Discovery of a small-molecule inhibitor and cellular probe of Keap1–Nrf2 protein–protein interaction

A high-throughput screen (HTS) of the MLPCN library using a homogenous fluorescence polarization assay identified a small molecule as a first-in-class direct inhibitor of Keap1–Nrf2 protein–protein interaction. The HTS hit has three chiral centers; a combination of flash and chiral chromatographic separation demonstrated that Keap1-binding activity resides predominantly in one stereoisomer (SRS)-5 designated as ML334 (LH601A), which is at least 100× more potent than the other stereoisomers. The stereochemistry of the four cis isomers was assigned using X-ray crystallography and confirmed using stereospecific synthesis. (SRS)-5 is functionally active in both an ARE gene reporter assay and an Nrf2 nuclear translocation assay. The stereospecific nature of binding between (SRS)-5 and Keap1 as well as the preliminary but tractable structure–activity relationships support its use as a lead for our ongoing optimization     

Signatures of diversifying selection at EST‐SSR loci and association with climate in natural Eucalyptus populations

Understanding the environmental parameters that drive adaptation among populations is important in predicting how species may respond to global climatic changes and how gene pools might be managed to conserve adaptive genetic diversity. Here, we used Bayesian F_ST outlier tests and allele–climate association analyses to reveal two Eucalyptus EST‐SSR loci as strong candidates for diversifying selection in natural populations of a southwestern Australian forest tree, Eucalyptus gomphocephala (Myrtaceae). The Eucalyptus homolog of a CONSTANS‐like gene was an F_ST outlier, and allelic variation showed significant latitudinal clinal associations with annual and winter solar radiation, potential evaporation, summer precipitation and aridity. A second F_ST outlier locus, homologous to quinone oxidoreductase, was significantly associated with measures of temperature range, high summer temperature and summer solar radiation, with important implications for predicting the effect of temperature on natural populations in the context of climate change. We complemented these data with investigations into neutral population genetic structure and diversity throughout the species range. This study provides an investigation into selection signatures at gene‐homologous EST‐SSRs in natural Eucalyptus populations, and contributes to our understanding of the relationship between climate and adaptive genetic variation, informing the conservation of both putatively neutral and adaptive components of genetic diversity.     

Sensitivity of continental United States atmospheric budgets of oxidized and reduced nitrogen to dry deposition parametrizations

Reactive nitrogen (N_r) is removed by surface fluxes (air–surface exchange) and wet deposition. The chemistry and physics of the atmosphere result in a complicated system in which competing chemical sources and sinks exist and impact that removal. Therefore, uncertainties are best examined with complete regional chemical transport models that simulate these feedbacks. We analysed several uncertainties in regional air quality model resistance analogue representations of air–surface exchange for unidirectional and bi-directional fluxes and their effect on the continental N_r budget. Model sensitivity tests of key parameters in dry deposition formulations showed that uncertainty estimates of continental total nitrogen deposition are surprisingly small, 5 per cent or less, owing to feedbacks in the chemistry and rebalancing among removal pathways. The largest uncertainties (5%) occur with the change from a unidirectional to a bi-directional NH₃ formulation followed by uncertainties in bi-directional compensation points (1–4%) and unidirectional aerodynamic resistance (2%). Uncertainties have a greater effect at the local scale. Between unidirectional and bi-directional formulations, single grid cell changes can be up to 50 per cent, whereas 84 per cent of the cells have changes less than 30 per cent. For uncertainties within either formulation, single grid cell change can be up to 20 per cent, but for 90 per cent of the cells changes are less than 10 per cent.     

Foraging area fidelity for Kemp's ridleys in the Gulf of Mexico

For many marine species, locations of key foraging areas are not well defined. We used satellite telemetry and switching state‐space modeling (SSM) to identify distinct foraging areas used by Kemp's ridley turtles (Lepidochelys kempii) tagged after nesting during 1998–2011 at Padre Island National Seashore, Texas, USA (PAIS; N = 22), and Rancho Nuevo, Tamaulipas, Mexico (RN; N = 9). Overall, turtles traveled a mean distance of 793.1 km (±347.8 SD) to foraging sites, where 24 of 31 turtles showed foraging area fidelity (FAF) over time (N = 22 in USA, N = 2 in Mexico). Multiple turtles foraged along their migratory route, prior to arrival at their “final” foraging sites. We identified new foraging “hotspots” where adult female Kemp's ridley turtles spent 44% of their time during tracking (i.e., 2641/6009 tracking days in foraging mode). Nearshore Gulf of Mexico waters served as foraging habitat for all turtles tracked in this study; final foraging sites were located in water <68 m deep and a mean distance of 33.2 km (±25.3 SD) from the nearest mainland coast. Distance to release site, distance to mainland shore, annual mean sea surface temperature, bathymetry, and net primary production were significant predictors of sites where turtles spent large numbers of days in foraging mode. Spatial similarity of particular foraging sites selected by different turtles over the 13‐year tracking period indicates that these areas represent critical foraging habitat, particularly in waters off Louisiana. Furthermore, the wide distribution of foraging sites indicates that a foraging corridor exists for Kemp's ridleys in the Gulf. Our results highlight the need for further study of environmental and bathymetric components of foraging sites and prey resources contained therein, as well as international cooperation to protect essential at‐sea foraging habitats for this imperiled species.     

Early Proliferation Does Not Prevent the Loss of Oligodendrocyte Progenitor Cells during the Chronic Phase of Secondary Degeneration in a CNS White Matter Tract

Partial injury to the central nervous system (CNS) is exacerbated by additional loss of neurons and glia via toxic events known as secondary degeneration. Using partial transection of the rat optic nerve (ON) as a model, we have previously shown that myelin decompaction persists during secondary degeneration. Failure to repair myelin abnormalities during secondary degeneration may be attributed to insufficient OPC proliferation and/or differentiation to compensate for loss of oligodendrocyte lineage cells (oligodendroglia). Following partial ON transection, we found that sub-populations of oligodendroglia and other olig2+ glia were differentially influenced by injury. A high proportion of NG2+/olig2–, NG2+/olig2+ and CC1−/olig2+ cells proliferated (Ki67+) at 3 days, prior to the onset of death (TUNEL+) at 7 days, suggesting injury-related cues triggered proliferation rather than early loss of oligodendroglia. Despite this, a high proportion (20%) of the NG2+/olig2+ OPCs were TUNEL+ at 3 months, and numbers remained chronically lower, indicating that proliferation of these cells was insufficient to maintain population numbers. There was significant death of NG2+/olig2– and NG2−/olig2+ cells at 7 days, however population densities remained stable, suggesting proliferation was sufficient to sustain cell numbers. Relatively few TUNEL+/CC1+ cells were detected at 7 days, and no change in density indicated that mature CC1+ oligodendrocytes were resistant to secondary degeneration in vivo. Mature CC1+/olig2– oligodendrocyte density increased at 3 days, reflecting early oligogenesis, while the appearance of shortened myelin internodes at 3 months suggested remyelination. Taken together, chronic OPC decreases may contribute to the persistent myelin abnormalities and functional loss seen in ON during secondary degeneration.     

Lung Surfactant Levels are Regulated by Ig-Hepta/GPR116 by Monitoring Surfactant Protein D

Lung surfactant is a complex mixture of lipids and proteins, which is secreted from the alveolar type II epithelial cell and coats the surface of alveoli as a thin layer. It plays a crucial role in the prevention of alveolar collapse through its ability to reduce surface tension. Under normal conditions, surfactant homeostasis is maintained by balancing its release and the uptake by the type II cell for recycling and the internalization by alveolar macrophages for degradation. Little is known about how the surfactant pool is monitored and regulated. Here we show, by an analysis of gene-targeted mice exhibiting massive accumulation of surfactant, that Ig-Hepta/GPR116, an orphan receptor, is expressed on the type II cell and sensing the amount of surfactant by monitoring one of its protein components, surfactant protein D, and its deletion results in a pulmonary alveolar proteinosis and emphysema-like pathology. By a coexpression experiment with Sp-D and the extracellular region of Ig-Hepta/GPR116 followed by immunoprecipitation, we identified Sp-D as the ligand of Ig-Hepta/GPR116. Analyses of surfactant metabolism in Ig-Hepta^+/+ and Ig-Hepta^−/− mice by using radioactive tracers indicated that the Ig-Hepta/GPR116 signaling system exerts attenuating effects on (i) balanced synthesis of surfactant lipids and proteins and (ii) surfactant secretion, and (iii) a stimulating effect on recycling (uptake) in response to elevated levels of Sp-D in alveolar space.     

High IKZF1/3 protein expression is a favorable prognostic factor for survival of relapsed/refractory multiple myeloma patients treated with lenalidomide

The aim of this study is to assess nucleoprotein expression of IKZF1/3 in patients with relapsed/refractory multiple myeloma (MM) who received lenalidomide-based therapy and correlated them with their clinical outcomes. A total of 50 patients diagnosed with MM were entered in the study with the median follow-up of 86.4 months. By immunohistochemistry (IHC), IKZF1 and IKZF3 were expressed in 72 and 58% of the cases, respectively. IKZF1 and IKZF3 expressions were associated with longer median progression free survival (P?=?0.0029 and P?<?0.0001) and overall survival (P?=?0.0014 and P?<?0.0001). IKZF3 expression also appears predicted a favorable response to the lenalidomide-based therapy.     

Association of DNA Methylation at CPT1A Locus with Metabolic Syndrome in the Genetics of Lipid Lowering Drugs and Diet Network (GOLDN) Study

In this study, we conducted an epigenome-wide association study of metabolic syndrome (MetS) among 846 participants of European descent in the Genetics of Lipid Lowering Drugs and Diet Network (GOLDN). DNA was isolated from CD4+ T cells and methylation at ~470,000 cytosine-phosphate-guanine dinucleotide (CpG) pairs was assayed using the Illumina Infinium HumanMethylation450 BeadChip. We modeled the percentage methylation at individual CpGs as a function of MetS using linear mixed models. A Bonferroni-corrected P-value of 1.1 x 10⁻⁷ was considered significant. Methylation at two CpG sites in CPT1A on chromosome 11 was significantly associated with MetS (P for cg00574958 = 2.6x10^-14 and P for cg17058475 = 1.2x10^-9). Significant associations were replicated in both European and African ancestry participants of the Bogalusa Heart Study. Our findings suggest that methylation in CPT1A is a promising epigenetic marker for MetS risk which could become useful as a treatment target in the future.     

The PDZ Protein Na+/H+ Exchanger Regulatory Factor-1 (NHERF1) Regulates Planar Cell Polarity and Motile Cilia Organization

Directional flow of the cerebrospinal fluid requires coordinated movement of the motile cilia of the ependymal epithelium that lines the cerebral ventricles. Here we report that mice lacking the Na⁺/H⁺ Exchanger Regulatory Factor 1 (NHERF1/Slc9a3r1, also known as EBP50) develop profound communicating hydrocephalus associated with fewer and disorganized ependymal cilia. Knockdown of NHERF1/slc9a3r1 in zebrafish embryos also causes severe hydrocephalus of the hindbrain and impaired ciliogenesis in the otic vesicle. Ultrastructural analysis did not reveal defects in the shape or organization of individual cilia. Similar phenotypes have been described in animals with deficiencies in Wnt signaling and the Planar Cell Polarity (PCP) pathway. We show that NHERF1 binds the PCP core genes Frizzled (Fzd) and Vangl. We further show that NHERF1 assembles a ternary complex with Fzd4 and Vangl2 and promotes translocation of Vangl2 to the plasma membrane, in particular to the apical surface of ependymal cells. Taken together, these results strongly support an important role for NHERF1 in the regulation of PCP signaling and the development of functional motile cilia.     

A high throughput substrate binding assay reveals hexachlorophene as an inhibitor of the ER-resident HSP70 chaperone GRP78

Glucose-regulated protein 78 (GRP78) is the ER resident 70 kDa heat shock protein 70 (HSP70) and has been hypothesized to be a therapeutic target for various forms of cancer due to its role in mitigating proteotoxic stress in the ER, its elevated expression in some cancers, and the correlation between high levels for GRP78 and a poor prognosis. Herein we report the development and use of a high throughput fluorescence polarization-based peptide binding assay as an initial step toward the discovery and development of GRP78 inhibitors. This assay was used in a pilot screen to discover the anti-infective agent, hexachlorophene, as an inhibitor of GRP78. Through biochemical characterization we show that hexachlorophene is a competitive inhibitor of the GRP78-peptide interaction. Biological investigations showed that this molecule induces the unfolded protein response, induces autophagy, and leads to apoptosis in a colon carcinoma cell model, which is known to be sensitive to GRP78 inhibition.