Genomics Core

Florida City, FL, United States

Genomics Core

Florida City, FL, United States
SEARCH FILTERS
Time filter
Source Type

Prlic A.,University of California at San Diego | Yates A.,European Bioinformatics Institute | Bliven S.E.,University of California at San Diego | Rose P.W.,University of California at San Diego | And 12 more authors.
Bioinformatics | Year: 2012

Motivation: BioJava is an open-source project for processing of biological data in the Java programming language. We have recently released a new version (3.0.5), which is a major update to the code base that greatly extends its functionality.Results: BioJava now consists of several independent modules that provide state-of-the-art tools for protein structure comparison, pairwise and multiple sequence alignments, working with DNA and protein sequences, analysis of amino acid properties, detection of protein modifications and prediction of disordered regions in proteins as well as parsers for common file formats using a biologically meaningful data model. © The Author 2012. Published by Oxford University Press. All rights reserved.


News Article | April 20, 2016
Site: www.nature.com

Mouse TT2 ES cells were cultured on gelatin coating plates with recombinant LIF. ES cells were grown in DMEM supplemented with 15% fetal bovine serum, 1% non-essential amino acids, 2 mM l-glutamine, 1,000 units of mLIF (EMD Millipore), 0.1 mM β-mercaptoethanol (Sigma) and antibiotics. A doxycycline (Dox)-inducible Cas9–eGFP ES cell line was established with TT2 ESC. Guide RNA oligos (5′-accgAGTGCCTCTGGCATCCCGGG-3′, 5′-aaacCCCGGGATGCCAGAGGCACT-3′) were annealed and cloned into a pLKO.1-based construct (Addgene: 52628). Guide RNA virus was made in 293FT cells and infected inducible Cas9 ES cells. ES cells were first selected with Puromycin (1 μg ml−1) for two days, and Dox (0.5 μg ml−1) was added to induce Cas9–eGFP expression for 24 h. ES cells were then seeded at low density to obtain single-derived colonies. Then, 72 ES cell colonies were randomly picked up and screened by PCR-enzyme digestion that is illustrated in Extended Data Fig. 3a. PCR screening primers flanking guide RNA sequence were designed as following: 5′-AGGCAGATTTCTGAGTTCAAGG-3′ and 5′-TTTAGTCATGTGCTTGTCCAGG-3′. PCR products were digested by XmaI overnight at 37 degrees and separated on 2% agarose gel. A total of 8 mutants from which PCR products show resistance to XmaI digestion were subjected to DNA sequencing. Clones that harbour deletion and coding frame shift (premature termination mutation) were expanded and used in this study. Human Alkbh–Flag DNA sequence was inserted into pCW lenti-virus based vector (puromycin or hygromycin resistance). The amino acid of D233 was mutated to A by QuickChange Site-Directed Mutagenesis (QuikChange II XL Site-Directed Mutagenesis Kit, number 200521, Agilent) according to the manual. For Alkbh1 rescue experiment, wild-type and D233A mutated Alkbh1 constructs were introduced to Alkbh1 knockout ES cells, pCW-Hygromycin was chosen as control. After infections, the cells were selected with hygromycin at 200 μg ml−1 for 4 days, and then the cells were expanded to isolate genomic DNA for N6-mA dot blotting or other tests. The 293FT cells were transfected with pCW-hAlkbh1 and pCW-hAlkbh1-D233A mutant plasmids along with package plasmids of pMD2.G and pSPAX2. Culture medium was changed 10 h after transfection. The viruses were collected and concentrated 24 and 48 h after transfecction according to manufacturer’s instructions (Lenti-X Concentrator, Clontech). To establish stable expression of hAlkbh1 and hAlkbh1-D233A cell lines, 293FT cells were infected the corresponding virus, and then select with puromycin at 1 μg ml−1 for 4 days. The stable cell lines of hAlkbh1-293FT and D233A-293FT were expanded to purify the proteins according to the previous reported method with some modifications34. Briefly, M2 Flag antibody was added to the nuclear extract and incubated overnight, and then Dynabeads M-280 (sheep anti-mouse IgG, from Life Technology) was added to the above solution and incubated for 3–4 h. Subsequently, the beads were separated from the solution and washed clean with washing buffer34. Finally, the beads were eluted with 3× Flag peptides, followed by standard chromatography purification to 95% purity. Proteins were analysed by mass spectrometry. Demethylation assays were performed in 50 μl volume, which contained 50 pmol of DNA oligos and 500 ng recombinant ALKBH1 (or D233A mutant) protein. The reaction mixture also consisted of 50  μM KCl, 1mM MgCl , 50 μM HEPES (pH = 7.0), 2 mM ascorbic acid, 1 mM-KG, and 1 mM (NH ) Fe(SO ) .6H O. Reactions were performed at 37 degrees for 1 h and then stopped with EDTA followed by heating at 95 degrees for 5 min. Then the reaction product was subjected to dot blotting. Substrate sequences are listed in Supplementary Table 2. First, DNA samples were denatured at 95 degrees for 5 min, cooled down on ice, neutralized with 10% vol of 6.6 M ammonium acetate. Samples were spotted on the membrane (Amersham Hybond-N+, GE) and air dry for 5 min, then UV-crosslink (2× auto-crosslink, 1800 UV Stratalinker, STRATAGENE). Membranes were blocked in blocking buffer (5% milk, 1% BSA, PBST) for 2 h at room temperature, incubated with 6mA antibodies (202-003, Synaptic Systems, 1:1000) overnight at 4 degrees. After 5 washes, membranes were incubated with HRP linked secondary anti-rabbit IgG antibody (1:5,000, Cell Signaling 7074S) for 30 min at room temperature. Signals were detected with ECL Plus Western Blotting Reagent Pack (GE Healthcare). DNA samples were purified by standard N-ChIP protocol. 5 μg anti-H2A.X antibodies were used per 10 million cells. DNA (250 ng) from ChIP pull-down were converted to SMRTbell templates using the PacBio RS DNA Template Preparation Kit 1.0 (PacBio catalogue number 100-259-100) following manufacturer’s instructions. Control samples were amplified by PCR (18 cycles). In brief, samples were end-repaired and ligated to blunt adaptors. Exonuclease incubation was carried out in order to remove all unligated adapters. Samples were extracted twice (0.6× AMPure beads) and the final ‘SMRTbells’ were eluted in 10 μl embryoid bodies. Final quantification was carried out on an Agilent 2100 Bioanalyzer with 1 μl of library. The amount of primer and polymerase required for the binding reaction was determined using the SMRTbell concentration (ng μl−1) and insert size previously determined using the manufacturer-provided calculator. Primers were annealed and polymerase was bound using the DNA/Polymerase Binding Kit P4 (PacBio catalogue number 100-236-500) and sequenced using DNA sequencing reagent 2.0 (PacBio catalogue number 100-216-400). Sequencing was performed on PacBio RS II sequencer using SMRT Cell 8Pac V3 (PacBio catalogue number 100-171-800). In all sequencing runs, a 240 min movie was captured for each SMRT Cell loaded with a single binding complex. Base modification was detected using SMRT Analysis 2.3.0 (Pacific Biosciences), which uses previously published methods for identifying modified bases based on inter-pulse duration ratios in the sequencing data35. All calculations used the Mus musculus mm10 genome as a reference. For the detection of modified bases in individual samples, the RS_Modification_Detection.1 protocol was used with the default parameters. Modifications were only called if the computed modification QV was better than 20, corresponding to P < 0.01 (versus in silico model, Welch’s t-test). The in silico model considers the IPDs from the eight nucleotides 5′ through the three nucleotides 3′ of the site in question. Only the sites with a sequencing coverage higher than 25 fold were used for subsequent analyses. To assess the significance of the overlap between N6-mA sites by SMRT-ChIP and peaks from DIP-seq, intersection with DIP-seq peaks was analysed for each of the N6-mA site called by SMRT-ChIP. To assess if the overlap is higher than expected by random chance, a permutation based approach was used, in which we randomly shuffle the original mapping between “As” that meet coverage cutoff and their corresponding QV scores, and estimated the expected overlap by random chance. As preparation for PacBio RS II sequencing, these relatively short DNA fragments (200–1,000 base pairs on average) were made topologically circular, allowing each base to be read many times by a single sequencing polymerase. Thus, the coverage requirement for modification detection was achieved both by sequencing different fragments pulled down from the same genomic regions and by sequencing the same fragment with many passes. Of note, the SMRT-ChIP approach did not identify more N6-mA sites in Alkbh1 knockout cells than wild-type cells. Although the exact reason remain to be identified, our analysis showed that much fewer adenines are sequenced at a comparable coverage in Alkbh1 knockout cells than wild-type cells (Extended Data Fig. 5c and Extended Data Fig. 1b), presumably due to the difficulty of using native ChIP approach to isolate H2A.X-deposition regions from Alkbh1 knockout cells because of heterochromatinization. Genomic DNA from wild-type or knockout ES cells was purified with DNeasy kit (QIAGEN, 69504). For each sample, 5 μg DNA was sonicated to 200–500 bp with Bioruptor. Then, adaptors were ligated to genomic DNA fragments following the Illumina protocol. The ligated DNA fragments were denatured at 95 degree for 5 min. Then, the single-stranded DNA fragments were immunoprecipitated with 6 mA antibodies (5 μg for each reaction, 202-003, Synaptic Systems) overnight at 4 degrees. N6-Me-dA enriched DNA fragments were purified according to the Active Motif hMeDIP protocol. IP DNA and input DNA were PCR amplified with Illumina indexing primers. The same volume WT and KO DNA samples were subjected to multiplexed library construction and sequencing with Illumina HiSeq2000. After sequencing and filter, high quality raw reads were aligned to the mouse genome (UCSC, mm10) with bowtie (2.2.4, default)36. By default, bowtie searches for multiple alignments and only reports the best match; for repeat sequences, such as transposons, bowtie reports the best matched locus or random one from the best-matched loci. After alignment, N6-mA enriched regions were called with SICER (version 1.1, FDR <1.0 × 10−15, input DNA as control)37. Higher FDR cut-off could not further reduce N6-mA peak number. MACS2 was also used for peak calling, which generated similar results as SICER. Part of the data analysis was done by in-house customized scripts in R, Python or Perl. Genomic DNA samples from mouse fibroblast cells (where the endogenous N6-mA level is undetectable) were spiked with increasing amount of N6-mA-containing, or unmodified (control), oligonucleotides, and the N6-mA levels were determined by qPCR approach after DIP and library construction. Followed manufacture’s protocol (Active Motif 5mC MeDIP kit). The 5 mC data processed with MEDIPS in Bioconductor, and in-house scripts in R, Python or Perl. Native chromatin immunoprecipitation (N-ChIP) assay was performed as previously described. 10 million ES cells were used for each ChIP and massive parallel sequencing (ChIP-seq) experiment. Cell fractionation and chromatin pellet isolation were performed as described. Chromatin pellets were briefly digested with micrococcal nuclease (New England BioLabs) and the mononucleosomes were monitored by electrophoresis. Co-purified DNA molecules were isolated and quantified (100–200 ng for sequencing). Co-purified DNA and whole cell extraction (WCE) input genomic DNA were subject to library construction, cluster generation and next-generation sequencing (Illumina HiSeq 2000). The output sequencing reads were filtered and pre-analyzed with Illumina standard workflow. After filtration, the qualified tags (in fastq format) were aligned to the mouse genome (UCSC, mm10) with bowtie (2.2.4, default)36. Then, these aligned reads were used for peak calling with the SICER algorithm (input control was used as control in peak calling). H3K4Me1 and H3K27Ac ChIP-seq data were aligned to mouse genome (mm10) and peaks were called with SICER. H3K4Me1 and H3K27Ac enriched regions were defined as enhancers. Then, RSEG38 (mode 3) was to call the H3K27Ac differentiated regions. Decommissioned enhancers in KO cells are determined by H3K27Ac downregulation (compared to wild-type cells). Native ChIP-qPCR assay was used to validate H4K4Me3 at levels on gene promoters (Extended Data Fig. 8). All procedures were similar to what has been described in ChIP-seq experiments, except that the co-purified DNA molecules were diluted and subject to qPCR (histone H3K4Me3 antibodies: Abcam Ab8580). Real-time PCR was performed with SybrGreen Reagent (Qiagen, QuantiTect SYBR Green PCR Kit, Cat: 204143) and quantified by a CFX96 system (BioRAD, Inc.). RNA was extracted with miRNeasy kit (QIAGEN, 217004) and standard RNA protocol. The quality of RNA samples was measured using the Agilent Bioanalyzer. Then, RNA was prepared for sequencing using standard Illumina ‘TruSeq’ single-end stranded or ‘Pair-End’ mRNA-seq library preparation protocols. 50 bp of single-end and 100 bp of pair-end sequencing were performed on an Illumina HiSeq 2000 instrument at Yale Stem Cell Center Genomics Core. RNA-seq reads were aligned to mm9 with splicing sites library with Tophat39 (2.0.4, default parameters). The gene model and FPKM were obtained from Cufflink2. The differentially expressed genes were identified by Cuffdiff40 (2.0.0, default parameters). To make sure the normalization is appropriate, the data were also analysed with DESeq2 (default parameters), which generated similar results (Extended Data Fig. 4b). For transposons analysis, unique best alignment reads were used (alignment with bowtie (0.12.9), -m 1; or BWA) and calculated RPKM for each subfamily. For qPCR, the cDNA libraries were generated with First-strand synthesis kit (Invitrogen). Real-time PCR was performed with SybrGreen Reagent (Qiagen, QuantiTect SYBR Green PCR Kit, Cat: 204143) and quantified by a CFX96 system (BioRAD, Inc.). For Fig. 3d, the specific loci L1Md elements primers were designed and optimized based on ref. 27. For embryoid body differentiation experiment, feeder-free cultured ES cells were treated with 0.5% trypsin-EDTA free solution and resuspended with culture medium and counted. Then, cells were seeded at 200,000 cells per ml to Petri dishes with embryoid body differentiation medium (ESC medium without LIF and beta-ME). Medium was changed every 2 days. Histones were isolated in biological triplicate from wild-type and Alkbh1 knockout cells by acid-extraction and resolved/visualized by SDS–PAGE/Coomassie staining. The low molecular weight region of the gel corresponding to core histones was excised and de-stained. The excised gel region containing the histones was treated with d6-acetic anhydride to convert unmodified lysine resides to heavy acetylated lysines (45 Da mass addition) as reported in ref. 41. Following d6-acetic anhydride treatment, the gel region was subjected to in-gel trypsin digestion. Histone peptides were analysed with a Thermo Velos Orbitrap mass spectrometer coupled to a Waters nanoACQUITY LC system as detailed in ref. 42. Tandem mass spectrometric data was searched with Mascot for the following possible modifications: heavy lysine acetylation, lysine acetylation, lysine monomethylation, lysine dimethylation and lysine trimethylation. For each biological replicate, histone H2A was identified with 100% sequence coverage across K118/119 that revealed predominately no detectable lysine methylation DNA was digested with DNA Degradase Plus (Zymo Research) by following the manufacturer’s instructions with small modification. Briefly, the digestion reaction was carried out at 37 °C for 70 min in a 25 μl final volume containing 5 units of DNA Degradase Plus and 5 fMol of internal standard. Following digestion, reaction mixture was diluted to 110 μl and the digested DNA solution was filtered with a Pall NanoSep 3kDa filter (Port Washington, NY) at 8,000 r.p.m. for 15 min. After centrifugal filtration, the digested DNA solution was injected onto an Agilent 1200 HPLC fraction collection system equipped with a diode-array detector (Agilent Technologies, Santa Clara, CA). Analytes were separated by reversed-phase liquid chromatography using an Atlantis C T3 (150 × 4.6 mm, 3 μm) column. The column temperature was kept at 30 °C. For the purification of N6-mA, the mobile phases were water with 0.1% acetic acid (A) and acetonitrile with 0.1% acetic acid (B). The flow rate was 1.0 ml min−1 with a starting condition of 2% B, which was held for 5 min, followed by a linear gradient of 4% B at 20 min, 10% B at 30 min, followed by 6 min at 80% B, then re-equilibration at the starting conditions for 20 min. dA and 6-Me-dA eluted with retention times of 14.7 and 27.0 min, respectively. The amount of dA in samples was quantitated by the UV peak area (λ = 254 nm) at the corresponding retention time using a calibration curve ranging from 0.2 to 5 nMol dA on column. For the simultaneous purification of N3-Me-dC, N1-Me-dA, N3-Me-dA, N6-Me-dA and dA, the mobile phases were water with 5 mM ammonium acetate (A) and acetonitrile (B). The flow rate was 0.45 ml min−1 and the gradient elution program was set at following conditions: 0 min, 1% B; 2 min, 1% B; 40 min, 4% B; 60 min, 30% B; 65 min, 30% B; 65.5 min, 1% B, and 75 min, 1% B. N3-Me-dC, N1-Me-dA, N3-Me-dA, N6-Me-dA and dA eluted with retention times of 24.8, 25.0, 22.0, 60.2 and 54.2 min, respectively. The amount of dA in samples was quantitated by the UV peak area (λ = 254 nm) at the corresponding retention time using a calibration curve ranging from 0.9 to 7.2 nMol dA on the column. HPLC fractions containing target analyte were dried in a SpeedVac and reconstituted in 22 μl of D.I. water before LC-MS/MS analysis. LC-MS-MS analysis of N3-Me-dC, N1-Me-dA, N3-Me-dA and N6-Me-dA was performed on Ultra Performance Liquid Chromatography system from Waters Corporation (Milford, MA) coupled to TSQ Quantum Ultra triple-stage quadrupole mass spectrometer (Thermo Scientific, San Jose, CA). 20 μl of sample was introduced into mass spectrometry through a 100 mm × 2.1 mm HSS T3 column (Waters) at flow rate of 0.15 ml/min. Mobile phases were comprised of water with 0.1% formic acid (A) or acetonitrile (B). Elution gradient condition was set as following: 0 min, 1%B; 3 min, 1%B; 15 min, 7.5%B; 15.5 min, 1%B; 20 min, 1%B. Ionization was operated in positive mode and analytes were detected in selected reaction monitoring (SRM) mode. Specifically, 6-Me-dA and its internal standard were detected by monitoring transition ions of m/z = 266.1 to m/z = 150.1 and m/z = 271.1 to m/z = 155.1, respectively. Similarly, N3-Me-dC, N1-Me-dA and N3-Me-dA was detected by monitoring transition ions of m/z = 242.1 to m/z = 126.1, m/z = 266.1 to m/z = 150.1 and m/z = 266.1 to m/z = 150.1, respectively. Mass spectrometry conditions were set as following: source voltage, 3,000 V; temperature of ion transfer tube, 280 °C; skimmer offset, 0; scan speed, 75 ms; scan width, 0.7 m/z; Q1 and Q3 peak width, 0.7 m/z; collision energy, 17 eV; collision gas (argon), 1.5 arbitrary units. For quantification of N6-Me-dA, the linear calibration curves ranging from 1.5 to 750 fMol, were obtained using the ratio of integrated peak area of the analytical standard over that of the internal standard. The linear calibration curves for analysis of N3-Me-dC, N1-Me-dA and N3-Me-dA were obtained using integrated peak area of the analytical standard. N3-Me-dA is not commercial available and was prepared from the reaction between 3-methyladenine and deoxythymidine in the presence of nucleoside deoxyribosyltransferase II. The chemical identity of purified N3-Me-dA was confirmed by using an Agilent 1200 series Diode Array Detector (DAD) HPLC system coupled with Agilent quadrupole-time-of-flight (QTOF)-MS (Agilent Technologies, Santa Clara, CA). Electrospray ionization (ESI)-MS-MS spectrum of N3-Me-dA was obtained by in source fragmentation. One product ion was observed from MS/MS spectra of the protonated precursor ion of N3-Me-dA, resulting from the loss of the deoxyribosyl group. The accurate masses for parent and fragment ion are m/z = 266.1253 and m/z = 150.0774, with mass error 0.4 p.p.m. and 3.8 p.p.m., respectively. The method sensitivity for N3-Me-dC, N1-Me-dA, N3-Me-dA and N6-Me-dA was detected at 1.0 fmol, 1.6 fmol, 1.0 fmol and 1.6 fmol, respectively. In order to confirm the chemical identity of the N6-Me-dA isolated from HLPC purification, HPLC fractions containing N6-Me-dA was analysed by HPLC-QTOF-MS/MS. The chemical identity of N6-Me-dA in HPLC fractions was characterized on an Agilent 1200 series Diode Array Detector (DAD) HPLC system coupled with Agilent quadrupole-time-of-flight (QTOF)-MS (Agilent Technologies, Santa Clara, CA). HPLC separation was carried out on a C18 reverse phase column (Waters Atlantis T3, 3  μM, 150 mm × 2.1 mm) with a flow rate at 0.15 ml min−1 and mobile phase A (0.05% acetic acid in water) and B (acetonitrile). The gradient elution program was set at following conditions: 0 min, 1% B; 2 min, 1% B; 15 min, 30% B; 15.5 min, 1% B; and 25 min, 1% B. N6-Me-dA was eluted with retention times of 12.7 min. The electrospray ion source in positive mode with the following conditions were used: gas temperature, 200 °C; drying gas flow, 12 litres per min; nebulizer, 35 psi; Vcap, 4000 V; fragmentor, 175 V; skimmer, 67 V. Electrospray ionization (ESI)-MS-MS spectrum of N6-Me-dA isolated from genomic DNA was obtained by in source fragmentation. One product ion was observed from MS/MS spectra of the protonated precursor ion of N6-Me-dA, resulting from the loss of the deoxyribosyl group. The accurate masses for parent and fragment ion are m/z = 266.1245 and m/z = 150.0775, with mass error 3.0 p.p.m. and 3.1 p.p.m., respectively. The same MS/MS fragmentation spectra was obtained from analytical standard of N6-Me-dA. For in vitro demethylation assay, sample was treated with EDTA to remove Fe2+. The mixture was transferred to Amicon Ultra Centrifugal Filter (EMD Millipore Corporation, 10K MWCO), followed by spin at 11,000 r.p.m. and 4 °C for 14 min. The concentrated sample was wash three times by adding 500 μl DI-H2O, followed spin at 11,000 r.p.m. and 4 °C for 14 min. The washed sample was digested with DNA Degradase Plus (Zymo Research) by following manufacturer’s instruction with small modification. Briefly, the digestion reaction was carried out at 37 °C for 60 min in 60 μl final volume containing 0.17 units per μl of DNA Degradase Plus and 50 fmol of Internal Standard of N6-Me-dA. Following digestion, reaction mixture was filtered with a Pall NanoSep 3kDa filter (Port Washington, NY) at 10000g and room temperature for 10 min to remove enzyme. The LC-MS/MS conditions for the quantification of dA and N6-Me-dA were set the same as those for quantification of N6-Me-dA in in vivo samples. The linear calibration curves for quantification of dA and N6-Me-dA was obtained using the ratio of integrated peak area of the analytical standard over that of the internal standard of N6-Me-dA.


News Article | February 27, 2017
Site: www.rdmag.com

In movies and TV shows, dolphins are often portrayed as heroes who save humans through remarkable feats of strength and tenacity. Now dolphins could save the day for humans in real life, too - with the help of emerging technology that can measure thousands of proteins and an improved database full of genetic data. "Dolphins and humans are very, very similar creatures," said NIST's Ben Neely, a member of the Marine Biochemical Sciences Group and the lead on a new project at the Hollings Marine Laboratory, a research facility in Charleston, South Carolina that includes the National Institute of Standards and Technology (NIST) as one of its partner institutions. "As mammals, we share a number of proteins and our bodies function in many similar ways, even though we are terrestrial and dolphins live in the water all their lives." Neely and his colleagues have just finished creating a detailed, searchable index of all the proteins found in the bottlenose dolphin genome. A genome is the complete set of genetic material present in an organism. Neely's project is built on years of marine mammal research and aims to provide a new level of bioanalytical measurements. The results of this work will aid wildlife biologists, veterinary professionals and biomedical researchers. Protein Maps Could Help Dolphins and Humans Although a detailed map of the bottlenose dolphin (Tursiops truncatus) genome was first compiled in 2008, recent technological breakthroughs enabled the creation of a new, more exhaustive map of all of the proteins produced by the dolphins' DNA. Neely led the process to generate the new genome with the help of colleagues at the Hollings Marine Laboratory. For this project, the initial genomic sequencing and assembly were completed by Dovetail Genomics, a private U.S.-based company. Next, the genome was annotated by the National Center for Biotechnology Information at the National Library of Medicine (NCBI) using previously deposited data generated in large part by the National Oceanic and Atmospheric Administration's National Centers for Coastal Ocean Science Marine Genomics Core. "Once you can identify all of the proteins and know their amounts as expressed by the genome," Neely explained, "you can figure out what's going on in the bottlenose dolphin's biological systems in this really detailed manner." Neely's study is part of an emerging field called proteomics. In the case of dolphins, proteomic work has a wide variety of potential applications. The zoo and aquarium industry, which generates revenues of approximately $16 billion a year, could use it to improve the care of bottlenose dolphins. In addition, improved dolphin proteomics could improve assessments of wild dolphin populations, and provide an immense amount of data on environmental contaminants and the safety and health of the world's oceanic food web. Comparing the proteins of humans and these other mammals is already providing researchers with a wealth of new information about how the human body works. Those findings could eventually be used to develop new, more precise treatment methods for common medical problems. As marine mammals descend, they shut off the blood flow to many of their organs, which has long puzzled and intrigued biologists. In contrast, if blood stops flowing to the organs of a human's body for even a few seconds, the result can be a stroke, kidney failure, or even death. Studies have recently revealed that lesser-known proteins in the blood of marine mammals may be playing a big role in the dives by protecting bottlenose dolphins' kidneys and hearts from damage when blood flow and oxygen flow start and stop repeatedly during those underwater forays. One of these proteins is known as vanin-1. Humans produce vanin-1, but in much smaller amounts. Researchers would like to gather more information on whether or not elevating levels of vanin-1 may offer protection to kidneys. "There's this gap in the knowledge about genes and the proteins they make. We are missing a huge piece of the puzzle in how these animals do what they do," said Mike Janech from the Medical University of South Carolina. His group has been researching vanin-1 (link is external) and has identified numerous other potential biomedical applications for the dolphin genome just created by NIST. "Genes carry the information of life," Janech said. "But proteins execute the functions." Vanin-1 is just one example of how genomic information about this mammalian cousin might prove useful. There may be hundreds of other similar applications, including some related to the treatment of high blood pressure and diabetes. This represents another avenue for biomimicry, which seeks solutions to human problems by examining and imitating nature's patterns and strategies. In the past, biomimicry was solely focused on the structural aspects of animal body parts such as arms and legs or functional patterns of things like noses and sniffing. But as the study of DNA has evolved, so too has our ability to examine the things happening at the most minute levels within another mammal's body. "We are now entering what could be called the post-model-organism era," Neely said. Instead of looking only for a structure to model, imitate or learn from, scientists are looking at the complete molecular landscape of genes and proteins of these creatures for model processes, too. "With abundant genomic resources it is now possible to study non-model organisms with similar molecular machinery in order to tackle difficult biomedical problems." To gather the needed protein information, Neely and his team used a specimen provided by the National Marine Mammal Tissue Bank (link is external) (NMMTB), the longest running project of NIST's Marine Environmental Specimen Bank. Half of the approximately 4,000 marine mammal specimens in the NMMTB are collected as a part of the Marine Mammal Health and Stranding Response Program (link is external). The specimen provided for Neely's study was known to originate very close to the Hollings Marine Lab. The new, state-of-the-art genome immediately began providing new biochemical insights. Studies at NIST are ongoing to validate the updated protein maps using an ultra-high-resolution tribrid mass spectrometer, which is the most powerful tool available to identify and quantify proteins. Other Mammal Proteins Seem Promising, Too Neely said the results demonstrate the utility of re-mapping genomes with the improved bioanalytical capabilities provided by new genomic sequencing technology coupled to high-resolution mass spectrometers. The data from this project will also be available in the public domain so that the results will be easy for others to access and use for diverse applications and research. This is the first of many such projects to be undertaken by the Charleston group whereby new analytical techniques could be applied to marine animals. Studying other diving marine mammals can improve our understanding of the molecular mechanisms involved in diving. Also, sea lion proteins may have much to tell us about metastatic cancer, which especially intrigues Neely and his colleagues. As a research chemist, Neely says he has not really spent much time before now observing marine mammals as a part of his work hours. He does encounter dolphins when he goes out surfing along the Carolina coastline, though. "It's amazing to think that we are at a point where cutting-edge research in marine mammals can directly advance human biomedical discoveries," he said.


News Article | February 23, 2017
Site: www.eurekalert.org

In movies and TV shows, dolphins are often portrayed as heroes who save humans through remarkable feats of strength and tenacity. Now dolphins could save the day for humans in real life, too - with the help of emerging technology that can measure thousands of proteins and an improved database full of genetic data. "Dolphins and humans are very, very similar creatures," said NIST's Ben Neely, a member of the Marine Biochemical Sciences Group and the lead on a new project at the Hollings Marine Laboratory, a research facility in Charleston, South Carolina that includes the National Institute of Standards and Technology (NIST) as one of its partner institutions. "As mammals, we share a number of proteins and our bodies function in many similar ways, even though we are terrestrial and dolphins live in the water all their lives." Neely and his colleagues have just finished creating a detailed, searchable index of all the proteins found in the bottlenose dolphin genome. A genome is the complete set of genetic material present in an organism. Neely's project is built on years of marine mammal research and aims to provide a new level of bioanalytical measurements. The results of this work will aid wildlife biologists, veterinary professionals and biomedical researchers. Although a detailed map of the bottlenose dolphin (Tursiops truncatus) genome was first compiled in 2008, recent technological breakthroughs enabled the creation of a new, more exhaustive map of all of the proteins produced by the dolphins' DNA. Neely led the process to generate the new genome with the help of colleagues at the Hollings Marine Laboratory. For this project, the initial genomic sequencing and assembly were completed by Dovetail Genomics, a private U.S.-based company. Next, the genome was annotated by the National Center for Biotechnology Information at the National Library of Medicine (NCBI) using previously deposited data generated in large part by the National Oceanic and Atmospheric Administration's National Centers for Coastal Ocean Science Marine Genomics Core. "Once you can identify all of the proteins and know their amounts as expressed by the genome," Neely explained, "you can figure out what's going on in the bottlenose dolphin's biological systems in this really detailed manner." Neely's study is part of an emerging field called proteomics. In the case of dolphins, proteomic work has a wide variety of potential applications. The zoo and aquarium industry, which generates revenues of approximately $16 billion a year, could use it to improve the care of bottlenose dolphins. In addition, improved dolphin proteomics could improve assessments of wild dolphin populations, and provide an immense amount of data on environmental contaminants and the safety and health of the world's oceanic food web. Comparing the proteins of humans and these other mammals is already providing researchers with a wealth of new information about how the human body works. Those findings could eventually be used to develop new, more precise treatment methods for common medical problems. As marine mammals descend, they shut off the blood flow to many of their organs, which has long puzzled and intrigued biologists. In contrast, if blood stops flowing to the organs of a human's body for even a few seconds, the result can be a stroke, kidney failure, or even death. Studies have recently revealed that lesser-known proteins in the blood of marine mammals may be playing a big role in the dives by protecting bottlenose dolphins' kidneys and hearts from damage when blood flow and oxygen flow start and stop repeatedly during those underwater forays. One of these proteins is known as vanin-1. Humans produce vanin-1, but in much smaller amounts. Researchers would like to gather more information on whether or not elevating levels of vanin-1 may offer protection to kidneys. "There's this gap in the knowledge about genes and the proteins they make. We are missing a huge piece of the puzzle in how these animals do what they do," said Mike Janech from the Medical University of South Carolina. His group has been researching vanin-1 (link is external) and has identified numerous other potential biomedical applications for the dolphin genome just created by NIST. "Genes carry the information of life," Janech said. "But proteins execute the functions." Vanin-1 is just one example of how genomic information about this mammalian cousin might prove useful. There may be hundreds of other similar applications, including some related to the treatment of high blood pressure and diabetes. This represents another avenue for biomimicry, which seeks solutions to human problems by examining and imitating nature's patterns and strategies. In the past, biomimicry was solely focused on the structural aspects of animal body parts such as arms and legs or functional patterns of things like noses and sniffing. But as the study of DNA has evolved, so too has our ability to examine the things happening at the most minute levels within another mammal's body. "We are now entering what could be called the post-model-organism era," Neely said. Instead of looking only for a structure to model, imitate or learn from, scientists are looking at the complete molecular landscape of genes and proteins of these creatures for model processes, too. "With abundant genomic resources it is now possible to study non-model organisms with similar molecular machinery in order to tackle difficult biomedical problems." To gather the needed protein information, Neely and his team used a specimen provided by the National Marine Mammal Tissue Bank (link is external) (NMMTB), the longest running project of NIST's Marine Environmental Specimen Bank. Half of the approximately 4,000 marine mammal specimens in the NMMTB are collected as a part of the Marine Mammal Health and Stranding Response Program (link is external). The specimen provided for Neely's study was known to originate very close to the Hollings Marine Lab. The new, state-of-the-art genome immediately began providing new biochemical insights. Studies at NIST are ongoing to validate the updated protein maps using an ultra-high-resolution tribrid mass spectrometer, which is the most powerful tool available to identify and quantify proteins. Neely said the results demonstrate the utility of re-mapping genomes with the improved bioanalytical capabilities provided by new genomic sequencing technology coupled to high-resolution mass spectrometers. The data from this project will also be available in the public domain so that the results will be easy for others to access and use for diverse applications and research. This is the first of many such projects to be undertaken by the Charleston group whereby new analytical techniques could be applied to marine animals. Studying other diving marine mammals can improve our understanding of the molecular mechanisms involved in diving. Also, sea lion proteins may have much to tell us about metastatic cancer, which especially intrigues Neely and his colleagues. As a research chemist, Neely says he has not really spent much time before now observing marine mammals as a part of his work hours. He does encounter dolphins when he goes out surfing along the Carolina coastline, though. "It's amazing to think that we are at a point where cutting-edge research in marine mammals can directly advance human biomedical discoveries," he said.


News Article | February 23, 2017
Site: phys.org

"Dolphins and humans are very, very similar creatures," said NIST's Ben Neely, a member of the Marine Biochemical Sciences Group and the lead on a new project at the Hollings Marine Laboratory, a research facility in Charleston, South Carolina that includes the National Institute of Standards and Technology (NIST) as one of its partner institutions. "As mammals, we share a number of proteins and our bodies function in many similar ways, even though we are terrestrial and dolphins live in the water all their lives." Neely and his colleagues have just finished creating a detailed, searchable index of all the proteins found in the bottlenose dolphin genome. A genome is the complete set of genetic material present in an organism. Neely's project is built on years of marine mammal research and aims to provide a new level of bioanalytical measurements. The results of this work will aid wildlife biologists, veterinary professionals and biomedical researchers. Protein Maps Could Help Dolphins and Humans Although a detailed map of the bottlenose dolphin (Tursiops truncatus) genome was first compiled in 2008, recent technological breakthroughs enabled the creation of a new, more exhaustive map of all of the proteins produced by the dolphins' DNA. Neely led the process to generate the new genome with the help of colleagues at the Hollings Marine Laboratory. For this project, the initial genomic sequencing and assembly were completed by Dovetail Genomics , a private U.S.-based company. Next, the genome was annotated by the National Center for Biotechnology Information at the National Library of Medicine (NCBI) using previously deposited data generated in large part by the National Oceanic and Atmospheric Administration's National Centers for Coastal Ocean Science Marine Genomics Core. "Once you can identify all of the proteins and know their amounts as expressed by the genome," Neely explained, "you can figure out what's going on in the bottlenose dolphin's biological systems in this really detailed manner." Neely's study is part of an emerging field called proteomics. In the case of dolphins, proteomic work has a wide variety of potential applications. The zoo and aquarium industry, which generates revenues of approximately $16 billion a year, could use it to improve the care of bottlenose dolphins. In addition, improved dolphin proteomics could improve assessments of wild dolphin populations, and provide an immense amount of data on environmental contaminants and the safety and health of the world's oceanic food web. Comparing the proteins of humans and these other mammals is already providing researchers with a wealth of new information about how the human body works. Those findings could eventually be used to develop new, more precise treatment methods for common medical problems. As marine mammals descend, they shut off the blood flow to many of their organs, which has long puzzled and intrigued biologists. In contrast, if blood stops flowing to the organs of a human's body for even a few seconds, the result can be a stroke, kidney failure, or even death. Studies have recently revealed that lesser-known proteins in the blood of marine mammals may be playing a big role in the dives by protecting bottlenose dolphins' kidneys and hearts from damage when blood flow and oxygen flow start and stop repeatedly during those underwater forays. One of these proteins is known as vanin-1. Humans produce vanin-1, but in much smaller amounts. Researchers would like to gather more information on whether or not elevating levels of vanin-1 may offer protection to kidneys. "There's this gap in the knowledge about genes and the proteins they make. We are missing a huge piece of the puzzle in how these animals do what they do," said Mike Janech from the Medical University of South Carolina. His group has been researching vanin-1 and has identified numerous other potential biomedical applications for the dolphin genome just created by NIST. "Genes carry the information of life," Janech said. "But proteins execute the functions." Vanin-1 is just one example of how genomic information about this mammalian cousin might prove useful. There may be hundreds of other similar applications, including some related to the treatment of high blood pressure and diabetes. This represents another avenue for biomimicry, which seeks solutions to human problems by examining and imitating nature's patterns and strategies. In the past, biomimicry was solely focused on the structural aspects of animal body parts such as arms and legs or functional patterns of things like noses and sniffing. But as the study of DNA has evolved, so too has our ability to examine the things happening at the most minute levels within another mammal's body. "We are now entering what could be called the post-model-organism era," Neely said. Instead of looking only for a structure to model, imitate or learn from, scientists are looking at the complete molecular landscape of genes and proteins of these creatures for model processes, too. "With abundant genomic resources it is now possible to study non-model organisms with similar molecular machinery in order to tackle difficult biomedical problems." To gather the needed protein information, Neely and his team used a specimen provided by the National Marine Mammal Tissue Bank (NMMTB), the longest running project of NIST's Marine Environmental Specimen Bank. Half of the approximately 4,000 marine mammal specimens in the NMMTB are collected as a part of the Marine Mammal Health and Stranding Response Program . The specimen provided for Neely's study was known to originate very close to the Hollings Marine Lab. The new, state-of-the-art genome immediately began providing new biochemical insights. Studies at NIST are ongoing to validate the updated protein maps using an ultra-high-resolution tribrid mass spectrometer, which is the most powerful tool available to identify and quantify proteins. Other Mammal Proteins Seem Promising, Too Neely said the results demonstrate the utility of re-mapping genomes with the improved bioanalytical capabilities provided by new genomic sequencing technology coupled to high-resolution mass spectrometers. The data from this project will also be available in the public domain so that the results will be easy for others to access and use for diverse applications and research. This is the first of many such projects to be undertaken by the Charleston group whereby new analytical techniques could be applied to marine animals. Studying other diving marine mammals can improve our understanding of the molecular mechanisms involved in diving. Also, sea lion proteins may have much to tell us about metastatic cancer, which especially intrigues Neely and his colleagues. As a research chemist, Neely says he has not really spent much time before now observing marine mammals as a part of his work hours. He does encounter dolphins when he goes out surfing along the Carolina coastline, though. "It's amazing to think that we are at a point where cutting-edge research in marine mammals can directly advance human biomedical discoveries," he said. Explore further: Researchers probing the beneficial secrets in dolphins' proteins


PubMed | Genomics Core, Emory University, University of Pennsylvania, Children's Hospital of Philadelphia and 3 more.
Type: | Journal: Human genome variation | Year: 2016

The 22q11.2 deletion syndrome is the most common microdeletion disorder, with wide phenotypic variability. To investigate variation within the non-deleted allele we performed targeted resequencing of the 22q11.2 region for 127 patients, identifying multiple deletion sizes, including two deletions with atypical breakpoints. We cataloged ~12,000 hemizygous variant positions, of which 84% were previously annotated. Within the coding regions 95 non-synonymous variants, three stop gains, and two frameshift insertions were identified, some of which we speculate could contribute to atypical phenotypes. We also catalog tolerability of 22q11 gene mutations based on related autosomal recessive disorders in man, embryonic lethality in mice, cross-species conservation and observations that some genes harbor more or less variants than expected. This extensive catalog of hemizygous variants will serve as a blueprint for future experiments to correlate 22q11DS variation with phenotype.


News Article | September 7, 2016
Site: www.nature.com

All zebrafish work was performed according to standard protocols approved by The University of Chicago (ACUP #72074). No statistical methods were used to predetermine sample size. The experiments were not randomized and the investigators were not blinded to allocation during experiments and outcome assessment. In situ hybridization for the hox13, Cre, and1 and shha genes were performed according to standard protocols29 after fixation in 4% paraformaldehyde overnight at 4 °C. Probes for hox13 and shha were as previously described18. Primers to clone Cre and and1 into vectors can be found in Extended Data Tables 1 and 2. Specimens were visualized on a Leica M205FA microscope. In order to create a destination vector for lineage tracing, we first designed a random sequence of 298 bp that contained a SmaI site to be used in downstream cloning. This sequence was ordered as a gBlocks fragment (IDT) and ligated into the pCR8/GW/TOPO TA cloning vector (Invitrogen). We then performed a Gateway LR reaction according to the manufacturers specifications between this entry vector and pXIG–cFos–GFP, which abolished an NcoI site present in the gateway cassette and introduced a SmaI site. We then removed the GFP gene with NcoI and BglII of the destination vector and ligated in Cre with (primers in Extended Data Table 1), using the ‘pCR8GW–Cre–pA–FRT–kan–FRT’ (kind gift of M. L. Suster, Sars International Center for Marine Molecular Biology, University of Bergen, Bergen, Norway) as a template for Cre PCR and Platinum Taq DNA polymerase High Fidelity (Invitrogen). In order to add a late phase enhancer to this vector, we first ordered four identical oligos (IDT gBlocks) of the core e16 sequence from gar, each flanked by different restriction sites. Each oligo was then ligated into pCR8/GW/TOPO, and sequentially cloned via restriction sites into a single pCR8/GW/TOPO vector. This entry vector was used a template to PCR the final Lo-e16x4 sequence and ligate it into the Cre destination vector using XhoI and SmaI, creating Lo-e16x4–Cre. The early phase enhancer Dr-CNS65x3 was cloned into the destination vector using the same strategy. Final vectors were confirmed by sequencing. A full list of sequences and primers used can be found in Extended Data Table 1. *AB zebrafish embryos were collected from natural spawning and injected according to the Tol2 system as described previously21. Transposase RNA was synthesized from the pCS2-zT2TP vector using the mMessage mMachine SP6 kit (Ambion)21. All injected embryos were raised to sexual maturity according to standard protocols. Adult F0 fish were outcrossed to wild-type *AB, and the total F1 clutch was lysed and DNA isolated at 24 hpf for genotyping (see Extended Data Table 1 for primers) to confirm germline transmission of Cre plasmids in the F0 founders. Multiple founders were identified and tested for the strongest and most consistent expression via antibody staining and in situ hybridization. One founder fish was identified as best, and all subsequent experiments were performed using offspring of this individual fish. Founder Lo-e16x4–Cre and Dr-CNS65x3–Cre fish were crossed to the Tg(ubi:Switch) line (kind gift from L. I. Zon). Briefly, this line contains a construct in which a constitutively active promoter (ubiquitin) drives expression of a loxP flanked GFP protein in all cells of the fish assayed. When Cre is introduced, the GFP gene is removed and the ubiquitin promoter is exposed to mCherry, thus permanently labelling the cell. We crossed our founder Cre fish to Tg(ubi:Switch) and fixed progeny at different time points to track cell fate. In order to detect the mCherry signal, embryos or adults were fixed overnight in 4% paraformaldehyde and subsequently processed for whole-mount antibody staining according to standard protocols30 using the following antibodies and dilutions: 1st rabbit anti-mCherry/DsRed (Clontech #632496) at 1:250, 1st mouse anti-Zns-5 (Zebrafish International Resource Center, USA) at 1:200, 2nd goat anti-rabbit Alexa Fluor 546 (Invitrogen #A11071) at 1:400, 2nd goat anti-mouse Alexa 647 (Invitrogen #A21235) at 1:400. Stained zebrafish were mounted under a glass slide and visualized using an LSM 710 confocal microscope (Organismal Biology and Anatomy, the University of Chicago). Antibody stains on adult zebrafish (90 dpf) fins were imaged on a Leica SP5 II tandem scanner AOBS Laser Scanning Confocal (the University of Chicago Integrated Light Microscopy Core Facility). Two mutations were simultaneously introduced into the first exon of each hox13 gene by CRISPR/Cas9 system as previously described in Xenopus tropicalis31. Briefly, two gRNAs that match the sequence of exon 1 of each hox13 gene were designed by ZiFiT (http://zifit.partners.org/ZiFiT/). To synthesize gRNAs, forward and reverse oligonucleotides that are unique for individual target sequences were synthesized by Integrated DNA Technologies, Inc. (IDT). Each oligonucleotide sequence can be found in Extended Data Table 2. Subsequently, each forward and reverse oligonucleotide were hybridized, and double stranded products were individually amplified by PCR with primers that include a T7 RNA promoter sequence, followed by purification by NucleoSpin Gel and PCR Clean-up Kit (Macherey-Nagel). Each gRNA was synthesized from the purified PCR products by in vitro transcription with the MEGAscript T7 Transcription kit (Ambion). Cas9 mRNA was synthesized by mMESSAGE mMACHINE SP6 Transcription Kit according to the manufacturer’s instructions (Ambion). Two gRNAs targeting exon 1 of each hox gene were injected with Cas9 mRNA into zebrafish eggs at the one-cell stage. We injected ~2 nl of the injection solution (5 μl solution containing 1,000 ng of each gRNA and 500 ng Cas9 diluted in nuclease-free water) into the single cell of the embryo. Injected embryos were raised to adulthood, and at three months were genotyped by extracting DNA from tail clips. Briefly, zebrafish were anaesthetized by Tricaine (0.004%) and tips of the tail fin (2–3 mm2) were removed and placed in an Eppendorf tube. The tissue was lysed in standard lysis buffer (10 mM Tris pH 8.2, 10 mM EDTA, 200 mM NaCl, 0.5% SDS, 200 μg/ml proteinase K) and DNA recovered by ethanol precipitation. Approximately 800-1,100 bp of exon 1 from each gene was amplified by PCR using the primers described in Extended Data Table 2. To determine whether mutations were present, PCR products were subjected to T7E1 (T7 endonuclease1) assay as previously reported32. After identification of mutant fish by T7E1 assay, detailed analysis of mutation patterns were performed by sequencing at the Genomics Core at the University of Chicago. Identified mutant fish were outcrossed to wild type to select frameshift mutations from mosaic mutational patterns and establish single heterozygous lines. Obtained embryos were raised to adults (~3 months), then analysed by T7E1 assay and sequenced. Among a variety of mutational patterns, fish that have frameshift mutations were used for assays as single heterozygous fish. We obtained several independent heterozygous mutant lines for each hox13 gene to compare the phenotype among different frameshift mutations. To obtain hoxa13a+/−, hoxa13b+/− double heterozygous mutant fish, each single heterozygous mutant line was crossed with the other mutant line. Offspring were analysed by T7E1 assay and sequenced after three months, and double heterozygous mutant fish were selected. To generate double homozygous hoxa13 mutant embryos and adult fish (hoxa13a−/−, hoxa13b−/−), double heterozygous fish (hoxa13a+/−, hoxa13b+/−) were crossed with each other. The ratio of each genotype from crossing heterozygous fish is summarized in Extended Data Table 4. After mutant lines were established, single (hoxa13a or hoxa13b) or double (hoxa13a, hoxa13b) mutant embryos and adult fish were genotyped by PCR for each analysis. Primer sequences for PCR are listed in Extended Data Table 2. To identify an 8 bp deletion in exon 1 of hoxa13a, the PCR product was treated by Ava1 at 37 °C for 2 h, because the 8 bp deletion produces a new Ava1 site in the PCR product (‘zebra hoxa13a_8 bp del’ primers, wild type; 231 bp, mutant; 111 bp and 119 bp). Final product size was confirmed by 3% agarose gel electrophoresis. To identify a 29 bp deletion in exon 1 of hoxa13a, the PCR product was confirmed by gel electrophoresis (‘zebra hoxa13a_29 bp del’ primers, wild type; 110 bp, mutant; 81 bp). To identify a 14 bp insertion in exon 1 of hoxa13b, the PCR product was treated by Bcc1 at 37°C for 2 h, because the 14 bp insertion produces a new Bcc1 site in the PCR product (‘zebra hoxa13b_14 bp ins’ primers, wild type; 98 bp, mutant; 53 bp + 57 bp). The final product size was confirmed by 3% agarose gel electrophoresis. The details of the mutant sequence are summarized in Extended Data Table 3a–c. Two gRNAs targeting exon 1 of hoxa13b and two gRNAs targeting exon 1 of hoxd13a were injected with Cas9 mRNA into zebrafish one-cell eggs that were obtained from crossing hoxa13a+/− and hoxa13a+/−, hoxa13b+/−, hoxd13a+/− (gRNAs were same as that were used to establish single hox13 knockout fishes and found in Extended Data Table 2). Injected eggs were raised to adult fish and genotyped by extracting DNA from tail fins. PCR products of each hox13 gene were cloned into PCRIITOPO (Invitrogen) and deep sequencing was performed (Genomic Core, the University of Chicago). At four months old, skeletal staining and CT scanning were performed to analyse the effect of triple gene deletions. The knockout ratios of each hox13 allele were calculated from the results of deep sequencing. Embryos were obtained by crossing hoxa13a+/−, hoxa13b+/− to each other and raised to 72 hpf or 96 hpf. After fixation by 4% PFA for 15 h, caudal halves were used for PCR genotyping. Pectoral fins of wild type and hoxa13a−/−, hoxa13b−/− were detached from the embryonic body and placed horizontally on glass slides. The fins were photographed with a Leica M205FA microscope, and the fin fold length along the proximodistal axis at the centre of the fin was measured using ImageJ. The resulting data were analysed by t-test comparing the means. Embryos were obtained by crossing hoxa13a+/−, hoxa13b+/− to each other and raised to 96 hpf. After fixation by 4% PFA for 15 h, caudal halves were used for PCR genotyping. Wild type and hoxa13a−/−, hoxa13b−/− embryos were stained by DAPI (1:4,000 in PBS-0.1% Triton) for 3 h and washed for 3 h by PBS−0.1% Triton. Pectoral fins were detached from the embryonic body, placed on glass slides and covered by a coverslip. The DAPI signal was detected by Zeiss LSM 710 (Organismal Biology and Anatomy, the University of Chicago). Individual nuclei were manually marked using Adobe Illustrator and the number of nuclei was counted. The data were analysed by t-test comparing the means. Skeletal staining was performed as previously described33. Briefly, fish were fixed in 10% neutral-buffered formalin overnight. After washing with milli-Q water, solutions were substituted by 70% EtOH in a stepwise fashion and then by 30% acetic acid/70% EtOH. Cartilage was stained with 0.02% alcian blue in 30% acetic acid/70% EtOH overnight. After washingwith milli-Q water, the solution was changed to a 30% saturated sodium borate solution and incubated overnight. Subsequently, specimens were immersed in 1% trypsin/30% saturated sodium borate and incubated at room temperature overnight. Following a milli-Q water wash, specimens were transferred into a 1% KOH solution containing 0.005% Alzarin Red S. The next day, specimens were washed with milli-Q water and subjected to glycerol substitution. Three replicates for each genotype were investigated. After skeletal staining, girdles and pectoral fins were manually separated from the body. Girdles and fins were stained with 0.5% PMA (phosphomolybdic acid) in milli-Q water for 16 h and followed by washes with milli-Q water. Specimens were placed into 1.5 ml Eppendorf tubes with water and kept overnight to settle in the tubes. The next day, tubes containing specimens were set and scanned with the UChicago PaleoCT scanner (GE Phoenix v/tome/x 240kv/180kv scanner) (http://luo-lab.uchicago.edu/paleoCT.html), at 50 kVp, 160 μA, no filtration, 5×-averaging, exposure timing of 500 ms per image, and a resolution of 8 μm per slice (512 μm3 per voxel). Scanned images were analysed and segmented using Amira 3D Software 6.0 (FEI). Three replicates for single and double homozygotes and five for mosaic triple knockout were investigated.


PubMed | Genomics Core, From the Genetics of Development and Disease Branch, Hirosaki University, From the Genetics of Development and Disease Branch. and 2 more.
Type: Journal Article | Journal: The Journal of biological chemistry | Year: 2016

Sphingosine-1-phosphate (S1P) is a sphingolipid metabolite that regulates basic cell functions through metabolic and signaling pathways. Intracellular metabolism of S1P is controlled, in part, by two homologous S1P phosphatases (SPPases), 1 and 2, which are encoded by the Sgpp1 and Sgpp2 genes, respectively. SPPase activity is needed for efficient recycling of sphingosine into the sphingolipid synthesis pathway. SPPase 1 is important for skin homeostasis, but little is known about the functional role of SPPase 2. To identify the functions of SPPase 2 in vivo, we studied mice with the Sgpp2 gene deleted. In contrast to Sgpp1(-/-) mice, Sgpp2(-/-) mice had normal skin and were viable into adulthood. Unexpectedly, WT mice expressed Sgpp2 mRNA at high levels in pancreatic islets when compared with other tissues. Sgpp2(-/-) mice had normal pancreatic islet size; however, they exhibited defective adaptive -cell proliferation that was demonstrated after treatment with either a high-fat diet or the -cell-specific toxin, streptozotocin. Importantly, -cells from untreated Sgpp2(-/-) mice showed significantly increased expression of proteins characteristic of the endoplasmic reticulum stress response compared with -cells from WT mice, indicating a basal islet defect. Our results show that Sgpp2 deletion causes -cell endoplasmic reticulum stress, which is a known cause of -cell dysfunction, and reveal a juncture in the sphingolipid recycling pathway that could impact the development of diabetes.


Shannahan J.H.,University of North Carolina at Chapel Hill | Nyska A.,Tel Aviv University | Cesta M.,National Health Research Institute | Schladweiler M.C.J.,Genomics Core | And 5 more authors.
Environmental Health Perspectives | Year: 2012

Background: Surface-available iron (Fe) is proposed to contribute to asbestos-induced toxicity through the production of reactive oxygen species. Objective: Our goal was to evaluate the hypothesis that rat models of cardiovascular disease with coexistent Fe overload would be increasingly sensitive to Libby amphibole (LA)-induced subchronic lung injury. Methods: Male healthy Wistar Kyoto (WKY), spontaneously hypertensive (SH), and SH heart failure (SHHF) rats were intratracheally instilled with 0.0, 0.25, or 1.0 mg LA (with saline as the vehicle). We examined bronchoalveolar lavage fluid (BALF) and histological lung sections after 1 week, 1 month, or 3 months for pulmonary biomarkers and pathology. SHHF rats were also assessed at 6 months for pathological changes. Results: All animals developed concentration-and time-dependent interstitial fibrosis. Time-dependent Fe accumulation occurred in LA-laden macrophages in all strains but was exacerbated in SHHF rats. LA-exposed SHHF rats developed atypical hyperplastic lesions of bronchiolar epithelial cell origin at 3 and 6 months. Strain-related baseline differences existed in gene expression at 3 months, with persistent LA effects in WKY but not SH or SHHF rats. LA exposure altered genes for a number of pathways, including inflammation, immune regulation, and cell-cycle control. Cell-cycle control genes were inhibited after LA exposure in SH and SHHF but not WKY rats, whereas tumor suppressor genes were induced only in WKY rats. The inflammatory gene expression also was apparent only in WKY rats. Conclusion: These data show that in Fe-overload conditions, progressive Fe accumulation occurs in fiber-laden macrophages within LA-induced lesions. Fe overload does not appear to contribute to chronic inflammation, and its role in hyperplastic lesion development requires further examination.


PubMed | Stem Cell and Microenvironment Laboratory, Genomics Core, Weill Cornell Medicine Qatar and Montpellier University
Type: Journal Article | Journal: Oncotarget | Year: 2016

Recently, a class of endogenous species of RNA called circular RNA (circRNA) has been shown to regulate gene expression in mammals and their role in cellular function is just beginning to be understood. To investigate the role of circRNAs in ovarian cancer, we performed paired-end RNA sequencing of primary sites, peritoneal and lymph node metastases from three patients with stage IIIC ovarian cancer. We developed an in-house computational pipeline to identify and characterize the circRNA expression from paired-end RNA-Seq libraries. This pipeline revealed thousands of circular isoforms in Epithelial Ovarian Carcinoma (EOC). These circRNAs are enriched for potentially effective miRNA seed matches. A significantly larger number of circRNAs are differentially expressed between tumor sites than mRNAs. Circular and linear expression exhibits an inverse trend for many cancer related pathways and signaling pathways like NFkB, PI3k/AKT and TGF- typically activated for mRNA in metastases are inhibited for circRNA expression. Further, circRNAs show a more robust expression pattern across patients than mRNA forms indicating their suitability as biomarkers in highly heterogeneous cancer transcriptomes. The consistency of circular RNA expression may offer new candidates for cancer treatment and prognosis.

Loading Genomics Core collaborators
Loading Genomics Core collaborators