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Niemann-Pick C1 protein (NPC1) is a late-endosomal membrane protein involved in trafficking of LDL-derived cholesterol, Niemann-Pick disease type C, and Ebola virus infection. NPC1 contains 13 transmembrane segments (TMs), five of which are thought to represent a "sterol-sensing domain" (SSD). Although present also in other key regulatory proteins of cholesterol biosynthesis, uptake, and signaling, the structure and mechanism of action of the SSD are unknown. Here we report a crystal structure of a large fragment of human NPC1 at 3.6 angstrom resolution, which reveals internal twofold pseudosymmetry along TM 2-13 and two structurally homologous domains that protrude 60 angstrom into the endosomal lumen. Strikingly, NPC1's SSD forms a cavity that is accessible from both the luminal bilayer leaflet and the endosomal lumen; computational modeling suggests that this cavity is large enough to accommodate one cholesterol molecule. We propose a model for NPC1 function in cholesterol sensing and transport.

Changes in neural circuits after experience-dependent plasticity are brought about by the formation of new circuits via axonal growth and pruning. Here, using a combination of electrophysiology, adeno-associated virus-delivered fluorescent proteins, analysis of mutant mice, and two-photon microscopy, we follow long-range horizontally projecting axons in primary somatosensory cortex before and after selective whisker plucking. Whisker plucking induces axonal growth and pruning of horizontal projecting axons from neurons located in the surrounding intact whisker representations. We report that amyloid precursor protein is crucial for axonal pruning and contributes in a cell autonomous way.

In vesicular transport pathways, membrane proteins and lipids are internalized, externalized or transported within cells, not by bulk diffusion of single molecules, but embedded in the membrane of small vesicles or thin tubules. The formation of these 'transport carriers' follows sequential events: membrane bending, fission from the donor compartment, transport and eventually fusion with the acceptor membrane. A similar sequence is involved during the internalization of drug or gene carriers inside cells. These membrane-shaping events are generally mediated by proteins binding to membranes. The mechanisms behind these biological processes are actively studied both in the context of cell biology and biophysics. Bin/amphiphysin/Rvs (BAR) domain proteins are ideally suited for illustrating how simple soft matter principles can account for membrane deformation by proteins. We review here some experimental methods and corresponding theoretical models to measure how these proteins affect the mechanics and the shape of membranes. In more detail, we show how an experimental method employing optical tweezers to pull a tube from a giant vesicle may give important quantitative insights into the mechanism by which proteins sense and generate membrane curvature and the mechanism of membrane scission. This article is part of the themed issue 'Soft interfacial materials: from fundamentals to formulation'.

Chordoma is a rare primary bone neoplasm that is resistant to standard chemotherapies. Despite aggressive surgical management, local recurrence and metastasis is not uncommon. To identify the specific genetic aberrations that play key roles in chordoma pathogenesis, we utilized a genome-wide high-resolution SNP-array and next generation sequencing (NGS)-based molecular profiling platform to study 24 patient samples with typical histopathologic features of chordoma. Matching normal tissues were available for 16 samples. SNP-array analysis revealed nonrandom copy number losses across the genome, frequently involving 3, 9p, 1p, 14, 10, and 13. In contrast, copy number gain is uncommon in chordomas. Two minimum deleted regions were observed on 3p within a similar to 8 Mb segment at 3p21.1-p21.31, which overlaps SETD2, BAP1 and PBRM1. The minimum deleted region on 9p was mapped to CDKN2A locus at 9p21.3, and homozygous deletion of CDKN2A was detected in 5/22 chordomas (similar to 23%). NGS-based molecular profiling demonstrated an extremely low level of mutation rate in chordomas, with an average of 0.5 mutations per sample for the 16 cases with matched normal. When the mutated genes were grouped based on molecular functions, many of the mutation events (similar to 40%) were found in chromatin regulatory genes. The combined copy number and mutation profiling revealed that SETD2 is the single gene affected most frequently in chordomas, either by deletion or by mutations. Our study demonstrated that chordoma belongs to the C-class (copy number changes) tumors whose oncogenic signature is non-random multiple copy number losses across the genome and genomic aberrations frequently alter chromatin regulatory genes. (C) 2016 Wiley Periodicals, Inc.

Despite the extensive use of doxycycline in tetracycline-inducible rodent models, little is known regarding its stability in feed or water or the most effective route or dose. We assessed the concentrations of doxycycline in reverse-osmosis-purified (RO; pH 6.0) and acidified RO (pH 2.6) water in untinted or green-tinted bottles. Doxycycline remained stable in all groups for 7 d and in acidified water in untinted bottles for 14 d. Fungal growth occurred in nonacidified water in tinted and untinted bottles by 12 and 14 d, respectively, and in tinted bottles containing acidified water on day 14, but not in untinted bottles with acidified water. Doxycycline concentrations were also assessed before and at various points after the pelleting of feed from 2 vendors. Each batch was divided for storage at 4 degrees C, at room temperature, or within ventilated mouse isolator cages and then sampled monthly for 6 mo. Drying caused the greatest decline in doxycycline concentration, whereas.-irradiation plus shipping and storage condition had minimal effect. Two mouse lines with tetracycline-inducible promoters received 25, 150, or 467 mu g/mL or 2 mg/mL doxycycline in water and 200 or 625 ppm in feed before analysis of GFP expression. GFP was expressed in Rosa-rtTA2 mice at 150 mu g/mL, whereas Cags-rtTA3 mice required 25 mu g/mL. These studies indicate that 1) doxycycline-compounded feed can be handled in the same manner as standard rodent feed, 2) tinted water bottles are not necessary for maintaining drug concentrations, and 3) concentrations lower than those used typically may be effective in lines with tetracycline-inducible promoters.

Amyloidosis is a major problem in over one hundred diseases, including Alzheimer's disease (AD). Using the iDISCO visualization method involving targeted molecular labeling, tissue clearing, and light-sheet microscopy, we studied plaque formation in the intact AD mouse brain at up to 27 months of age. We visualized amyloid plaques in 3D together with tau, microglia, and vasculature. Volume imaging coupled to automated detection and mapping enables precise and fast quantification of plaques within the entire intact mouse brain. The present methodology is also applicable to analysis of frozen human brain samples without specialized preservation. Remarkably, amyloid plaques in human brain tissues showed greater 3D complexity and surprisingly large three-dimensional amyloid patterns, or TAPs. The ability to visualize amyloid in 3D, especially in the context of their micro-environment, and the discovery of large TAPs may have important scientific and medical implications.

Cocaine's enhancement of dopamine signaling is crucial for its rewarding effects but its serotonergic effects are also relevant. Here we examined the role of the protein p11, which recruits serotonin 5HT(1B) and 5HT(4) receptors to the cell surface, in cocaine reward. For this purpose we tested wild-type (WT) and p11 knockout (KO) male and female mice for cocaine conditioned place preference (CPP) and its cocaine-induced reinstatement at different abstinence times, after 8 days of extinction and 28 days of being home-caged. All mice showed significant cocaine CPP. Among males, p11KO showed lower CPP than WT; this difference was also evident after 28 days of home-cage abstinence. In contrast, in females there were no CPP differences between p11KO and WT mice at any time point tested. Cocaine priming after the 28-day home-cage abstinence period also resulted in lower cocaine conditioned motor activity in both male and female p11KO mice. These results suggest that cocaine CPP and its persistence during extinction and reinstatement are modulated in a sex-differentiated manner by p11. The lack of protein p11 confers protection from CPP on male, but not female mice, immediately after cocaine conditioning as well as after prolonged abstinence, but not after short-term withdrawal. (C) 2016 Wiley Periodicals, Inc.

Novel ideas are needed to increase adherence to antihypertensive medication. The current study used data from the Counseling African Americans to Control Hypertension (CAATCH) study, a sample of 442 hypertensive African Americans, to investigate the mediating effects of expectation of hypertension care, social support, hypertension knowledge, and medication adherence, adjusting for age, sex, number of medications, diabetes, education, income, employment, insurance status, and intervention. Sixty-six percent of patients had an income of $20,000 or less and 56% had a high school education or less, with a mean age of 57 years. Greater expectation of care was associated with greater medication adherence (P=.007), and greater social support was also associated with greater medication adherence (P=.046). Analysis also showed that expectation of care mediated the relationship between hypertension knowledge and medication adherence (P<.05). Expectation of care and social support are important factors for developing interventions to increase medication adherence among blacks.

High-frequency point mutations of genes encoding his tones have been identified recently as novel drivers in a number of tumors. Specifically, the H3K36M/I mutations were shown to be oncogenic in chondroblastomas and undifferentiated sarcomas by inhibiting H3K36 methyltransferases, including SETD2. Here we report the crystal structures of the SETD2 catalytic domain bound to H3K36M or H3K36I peptides with SAH (5-adenosylhomocysteine). In the complex structure, the catalytic domain adopts an open conformation, with the K36M/I peptide snuggly positioned in a newly formed substrate channel. Our structural and biochemical data reveal the molecular basis underying oncohistone recognition by and inhibition of SETD2.