Chitosan-Hyaluronic acid hybrid vectors for retinal gene therapy

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Sustained gene expression in the retina by improved episomal vectors

Gene and cellular therapies are nowadays part of therapeutic strategies for the treatment of diverse pathologies. The drawbacks associated with gene therapy-low levels of transgene expression, vector loss during mitosis, and gene silencing-need to be addressed. The pEPI-1 and pEPito family of vectors was developed to overcome these limitations. It contains a scaffold/matrix attachment region, which anchors its replication to cell division in eukaryotic cells while in an extrachromosomal state and is less prone to silencing, due to a lower number of CpG motifs. Recent success showed that ocular gene therapy is an important tool for the treatment of several diseases, pending the overcome of the aforementioned limitations. To achieve sustained gene delivery in the retina, we evaluated several vectors based on pEPito and pEPI-1 for their ability to sustain transgene expression in retinal cells. These vectors stably transfected and replicated in retinal pigment epithelial (RPE) cells. Expression levels were promoter dependent with constitutive promoters cytomegalovirus immediate early promoter (CMV) and human CMV enhancer/human elongation factor 1 alpha promoter yielding the highest levels of transgene expression compared with the retina-specific RPE65 promoter. When injected in C57Bl6 mice, transgene expression was sustained for at least 32 days. Furthermore, the retina-specific RPE65 promoter showed higher efficiency in vivo compared to in vitro. In this study, we demonstrate that by combining tissue-specific promoters with a mitotic stable system, less susceptible to epigenetic silencing such as pEPito-based plasmids, we can achieve prolonged gene expression and a sustained therapeutic effect.Fundacao para a Ciencia e Tecnologia, Portugal [PEst/OE/EQB-LA 0023/2013, SFRH/BD/76873/2011, SFRH/BD/70318/2010, PTDC/SAU/BEB/098475/2008]; European Union [PIRG-GA-2009-249314

pEPito-driven PEDF Expression Ameliorates Diabetic Retinopathy Hallmarks

Diabetic retinopathy (DR) is one of the major complications of diabetes mellitus. It is characterized by retinal microvascular changes caused by chronic exposure to hyperglycemia, leading to low tissue oxygenation and ultimately to neovascularization. Laser photocoagulation and vitrectomy are the most efficient treatments for DR, but display severe side effects such as the destruction of the healthy retina. Another clinical approach uses antiangiogenic agents to prevent and delay progression of neovascularization, but these require recurrent local administrations that increase the possibility of retinal detachment, vitreous hemorrhage, and cataract formation. Studies in human diabetic retinas have revealed an imbalance between proangiogenic factors such as the vascular endothelial growth factor (VEGF) and antiangiogenic factors, such as pigment epithelial-derived factor (PEDF). This imbalance favors pathological angiogenesis contributing to DR, and can constitute a therapeutic target. Gene therapy was recently shown to be an adequate intervention for long-term treatment of several retinal pathologies. We have previously shown the newly engineered episomal vector pEPito to be able of sustained gene expression in the mouse retina. We here show that pEPito was able to overexpress PEDF for up to three months, both in in vitro cultures of human retinal pigment epithelial cells and in the retina of diabetic mice after a single subretinal injection. In vivo, in parallel with the increase in PEDF we observed a decrease in VEGF levels in injected compared with noninjected eyes and a significant effect on two hallmarks of DR: reduction of glucose transport (by glucose transporter GLUT1), and reduction of inflammation by decreased reactivity of microglia. Jointly, these results point to a significant therapeutic potential of gene therapy with pEPito-PEDF for the treatment of DR

GLUT1 activity contributes to the impairment of PEDF secretion by the RPE

Purpose: In this study, we aimed to understand whether glucose transporter 1 (GLUT1) activity affects the secretion capacity of antiangiogenic factor pigment epithelium-derived factor (PEDF) by the RPE cells, thus explaining the reduction in PEDF levels observed in patients with diabetic retinopathy (DR).Methods: Analysis of GLUT1 expression, localization, and function was performed in vitro in RPE cells (D407) cultured with different glucose concentrations, corresponding to non-diabetic (5 mM of glucose) and diabetic (25 mM of glucose) conditions, further subjected to normoxia or hypoxia. The expression of PEDF was also evaluated in the secretome of the cells cultured in these conditions. Analysis of GLUT1 and PEDF expression was also performed in vivo in the RPE of Ins2(Akita) diabetic mice and age-matched wild-type (WT) controls.Results: We observed an increase in GLUT1 under hypoxia in a glucose-dependent manner, which we found to be directly associated with the translocation and stabilization of GLUT1 in the cell membrane. This stabilization led to an increase in glucose uptake by RPE cells. This increase was followed by a decrease in PEDF expression in RPE cells cultured in conditions that simulated DR. Compared with non-diabetic WT mice, the RPE of Ins2Akita mice showed increased GLUT1 overexpression with a concomitant decrease in PEDF expression.Conclusions: Collectively, our data show that expression of GLUT1 is stimulated by hyperglycemia and low oxygen supply, and this overexpression was associated with increased activity of GLUT1 in the cell membrane that contributes to the impairment of the RPE secretory function of PEDF

Overlooked gall-inducing moths revisited, with the description of Andescecidium parrai gen. et sp. n. and Oliera saizi sp. n. from Chile (Lepidoptera, Cecidosidae)

There are still many gall systems associated with larvae of Lepidoptera in which the true gall-inducers have not been identified to species. Reports on misidentification of gall inducers have been recurrent for these galls, particularly in complex gall-systems that may include inquilines, kleptoparasites, and cecidophages, among other feeding guilds such as predators and parasitoid wasps. Here we describe and illustrate the adults, larvae, pupae and galls, based on light and scanning microscopy, of Andescecidium parrai gen. et sp. n. and Oliera saizi sp. n., two sympatric cecidosid moths that are associated with Schinus polygamus (Cav.) Cabrera (Anacardiaceae) in central Chile. Adults, immatures, and galls of the former did not conform to any known cecidosid genus. Galls of A. parrai are external, spherical, and conspicuous, being known for more than one century. However, their induction has been mistakenly associated with either unidentified Coleoptera (original description) or Oliera argentinana Br糨es (recently), a distinct cecidosid species with distribution restricted to the eastern Andes. Galls of O. saizi had been undetected, as they are inconspicuous. They occur under the bark within swollen stems, and may occur on the same plant, adjacent to those of A. parrai. We also propose a time-calibrated phylogeny using sequences from mitochondrial and nuclear loci, including specimens of the new proposed taxa. Thus in addition to clarifying the taxonomy of the Chilean cecidosid species we also tested their monophyly in comparison to congeneric species and putative specimens of all genera of Neotropical and African cecidosids.Fil: Silva, Gabriela T.. Universidade Federal do Rio Grande do Sul; BrasilFil: Moreira, Gilson R. P.. Universidade Federal do Rio Grande do Sul; BrasilFil: Vargas, Héctor A.. Universidad de Tarapacá de Arica; ChileFil: Gonçalves, Gislene L.. Universidade Federal do Rio Grande do Sul; Brasil. Universidad de Tarapacá de Arica; ChileFil: Mainardi, Marina D.. Universidade Federal do Rio Grande do Sul; BrasilFil: San Blas, Diego German. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de La Pampa; ArgentinaFil: Davis, Donald. National Museum of Natural History; Estados Unido

Yeast as a model organism for studying the evolution of non-standard genetic codes

During the last 30 years, a number of alterations to the standard genetic code have been uncovered both in prokaryotes and eukaryotic nuclear and mitochondrial genomes. But, the study of the evolutionary pathways and molecular mechanisms of codon identity redefinition has been largely ignored due to the assumption that non-standard genetic codes can only evolve through neutral evolutionary mechanisms and that they have no functional significance. The recent discovery of a genetic code change in the genus Candida that evolved through an ambiguous messenger RNA decoding mechanism is bringing that naive assumption to an abrupt end by showing, in a rather dramatic way, that genetic code changes have profound physiological and evolutionary consequences for the species that redefine codon identity. In this paper, the recent data on the evolution of the Candida genetic code are reviewed and an experimental framework based on forced evolution, molecular genetics and comparative and functional genomics methodologies is put forward for the study of non-standard genetic codes and genetic code ambiguity in general. Additionally, the importance of using Saccharomyces cerevisiae as a model organism for elucidating the evolutionary pathway of the Candida and other genetic code changes is emphasised.publishe

Polyphenol metabolite pyrogallol-o-sulfate decreases microglial activation and vegf in retinal pigment epithelium cells and diabetic mouse retina

Funding Information: The authors acknowledge Fundacao para a Ciencia e Tecnologia for funding (PD/BD/114251/2016 scholarship to D.F. Santos; EXPL-BIM-MEC-1433-2013; PTDC/BTM/ORG/28121/2017 grants to G.A.Silva) and iNOVA4Health ? UIDB/Multi/04462/2020, a program financially supported by Funda??o para a Ci?ncia e Tecnologia/Ministerio da Educacao e Ciencia through national funds and co-funded by FEDER under the PT2020 Partnership Agreement, is also acknowledged. C.N.S. also acknowledges the European Research Council (ERC) under the European Union?s Horizon 2020 Research and Innovation Programme under Grant Agreement No. 804229. Publisher Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland.(Poly)phenol-derived metabolites are small molecules resulting from (poly)phenol metab-olization after ingestion that can be found in circulation. In the last decade, studies on the impact of (poly)phenol properties in health and cellular metabolism accumulated evidence that (poly)phenols are beneficial against human diseases. Diabetic retinopathy (DR) is characterized by inflammation and neovascularization and targeting these is of therapeutic interest. We aimed to study the effect of pyrogallol-O-sulfate (Pyr-s) metabolite in the expression of proteins involved in retinal glial acti-vation, neovascularization, and glucose transport. The expression of PEDF, VEGF, and GLUT-1 were analyzed upon pyrogallol-O-sulfate treatment in RPE cells under high glucose and hypoxia. To test its effect on a diabetic mouse model, Ins2Akita mice were subjected to a single intraocular injection of the metabolite and the expression of PEDF, VEGF, GLUT-1, Iba1, or GFAP measured in the neural retina and/or retinal pigment epithelium (RPE), two weeks after treatment. We observed a significant decrease in the expression of pro-angiogenic VEGF in RPE cells. Moreover, pyrogallol-O-sulfate significantly decreased the expression of microglial marker Iba1 in the diabetic retina at different stages of disease progression. These results highlight the potential pyrogallol-O-sulfate metabolite as a preventive approach towards DR progression, targeting molecules involved in both inflammation and neovascularization.publishersversionpublishe

Altered bone microarchitecture in a type 1 diabetes mouse model Ins2 Akita

Type 1 diabetes mellitus (T1DM) has been associated to several cartilage and bone alterations including growth retardation, increased fracture risk, and bone loss. To determine the effect of long term diabetes on bone we used adult and aging Ins2(Akita) mice that developed T1DM around 3-4 weeks after birth. Both Ins2(Akita) and wild-type (WT) mice were analyzed at 4, 6, and 12 months to assess bone parameters such as femur length, growth plate thickness and number of mature and preapoptotic chondrocytes. In addition, bone microarchitecture of the cortical and trabecular regions was measured by microcomputed tomography and gene expression of Adamst-5, Col2, Igf1, Runx2, Acp5, and Oc was quantified by quantitative real-time polymerase chain reaction. Ins2(Akita) mice showed a decreased longitudinal growth of the femur that was related to decreased growth plate thickness, lower number of chondrocytes and to a higher number of preapoptotic cells. These changes were associated with higher expression of Adamst-5, suggesting higher cartilage degradation, and with low expression levels of Igf1 and Col2 that reflect the decreased growth ability of diabetic mice. Ins2(Akita) bone morphology was characterized by low cortical bone area (Ct.Ar) but higher trabecular bone volume (BV/TV) and expression analysis showed a downregulation of bone markers Acp5, Oc, and Runx2. Serum levels of insulin and leptin were found to be reduced at all-time points Ins2(Akita). We suggest that Ins2(Akita) mice bone phenotype is caused by lower bone formation and even lower bone resorption due to insulin deficiency and to a possible relation with low leptin signaling.F. R. Carvalho and S. M. Calado acknowledge the financial support from the Portuguese Foundation for Science and Technology (FCT) through Ph.D. fellowships SFRH/BD/76429/2011 and SFRH/BD/76873/2011, respectively. This study was funded in part by CCMAR funding from European Regional Development Fund (ERDF) under COMPETE Program and through FCT under PEst-C/MAR/LA0015/2011 project and through UID/Multi/04326/2013 project. GA Silva was funded by (PIRG05-GA-2009-249314-EyeSee) and Research Center Grant UID/BIM/04773/2013 to CBMR

Altered bone microarchitecture in a type 1 diabetes mouse model Ins2 (Akita)

Type 1 diabetes mellitus (T1DM) has been associated to several cartilage and bone alterations including growth retardation, increased fracture risk, and bone loss. To determine the effect of long term diabetes on bone we used adult and aging Ins2 Akita mice that developed T1DM around 3-4 weeks after birth. Both Ins2 Akita and wild-type (WT) mice were analyzed at 4, 6, and 12 months to assess bone parameters such as femur length, growth plate thickness and number of mature and preapoptotic chondrocytes. In addition, bone microarchitecture of the cortical and trabecular regions was measured by microcomputed tomography and gene expression of Adamst-5, Col2, Igf1, Runx2, Acp5, and Oc was quantified by quantitative real-time polymerase chain reaction. Ins2 Akita mice showed a decreased longitudinal growth of the femur that was related to decreased growth plate thickness, lower number of chondrocytes and to a higher number of preapoptotic cells. These changes were associated with higher expression of Adamst-5, suggesting higher cartilage degradation, and with low expression levels of Igf1 and Col2 that reflect the decreased growth ability of diabetic mice. Ins2 Akita bone morphology was characterized by low cortical bone area (Ct.Ar) but higher trabecular bone volume (BV/TV) and expression analysis showed a downregulation of bone markers Acp5, Oc, and Runx2. Serum levels of insulin and leptin were found to be reduced at all-time points Ins2 Akita . We suggest that Ins2 Akita mice bone phenotype is caused by lower bone formation and even lower bone resorption due to insulin deficiency and to a possible relation with low leptin signaling.SFRH/BD/76873/2011/ SFRH/BD/76429/2011/ PEst-C/MAR/LA0015/2011info:eu-repo/semantics/publishedVersio