A single historical substitution drives an increase in acetylcholine receptor complexity.

Human adult muscle-type acetylcholine receptors are heteropentameric ion channels formed from four different, but evolutionarily related, subunits. These subunits assemble with a precise stoichiometry and arrangement such that two chemically distinct agonist-binding sites are formed between specific subunit pairs. How this subunit complexity evolved and became entrenched is unclear.

Building the vertebrate codex using the gene breaking protein trap library.

One key bottleneck in understanding the human genome is the relative under-characterization of 90% of protein coding regions. We report a collection of 1200 transgenic zebrafish strains made with the gene-break transposon (GBT) protein trap to simultaneously report and reversibly knockdown the tagged genes. Protein trap-associated mRFP expression shows previously undocumented expression of 35% and 90% of cloned genes at 2 and 4 days post-fertilization, respectively.

Back to the future: Rational maps for exploring acetylcholine receptor space and time.

Global functions of nicotinic acetylcholine receptors, such as subunit cooperativity and compatibility, likely emerge from a network of amino acid residues distributed across the entire pentameric complex. Identification of such networks has stymied traditional approaches to acetylcholine receptor structure and function, likely due to the cryptic interdependency of their underlying amino acid residues. An emerging evolutionary biochemistry approach, which traces the evolutionary history of acetylcholine receptor subunits, allows for rational mapping of acetylcholine receptor sequence space, and offers new hope for uncovering the amino acid origins of these enigmatic properties.

Ancestral Reconstruction Approach to Acetylcholine Receptor Structure and Function.

Acetylcholine receptors (AChRs) are members of a superfamily of proteins called pentameric ligand-gated ion channels, which are found in almost all forms of life and thus have a rich evolutionary history. Muscle-type AChRs are heteropentameric complexes assembled from four related subunits (α, β, δ, and ɛ). Here we reconstruct the amino acid sequence of a β subunit ancestor shared by humans and cartilaginous fishes (i.

Antitumor effect of FGFR inhibitors on a novel cholangiocarcinoma patient derived xenograft mouse model endogenously expressing an FGFR2-CCDC6 fusion protein.

Cholangiocarcinoma is a highly lethal cancer with limited therapeutic options. Recent genomic analysis of cholangiocarcinoma has revealed the presence of fibroblast growth factor receptor 2 (FGFR2) fusion proteins in up to 13% of intrahepatic cholangiocarcinoma (iCCA). FGFR fusions have been identified as a novel oncogenic and druggable target in a number of cancers.

Activation of P-TEFb by Androgen Receptor-Regulated Enhancer RNAs in Castration-Resistant Prostate Cancer.

The androgen receptor (AR) is required for castration-resistant prostate cancer (CRPC) progression, but the function and disease relevance of AR-bound enhancers remain unclear. Here, we identify a group of AR-regulated enhancer RNAs (e.g.

Allele-Specific Quantitative PCR for Accurate, Rapid, and Cost-Effective Genotyping.

Customizable endonucleases such as transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) enable rapid generation of mutant strains at genomic loci of interest in animal models and cell lines. With the accelerated pace of generating mutant alleles, genotyping has become a rate-limiting step to understanding the effects of genetic perturbation. Unless mutated alleles result in distinct morphological phenotypes, mutant strains need to be genotyped using standard methods in molecular biology.

FusX: A Rapid One-Step Transcription Activator-Like Effector Assembly System for Genome Science.

Transcription activator-like effectors (TALEs) are extremely effective, single-molecule DNA-targeting molecular cursors used for locus-specific genome science applications, including high-precision molecular medicine and other genome engineering applications. TALEs are used in genome engineering for locus-specific DNA editing and imaging, as artificial transcriptional activators and repressors, and for targeted epigenetic modification. TALEs as nucleases (TALENs) are effective editing tools and offer high binding specificity and fewer sequence constraints toward the targeted genome than other custom nuclease systems.

Protein-Trap Insertional Mutagenesis Uncovers New Genes Involved in Zebrafish Skin Development, Including a Neuregulin 2a-Based ErbB Signaling Pathway Required during Median Fin Fold Morphogenesis.

Skin disorders are widespread, but available treatments are limited. A more comprehensive understanding of skin development mechanisms will drive identification of new treatment targets and modalities. Here we report the Zebrafish Integument Project (ZIP), an expression-driven platform for identifying new skin genes and phenotypes in the vertebrate model Danio rerio (zebrafish).

Human trafficking: crime in our own backyard.

Imagine for a moment being in a small, cold, dark, and dirty room. You haven't seen your family in months and you're not sure if you ever will again. When the drugs that you've given begin to wear off, you feel hunger pangs because you haven't eaten anything in more hours than you can count.

Heat shock protein 27 attenuates neointima formation and accelerates reendothelialization after arterial injury and stent implantation: importance of vascular endothelial growth factor up-regulation.

Elevated serum heat shock protein 27 (HSP27) levels are atheroprotective; however, the role of HSP27 after arterial injury is unknown. Human endothelial progenitor cells (EPCs) were treated with recombinant (r)HSP27 (50 μg/ml) or its inactive C1 terminus, and gene expression was characterized before functional studies were performed in vitro and in vivo. Vascular endothelial growth factor (VEGF) was markedly up-regulated by rHSP27 (10- and 6-fold increases in mRNA and secretion, respectively).

Sclerostin alters serum vitamin D metabolite and fibroblast growth factor 23 concentrations and the urinary excretion of calcium.

Inactivating mutations of the SOST (sclerostin) gene are associated with overgrowth and sclerosis of the skeleton. To determine mechanisms by which increased amounts of calcium and phosphorus are accreted to enable enhanced bone mineralization in the absence of sclerostin, we measured concentrations of calciotropic and phosphaturic hormones, and urine and serum calcium and inorganic phosphorus in mice in which the sclerostin (sost) gene was replaced by the β-D-galactosidase (lacZ) gene in the germ line. Knockout (KO) (sost(-/-)) mice had increased bone mineral density and content, increased cortical and trabecular bone thickness, and greater net bone formation as a result of increased osteoblast and decreased osteoclast surfaces compared with wild-type (WT) mice.

Chronic over-expression of heat shock protein 27 attenuates atherogenesis and enhances plaque remodeling: a combined histological and mechanical assessment of aortic lesions.

Aims: Expression of Heat Shock Protein-27 (HSP27) is reduced in human coronary atherosclerosis. Over-expression of HSP27 is protective against the early formation of lesions in atherosclerosis-prone apoE(-/-) mice (apoE(-/-)HSP27(o/e)) - however, only in females. We now seek to determine if chronic HSP27 over-expression is protective in a model of advanced atherosclerosis in both male and female apoE(-/-) mice.

Seed storage proteins of the globulin family are cleaved post-translationally in wheat embryos.

Background: The 7S globulins are plant seed storage proteins that have been associated with the development of a number of human diseases, including peanut allergy. Immune reactivity to the wheat seed storage protein globulin-3 (Glo-3) has been associated with the development of the autoimmune disease type 1 diabetes in diabetes-prone rats and mice, as well as in a subset of human patients.

Findings: The present study characterized native wheat Glo-3 in salt-soluble wheat seed protein extracts.

Research resource: whole transcriptome RNA sequencing detects multiple 1α,25-dihydroxyvitamin D(3)-sensitive metabolic pathways in developing zebrafish.

The biological role of vitamin D receptors (VDR), which are abundantly expressed in developing zebrafish (Danio rerio) as early as 48 h after fertilization, and before the development of a mineralized skeleton and mature intestine and kidney, is unknown. We probed the role of VDR in developing zebrafish biology by examining changes in expression of RNA by whole transcriptome shotgun sequencing (RNA-seq) in fish treated with picomolar concentrations of the VDR ligand and hormonal form of vitamin D(3), 1α,25-dihydroxyvitamin D(3) [1α,25(OH)(2)D(3))].We observed significant changes in RNAs of transcription factors, leptin, peptide hormones, and RNAs encoding proteins of fatty acid, amino acid, xenobiotic metabolism, receptor-activator of NFκB ligand (RANKL), and calcitonin-like ligand receptor pathways.

Expression of sclerostin in the developing zebrafish (Danio rerio) brain and skeleton.

Sclerostin is a highly conserved, secreted, cystine-knot protein which regulates osteoblast function. Humans with mutations in the sclerostin gene (SOST), manifest increased axial and appendicular skeletal bone density with attendant complications. In adult bone, sclerostin is expressed in osteocytes and osteoblasts.

Attenuation of atherogenesis via the anti-inflammatory effects of the selective estrogen receptor beta modulator 8β-VE2.

Background: Recent studies suggest that modulation of estrogen receptor β (ERβ) may play a crucial role in maintaining vascular homeostasis. We hypothesized that selective ERβ activation will attenuate atherogenesis via anti-inflammatory mechanisms.

Methods And Results: Atherosclerosis-prone apoE mice were ovariectomized and then fed a high-cholesterol diet with daily subcutaneous injections of the highly selective and potent ERβ agonist (8β-VE2) for 5 weeks.

Determination of serum and plasma sclerostin concentrations by enzyme-linked immunoassays.

Background: Sclerostin alters bone formation. The precise and reproducible measurement of sclerostin concentrations in biological samples is important for assessment of metabolic bone disease. We determined sclerostin concentrations in serum and plasma using two commercially available ELISA.

Heat shock protein 27 protects against atherogenesis via an estrogen-dependent mechanism: role of selective estrogen receptor beta modulation.

Objective: We recently identified HSP27 as an atheroprotective protein that acts extracellularly to prevent foam cell formation and atherogenesis in female but not male mice, where serum levels of HSP27 were increased and inversely correlated with degree of lesion burden. In the current study we sought to determine whether estrogens are required for the observed atheroprotective benefits of HSP27 as well as its extracellular release.

Methods And Results: In vitro estrogens prompted the release of HSP27 from macrophages in an ERbeta specific manner that involved exosomal trafficking.

Extracellular release of the atheroprotective heat shock protein 27 is mediated by estrogen and competitively inhibits acLDL binding to scavenger receptor-A.

We recently identified heat shock protein 27 (HSP27) as an estrogen receptor beta (ERbeta)-associated protein and noted its role as a biomarker for atherosclerosis. The current study tests the hypothesis that HSP27 is protective against the development of atherosclerosis. HSP27 overexpressing (HSP27o/e) mice were crossed to apoE-/- mice that develop atherosclerosis when fed a high-fat diet.