Microsatellite documentation of male-mediated outcrossing between inbred laboratory strains of the self-fertilizing mangrove killifish (Kryptolebias marmoratus

Abstract Primers for 36 microsatellite loci were developed and employed to characterize genetic stocks and detect possible outcrossing between highly inbred laboratory strainsof the self-fertilizing mangrove killifish, Kryptolebias marmoratus. From attempted crosses involving hermaphrodites from particular geographic strains and gonochoristic males from others, 2 among a total of 32 surveyed progenies (6.2%) displayed multilocus heterozygosity clearly indicative of interstrain gametic syngamy. One of these outcross hybrids was allowed to resume self-fertilization, and microsatellite assays of progeny showed that heterozygosity decreased by approximately 50% after one generation, as expected. Although populations of K. marmoratus consist mostly of synchronous hermaphrodites with efficient mechanisms of internal self-fertilization, these laboratory findings experimentally confirm that conspecific males can mediate occasional outcross events and that this process can release extensive genic heterozygosity. The mangrovekillifish,Kryptolebias(formerlyRivulus)marmoratus, is the only vertebrate species known to reproduce uniparentally by internal self-fertilization Earlier molecular techniques including multilocus DNA fingerprinting have revealed extensive genetic variation in some natural populations of K. marmoratus Materials and Methods Microsatellite Development To enrich for microsatellites in a genomic library for K. marmoratus, we employed a modified version of a hybridization The hybridization solution was mixed with magnetic streptavidin beads (Invitrogen, Carlsbad, CA), and hybridized fragments were captured on a magnetic block. Enriched DNA, recovered by precipitation, was polymerase chain reaction (PCR) amplified (25 ll total reaction volume) under the following conditions: 10 mM Tris-HCl pH 9.0, 50 mM KCl, 0.1% Triton X-100, 2.0 mM MgCl 2 , 25.0 lg/ml bovine serum albumin, 0.2 mM each dNTP, 0.5 lM SuperSNX24 forward primer, and 0.5 units Taq DNA Polymerase (Promega, Madison, WI). The PCR product was ligated into a PCR 2.1-TOPO vector and transformed into One Shot Top10 Chemically Competent Escherichia coli cells. Positive colonies were screened for b-galactosidase activity using materials in the TOPO TA cloning kit (Invitrogen; Carlsbad, CA). Insert sizes were verified from positive colonies by PCR amplification with the M13 forward (À20) and reverse (À27) primers (0.5 lM final concentration). Inserts !500 bp were purified using ExoSAP-IT (United States Biochemicals, Cleveland, OH) and sequenced with Big Dye chemistry (version 3.1, Applied Biosystems, Foster City, CA) using M13 forward or reverse primer on an ABI 3100 Genetic Analyzer equipped with 80-cm capillaries. Sequences were edited using Sequencher version 4.1.4 (Gene Codes Corporation, Ann Arbor, MI), and primers flanking microsatellite regions were developed using OligoAnalyzer 3.0 (Integrated DNA Technologies, Coralville, IA; http://www. idtdna.com/Scitools/Applications/Primerquest/). Primers were tested by amplifying genomic DNA from adult K. marmoratus specimens in laboratory strains initiated many generations ago from single hermaphroditic specimens from Florida, Belize, or Honduras. PCR conditions were optimized for each primer pair, and one primer from each pair was labeled at the 5#-end in either of 2 ways: directly with a 6-FAM or HEX fluorophore (Integrated DNA Technologies) or by attaching a reverse tag (5#-GGAAACAGCTATGACCATG-3#) for tailed PCR with an M13 primer labeled with a 6-FAM, HEX, or NED (Applied Biosystems) fluorophore. The 36 sequences from which primers were developed to amplify microsatellite loci were deposited to GenBank (accession numbers DQ335412-DQ335447). Laboratory Crosses Crossing experiments involved highly inbred laboratory lines each propagated generation after generation by a single selffertilizing hermaphrodite (herm) and each tracing back to a single wild herm originally collected at Utila Island, Honduras (strains Hon2 and Hon7), Belize (Bel50.91), Florida's Everglades National Park (ENP12), or Brevard County, Florida (CCHA). Each attempted cross involved placing an old (nearly senescent) herm with a secondary male in a small culture dish containing 13% saltwater solution DNA Extractions High molecular weight genomic DNA samples were isolated using a standard guanidinium isothiocyanate (GIT) chaotropic salt procedure Genotypic Assays Amplifications of microsatellite loci with fluorescently labeled PCR primers were performed in a 12.5-ll reaction volume containing 1 ll of purified genomic DNA, PCR Master Mix (Promega), and 0.5 lM of both forward and reverse primers. Tailed PCRs were performed in a 10-ll volume containing 1 ll of purified genomic DNA, 10 mM Tris-HCl pH 9.0, 50 mM KCl, 0.1% Triton X-100, 1.5 mM MgCl 2 , 25.0 lg/ml bovine serum albumin, 0.2 mM each dNTP, 0.25 lM M13-labeled primer, 0.25 lM locus-specific primer, 0.025 lM tailed locus-specific primer, and 0.4 units Taq DNA Polymerase (Promega). PCR products (1.5 ll) were mixed with 2.5 ll deionized formamide, 0.5 ll of either GeneScan-400HD [ROX] or GeneScan-500[ROX] internal lane standard, and 0.5 ll loading dye. Samples were denatured in a 95°C heating block for 3-5 min and chilled on ice before being loaded onto 5% acrylamide gels. Samples were electrophoresed on an ABI 377 DNA Sequencer at 3000 V for 2 h at 55°C. Alleles were sized using the software packages GeneScan version

Fine Definition of the CXCR4-Binding Region on the V3 Loop of Feline Immunodeficiency Virus Surface Glycoprotein

The chemokine receptor CXCR4 is shared by primary and laboratory-adapted strains of feline immunodeficiency virus (FIV) for viral entry. Our previous studies implicated a contiguous nine-amino-acid region of the V3 loop of the FIV envelope surface as important in CXCR4 binding and virus entry. The binding is specific for CXCR4 since it can be inhibited by AMD3100, a selective CXCR4 inhibitor. Additional site-directed mutagenesis was used to further reveal the key residues. Binding studies indicated that basic residues R395, K397, R399 as well as N398 are critical for CXCR4 binding. The effect of other amino acid residues on receptor binding depends on the type of amino acid residue substituted. The binding study results were confirmed on human CXCR4-expressing SupT1 cells and correlated with entry efficiency using a virus entry assay. Amino acid residues critical for CXCR4 are not critical for interactions with the primary binding receptor CD134, which has an equivalent role as CD4 for HIV-1 binding. The ELISA results show that W394 and W400 are crucial for the recognition by neutralizing anti-V3 antibodies. Since certain strains of HIV-1 also use CXCR4 as the entry receptor, the findings make the feline model attractive for development of broad-based entry antagonists and for study of the molecular mechanism of receptor/virus interactions

Does Proximity to Retailers Influence Alcohol and Tobacco Use Among Latino Adolescents?

Despite decades of research surrounding determinants of alcohol and tobacco (A&T) use among adolescents, built environment influences have only recently been explored. This study used ordinal regression on 205 Latino adolescents to explore the influence of the built environment (proximity to A&T retailers) on A&T use, while controlling for recognized social predictors. The sample was 45% foreign-born. A&T use was associated with distance from respondents’ home to the nearest A&T retailer (−), acculturation (+), parents’ consistent use of contingency management (−), peer use of A&T (+), skipping school (+), attending school in immediate proximity to the US/Mexico border (+), and the interaction between the distance to the nearest retailer and parents’ consistent use of contingency management (+). The association between decreasing distance to the nearest A&T retailer and increased A&T use in Latino adolescents reveals an additional risk behavior determinant in the US–Mexico border region

Pervasive Sharing of Genetic Effects in Autoimmune Disease

Genome-wide association (GWA) studies have identified numerous, replicable, genetic associations between common single nucleotide polymorphisms (SNPs) and risk of common autoimmune and inflammatory (immune-mediated) diseases, some of which are shared between two diseases. Along with epidemiological and clinical evidence, this suggests that some genetic risk factors may be shared across diseases—as is the case with alleles in the Major Histocompatibility Locus. In this work we evaluate the extent of this sharing for 107 immune disease-risk SNPs in seven diseases: celiac disease, Crohn's disease, multiple sclerosis, psoriasis, rheumatoid arthritis, systemic lupus erythematosus, and type 1 diabetes. We have developed a novel statistic for Cross Phenotype Meta-Analysis (CPMA) which detects association of a SNP to multiple, but not necessarily all, phenotypes. With it, we find evidence that 47/107 (44%) immune-mediated disease risk SNPs are associated to multiple—but not all—immune-mediated diseases (SNP-wise PCPMA<0.01). We also show that distinct groups of interacting proteins are encoded near SNPs which predispose to the same subsets of diseases; we propose these as the mechanistic basis of shared disease risk. We are thus able to leverage genetic data across diseases to construct biological hypotheses about the underlying mechanism of pathogenesis

Transcriptomic analysis of the temporal host response to skin infestation with the ectoparasitic mite Psoroptes ovis

<p>Abstract</p> <p>Background</p> <p>Infestation of ovine skin with the ectoparasitic mite <it>Psoroptes ovis </it>results in a rapid cutaneous immune response, leading to the crusted skin lesions characteristic of sheep scab. Little is known regarding the mechanisms by which such a profound inflammatory response is instigated and to identify novel vaccine and drug targets a better understanding of the host-parasite relationship is essential. The main objective of this study was to perform a combined network and pathway analysis of the <it>in vivo </it>skin response to infestation with <it>P. ovis </it>to gain a clearer understanding of the mechanisms and signalling pathways involved.</p> <p>Results</p> <p>Infestation with <it>P. </it>ovis resulted in differential expression of 1,552 genes over a 24 hour time course. Clustering by peak gene expression enabled classification of genes into temporally related groupings. Network and pathway analysis of clusters identified key signalling pathways involved in the host response to infestation. The analysis implicated a number of genes with roles in allergy and inflammation, including pro-inflammatory cytokines (<it>IL1A, IL1B, IL6, IL8 </it>and <it>TNF</it>) and factors involved in immune cell activation and recruitment (<it>SELE, SELL, SELP, ICAM1, CSF2, CSF3, CCL2 </it>and <it>CXCL2</it>). The analysis also highlighted the influence of the transcription factors NF-kB and AP-1 in the early pro-inflammatory response, and demonstrated a bias towards a Th2 type immune response.</p> <p>Conclusions</p> <p>This study has provided novel insights into the signalling mechanisms leading to the development of a pro-inflammatory response in sheep scab, whilst providing crucial information regarding the nature of mite factors that may trigger this response. It has enabled the elucidation of the temporal patterns by which the immune system is regulated following exposure to <it>P. ovis</it>, providing novel insights into the mechanisms underlying lesion development. This study has improved our existing knowledge of the host response to <it>P. ovis</it>, including the identification of key parallels between sheep scab and other inflammatory skin disorders and the identification of potential targets for disease control.</p

Genome-wide scan reveals association of psoriasis with IL-23 and NF-κB pathways

Psoriasis is a common immune-mediated disorder that affects the skin, nails and joints. To identify psoriasis susceptibility loci, we genotyped 438,670 SNPs in 1,409 psoriasis cases and 1,436 controls of European ancestry. We followed up 21 promising SNPs in 5,048 psoriasis cases and 5,041 controls. Our results provide strong support for the association of at least seven genetic loci and psoriasis (each with combined P less than 5 × 10−8). Loci with confirmed association include HLA-C, three genes involved in IL-23 signaling (IL23A, IL23R, IL12B), two genes that act downstream of TNF-α and regulate NF-κB signaling (TNIP1, TNFAIP3) and two genes involved in the modulation of Th2 immune responses (IL4, IL13). Although the proteins encoded in these loci are known to interact biologically, we found no evidence for epistasis between associated SNPs. Our results expand the catalog of genetic loci implicated in psoriasis susceptibility and suggest priority targets for study in other auto-immune disorders

Molecular characterization and clinical relevance of metabolic expression subtypes in human cancers.

Metabolic reprogramming provides critical information for clinical oncology. Using molecular data of 9,125 patient samples from The Cancer Genome Atlas, we identified tumor subtypes in 33 cancer types based on mRNA expression patterns of seven major metabolic processes and assessed their clinical relevance. Our metabolic expression subtypes correlated extensively with clinical outcome: subtypes with upregulated carbohydrate, nucleotide, and vitamin/cofactor metabolism most consistently correlated with worse prognosis, whereas subtypes with upregulated lipid metabolism showed the opposite. Metabolic subtypes correlated with diverse somatic drivers but exhibited effects convergent on cancer hallmark pathways and were modulated by highly recurrent master regulators across cancer types. As a proof-of-concept example, we demonstrated that knockdown of SNAI1 or RUNX1—master regulators of carbohydrate metabolic subtypes-modulates metabolic activity and drug sensitivity. Our study provides a system-level view of metabolic heterogeneity within and across cancer types and identifies pathway cross-talk, suggesting related prognostic, therapeutic, and predictive utility

Population Genetic Divergence Of Isle Royale Pearl Dace, Margarita margariscus (Cyprinidae).

In 1949, Hubbs and Lagler described morphological variation among pearl dace (Margariscus margarita) of inland lakes on Isle Royale, Michigan. For Harvey Lake, Hubbs and Lagler, proposed that pearl dace were sufficiently morphologically distinct to warrant subspecific status. They argued that divergence of the Harvey Lake pearl dace was due to allopatric differentiation in isolation from lower elevation lakes. Harvey Lake has been isolated by elevation from lower elevation lakes for approximately 10 to 15 thousand years. No genetic studies have been done on Isle Royale pearl dace to evaluate this hypothesis to date. Here we report the analysis of Margariscus margarita populations using a limited battery of microsatellite loci to assess the extent of and genetic divergence among the Harvey Lake and other Isle Royale lowland lake populations. Microsatellite loci were analyzed using PCR primers developed for the non-congener, longnose dace (Rhinichthys cataractae). Statistical analyses of allele frequency data indicate genetic differentiation among all Isle Royale pearl dace populations inclusive of both Harvey Lake and lowland populations concurring with Hubbs and Lagler’s hypothesis that the Harvey Lake population is genetically divergent. Results also indicate that the lowland Isle Royale populations are apparently equally divergent from one another