Exposure to the complement C5b-9 complex sensitizes 661W photoreceptor cells to both apoptosis and necroptosis.

The loss of photoreceptors is the defining characteristic of many retinal degenerative diseases, but the mechanisms that regulate photoreceptor cell death are not fully understood. Here we have used the 661W cone photoreceptor cell line to ask whether exposure to the terminal complement complex C5b-9 induces cell death and/or modulates the sensitivity of these cells to other cellular stressors. 661W cone photoreceptors were exposed to complete normal human serum following antibody blockade of CD59. Apoptosis induction was assessed morphologically, by flow cytometry, and on western blotting by probing for cleaved PARP and activated caspase-3. Necroptosis was assessed by flow cytometry and Sirtuin 2 inhibition using 2-cyano-3-[5-(2,5-dichlorophenyl)-2-furyl]-N-5-quinolinylacrylamide (AGK2). The sensitivity of 661W cells to ionomycin, staurosporine, peroxide and chelerythrine was also investigated, with or without prior formation of C5b-9. 661W cells underwent apoptotic cell death following exposure to C5b-9, as judged by poly(ADP-ribose) polymerase 1 cleavage and activation of caspase-3. We also observed apoptotic cell death in response to staurosporine, but 661W cells were resistant to both ionomycin and peroxide. Interestingly, C5b-9 significantly increased 661W sensitivity to staurosporine-induced apoptosis and necroptosis. These studies show that low levels of C5b-9 on 661W cells can induce apoptosis, and that C5b-9 specifically sensitizes 661W cells to certain apoptotic and necroptotic pathways. Our observations provide new insight into the potential role of the complement system in photoreceptor loss, with implications for the molecular aetiology of retinal disease.

Regulation of retinal pigment epithelial cell phenotype by Annexin A8.

The retinoic acid derivative fenretinide (FR) is capable of transdifferentiating cultured retinal pigment epithelial (RPE) cells towards a neuronal-like phenotype, but the underlying mechanisms are not understood. To identify genes involved in this process we performed a microarray analysis of RPE cells pre- and post-FR treatment, and observed a marked down-regulation of AnnexinA8 (AnxA8) in transdifferentiated cells. To determine whether AnxA8 plays a role in maintaining RPE cell phenotype we directly manipulated AnxA8 expression in cultured and primary RPE cells using siRNA-mediated gene suppression, and over-expression of AnxA8-GFP in conjunction with exposure to FR. Treatment of RPE cells with AnxA8 siRNA recapitulated exposure to FR, with cell cycle arrest, neuronal transdifferentiation, and concomitant up-regulation of the neuronal markers calretinin and calbindin, as assessed by real-time PCR and immunofluorescence. In contrast, AnxA8 transient over-expression in ARPE-19 cells prevented FR-induced differentiation. Ectopic expression of AnxA8 in AnxA8-depleted cells led to decreased neuronal marker staining, and normal cell growth as judged by phosphohistone H3 staining, cell counting and cleaved caspase-3 levels. These data show that down-regulation of AnxA8 is both necessary and sufficient for neuronal transdifferentiation of RPE cells and reveal an essential role for AnxA8 as a key regulator of RPE phenotype.

The Regional Hydrologic Extremes Assessment System: A software framework for hydrologic modeling and data assimilation.

The Regional Hydrologic Extremes Assessment System (RHEAS) is a prototype software framework for hydrologic modeling and data assimilation that automates the deployment of water resources nowcasting and forecasting applications. A spatially-enabled database is a key component of the software that can ingest a suite of satellite and model datasets while facilitating the interfacing with Geographic Information System (GIS) applications. The datasets ingested are obtained from numerous space-borne sensors and represent multiple components of the water cycle. The object-oriented design of the software allows for modularity and extensibility, showcased here with the coupling of the core hydrologic model with a crop growth model. RHEAS can exploit multi-threading to scale with increasing number of processors, while the database allows delivery of data products and associated uncertainty through a variety of GIS platforms. A set of three example implementations of RHEAS in the United States and Kenya are described to demonstrate the different features of the system in real-world applications.

Hepatitis B virus basal core promoter mutations show lower replication fitness associated with cccDNA acetylation status.

In chronic hepatitis B virus (HBV) infection, variants with mutations in the basal core promoter (BCP) and precore region predominate and associate with more severe disease forms. Studies on their effect on viral replication remain controversial. Increasing evidence shows that epigenetic modifications of cccDNA regulate HBV replication and disease outcome. Here we determined the transcription and viral replication efficiency of well-defined BCP and precore mutations and their effect on cccDNA epigenetic control. HBV monomers bearing BCP mutations A1762T/G1764A and A1762T/G1764A/C1766T, and precore mutations G1896A, G1899A and G1896A/G1899A, were transfected into HepG2 cells using a plasmid-free approach. Viral RNA transcripts were detected by Northern blot hybridization and RT PCR, DNA replicative intermediates by Southern blotting and RT PCR, and viral release was measured by ELISA. Acetylation of cccDNA-bound histones was assessed by Chromatin ImmunoPrecipitation (ChIP) assay and methylation of cccDNA by bisulfite sequencing. BCP mutations resulted in low viral release, mRNA transcription and pgRNA/cccDNA ratios that paralleled the acetylation of cccDNA-bound H4 histone and inversely correlated with the HDAC1 recruitment onto cccDNA. Independently of the mutations, cccDNA was a target for methylation, accompanied by the upregulation of DNMT1 expression and DNMT1 recruitment onto cccDNA. Our results suggest that BCP mutations decrease viral replication capacity possibly by modulating the acetylation and deacetylation of cccDNA-bound histones while precore mutations do not have a significant effect on viral replication. These data provide evidence that epigenetic factors contribute to the regulation of HBV viral replication.

Assay-dependent phytotoxicity of nanoparticles to plants.

The effects of five nanomaterials (multiwalled carbon nanotubes [MWCNTs], Ag, Cu, ZnO, Si) and their corresponding bulk counterparts on seed germination, root elongation, and biomass of Cucurbita pepo (zucchini) were investigated. The plants were grown in hydroponic solutions amended with nanoparticles or bulk material suspensions at 1000 mg/L. Seed germination was unaffected by any of the treatments, but Cu nanoparticles reduced emerging root length by 77% and 64% relative to unamended controls and seeds exposed to bulk Cu powder, respectively. During a 15-day hydroponic trial, the biomass of plants exposed to MWCNTs and Ag nanoparticles was reduced by 60% and 75%, respectively, as compared to control plants and corresponding bulk carbon and Ag powder solutions. Although bulk Cu powder reduced biomass by 69%, Cu nanoparticle exposure resulted in 90% reduction relative to control plants. Both Ag and Cu ion controls (1-1000 mg/L) and supernatant from centrifuged nanoparticle solutions (1000 mg/L) indicate that half the observed phytotoxicity is from the elemental nanoparticles themselves. The biomass and transpiration volume of zucchini exposed to Ag nanoparticles or bulk powder at 0-1000 mg/mL for 17 days was measured. Exposure to Ag nanoparticles at 500 and 100 mg/L resulted in 57% and 41% decreases in plant biomass and transpiration, respectively, as compared to controls or to plants exposed to bulk Ag. On average, zucchini shoots exposed to Ag nanoparticles contained 4.7 greater Ag concentration than did the plants from the corresponding bulk solutions. These findings demonstrate that standard phytotoxicity tests such as germination and root elongation may not be sensitive enough or appropriate when evaluating nanoparticle toxicity to terrestrial plant species.