Journal: PLoS ONE

Article Title: Functional characterization of zebrafish orthologs of the human Beta 3-Glucosyltransferase B3GLCT gene mutated in Peters Plus Syndrome

doi: 10.1371/journal.pone.0184903

Figure Lengend Snippet: (A) The two zebrafish orthologs of B3GLCT show overall similar exonic arrangement. The number of each exon is located within each box and the size of the exon (in base pairs) is shown above each exon. The 5’ and 3’ UTRs are indicated preceding the first ATG and following the stop codon ( TAA/TAG ). White indicates the N-terminal signal sequence, light grey indicates the stem region and dark grey indicates the catalytic domain. The vertical black bar in exon 12 of each gene indicates the location of nucleotides encoding for the catalytic tri-aspartic acid residues. Horizontal lines underneath the zebrafish genes indicate previously annotated sequence and sequence identified in this study. (B) Schematic of genomic context for B3GLCT/b3glct. (C) Multiple species alignment of B3GLCT orthologs from human (NP_919299), mouse (NP_001074673), Xenopus (NP_001072551), and zebrafish. Blue bar indicates signal peptide, green indicates stem region and orange indicates catalytic core. Grey shading of amino acids indicates conservation. The DxD motif is boxed in red.

Article Snippet: The plasmids encoding for TALENs targeting exon 12 of b3glcta or b3glctb were digested using SmaI (New England Biolabs, Ipswich, MA, USA); the linearized plasmids were purified and used as a template for mRNA synthesis using the T7 RNA polymerase.

Techniques: Sequencing

Journal: PLoS ONE

Article Title: Functional characterization of zebrafish orthologs of the human Beta 3-Glucosyltransferase B3GLCT gene mutated in Peters Plus Syndrome

doi: 10.1371/journal.pone.0184903

Figure Lengend Snippet: (A) Schematic of b3glct genes indicating TALEN target sites (exon 1 and 12 for b3glcta and exon 12 for b3glctb , black arrows). The predicted protein product resulting from TALEN mediated disruption is shown. Editing events in the first exon of b3glcta are predicted to disrupt nearly the entire coding region of the transcript. For both b3glcta and b3glctb , editing in the 12 th exon is predicted to result in loss of most of the catalytic domain including the catalytic core and KDEL-like ER retention signal. Blue (SP)- Signal Peptide, Green (SR)- Stem Region, Orange (CD)- Catalytic Domain. (B) Images of zebrafish embryos at 5-dpf and adult zebrafish showing no gross morphological defects associated with loss of b3glct . (C) Functional evaluation of wild-type and mutant b3glct by in vitro β3-glucosyltransferase assays. Left panel- the endogenous β3-glucosyltransferase activity toward O -fucosylated TSR3 is dependent on the amount of protein in the wild type zebrafish homogenate; control reaction with 10 μg homogenate was performed using unmodified TSR3. Right panel- the endogenous β3-glucosyltransferase activity toward O -fucosylated TSR3 in the homogenate of double homozygous b3glct embryos is profoundly reduced compared with that in the wild type zebrafish homogenate; control reactions were performed using unmodified TSR3. Assays were performed in triplicate. Error bars indicate s.d.

Article Snippet: The plasmids encoding for TALENs targeting exon 12 of b3glcta or b3glctb were digested using SmaI (New England Biolabs, Ipswich, MA, USA); the linearized plasmids were purified and used as a template for mRNA synthesis using the T7 RNA polymerase.

Techniques: Functional Assay, Mutagenesis, In Vitro, Activity Assay

Journal: Journal of neurochemistry

Article Title: Murine dopaminergic Müller cells restore motor function in a model of Parkinson's disease.

doi: 10.1111/jnc.12475

Figure Lengend Snippet: Fig. 3 Cultured monkey M€uller cells also exhibit functional dopaminergic features. (a) Representative cultured M€uller cells from adult monkey retina (Cebus apella) show glutamine synthetase (GS) and nuclear receptor-related factor 1 (Nurr1)-immunoreactivity after 48 h treatment with 10 lM forskolin (FK) (scale bar = 20 lm). (b) Western blot analysis confirms tyrosine hydroxylase (TH) and M€uller-specific marker cellular retinaldehyde binding protein (CRALBP) expression in these cells. Recombinant TH was used for comparison (rTH). (c) HPLC-ED analysis shows detectable dopamine levels in control and FK-treated cultures. One hour pre-incubation with

Article Snippet: Samples were pre-incubated with 5% bovine serum albumin for 60 min, followed by overnight incubation with primary antibodies for Nurr1 (1 : 500; Santa Cruz Biotechnology, Dallas, TX, USA), TH (1 : 1000; Millipore Corporation, Bedford, MA, USA), glutamine synthase (GS – 1 : 100; Abcam, Cambridge, UK), CRALBP (1 : 250; Abcam).

Techniques: Cell Culture, Functional Assay, Western Blot, Marker, Binding Assay, Expressing, Recombinant, Comparison, Control, Incubation

Journal: Acta biomaterialia

Article Title: Synthesis and evaluation of dual crosslinked alginate microbeads

doi: 10.1016/j.actbio.2017.10.046

Figure Lengend Snippet: Live/Dead imaging of encapsulated MIN6 cells in dual crosslinked alginate hydrogels in vitro. Total number of cells appear to decrease from day 1 to day 10 for all groups tested. Dashed lines represents margins of the beads. Scale bar represents 200 μm.

Article Snippet: Cell viability and function MIN6 cells (AddexBio) were encapsulated within beads to evaluate toxicity of the crosslinked alginate.

Techniques: Imaging, In Vitro

Journal: Acta biomaterialia

Article Title: Synthesis and evaluation of dual crosslinked alginate microbeads

doi: 10.1016/j.actbio.2017.10.046

Figure Lengend Snippet: Insulin secretion of MIN6 cells encapsulated within ionically and covalently crosslinked alginate microbeads plotted both as A) fold change per time and B) concentration (ng/ml) per time. Insulin secretion increased with time for all formulations. *Denotes statistical difference (p ≤ .05) for Alg when compared to time 0. #Denotes statistical difference (p ≤.05) for 1.12 MethAlg when compared to time 0. +Denotes statistical difference (p ≤ .05) for 3.95 MethAlg when compared to time 0.

Article Snippet: Cell viability and function MIN6 cells (AddexBio) were encapsulated within beads to evaluate toxicity of the crosslinked alginate.

Techniques: Concentration Assay

Journal: Annual review of genomics and human genetics

Article Title: Defining the Clinical Value of a Genomic Diagnosis in the Era of Next-Generation Sequencing

doi: 10.1146/annurev-genom-083115-022348

Figure Lengend Snippet: Sequencing details for seven large genome-scale studies

Article Snippet: Exclusion of critical details coupled with inherently unique populations reported in each publication virtually guarantees a lack of reproducibility among these studies. table ft1 table-wrap mode="anchored" t5 caption a7 Study type Exome (research study) Exome (clinical laboratory) Genome Study Zhu et al. (136) NCGENES a Lee et al. (68) Yang et al. (134) Farwell et al. (39) Retterer et al. (99) Taylor et al. (120) Capture Illumina TruSeq Exome Enrichment kit (65 Mb) Roche NimbleGen SeqCap EZ Human Exome Library kit Agilent SureSelect Human All Exon kit (50 Mb) Agilent SureSelect XT Customized Agilent SureSelect Human All Exon V2 kit (50 Mb) Roche NimbleGen SeqCap EZ HGSC VCRome (version 2.1) Agilent SureSelect Target Enrichment System Roche NimbleGen SeqCap EZ HGSC VCRome (version 2.0) Agilent SureSelect Human All Exon V4 kit (50 Mb) None Sequencing platform Illumina HiSeq 2000 Illumina HiSeq 2000 and 2500 Illumina HiSeq 2000 (50 bp paired-end) or 2500 (100 bp paired-end) Illumina Genome Analyzer IIx (100 bp paired-end) or HiSeq 2000 Not specified Illumina HiSeq 2000 or 2500 (100 bp paired-end) Illumina HiSeq 2000 or 2500 (100 bp) Alignment software BWA (version 0.5.10) BWA NovoAlign (version 2.07.15b) GATK (version 1.1-33) BWA (version 0.5.10) Not specified BWA BWA (version 0.5.6) Stampy (versions 1.0.12–1.0.22) Variant caller GATK (version 1.6-11) Annotation SnpEff (version 3.3) GATK VAX Internal annotation databases CASAVA Pindel SAMtools (version 0.1.18) Platypus (version 0.1.9) Minimum coverage 10x 30x 10x 20x 10x 10x 22x Variant filtering Allele frequency, inheritance pattern, variant type, coverage, and quality score Disease-based gene lists, allele frequency, variant type, coverage, and quality score MySQL 5.2 database for filtering by variant type and MAF (analyzing variants with a MAF of <1%) Cassandra (BCM software) Stepwise filtering by variant type, MAF, and family history using the FIND bioinformatics program Phenotype-driven gene lists (HPO and HGMD), inheritance patterns, variant type, phenotype, and population frequencies Variant functional impact, frequency in the population, inheritance patterns and, statistical evidence for association (when applicable) Open in a separate window Abbreviations: bp, base pair; BWA, Burrows-Wheeler Aligner; CASAVA, Consensus Assessment of Sequence and Variation; FIND, Family History Inheritance-Based Detection; GATK, Genome Analysis Toolkit; HGMD, Human Gene Mutation Database; HPO, Human Phenotype Ontology; MAF, minor allele frequency; NA, not applicable; NCGENES, North Carolina Clinical Genomic Evaluation by Next-Generation Exome Sequencing; VAX, Variant Annotator X. a J.S.

Techniques: Sequencing, Software, Variant Assay, Functional Assay