Chromosome 10q26-driven age-related macular degeneration is associated with reduced levels of HTRA1 in human retinal pigment epithelium

Genome-wide association studies have identified the chromosome 10q26 (Chr10) locus, which contains the age-related maculopathy susceptibility 2 (ARMS2) and high temperature requirement A serine peptidase 1 (HTRA1) genes, as the strongest genetic risk factor for age-related macular degeneration (AMD) [L.G. Fritsche et al., Annu. Rev. Genomics Hum. Genet. 15, 151–171, (2014)]. To date, it has been difficult to assign causality to any specific single nucleotide polymorphism (SNP), haplotype, or gene within this region because of high linkage disequilibrium among the disease-associated variants [J. Jakobsdottir et al. Am. J. Hum. Genet. 77, 389–407 (2005); A. Rivera et al. Hum. Mol. Genet. 14, 3227–3236 (2005)]. Here, we show that HTRA1 messenger RNA (mRNA) is reduced in retinal pigment epithelium (RPE) but not in neural retina or choroid tissues derived from human donors with homozygous risk at the 10q26 locus. This tissue-specific decrease is mediated by the presence of a noncoding, cis-regulatory element overlapping the ARMS2 intron, which contains a potential Lhx2 transcription factor binding site that is disrupted by risk variant rs36212733. HtrA1 protein increases with age in the RPE–Bruch’s membrane (BM) interface in Chr10 nonrisk donors but fails to increase in donors with homozygous risk at the 10q26 locus. We propose that HtrA1, an extracellular chaperone and serine protease, functions to maintain the optimal integrity of the RPE–BM interface during the aging process and that reduced expression of HTRA1 mRNA and protein in Chr10 risk donors impairs this protective function, leading to increased risk of AMD pathogenesis. HtrA1 augmentation, not inhibition, in high-risk patients should be considered as a potential therapy for AMD

Framing REDD+ in the Brazilian national media: how discourses evolved amid global negotiation uncertainties

Reducing emissions from deforestation and degradation (REDD+) in tropical countries is an important and contested element of the post-Kyoto climate regime. For policy options which generate controversy between diverse actor groups, such as REDD+, mass media plays an important role in defining and supporting policy possibilities. Analysis of the way in which national media frames issues of climate change and deforestation can offer insights into the nature of the contested domains of the REDD+ policy process. Here, we examine the Brazilian national media discourses surrounding REDD+ because it contributes to setting the tone of policy debates at the federal level. Specifically, we ask the following: (i) How was REDD+ portrayed in the Brazilian national print media and whose opinions and perceptions were represented? and (ii) How have media frames on REDD+ in the national print media changed over time? Our results contribute with new knowledge for understanding the observed progress of REDD+ in Brazil. We identify two main themes that dominate the focus in the national media coverage of REDD+, specifically “politics and policymaking” (representing half the coverage) and “economics and market” (with over a third). Results show that discussions around carbon markets were amongst the most contested and that optimism in relation to REDD+ effectiveness declined over time. The analysis suggests that positions adopted on the national REDD+ strategy were shaped by state and federal collision of interests. We demonstrate an evolution of national concerns from an initial focus on efficiency (e.g. finance and carbon markets) to a recentred focus on equity issues (e.g. implementation of safeguards). We conclude with some thoughts on the implications of these features for REDD+ interventions and implementation in Brazil

Non-Integrative Lentivirus Drives High-Frequency cre-Mediated Cassette Exchange in Human Cells

Recombinase mediated cassette exchange (RMCE) is a two-step process leading to genetic modification in a specific genomic target sequence. The process involves insertion of a docking genetic cassette in the genome followed by DNA transfer of a second cassette flanked by compatible recombination signals and expression of the recombinase. Major technical drawbacks are cell viability upon transfection, toxicity of the enzyme, and the ability to target efficiently cell types of different origins. To overcome such drawbacks, we developed an RMCE assay that uses an integrase-deficient lentivirus (IDLV) vector in the second step combined with promoterless trapping of double selectable markers. Additionally, recombinase expression is self-limiting as a result of the exchangeable reaction, thus avoiding toxicity. Our approach provides proof-of-principle of a simple and novel strategy with expected wide applicability modelled on a human cell line with randomly integrated copies of a genetic landing pad. This strategy does not present foreseeable limitations for application to other cell systems modified by homologous recombination. Safety, efficiency, and simplicity are the major advantages of our system, which can be applied in low-to-medium throughput strategies for screening of cDNAs, non-coding RNAs during functional genomic studies, and drug screening