Identification of fluoxetine as a direct NLRP3 inhibitor to treat atrophic macular degeneration.

The atrophic form of age-related macular degeneration (dry AMD) affects nearly 200 million people worldwide. There is no Food and Drug Administration (FDA)-approved therapy for this disease, which is the leading cause of irreversible blindness among people over 50 y of age. Vision loss in dry AMD results from degeneration of the retinal pigmented epithelium (RPE). RPE cell death is driven in part by accumulation of Alu RNAs, which are noncoding transcripts of a human retrotransposon. Alu RNA induces RPE degeneration by activating the NLRP3-ASC inflammasome. We report that fluoxetine, an FDA-approved drug for treating clinical depression, binds NLRP3 in silico, in vitro, and in vivo and inhibits activation of the NLRP3-ASC inflammasome and inflammatory cytokine release in RPE cells and macrophages, two critical cell types in dry AMD. We also demonstrate that fluoxetine, unlike several other antidepressant drugs, reduces Alu RNA-induced RPE degeneration in mice. Finally, by analyzing two health insurance databases comprising more than 100 million Americans, we report a reduced hazard of developing dry AMD among patients with depression who were treated with fluoxetine. Collectively, these studies identify fluoxetine as a potential drug-repurposing candidate for dry AMD.

Direct Detection of Conserved Viral Sequences and Other Nucleic Acid Motifs with Solid-State Nanopores.

The rapid and reliable recognition of nucleic acid sequences is essential to a broad range of fields including genotyping, gene expression analysis, and pathogen screening. For viral detection in particular, the capability is critical for optimal therapeutic response and preventing disease transmission. Here, we report an approach for detecting identifying sequence motifs within genome-scale single-strand DNA and RNA based on solid-state nanopores. By designing DNA oligonucleotide probes with complementarity to target sequences within a target genome, we establish a protocol to yield affinity-tagged duplex molecules the same length as the probe only if the target is present. The product can subsequently be bound to a protein chaperone and analyzed quantitatively with a selective solid-state nanopore assay. We first use a model DNA genome (M13mp18) to validate the approach, showing the successful isolation and detection of multiple target sequences simultaneously. We then demonstrate the protocol for the detection of RNA viruses by identifying and targeting a highly conserved sequence within human immunodeficiency virus (HIV-1B).

Selenium-Dependent Read Through of the Conserved 3'-Terminal UGA Stop Codon of HIV-1 nef.

The HIV-1 nef gene terminates in a 3'-UGA stop codon, which is highly conserved in the main group of HIV-1 subtypes, along with a downstream potential coding region that could extend the nef protein by 33 amino acids, if readthrough of the stop codon occurs. Antisense tethering interactions (ATIs) between a viral mRNA and a host selenoprotein mRNA are a potential viral strategy for the capture of a host selenocysteine insertion sequence (SECIS) element (Taylor et al, 2016) [1]. This mRNA hijacking mechanism could enable the expression of virally encoded selenoprotein modules, via translation of in-frame UGA stop codons as selenocysteine (SeC). Here we show that readthrough of the 3'-terminal UGA codon of nef occurs during translation of HIV-1 nef expression constructs in transfected cells. This was accomplished via fluorescence microscopy image analysis and flow cytometry of HEK 293 cells, transfected with engineered GFP reporter gene plasmid constructs, in which GFP can only be expressed by translational recoding of the UGA codon. SiRNA knockdown of thioredoxin reductase 1 (TR1) mRNA resulted in a 67% decrease in GFP expression, presumably due to reduced availability of the components involved in selenocysteine incorporation for the stop codon readthrough, thus supporting the proposed ATI. Addition of 20 nM sodium selenite to the media significantly enhanced stop codon readthrough in the pNefATI1 plasmid construct, by >100%, supporting the hypothesis that selenium is involved in the UGA readthrough mechanism.

A G-quadruplex nanoswitch in the SGK1 promoter regulates isoform expression by K+/Na+ balance and resveratrol binding.

BACKGROUND: High sodium intake can up-regulate the level of renal serum- and glucocorticoid-inducible kinase-1 (SGK1), which plays a pivotal role in controlling blood pressure via activation of the epithelial sodium channel (ENaC), which can lead to salt-sensitive hypertension. Increased potassium intake, or a vegetarian diet, counteracts salt-sensitive hypertension, but the underlying mechanisms are not fully understood. METHODS: Bioinformatics and molecular modeling were used to identify G-quadruplex (G4) and their conformations in the SGK1 promoter. CD spectra and UV melting dynamics were measured to study the stability of G4 as influenced by potassium/sodium balance and resveratrol. RT-PCR and Western blot were employed to study the effects of potassium and resveratrol on the SGK1 isoform expression. RESULTS: The SGK1 gene encodes a G4 structure in the proximal upstream of promoter-2; the G4 structure is stabilized by potassium or resveratrol, but destabilized by sodium. Super-physiological levels of sodium stimulate the transcription of all SGK1 isoforms, whereas resveratrol or potassium supplementation inhibits the transcription of iso-2 and iso-3, but not iso-1. CONCLUSIONS: Stabilizing the G4 by potassium or resveratrol induces alternative promoter usage and/or pre-mRNA splicing in the transcription of SGK1. GENERAL SIGNIFICANCE: Potassium/sodium ion balance or resveratrol binding can act to regulate G4 molecular switches for controlling SGK1 gene expression, thereby presenting a new avenue for drug development.

Towards a Better Understanding of Computational Models for Predicting DNA Methylation Effects at the Molecular Level.

Human DNA is a very sensitive macromolecule and slight changes in the structure of DNA can have disastrous effects on the organism. When nucleotides are modified, or changed, the resulting DNA sequence can lose its information, if it is part of a gene, or it can become a problem for replication and repair. Human cells can regulate themselves by using a process known as DNA methylation. This methylation is vitally important in cell differentiation and expression of genes. When the methylation is uncontrolled, however, or does not occur in the right place, serious pathophysiological consequences may result. Excess methylation causes changes in the conformation of the DNA double helix. The secondary structure of DNA is highly dependent upon the sequence. Therefore, if the sequence changes slightly the secondary structure can change as well. These slight changes will then cause the doublestranded DNA to be more open and available in some places where large adductions can come in and react with the DNA base pairs. Computer models have been used to simulate a variety of biological processes including protein function and binding, and there is a growing body of evidence that in silico methods can shed light on DNA methylation. Understanding the anomeric effect that contributes to the structural and conformational flexibility of furanose rings through a combination of quantum mechanical and experimental studies is critical for successful molecular dynamic simulations.

Solid-State Nanopore Analysis of Diverse DNA Base Modifications Using a Modular Enzymatic Labeling Process.

Many regulated epigenetic elements and base lesions found in genomic DNA can both directly impact gene expression and play a role in disease processes. However, due to their noncanonical nature, they are challenging to assess with conventional technologies. Here, we present a new approach for the targeted detection of diverse modified bases in DNA. We first use enzymatic components of the DNA base excision repair pathway to install an individual affinity label at each location of a selected modified base with high yield. We then probe the resulting material with a solid-state nanopore assay capable of discriminating labeled DNA from unlabeled DNA. The technique features exceptional modularity via selection of targeting enzymes, which we establish through the detection of four DNA base elements: uracil, 8-oxoguanine, T:G mismatch, and the methyladenine analog 1,N6-ethenoadenine. Our results demonstrate the potential for a quantitative nanopore assessment of a broad range of base modifications.

Sequence-Specific Recognition of MicroRNAs and Other Short Nucleic Acids with Solid-State Nanopores.

The detection and quantification of short nucleic acid sequences has many potential applications in studying biological processes, monitoring disease initiation and progression, and evaluating environmental systems, but is challenging by nature. We present here an assay based on the solid-state nanopore platform for the identification of specific sequences in solution. We demonstrate that hybridization of a target nucleic acid with a synthetic probe molecule enables discrimination between duplex and single-stranded molecules with high efficacy. Our approach requires limited preparation of samples and yields an unambiguous translocation event rate enhancement that can be used to determine the presence and abundance of a single sequence within a background of nontarget oligonucleotides.

Nanopore Analysis of Single-Stranded Binding Protein Interactions with DNA.

We study the binding of E. coli single-stranded binding protein (SSB) to single-stranded DNA (ssDNA) using a solid-state nanopore assay. We find that saturated nucleoprotein complexes can be distinguished easily from free SSB, ssDNA, or double-stranded DNA individually and demonstrate that the high affinity of SSB for ssDNA can be exploited to achieve high-fidelity differentiation from duplex molecules in a mixture. We then study nucleoprotein filament formation by systematically varying the amount of SSB relative to ssDNA. We observe a concomitant shift in the mean amplitude of electrical events that is consistent with weakly cooperative binding. Finally, we compare circular and linearized ssDNA saturated with SSB and use the results to infer structural details of the nucleoprotein complex.

Detecting DNA depurination with solid-state nanopores.

Among the different types of DNA damage that occur endogenously in the cell, depurination is especially prevalent. These lesions can initiate mutagenesis and have been implicated in a variety of diseases, including cancer. Here, we demonstrate a new approach for the detection of depurination at the single-molecule scale using solid-state nanopores. We induce depurination in short duplex DNA using acidic conditions and observe that the presence of apurinic sites results in significantly slower dynamics during electrokinetic translocation. This procedure may be valuable as a diagnostic for in situ quantification of DNA depurination.

Selective detection and quantification of modified DNA with solid-state nanopores.

We demonstrate a solid-state nanopore assay for the unambiguous discrimination and quantification of modified DNA. Individual streptavidin proteins are employed as high-affinity tags for DNA containing a single biotin moiety. We establish that the rate of translocation events corresponds directly to relative concentration of protein-DNA complexes and use the selectivity of our approach to quantify modified oligonucleotides from among a background of unmodified DNA in solution.

Interpreting the conductance blockades of DNA translocations through solid-state nanopores.

Solid-state nanopore electrical signatures can be convoluted and are thus challenging to interpret. In order to better understand the origin of these conductance changes, we investigate the translocation of DNA through small, thin pores over a range of voltage. We observe multiple, discrete populations of conductance blockades that vary with applied voltage. To describe our observations, we develop a simple model that is applicable to solid-state nanopores generally. These results represent an important step toward understanding the dynamics of the electrokinetic translocation process.

Impact of heat treatment on size, structure, and bioactivity of elemental selenium nanoparticles.

BACKGROUND: Elemental selenium nanoparticles have emerged as a novel selenium source with the advantage of reduced risk of selenium toxicity. The present work investigated whether heat treatment affects the size, structure, and bioactivity of selenium nanoparticles. METHODS AND RESULTS: After a one-hour incubation of solution containing 80 nm selenium particles in a 90°C water bath, the nanoparticles aggregated into larger 110 nm particles and nanorods (290 nm × 70 nm), leading to significantly reduced bioavailability and phase II enzyme induction in selenium-deficient mice. When a solution containing 40 nm selenium nanoparticles was treated under the same conditions, the nanoparticles aggregated into larger 72 nm particles but did not transform into nanorods, demonstrating that the thermostability of selenium nanoparticles is size-dependent, smaller selenium nanoparticles being more resistant than larger selenium nanoparticles to transformation into nanorods during heat treatment. CONCLUSION: The present results suggest that temperature and duration of the heat process, as well as the original nanoparticle size, should be carefully selected when a solution containing selenium nanoparticles is added to functional foods.

Sequence- and target-independent angiogenesis suppression by siRNA via TLR3.

Clinical trials of small interfering RNA (siRNA) targeting vascular endothelial growth factor-A (VEGFA) or its receptor VEGFR1 (also called FLT1), in patients with blinding choroidal neovascularization (CNV) from age-related macular degeneration, are premised on gene silencing by means of intracellular RNA interference (RNAi). We show instead that CNV inhibition is a siRNA-class effect: 21-nucleotide or longer siRNAs targeting non-mammalian genes, non-expressed genes, non-genomic sequences, pro- and anti-angiogenic genes, and RNAi-incompetent siRNAs all suppressed CNV in mice comparably to siRNAs targeting Vegfa or Vegfr1 without off-target RNAi or interferon-alpha/beta activation. Non-targeted (against non-mammalian genes) and targeted (against Vegfa or Vegfr1) siRNA suppressed CNV via cell-surface toll-like receptor 3 (TLR3), its adaptor TRIF, and induction of interferon-gamma and interleukin-12. Non-targeted siRNA suppressed dermal neovascularization in mice as effectively as Vegfa siRNA. siRNA-induced inhibition of neovascularization required a minimum length of 21 nucleotides, a bridging necessity in a modelled 2:1 TLR3-RNA complex. Choroidal endothelial cells from people expressing the TLR3 coding variant 412FF were refractory to extracellular siRNA-induced cytotoxicity, facilitating individualized pharmacogenetic therapy. Multiple human endothelial cell types expressed surface TLR3, indicating that generic siRNAs might treat angiogenic disorders that affect 8% of the world's population, and that siRNAs might induce unanticipated vascular or immune effects.

Further insight into the impact of sodium selenite on selenoenzymes: high-dose selenite enhances hepatic thioredoxin reductase 1 activity as a consequence of liver injury.

Selenium (Se) at supranutritional levels can enhance the activity of glutathione S-transferase (GST), whose gene is a target of nuclear factor erythroid-2 related factor 2 (Nrf2). Recent studies indicated that the thioredoxin reductase 1 (TrxR1) gene could also be targeted by Nrf2. Thus, high-dose Se may stimulate TrxR1 provided it enhances GST activity. Indeed, one study found that Se at supranutritional levels transiently increased hepatic TrxR1 activity. However, another study reported that supranutritional Se had no such effect on hepatic TrxR1 activity. In view of this discrepancy, the present research investigated whether high-dose Se has any impact on hepatic TrxR1. Moreover, we investigated whether Se preferentially activates GST over TrxR1. We observed that when sodium selenite (SS) caused liver injury, both hepatic TrxR1 activity and hepatic GST activity increased. Further experiments indicated that SS increased hepatic GST activity at either toxic or high but non-toxic dose levels; however, increase in hepatic TrxR1 activity occurred only at toxic levels, suggesting that enhanced TrxR1 activity correlates with liver injury. To corroborate this, we showed that hepatotoxic agents, thioacetamide or carbon tetrachloride, caused marked increases in hepatic TrxR1 activity. In conclusion, high-dose SS indeed can enhance hepatic TrxR1 activity, but only on the condition that it causes liver injury. High-dose SS affects hepatic GST more readily than hepatic TrxR1. Thus, the cancer-preventive mechanism of Se at non-toxic supranutritional levels relies more on its modulation of GST rather than TrxR1, at least in liver tissue.

Inhibition of TNF-alpha induced ICAM-1, VCAM-1 and E-selectin expression by selenium.

The initiation of an atherosclerotic lesion involves an endothelial cell pro-inflammatory state that recruits leukocytes and promotes their movement across the endothelium. These processes require endothelial expression of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and endothelial-leukocyte adhesion molecule-1 (E-selectin). Tumor necrosis factor-alpha (TNF-alpha) is a powerful inducer of these adhesion molecules. Selenium status is known to affect the rate of atherosclerosis. These experiments tested whether selenium alters cytokine-induced expression of these adhesion molecules. Human umbilical vein endothelial cells (HUVECs) were pretreated for 24 h with sodium selenite (0-2 microM) and then treated with 0 or 50 U/ml TNF-alpha in the presence of 0-2 microM selenite. ICAM-1, VCAM-1 and E-selectin were detected by ELISA and their mRNAs were evaluated by Northern blots. Selenite significantly inhibited TNF-alpha-induced expression of each adhesion molecule in a dose-dependent manner and reduced the level of the respective mRNAs. Nuclear factor-kappa B (NF-kappa B) is required for transcription of these adhesion molecule genes. Western blot analysis revealed that selenite did not inhibit the translocation of the p65 subunit of NF-kappa B to the nucleus. In conclusion, these data indicate selenium can modulate cytokine-induced expression of ICAM-1, VCAM-1 and E-selectin in HUVECs without interfering with translocation of NF-kappa B.