Enhancement of Adeno-Associated Virus-Mediated Gene Therapy Using Hydroxychloroquine in Murine and Human Tissues.

The therapeutic effects of gene therapy using adeno-associated virus (AAV) vectors are dependent on the efficacy of viral transduction. Currently, we have reached the safe limits of AAV vector dose, beyond which damaging inflammatory responses are seen. To improve the efficacy of AAV transduction, we treated mouse embryonic fibroblasts, primate retinal pigment epithelial cells, and human retinal explants with hydroxychloroquine (HCQ) 1 h prior to transduction with an AAV2 vector encoding GFP driven by a ubiquitous CAG promoter. This led to a consistent increase in GFP expression, up to 3-fold, compared with vector alone. Comparing subretinal injections of AAV2.CAG.GFP vector alone versus co-injection with 18.75 μM HCQ in paired eyes in mice, mean GFP expression was 4.6-fold higher in retinae co-treated with HCQ without retinal toxicity. A comparative 5.9-fold effect was seen with an AAV8(Y733F).GRK1.GFP vector containing the photoreceptor-specific rhodopsin kinase promoter. While the mechanism of action remains to be fully elucidated, our data suggest that a single pulse of adjunctive HCQ could safely improve AAV transduction in vivo, thus providing a novel strategy for enhancing the clinical effects of gene therapy

Sustainable woody biomass production systems: a novel solution for energy, agriculture and the environment in Portugal

Bioenergy is a major current issue within the agriculture and energy sectors and the society in general. The environmental and social concerns raised by first generation biofuel crops increased the interest on woody biomass. Biomass production for direct conversion to heat and/or electricity is common in northern European countries, the USA, and, more recently, southern Europe, mainly based on short rotation woody crops (SRWC). These crops are renewable sources of energy, CO2 neutral, able to preserve biodiversity and water, and to contribute to socio-economic development. In Portugal, there is an increasing demand of renewable and sustainable sources of energy. Portugal also presents conditions to expand biomass production and use for energy, namely through SRWCs. There is, however, a lack of experience in this field. With the purpose of studying short rotation woody crops dedicated to energy production, we have launched in Bragança, Portugal, a research project aiming to develop technology for the sustainable production, processing, and use of woody biomass for energy. The goal is to analyse environmental and socio-economic aspects of these crops considering carbon cycle, conservation of water, soils, and biodiversity, as well as creation of wealth locally. It also aims to evaluate the potential of northeastern Portugal for biomass production and carbon sequestration. In this presentation we address the rational behind the project, the methodologies followed, and the results obtained during the first year of the study. We also present our perspectives on the role of woody biomass production at several scales and for several scenarios.Fundação para a Ciência e Tecnologia. (PTDC/AGR‐CFL/64500/2006

Seagrass connectivity on the west coast of Africa supports the hypothesis of grazer-mediated seed dispersal

Population connectivity influences the distribution of genetic diversity and divergence along a species range, as the likelihood of extinction or differentiation increases in isolated populations. However, there is still poor understanding of the processes mediating interpopulation dispersal in marine species that are sessile and lack planktonic life stages. One such case is the seagrass species Halodule wrightii, which produces basal seeds, although detached plants can drift. Along the tropical western coast of Africa, this species occurs in distant discontinuous habitats, raising the question of how interpopulation dispersal is mediated. The species is a key source of ecosystem functions including feeding large migratory grazers. This study aims to infer whether genetic differentiation of the seagrass H. wrightii along the western coast of Africa supports a hypothesis of predominant transportation of rafting seagrass by ocean currents, versus the hypothesis of biotic vectors of dispersal. Additional hypotheses were addressed concerning range-wide clonality and genetic diversity, assessed with microsatellite markers on populations of the west coast of Africa from Mauritania to Angola. Population genetic diversity and structure were compared with predictions from biophysical models of dispersal by oceanographic currents. The genetic data revealed low divergence among most populations, in strong contrast with predictions of very low probability of connectivity mediated by currents along the western African coastline. Moderate to high genotypic diversity showed important seed recruitment, but genetic and genotypic diversities were lower at range edges. Populations north and south of the equator were differentiated, and remarkably, so were neighboring equatorial populations despite their proximity. These results reveal independent sources of colonization of meadows in these islands, which are major habitat for migratory grazing green turtles, also supporting the hypothesis of biotically mediated seed transport. The importance of seagrass for conservation of endangered macrofauna has been widely reported; here we report evidence supporting the reciprocal role, that macrofauna protection can also plays a role in long-term survival and reproductive success of seagrass.Fundação para a Ciência e Tecnologia - FCTinfo:eu-repo/semantics/publishedVersio

Non-classical forms of pemphigus: pemphigus herpetiformis, IgA pemphigus, paraneoplastic pemphigus and IgG/IgA pemphigus

The pemphigus group comprises the autoimmune intraepidermal blistering diseases classically divided into two major types: pemphigus vulgaris and pemphigus foliaceous. Pemphigus herpetiformis, IgA pemphigus, paraneoplastic pemphigus and IgG/IgA pemphigus are rarer forms that present some clinical, histological and immunopathological characteristics that are different from the classical types. These are reviewed in this article. Future research may help definitively to locate the position of these forms in the pemphigus group, especially with regard to pemphigus herpetiformis and the IgG/ IgA pemphigus.Universidade Federal de São Paulo (UNIFESP), Escola Paulista de Medicina (EPM) Dermatology DepartmentUniversidade Federal de São Paulo (UNIFESP), Escola Paulista de Medicina (EPM) Dermatology and Pathology DepartmentsUNIFESP, EPM, Dermatology DepartmentUNIFESP, EPM, Dermatology and Pathology DepartmentsSciEL

Outcomes from elective colorectal cancer surgery during the SARS-CoV-2 pandemic

This study aimed to describe the change in surgical practice and the impact of SARS-CoV-2 on mortality after surgical resection of colorectal cancer during the initial phases of the SARS-CoV-2 pandemic

Inclusion of the Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element Enhances AAV2-Driven Transduction of Mouse and Human Retina

The woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) has been included in the transgene cassette of adeno-associated virus (AAV) in several gene therapy clinical trials, including those for inherited retinal diseases. However, the extent to which WPRE increases transgene expression in the retina is still unclear. To address this question, AAV2 vectors containing a reporter gene with and without WPRE were initially compared in vitro and subsequently in vivo by subretinal delivery in mice. In both instances, the presence of WPRE led to significantly higher levels of transgene expression as measured by fundus fluorescence, western blot, and immunohistochemistry. The two vectors were further compared in human retinal explants derived from patients undergoing clinically indicated retinectomy, where again the presence of WPRE resulted in an enhancement of reporter gene expression. Finally, an analogous approach using a transgene currently employed in a clinical trial for choroideremia delivered similar results both in vitro and in vivo, confirming that the WPRE effect is transgene independent. Our data fully support the inclusion of WPRE in ongoing and future AAV retinal gene therapy trials, where it may allow a therapeutic effect to be achieved at an overall lower dose of vector. Keywords: AAV2, WPRE, gene therapy, mouse retina, human retina, choroideremi

Impact of Vital Dyes on Cell Viability and Transduction Efficiency of AAV Vectors Used in Retinal Gene Therapy Surgery: An In Vitro and In Vivo Analysis

PURPOSE: Treatment of inherited retinal degenerations using adeno-associated viral (AAV) vectors involves delivery by subretinal injection. In the latter stages, alteration of normal anatomy may cause difficulty in visualizing the retinotomy, retinal detachment extension, and vector diffusion. Vital dyes may be useful surgical adjuncts, but their safety and impact on AAV transduction are largely unknown. METHODS: The effects of Sodium Fluorescein (SF), Membrane Blue (MB), and Membrane Blue Dual (DB) at a range of dilutions were assessed on human embryonic kidney cells in vitro using an AAV2-green fluorescent protein (GFP) reporter at different multiplicities of infection. Flow cytometry analysis was performed to assess both cell viability and transduction efficiency. The effect on quantitative (q)PCR titer was determined. Balanced salt solution (BSS) or dilute DB (1:5 in BSS) were delivered subretinally into left/right eyes of C57BL/6J mice (n = 12). Retinal structure and function were analyzed by optical coherence tomography, autofluorescence, dark-and light-adapted full-field electroretinography. RESULTS: DB and MB were not toxic at any concentration tested, SF only when undiluted. The presence of dyes did not adversely affect the genomic titer. DB even increased the values, due to presence of surfactant in the formulation. AAV2-GFP transduction efficiency was not reduced by the dyes. No structural and functional toxic effects were observed following subretinal delivery of DB. CONCLUSIONS: Only undiluted SF affected cell viability. No effects on qPCR titer and transduction efficiency were observed. DB does not appear toxic when delivered subretinally and improves titer accuracy. DB may therefore be a safe and helpful adjunct during gene therapy surgery. TRANSLATIONAL RELEVANCE: This paper might be of interest to the retinal gene therapy community: it is a "bench to bedside" research paper about the potential use of dyes as a surgical adjunct during the gene therapy surgery. We have tested the potential toxicity and impact on transduction efficiency in an in vitro and in vivo model