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Cruz-Orengo L.,University of Washington | Daniels B.P.,University of Washington | Dorsey D.,University of Washington | Basak S.A.,University of Washington | And 7 more authors.
Journal of Clinical Investigation | Year: 2014

Multiple sclerosis (MS) is an inflammatory disease of the CNS that is characterized by BBB dysfunction and has a much higher incidence in females. Compared with other strains of mice, EAE in the SJL mouse strain models multiple features of MS, including an enhanced sensitivity of female mice to disease; however, the molecular mechanisms that underlie the sex- and strain-dependent differences in disease susceptibility have not been described. We identified sphingosine-1-phosphate receptor 2 (S1PR2) as a sex- and strain-specific, disease-modifying molecule that regulates BBB permeability by destabilizing adherens junctions. S1PR2 expression was increased in disease-susceptible regions of the CNS of both female SJL EAE mice and female patients with MS compared with their male counterparts. Pharmacological blockade or lack of S1PR2 signaling decreased EAE disease severity as the result of enhanced endothelial barrier function. Enhanced S1PR2 signaling in an in vitro BBB model altered adherens junction formation via activation of Rho/ROCK, CDC42, and caveolin endocytosis-dependent pathways, resulting in loss of apicobasal polarity and relocation of abluminal CXCL12 to vessel lumina. Furthermore, S1PR2-dependent BBB disruption and CXCL12 relocation were observed in vivo. These results identify a link between S1PR2 signaling and BBB polarity and implicate S1PR2 in sex-specific patterns of disease during CNS autoimmunity.

Poppler L.,Washington University in St. Louis | Cohen J.,Washington University in St. Louis | Dolen U.C.,Washington University in St. Louis | Schriefer A.E.,Genome Technology Access Center | And 4 more authors.
Aesthetic Surgery Journal | Year: 2015

Background Subclinical infections, manifest as biofilms, are considered an important cause of capsular contracture. Acellular dermal matrices (ADMs) are frequently used in revision surgery to prevent recurrent capsular contractures. Objective We sought to identify an association between capsular contracture and biofilm formation on breast prostheses, capsules, and ADMs in a tissue expander/implant (TE/I) exchange clinical paradigm. Methods Biopsies of the prosthesis, capsule, and ADM from patients (N = 26) undergoing TE/I exchange for permanent breast implant were evaluated for subclinical infection. Capsular contracture was quantified with Baker Grade and intramammary pressure. Biofilm formation was evaluated with specialized cultures, rtPCR, bacterial taxonomy, live:dead staining, and scanning electron microscopy (SEM). Collagen distribution, capsular histology, and ADM remodeling were quantified following fluorescent and light microscopy. Results Prosthetic devices were implanted from 91 to 1115 days. Intramammary pressure increased with Baker Grade. Of 26 patients evaluated, one patient had a positive culture and one patient demonstrated convincing evidence of biofilm morphology on SEM. Following PCR amplification 5 samples randomly selected for 16S rRNA gene sequencing demonstrated an abundance of suborder Micrococcineae, consistent with contamination. Conclusions Our data suggest that bacterial biofilms likely contribute to a proportion, but not all diagnosed capsular contractures. Biofilm formation does not appear to differ significantly between ADMs or capsules. While capsular contracture remains an incompletely understood but common problem in breast implant surgery, advances in imaging, diagnostic, and molecular techniques can now provide more sophisticated insights into the pathophysiology of capsular contracture. Level of Evidence 4 Therapeutic. © 2015 The American Society for Aesthetic Plastic Surgery, Inc.

van Meel E.,University of Washington | Wegner D.J.,University of Washington | Cliften P.,Genome Technology Access Center | Willing M.C.,University of Washington | And 3 more authors.
BMC Medical Genetics | Year: 2013

Background: Methionyl-tRNA synthetase (MARS) catalyzes the ligation of methionine to its cognate transfer RNA and therefore plays an essential role in protein biosynthesis.Methods: We used exome sequencing, aminoacylation assays, homology modeling, and immuno-isolation of transfected MARS to identify and characterize mutations in the methionyl-tRNA synthetase gene (MARS) in an infant with an unexplained multi-organ phenotype.Results: We identified compound heterozygous mutations (F370L and I523T) in highly conserved regions of MARS. The parents were each heterozygous for one of the mutations. Aminoacylation assays documented that the F370L and I523T MARS mutants had 18 ± 6% and 16 ± 6%, respectively, of wild-type activity. Homology modeling of the human MARS sequence with the structure of E. coli MARS showed that the F370L and I523T mutations are in close proximity to each other, with residue I523 located in the methionine binding pocket. We found that the F370L and I523T mutations did not affect the association of MARS with the multisynthetase complex.Conclusion: This infant expands the catalogue of inherited human diseases caused by mutations in aminoacyl-tRNA synthetase genes. © 2013 van Meel et al.; licensee BioMed Central Ltd.

Genome Technology Access Center | Entity website

GTAC makes DNA technologies accessible to scientistsLearn More. The Genome Technology Access Center was established by the Department of Genetics at Washington University in St ...

Genome Technology Access Center | Entity website

Service Time Time to Process Samples Library Preparation From sample drop-off to beginning of processing:3-5weeks Wet-lab time: 3-7 days depending on prep type/sample size Sequencing From sample drop-off to beginning of processing:1-3 weeks Sequencing time: 1-4 days for Single-End reads, 2 days for Paired-End reads Data Analysis: 7 days for RNA-seq, 2-4 daysother standardanalyses Microarray From sample drop-off to beginning of processing:1-2 weeks Wet-lab processing: 4 days Data Analysis: 1 day Note: These times are estimates based on average experiment sizes and standard analyses. Average Experiment Sizes: 12-24 samples for microarray 12-24 samples for library prep and sequencing 48-96 samples for PCR Standard Analyses: Demultiplexing Alignment Snp Calling ChIP-Seq Peak detection Gene Expression Last updated on July 7, 2016

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