In vivo, retinal degeneration was induced by intravenous injection of NaIO3 (20 mg/kg) and subretinal fibrosis by laser-induced choroidal neovascularization. Increased SA-ßgal staining and p16 and p21 expression was observed after DOX- or H2O2-induced senescence and mini Cry significantly decreased senescence-positive cells. The expression of mitochondrial biogenesis proteins PGC-1 and mTFA increased with senescence, and mini Cry reduced expression significantly. Senescent RPE cells were metabolically active, as evidenced by significantly enhanced oxidative phosphorylation and anaerobic glycolysis, mini Cry markedly reduced rates of respiration and glycolysis. Senescent RPE cells maintain a proinflammatory phenotype characterized by significantly increased production of cytokines (IFN-Ë , TNF-α, IL1-α IL1-ß, IL-6, IL-8, IL-10), and VEGF-A; mini Cry significantly inhibited their secretion. We identified and localized senescent RPE cells for the first time in NaIO3-induced retinal degeneration and laser-induced subretinal fibrosis mouse models. We conclude that mini Cry significantly impairs stress-induced senescence by modulating mitochondrial biogenesis and fission proteins in RPE cells. Characterization of senescence could provide further understanding of the metabolic changes that accompany the senescent phenotype in ocular disease. Future studies in vivo may better define the role of senescence in AMD and the therapeutic potential of mini Cry as a senotherapeutic.
Assuntos
Degeneração Macular , Degeneração Retiniana , Animais , Senescência Celular , Modelos Animais de Doenças , Fibrose , Peróxido de Hidrogênio/farmacologia , Degeneração Macular/metabolismo , Camundongos , Estresse Oxidativo , Peptídeos/farmacologia , Degeneração Retiniana/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Cadeia B de alfa-Cristalina/genéticaRESUMO
The choriogenin H - EGFP transgenic medaka (Oryzias melastigma) has been used to test estrogenic substances and quantify estrogenic activity into 17ß-estradiol (E2) equivalency (EEQ). The method uses 8 eleutheroembryos in 2 ml solution per well and 3 wells per treatment in 24-well plates at 26 ± 1 °C for 24 ± 2 h, with subsequent measurements of induced GFP signal intensity. EEQ measurements are calculated using a E2 probit regression model with a coefficient of determination (R2) > 0.90. The selectivity was confirmed evaluating 27 known estrogenic and 5 known non-estrogenic compounds. Limit of quantitation (LOQ), recovery rate and bias were calculated to be 1 ng/ml EEQ, 104% and 4% respectively. Robustness analysis revealed exposure temperature is a sensitive parameter that should be kept at 26 ± 1 °C. The repeatability of intra- and inter-laboratories achieved CV < 30% for most tested food and cosmetics samples. The lot-lot stability was confirmed by the stable EEQ qualitative control (QC, 1 ng/mL E2) and calibration curve results. The stability of standard reagents, samples and sample extracts was also investigated. These data demonstrated this method to be an accurate indicator of estrogenic activity for both chemicals and extracts.
Assuntos
Animais Geneticamente Modificados/metabolismo , Proteínas do Ovo/análise , Estradiol/química , Oryzias/metabolismo , Precursores de Proteínas/análise , Animais , Animais Geneticamente Modificados/embriologia , Técnicas Biossensoriais , Extratos Celulares/química , Estradiol/metabolismo , Limite de Detecção , Oryzias/embriologia , Análise de RegressãoRESUMO
Historic industrial pollution of the Elizabeth River, Virginia resulted in polycyclic aromatic hydrocarbon (PAH) contamination in sediments. Atlantic killifish (Fundulus heteroclitus) inhabiting the Atlantic Wood (AW) industrial site adapted to complex PAH mixture at this Superfund site. Their embryos have proved highly resistant to cardiac abnormalities indicative of PAH toxicity. In this study, embryos spawned from adults collected at AW and King's Creek (KC), a reference site, were exposed at 24 h post fertilization (hpf) to Elizabeth River Sediment Extract (ERSE), a complex PAH mixture, in a range of concentrations (0, 5.04, 50.45, 100.90, 151.35, or 252.25 µg/L total PAHs). Embryos were processed for histology at 144 hpf to enable evaluations of hearts at tissue and cellular levels. Morphometry and severity scoring were used to evaluate the extent of alterations. Unexposed embryos were similar in both populations. ERSE exposure resulted in multiple changes to hearts of KC embryos but not AW. Alterations were particularly evident in KC embryos exposed to concentrations above 1% ERSE (50.45 µg/L), which had thinner ventricular walls and larger pericardial edema. Individuals with moderate pericardial edema maintained arrangement and proximity of heart chambers, but changes were seen in ventricular myocytes. Severe pericardial edema was prevalent in exposed KC embryos and typically resulted in tube heart formation. Ventricles of tube hearts had very thin walls composed of small, basophilic cells and lacked trabeculae. Edematous pericardial fluid contained small amounts of proteinaceous material, as did controls, and was free of cells. This fluid was primarily unstained, suggesting water influx due to increased permeability. The use of histological approaches provided more specific detail for tissue and cellular effects in hearts of embryos exposed to PAHs and enabled understanding of potential links to later life effects of early life exposure.
Assuntos
Fundulidae/fisiologia , Hidrocarbonetos Policíclicos Aromáticos/toxicidade , Poluentes Químicos da Água/toxicidade , Adaptação Fisiológica , Animais , Coração , Cardiopatias Congênitas , RiosRESUMO
Pericyte loss and deficient vascular platelet-derived growth factor receptor-ß (PDGFRß) signaling are prominent features of the blood-brain barrier breakdown described in Alzheimer's disease (AD) that can predict cognitive decline yet have never been studied in the retina. Recent reports using noninvasive retinal amyloid imaging, optical coherence tomography angiography, and histological examinations support the existence of vascular-structural abnormalities and vascular amyloid ß-protein (Aß) deposits in retinas of AD patients. However, the cellular and molecular mechanisms of such retinal vascular pathology were not previously explored. Here, by modifying a method of enzymatically clearing non-vascular retinal tissue and fluorescent immunolabeling of the isolated blood vessel network, we identified substantial pericyte loss together with significant Aß deposition in retinal microvasculature and pericytes in AD. Evaluation of postmortem retinas from a cohort of 56 human donors revealed an early and progressive decrease in vascular PDGFRß in mild cognitive impairment (MCI) and AD compared to cognitively normal controls. Retinal PDGFRß loss significantly associated with increased retinal vascular Aß40 and Aß42 burden. Decreased vascular LRP-1 and early apoptosis of pericytes in AD retina were also detected. Mapping of PDGFRß and Aß40 levels in pre-defined retinal subregions indicated that certain geometrical and cellular layers are more susceptible to AD pathology. Further, correlations were identified between retinal vascular abnormalities and cerebral Aß burden, cerebral amyloid angiopathy (CAA), and clinical status. Overall, the identification of pericyte and PDGFRß loss accompanying increased vascular amyloidosis in Alzheimer's retina implies compromised blood-retinal barrier integrity and provides new targets for AD diagnosis and therapy.
Assuntos
Doença de Alzheimer/patologia , Amiloidose/patologia , Encéfalo/patologia , Pericitos/patologia , Retina/patologia , Idoso , Idoso de 80 Anos ou mais , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Amiloidose/complicações , Barreira Hematoencefálica/patologia , Angiopatia Amiloide Cerebral/patologia , Cognição/fisiologia , Feminino , Humanos , MasculinoRESUMO
Humanin (HN) is a hydrophobic 24-amino acid peptide derived from mitochondrial DNA that modulates cellular responses to oxidative stress and protects human retinal pigment epithelium (RPE) cells from apoptosis. To solubilize HN, this report describes two genetically-encoded fusions between HN and elastin-like polypeptides (ELP). ELPs provide steric stabilization and/or thermo-responsive phase separation. Fusions were designed to either remain soluble or phase separate at the physiological temperature of the retina. Interestingly, the soluble fusion assembles stable colloids with a hydrodynamic radius of 39.1 nm at 37°C. As intended, the thermo-responsive fusion forms large coacervates (>1,000 nm) at 37°C. Both fusions bind human RPE cells and protect against oxidative stress-induction of apoptosis (TUNEL, caspase-3 activation). Their activity is mediated through STAT3; furthermore, STAT3 inhibition eliminates their protection. These findings suggest that HN polypeptides may facilitate cellular delivery of biodegradable nanoparticles with potential protection against age-related diseases, including macular degeneration.
Assuntos
Elastina , Células Epiteliais/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Nanopartículas/química , Estresse Oxidativo/efeitos dos fármacos , Peptídeos , Epitélio Pigmentado da Retina/metabolismo , Apoptose/efeitos dos fármacos , Células Cultivadas , Elastina/química , Elastina/farmacologia , Células Epiteliais/patologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/farmacologia , Degeneração Macular/metabolismo , Degeneração Macular/patologia , Peptídeos/química , Peptídeos/farmacologia , Epitélio Pigmentado da Retina/patologiaRESUMO
This session explored the effects of pollutants on One Health at the ecosystem level that included microbes, insects, fish, and humans. The concept of One Health seeks to synergize medical, veterinary, and other health science disciplines to more effectively advance human and animal health. Presentations explored the interactions of pesticides, pathogens, phytochemicals, and xenobiotic biotransformation in bee colony losses critical for food security (bees have been recently listed under the 2017 US Food and Drug Administration (FDA) veterinary feed directive); the role of pathology in identifying the effects of pollutants on fish as sentinels for human health; the effects in rats of per- and polyfluoroalkyl substances (PFAS) that can persist in the environment and contaminate drinking water; harmful algal blooms and toxin production leading to animal and human disease; and the processing of environmental carcinogens by intestinal microbiota.
Assuntos
Pesquisa Biomédica/métodos , Poluentes Ambientais/toxicidade , Modelos Animais , Saúde Única , Patologia , Animais , Congressos como Assunto , EcossistemaRESUMO
Thiram, a pesticide in the dithiocarbamate chemical family, is widely used to prevent fungal disease in seeds and crops. Its off-site movement to surface waters occurs and may place aquatic organisms at potential harm. Zebrafish embryos were used for investigation of acute (1â¯h) thiram exposure (0.001-10⯵M) at various developmental stages. Survival decreased at 1⯵M and 10⯵M and hatching was delayed at 0.1⯵M and 1⯵M. Notochord curvatures were seen at 0.1 and 1⯵M thiram when exposure was initiated at 2 and at 10 hpf. Similar notochord curvatures followed exposure to the known TPO inhibitor, methimazole (MMI). Changes were absent in embryos exposed at later stages, i.e., 12 hpf. In embryos exposed to 0.1 or 1⯵M at 10 hpf, levels of the thyroid enzyme, Deiodinase 3, increased by 12 hpf. Thyroid peroxide (TPO), important in T4 synthesis, decreased by 48 hpf in embryos exposed to 1⯵M at 10 hpf. Thiram toxicity was stage-dependent and early life stage exposure may be responsible for adverse effects seen later. These effects may be due to impacts on the thyroid via regulation of specific thyroid genes including TPO and Deiodinase 3.
Assuntos
Tiram/toxicidade , Glândula Tireoide/efeitos dos fármacos , Peixe-Zebra/fisiologia , Animais , Desenvolvimento Ósseo/efeitos dos fármacos , Edema/patologia , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Iodeto Peroxidase/genética , Iodeto Peroxidase/metabolismo , Larva/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Peixe-Zebra/anatomia & histologia , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
PURPOSE: We sought to characterize the angiofibrotic and apoptotic effects of vascular endothelial growth factor (VEGF)-inhibition on fibrovascular epiretinal membranes in eyes with traction retinal detachment because of proliferative diabetic retinopathy. METHODS: Membranes were excised from 20 eyes of 19 patients (10 randomized to intravitreal bevacizumab, 10 controls) at vitrectomy. Membranes were stained with antibodies targeting connective tissue growth factor (CTGF) or VEGF and colabeled with antibodies directed against endothelial cells (CD31), myofibroblasts, or retinal pigment epithelium markers. Quantitative and colocalization analyses of antibody labeling were obtained through immunofluorescence confocal microscopy. Masson trichrome staining, cell counting of hematoxylin and eosin sections, and terminal dUTP nick-end labeling staining were performed. RESULTS: High levels of fibrosis were observed in both groups. Cell apoptosis was higher (P = 0.05) in bevacizumab-treated membranes compared with controls. The bevacizumab group had a nonsignificant reduction in colocalization in CD31-CTGF and cytokeratin-VEGF studies compared with controls. Vascular endothelial growth factor in extracted membranes was positively correlated with vitreous levels of VEGF; CTGF in extracted membranes was negatively correlated with vitreous levels of CTGF. CONCLUSION: Bevacizumab suppresses vitreous VEGF levels, but does not significantly alter VEGF or CTGF in diabetic membranes that may be explained by high baseline levels of fibrosis. Bevacizumab may cause apoptosis within fibrovascular membranes.
Assuntos
Apoptose , Bevacizumab/administração & dosagem , Retinopatia Diabética/patologia , Membrana Epirretiniana/cirurgia , Retina/patologia , Vitrectomia/métodos , Actinas/biossíntese , Inibidores da Angiogênese/administração & dosagem , Proliferação de Células , Fator de Crescimento do Tecido Conjuntivo/biossíntese , Retinopatia Diabética/complicações , Retinopatia Diabética/tratamento farmacológico , Membrana Epirretiniana/complicações , Membrana Epirretiniana/patologia , Fibrose/patologia , Humanos , Injeções Intravítreas , Queratinas/biossíntese , Molécula-1 de Adesão Celular Endotelial a Plaquetas/biossíntese , Estudos Prospectivos , Receptores de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Retina/metabolismo , Fator A de Crescimento do Endotélio Vascular/biossínteseRESUMO
The goal of this study was to quantitatively assess retinal thickness using spectral domain optical coherence tomography (SD-OCT) after subretinal implantation of human embryonic stem cell-derived retinal pigment epithelium in a porcine model. The implant is called CPCB-RPE1 for the California Project to Cure Blindness-Retinal Pigment Epithelium 1. Data were derived from previous experiments on 14 minipigs that received either subretinal implantation of CPCB-RPE1 (n = 11) or subretinal bleb formation alone (sham; n = 3) using previously described methods and procedures (Brant Fernandes et al. Ophthalmic Surg Lasers Imaging Retina 47:342-51, 2016; Martynova et al. (2016) Koss et al. Graefes Arch Clin Exp Ophthalmol 254:1553-65, 2016; Hu et al. Ophthalmic Res 48:186-91, 2016; Martynova et al. ARVO Abstract 2016. SD-OCT retinal thickness (RT) and sublayer thickness over the implant were compared with topographically similar preimplantation regions as described previously Martynova et al. ARVO Abstract 2016. Imaging results were compared to postmortem histology using hematoxylin-eosin staining. RT overlying the CPCB-RPE1 postimplantation was not significantly different from preimplantation (308 ± 72 µm vs 292 ± 41 µm; p = 0.44). RT was not significantly different before and after implantation in any retinal sublayer at 1 month. Histology demonstrated grossly normal retinal anatomy as well as photoreceptor interdigitation with RPE.
Assuntos
Células-Tronco Embrionárias Humanas/transplante , Retina/diagnóstico por imagem , Epitélio Pigmentado da Retina/citologia , Tomografia de Coerência Óptica , Animais , California , Humanos , SuínosRESUMO
Bohai Bay, in the western region of northeastern China's Bohai Sea, receives water from large rivers containing various pollutants including dioxin-like compounds (DLCs). This study used the established zebrafish (Danio rerio) model, its known developmental toxicity endpoints and sensitive molecular analyses to evaluate sediments near and around an industrial effluent site in Bohai Bay. The primary objective was to assess the efficacy of rapid biological detection methods as an addition to chemical analyses. Embryos were exposed to various concentrations of sediment extracts as well as a 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD) positive control. Exposure to sediment extract nearest the discharge site (P1) resulted in the most severe- and highest rates of change in embryos and larvae, suggesting that DLC contaminated sediment probably did not occur much beyond it. P1 extract resulted in concentration dependent increases in mortality and pericardial edema. Its highest concentration caused up-regulation of P-450 (CYP)-1A1(CYP1A) mRNA expression at 72â¯h post fertilization (hpf), an increase in its expression in gill arches as observed by whole mount in situ hybridization, and an increased signal in the Tg(cyp1a: mCherry) transgenic line. The pattern and magnitude of response was very similar to that of TCDD and supported the presence of DLCs in these sediment samples. Follow-up chemical analysis confirmed this presence and identified H7CDF, O8CDF and O8CDD as the main components in P1 extract. This study validates the use of biological assays as a rapid, sensitive, and cost-effective method to evaluate DLCs and their effects in sediment samples. Additionally, it provides support for the conclusion that DLCs have limited remobilization capacity in marine sediments.
Assuntos
Dioxinas/análise , Monitoramento Ambiental/métodos , Sedimentos Geológicos/análise , Poluentes Químicos da Água/análise , Peixe-Zebra/metabolismo , Animais , China , Embrião não Mamífero/efeitos dos fármacos , Desenvolvimento Embrionário/efeitos dos fármacos , Monitoramento Ambiental/economiaRESUMO
Age-related macular degeneration (AMD) is a leading cause of blindness in the developed world. The retinal pigment epithelium (RPE) is a critical site of pathology in AMD. Oxidative stress plays a key role in the development of AMD. We generated a chimeric high-density lipoprotein (HDL), mimetic peptide named HM-10/10, with anti-oxidant properties and investigated its potential for the treatment of retinal disease using cell culture and animal models of RPE and photoreceptor (PR) degeneration. Treatment with HM-10/10 peptide prevented human fetal RPE cell death caused by tert-Butyl hydroperoxide (tBH)-induced oxidative stress and sodium iodate (NaIO3), which causes RPE atrophy and is a model of geographic atrophy in mice. We also show that HM-10/10 peptide ameliorated photoreceptor cell death and significantly improved retinal function in a mouse model of N-methyl-N-nitrosourea (MNU)-induced PR degeneration. Our results demonstrate that HM-10/10 protects RPE and retina from oxidant injury and can serve as a potential therapeutic agent for the treatment of retinal degeneration.
Assuntos
Lipoproteínas HDL/metabolismo , Peptídeos/farmacologia , Células Fotorreceptoras/efeitos dos fármacos , Células Fotorreceptoras/metabolismo , Degeneração Retiniana/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Animais , Apoptose , Caspase 3/metabolismo , Caspase 7/metabolismo , Modelos Animais de Doenças , Iodatos , Camundongos , Estresse Oxidativo/efeitos dos fármacos , Degeneração Retiniana/diagnóstico , Degeneração Retiniana/etiologia , Epitélio Pigmentado da Retina/patologia , Tomografia de Coerência ÓpticaRESUMO
Acamprosate is an FDA-approved medication for the treatment of alcoholism that is unfortunately only effective in certain patients. Although acamprosate is known to stabilize the hyper-glutamatergic state in alcoholism, pharmacological mechanisms of action in brain tissue remains unknown. To investigate the mechanism of acamprosate efficacy, the authors employ a pharmacoproteomics approach using an animal model of alcoholism, type 1 equilibrative nucleoside transporter (ENT1) null mice. The results demonstrate that acamprosate treatment significantly decreased both ethanol drinking and preference in ENT1 null mice compared to that of wild-type mice. Then, to elucidate acamprosate efficacy mechanism in ENT1 null mice, the authors utilize label-free quantification proteomics comparing both genotype and acamprosate treatment effects in the nucleus accumbens (NAc). A total of 1040 protein expression changes are identified in the NAc among 3634 total proteins detected. The proteomics and Western blot result demonstrate that acamprosate treatment decreased EAAT expression implicating stabilization of the hyper-glutamatergic condition in ENT1 null mice. Pathway analysis suggests that acamprosate treatment in ENT1 null mice seems to rescue glutamate toxicity through restoring of RTN4 and NF-κB medicated neuroimmune signaling compared to wild-type mice. Overall, pharmacoproteomics approaches suggest that neuroimmune restoration is a potential efficacy mechanism in the acamprosate treatment of certain sub-populations of alcohol dependent subjects.
Assuntos
Acamprosato/uso terapêutico , Dissuasores de Álcool/uso terapêutico , Alcoolismo/tratamento farmacológico , Modelos Animais de Doenças , Alcoolismo/genética , Alcoolismo/metabolismo , Animais , Transportador Equilibrativo 1 de Nucleosídeo/genética , Regulação da Expressão Gênica , Masculino , Camundongos , Camundongos Knockout , NF-kappa B/genética , NF-kappa B/metabolismo , Proteínas Nogo/genética , Proteínas Nogo/metabolismo , Proteômica , Transdução de Sinais , Resultado do TratamentoRESUMO
We previously described an in vitro model in which serous ovarian cystadenomas were transfected with SV40 large T antigen, resulting in loss of RB and P53 functions and thus mimicking genetic defects present in early high-grade serous extra-uterine Müllerian (traditionally called high-grade serous ovarian) carcinomas including those associated with the BRCA1 mutation carrier state. We showed that replicative aging in this cell culture model leads to a mitotic arrest at the spindle assembly checkpoint. Here we show that this arrest is due to a reduction in microtubule anchoring that coincides with decreased expression of the BUB1 kinase and of the phosphorylated form of its substrate, BUB3. The ensuing prolonged mitotic arrest leads to cohesion fatigue resulting in cell death or, in cells that recover from this arrest, in cytokinesis failure and polyploidy. Down-regulation of BRCA1 to levels similar to those present in BRCA1 mutation carriers leads to increased and uncontrolled microtubule anchoring to the kinetochore resulting in overcoming the spindle assembly checkpoint. Progression to anaphase under those conditions is associated with formation of chromatin bridges between chromosomal plates due to abnormal attachments to the kinetochore, significantly increasing the risk of cytokinesis failure. The dependence of this scenario on accelerated replicative aging can, at least in part, account for the site specificity of the cancers associated with the BRCA1 mutation carrier state, as epithelia of the mammary gland and of the reproductive tract are targets of cell-nonautonomous consequences of this carrier state on cellular proliferation associated with menstrual cycle progressions.
Assuntos
Proteína BRCA1/genética , Cistadenoma/genética , Citocinese/genética , Neoplasias Ovarianas/genética , Transdução de Sinais/genética , Proteína Supressora de Tumor p53/genética , Proteínas de Ciclo Celular/genética , Cromossomos/genética , Feminino , Humanos , Microtúbulos/genética , Mitose/genética , Poliploidia , Fuso Acromático/genéticaRESUMO
Although microplastic (MP) pollution in freshwater systems is gaining attention, our knowledge of its distribution in small waterbodies is scarce. Small waterbodies are freshwater habitats to many species, including amphibians, that are vulnerable to MP pollution. This study analyzed the distribution and characteristics of MPs in 25 small waterbodies from the Yangtze River Delta, China. MPs were detected in surface water, sediment, and tadpoles with abundances ranging from 0.48 to 21.52 items L-1, 35.76 to 3185.33 items kg-1, and 0 to 2.73 items individual-1 (0 to 168.48 items g-1), respectively. The dominant shape and polymer of MPs in water and tadpole samples were polyester (PES) fibers, and polypropylene (PP) fibers and fragments were dominant in sediment samples. In addition, MPs were primarily <0.5 mm in length in all samples. Tadpole length was positively correlated to the number of MPs detected. The abundance, shape, and polymer distribution of MPs in tadpoles resembled that of water rather than sediment, suggesting that tadpoles likely take up MPs from the surrounding water. This study demonstrated that MPs are abundant in these small waterbodies and are ingested by resident tadpoles. This may suggest a pathway of MP entry into aquatic and terrestrial food webs.
Assuntos
Rios , Poluentes Químicos da Água , Animais , China , Monitoramento Ambiental , Larva , PlásticosRESUMO
PURPOSE: To create new immunodeficient Royal College of Surgeons (RCS) rats by introducing the defective MerTK gene into athymic nude rats. METHODS: Female homozygous RCS (RCS-p+/RCS-p+) and male nude rats (Hsd:RH-Foxn1mu, mutation in the foxn1 gene; no T cells) were crossed to produce heterozygous F1 progeny. Double homozygous F2 progeny obtained by crossing the F1 heterozygotes was identified phenotypically (hair loss) and genotypically (RCS-p+ gene determined by PCR). Retinal degenerative status was confirmed by optical coherence tomography (OCT) imaging, electroretinography (ERG), optokinetic (OKN) testing, superior colliculus (SC) electrophysiology, and by histology. The effect of xenografts was assessed by transplantation of human embryonic stem cell-derived retinal pigment epithelium (hESC-RPE) and human-induced pluripotent stem cell-derived RPE (iPS-RPE) into the eye. Morphological analysis was conducted based on hematoxylin and eosin (H&E) and immunostaining. Age-matched pigmented athymic nude rats were used as control. RESULTS: Approximately 6% of the F2 pups (11/172) were homozygous for RCS-p+ gene and Foxn1mu gene. Homozygous males crossed with heterozygous females resulted in 50% homozygous progeny for experimentation. OCT imaging demonstrated significant loss of retinal thickness in homozygous rats. H&E staining showed photoreceptor thickness reduced to 1-3 layers at 12 weeks of age. Progressive loss of visual function was evidenced by OKN testing, ERG, and SC electrophysiology. Transplantation experiments demonstrated survival of human-derived cells and absence of apparent immune rejection. CONCLUSIONS: This new rat animal model developed by crossing RCS rats and athymic nude rats is suitable for conducting retinal transplantation experiments involving xenografts.
Assuntos
Modelos Animais de Doenças , Células-Tronco Embrionárias Humanas/transplante , Síndromes de Imunodeficiência/terapia , Células-Tronco Pluripotentes Induzidas/transplante , Distrofias Retinianas/terapia , Epitélio Pigmentado da Retina/transplante , Animais , Sobrevivência Celular , Eletrorretinografia , Feminino , Técnicas de Genotipagem , Sobrevivência de Enxerto/fisiologia , Células-Tronco Embrionárias Humanas/fisiologia , Humanos , Síndromes de Imunodeficiência/diagnóstico , Síndromes de Imunodeficiência/fisiopatologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Masculino , Fenótipo , Ratos , Ratos Nus , Retina/fisiopatologia , Distrofias Retinianas/diagnóstico , Distrofias Retinianas/fisiopatologia , Epitélio Pigmentado da Retina/fisiologia , Tomografia de Coerência Óptica , c-Mer Tirosina Quinase/genéticaRESUMO
Selenium (Se) is an essential element and its biological activity is related to its speciation. It is also well-known that in excess it can cause teratogenesis in fish and birds. In this study we compared dietary toxicity of elemental selenium nanoparticles (SeNPs) with selenite and selenomethionine (Se-Met). Japanese medaka (Oryzias latipes) was used as a laboratory model to determine Se effects on adults and their offspring. Adult females were individually exposed using a dry diet fortified with 0, 10 or 20⯵g/g of the three Se species for 7 days and then allowed to breed for 3 days. Fertilization rate and the proportion of malformed offspring were examined. The three Se diets led to significant increase in maternal tissue Se concentration in the order of Se-Met >>selenite > SeNP. However, in terms of proportion of malformed offspring, the effect of Se-Met = selenite > SeNP. The malformations included pericardial edema and craniofacial changes, which were typical for Se toxicity. The mismatch of maternal ovary Se concentration and proportion of malformed offspring suggested total Se concentration is a poor predictor of toxicity and teratogenesis. Comparing expression of four genes related to oxidative stress in maternal tissue also showed that there were significant differences in expression patterns between three Se diets in the order of selenite = SeNP > Se-Met. Our results showed that SeNPs cause similar toxicity as other Se species but require further study to elucidate the underlying mechanism.