1. Examination of Csr regulatory circuitry using epistasis analysis with RNA-seq (Epi-seq) confirms that CsrD affects gene expression via CsrA, CsrB and CsrC. Potts Anastasia H, Leng Yuanyuan, Babitzke Paul, Romeo Tony. Sci Rep 2018. 8:1. 5373. PMID 29599472
The Csr global regulatory system coordinates gene expression in response to metabolic status. This system utilizes the RNA binding protein CsrA to regulate gene expression by binding to transcripts of structural and regulatory genes, thus affecting their structure, stability, translation, and/or transcription elongation. CsrA activity is controlled by sRNAs, CsrB and CsrC, which sequester CsrA away from other transcripts. CsrB/C levels are partly determined by their rates of turnover, which requires CsrD to render them susceptible to RNase E cleavage. Previous epistasis analysis suggested that CsrD affects gene expression through the other Csr components, CsrB/C and CsrA. However, those conclusions were based on a limited analysis of reporters. Here, we reassessed the global behavior of the Csr circuitry using epistasis analysis with RNA seq (Epi-seq). Because CsrD effects on mRNA levels were entirely lost in the csrA mutant and largely eliminated in a csrB/C mutant under our experimental conditions, while the majority of CsrA effects persisted in the absence of csrD, the original model accounts for the global behavior of the Csr system. Our present results also reflect a more nuanced role of CsrA as terminal regulator of the Csr system than has been recognized.
2. Systematic reanalysis of genomic data improves quality of variant interpretation. Hiatt S M, Amaral M D, Bowling K M, Finnila C R, Thompson M L, Gray D E, Lawlor J M J, Cochran J Nicholas, Bebin E Martina, Brothers K B, East K M, Kelley W V, Lamb N E, Levy S E, Lose E J et al. Clin. Genet. 2018. X:X. X. PMID 29652076
As genomic sequencing expands, so does our knowledge of the link between genetic variation and disease. Deeper catalogs of variant frequencies improve identification of benign variants, while sequencing affected individuals reveals disease-associated variation. Accumulation of human genetic data thus makes reanalysis a means to maximize benefits of clinical sequencing. We implemented pipelines to systematically reassess sequencing data from 494 individuals with developmental disability. Reanalysis yielded pathogenic or likely pathogenic (P/LP) variants in six individuals who initially had no P/LP variants, a 16% increase to P/LP yield. We also downgraded three LP and six variants of uncertain significance (VUS) due to updated population frequency data. The likelihood of identifying a new P/LP variant increased over time, as ~22% of individuals who did not receive a P/LP variant at their original analysis subsequently did after three years. We show here that reanalysis and data sharing increase the diagnostic yield and accuracy of clinical sequencing.
3. Basal polarization of the immune responses to Streptococcus agalactiae susceptible and resistant tilapia (Oreochromis niloticus). Zhu Jiajie, Gan Xi, Ao Qiuwei, Shen Xiashuang, Tan Yun, Chen Ming, Luo Yongju, Wang Hui, Jiang Hesheng, Li Chao. Fish Shellfish Immunol. 2018. X:X. X. PMID 29454032
One of the highest priority areas for improvement is the development of effective strategies for decreasing disease mortality levels in aquaculture production, a better understanding of the components of the fish immune system and their functions in the context of pathogen invasion is needed. Tilapia is the most common fish in South China, and Streptococcus agalactiae has become the most serious disease problem for tilapia industry in China. Here, we profiled gene expression differences between tilapia differing in their susceptibility to S. agalactiae both basally (before infection) and at three early timepoints post-infection (5?h, 50?h, and 7?d). Between group comparisons revealed 5756 unique genes differentially expressed greater than 2-fold at one or more timepoints. And the resistant fish showed much more strong ability in pathogen recognition, antigen presentation, immune activation, while the susceptible fish showed fast activation of apoptosis. Taken together, the immune profiles expand our knowledge for molecular mechanisms for disease resistance, as well as provide solid molecular resources for further identification of the candidate markers for disease-resistant selection and evaluation of disease prevention and treatment options.
4. Phylogeny of a cosmopolitan family of morphologically conserved trapdoor spiders (Mygalomorphae, Ctenizidae) using Anchored Hybrid Enrichment, with a description of the new family, Cyclocosmidae. Godwin Rebecca L, Opatova Vera, Garrison Nicole L, Hamilton Chris A, Bond Jason E. Mol. Phylogenet. Evol. 2018. X:X. X. PMID 29656103
The mygalomorph family Ctenizidae has a world-wide distribution and currently contains nine genera and 130 species. However, the monophyly of this group has long been questioned on both morphological and molecular grounds. Here, we use Anchored Hybrid Enrichment (AHE) to gather hundreds of loci from across the genome for reconstructing the phylogenetic relationships among the nine genera and test the monophyly of the family. We also reconstruct the possible ancestral ranges of the cyclocosmid clade. Using AHE, we generate a supermatrix of 565 loci and 115,209bp for 27 individuals. For the first time, analyses using all nine genera produce results definitively establishing the non-monophyly of Ctenizidae. A lineage formed exclusively by representatives of South African Stasimopus was placed as the sister group to the remaining taxa in the tree, and the Mediterranean Cteniza and Cyrtocarenum were recovered with high support as sister to exemplars of Euctenizidae, Migidae, and Idiopidae. All the remaining genera-Bothriocyrtum, Conothele, Cyclocosmia, Hebestatis, Latouchia, and Ummidia-share a common ancestor. Based on these results, we elevate this clade to the level of family creating the new family Cyclocosmidae. Our results definitively establish both the non-monophyly of the Ctenizidae and non-validity of the subfamilies Ummidiinae and Ctenizinae. In order to establish the placement of the remaining three ctenizid genera, Cteniza, Cyrtocarenum, and Stasimopus, thorough analyses within the context of a complete mygalomorph phylogenetic framework are needed. We formally describe the new family Cyclocosmidae and infer that the family's most recent common ancestor was likely distributed in western North America and Asia.
5. Historical demography of common carp estimated from individuals collected from various parts of the world using the pairwise sequentially markovian coalescent approach. Yuan Zihao, Huang Wei, Liu Shikai, Xu Peng, Dunham Rex, Liu Zhanjiang. Genetica 2018. X:X. X. PMID 29299799
The inference of historical demography of a species is helpful for understanding species' differentiation and its population dynamics. However, such inference has been previously difficult due to the lack of proper analytical methods and availability of genetic data. A recently developed method called Pairwise Sequentially Markovian Coalescent (PSMC) offers the capability for estimation of the trajectories of historical populations over considerable time periods using genomic sequences. In this study, we applied this approach to infer the historical demography of the common carp using samples collected from Europe, Asia and the Americas. Comparison between Asian and European common carp populations showed that the last glacial period starting 100 ka BP likely caused a significant decline in population size of the wild common carp in Europe, while it did not have much of an impact on its counterparts in Asia. This was probably caused by differences in glacial activities in East Asia and Europe, and suggesting a separation of the European and Asian clades before the last glacial maximum. The North American clade which is an invasive population shared a similar demographic history as those from Europe, consistent with the idea that the North American common carp probably had European ancestral origins. Our analysis represents the first reconstruction of the historical population demography of the common carp, which is important to elucidate the separation of European and Asian common carp clades during the Quaternary glaciation, as well as the dispersal of common carp across the world.
7. Functional aspects of meningeal lymphatics in ageing and Alzheimer's disease. Da Mesquita Sandro, Louveau Antoine, Vaccari Andrea, Smirnov Igor, Cornelison R Chase, Kingsmore Kathryn M, Contarino Christian, Onengut-Gumuscu Suna, Farber Emily, Raper Daniel, Viar Kenneth E, Powell Romie D, Baker Wendy, Dabhi Nisha, Bai Robin et al. Nature 2018. X:X. X. PMID 30046111
Ageing is a major risk factor for many neurological pathologies, but its mechanisms remain unclear. Unlike other tissues, the parenchyma of the central nervous system (CNS) lacks lymphatic vasculature and waste products are removed partly through a paravascular route. (Re)discovery and characterization of meningeal lymphatic vessels has prompted an assessment of their role in waste clearance from the CNS. Here we show that meningeal lymphatic vessels drain macromolecules from the CNS (cerebrospinal and interstitial fluids) into the cervical lymph nodes in mice. Impairment of meningeal lymphatic function slows paravascular influx of macromolecules into the brain and efflux of macromolecules from the interstitial fluid, and induces cognitive impairment in mice. Treatment of aged mice with vascular endothelial growth factor C enhances meningeal lymphatic drainage of macromolecules from the cerebrospinal fluid, improving brain perfusion and learning and memory performance. Disruption of meningeal lymphatic vessels in transgenic mouse models of Alzheimer's disease promotes amyloid-? deposition in the meninges, which resembles human meningeal pathology, and aggravates parenchymal amyloid-? accumulation. Meningeal lymphatic dysfunction may be an aggravating factor in Alzheimer's disease pathology and in age-associated cognitive decline. Thus, augmentation of meningeal lymphatic function might be a promising therapeutic target for preventing or delaying age-associated neurological diseases.
8. Functional signaling and gene regulatory networks between the oocyte and the surrounding cumulus cells. Biase Fernando H, Kimble Katelyn M. BMC Genomics 2018. 19:1. 351. PMID 29747587
The maturation and successful acquisition of developmental competence by an oocyte, the female gamete, during folliculogenesis is highly dependent on molecular interactions with somatic cells. Most of the cellular interactions identified, thus far, are modulated by growth factors, ions or metabolites. We hypothesized that this interaction is also modulated at the transcriptional level, which leads to the formation of gene regulatory networks between the oocyte and cumulus cells. We tested this hypothesis by analyzing transcriptome data from single oocytes and the surrounding cumulus cells collected from antral follicles employing an analytical framework to determine interdependencies at the transcript level.
9. Transcriptome profiles in peripheral white blood cells at the time of artificial insemination discriminate beef heifers with different fertility potential. Dickinson Sarah E, Griffin Brock A, Elmore Michelle F, Kriese-Anderson Lisa, Elmore Joshua B, Dyce Paul W, Rodning Soren P, Biase Fernando H. BMC Genomics 2018. 19:1. 129. PMID 29426285
Infertility is a longstanding limitation in livestock production with important economic impact for the cattle industry. Female reproductive traits are polygenic and lowly heritable in nature, thus selection for fertility is challenging. Beef cattle operations leverage estrous synchronization in combination with artificial insemination (AI) to breed heifers and benefit from an early and uniform calving season. A couple of weeks following AI, heifers are exposed to bulls for an opportunity to become pregnant by natural breeding (NB), but they may also not become pregnant during this time period. Focusing on beef heifers, in their first breeding season, we hypothesized that: a- at the time of AI, the transcriptome of peripheral white blood cells (PWBC) differs between heifers that become pregnant to AI and heifers that become pregnant late in the breeding season by NB or do not become pregnant during the breeding season; and b- the ratio of transcript abundance between genes in PWBC classifies heifers according to pregnancy by AI, NB, or failure to become pregnant.
10. The sea lamprey germline genome provides insights into programmed genome rearrangement and vertebrate evolution. Smith Jeramiah J, Timoshevskaya Nataliya, Ye Chengxi, Holt Carson, Keinath Melissa C, Parker Hugo J, Cook Malcolm E, Hess Jon E, Narum Shawn R, Lamanna Francesco, Kaessmann Henrik, Timoshevskiy Vladimir A, Waterbury Courtney K M, Saraceno Cody, Wiedemann Leanne M et al. Nat. Genet. 2018. X:X. X. PMID 29358652
The sea lamprey (Petromyzon marinus) serves as a comparative model for reconstructing vertebrate evolution. To enable more informed analyses, we developed a new assembly of the lamprey germline genome that integrates several complementary data sets. Analysis of this highly contiguous (chromosome-scale) assembly shows that both chromosomal and whole-genome duplications have played significant roles in the evolution of ancestral vertebrate and lamprey genomes, including chromosomes that carry the six lamprey HOX clusters. The assembly also contains several hundred genes that are reproducibly eliminated from somatic cells during early development in lamprey. Comparative analyses show that gnathostome (mouse) homologs of these genes are frequently marked by polycomb repressive complexes (PRCs) in embryonic stem cells, suggesting overlaps in the regulatory logic of somatic DNA elimination and bivalent states that are regulated by early embryonic PRCs. This new assembly will enhance diverse studies that are informed by lampreys' unique biology and evolutionary/comparative perspective.
11. Hijacking a key chromatin modulator creates epigenetic vulnerability for Myc-driven cancer. Yang Zhenhua, Shah Kushani, Busby Theodore, Giles Keith, Khodadadi-Jamayran Alireza, Li Wei, Jiang Hao. J. Clin. Invest. 2018. X:X. X. PMID 29870403
While the genomic binding of MYC protein correlates with active epigenetic marks on chromatin, it remains largely unclear how major epigenetic mechanisms functionally impact the tumorigenic potential of MYC. Here we showed that compared to the catalytic subunits, the core subunits, including DPY30, of the major H3K4 methyltransferase complexes were frequently amplified in human cancers, and selectively upregulated in Burkitt lymphoma. We showed that DPY30 promoted expression of endogenous MYC, and was also functionally important for efficient binding of MYC to its genomic targets by regulating chromatin accessibility. Dpy30 heterozygosity did not affect normal animal physiology including life span, but significantly suppressed Myc-driven lymphomagenesis, as cells failed to combat oncogene-triggered apoptosis due to insufficient epigenetic modulation and expression of a subset of anti-apoptotic genes. Dpy30 reduction also greatly impeded MYC-dependent cellular transformation without affecting normal cell growth. These results suggest that MYC hijacks a major epigenetic pathway - H3K4 methylation - to facilitate its molecular activity in target binding and to coordinate its oncogenic program for efficient tumorigenesis, meanwhile creating "epigenetic vulnerability." DPY30 and the H3K4 methylation pathway are thus potential epigenetic targets for treating certain MYC-driven cancers.
12. O-GlcNAc transferase missense mutations linked to X-linked intellectual disability deregulate genes involved in cell fate determination and signaling. Selvan Nithya, George Stephan, Serajee Fatema J, Shaw Marie, Hobson Lynne, Kalscheuer Vera M, Prasad Nripesh, Levy Shawn E, Taylor Juliet, Afitmos Salim, Schwartz Charles E, Huq Ahm M, Gecz Jozef, Wells Lance. J. Biol. Chem. 2018. X:X. X. PMID 29769320
It is estimated that ~1% of the world's population has intellectual disability, with males affected more often than females. OGT is an X-linked gene encoding for the enzyme O-GlcNAc transferase (OGT), which carries out the reversible addition of N-Acetylglucosamine (GlcNAc) to Ser/Thr residues of its intracellular substrates. Three missense mutations in the tetratricopeptide (TPR) repeats of OGT have recently been reported to cause X-linked Intellectual Disability (XLID). Here we report the discovery of two additional novel missense mutations (c. 775 G>A, p. A259T and c. 1016 A>G, p. E339G) in the TPR domain of OGT that segregate with XLID in affected families. Characterization of all five of these XLID missense variants of OGT demonstrates modest declines in thermodynamic stability and/or activities of the variants. We engineered each of the mutations into a male human embryonic stem cell line using CRISPR/Cas9. Investigation of the global O-GlcNAc profile as well as OGT and OGA levels by Western blotting showed no gross changes in steady-state levels in the engineered lines. However, analyses of the differential transcriptomes of the OGT variant expressing stem cells revealed shared deregulation of genes involved in cell fate determination and LXR/RXR signaling, which has been implicated in neuronal development. Thus, here we reveal 2 additional mutations encoding residues in the TPR regions of OGT that appear causal for XLID and provide evidence that the relatively stable and active TPR variants may share a common, unelucidated, mechanism of altering gene expression profiles in human embryonic stem cells.
13. Ancient balancing selection on heterocyst function in a cosmopolitan cyanobacterium. Sano Emiko B, Wall Christopher A, Hutchins Patrick R, Miller Scott R. Nat Ecol Evol 2018. X:X. X. PMID 29335576
The conventional view of bacterial adaptation emphasizes the importance of rapidly evolved changes that are highly repeatable in response to similar environments and subject to loss in the absence of selection. Consequently, genetic variation is not expected to persist over long time scales for these organisms. Here, we show that a geographically widespread gene content polymorphism has surprisingly been maintained for tens of millions of years of diversification of the multicellular cyanobacterium Fischerella thermalis. The polymorphism affects gas permeability of the heterocyst-the oxygen-sensitive, nitrogen-fixing cell produced by these bacteria-and spatial variation in temperature favours alternative alleles due to thermodynamic effects on both heterocyst function and organism fitness at physiological temperature extremes. Whether or not ancient balancing selection plays a generally important role in the maintenance of microbial diversity remains to be investigated.
14. Discovery and validation of circulating biomarkers of colorectal adenoma by high-depth small RNA sequencing. Roberts Brian S, Hardigan Andrew A, Moore Dianna E, Ramaker Ryne C, Jones Angela L, Fitz-Gerald Meredith B, Cooper Gregory M, Wilcox C Mel, Kimberly Robert P, Myers Richard M. Clin. Cancer Res. 2018. X:X. X. PMID 29490987
Colorectal cancer (CRC) is the third most common cancer worldwide, causing ~700,000 deaths each year. The majority of CRCs begin as adenomas. Definitive screening for colorectal adenomas is currently accomplished through colonoscopy but, owing largely to costs and invasiveness, is typically limited to patient groups at higher risk by virtue of age or family history. We sought to determine if blood-based small RNA markers could detect colorectal adenoma.
15. Peripherally derived macrophages can engraft the brain independent of irradiation and maintain an identity distinct from microglia. Cronk James C, Filiano Anthony J, Louveau Antoine, Marin Ioana, Marsh Rachel, Ji Emily, Goldman Dylan H, Smirnov Igor, Geraci Nicholas, Acton Scott, Overall Christopher C, Kipnis Jonathan. J. Exp. Med. 2018. X:X. X. PMID 29643186
Peripherally derived macrophages infiltrate the brain after bone marrow transplantation and during central nervous system (CNS) inflammation. It was initially suggested that these engrafting cells were newly derived microglia and that irradiation was essential for engraftment to occur. However, it remains unclear whether brain-engrafting macrophages (beM?s) acquire a unique phenotype in the brain, whether long-term engraftment may occur without irradiation, and whether brain function is affected by the engrafted cells. In this study, we demonstrate that chronic, partial microglia depletion is sufficient for beM?s to populate the niche and that the presence of beM?s does not alter behavior. Furthermore, beM?s maintain a unique functional and transcriptional identity as compared with microglia. Overall, this study establishes beM?s as a unique CNS cell type and demonstrates that therapeutic engraftment of beM?s may be possible with irradiation-free conditioning regimens.
16. Endosymbiont genomes yield clues of tubeworm success. Li Yuanning, Liles Mark R, Halanych Kenneth M. ISME J 2018. X:X. X. PMID 30022157
Forty years after discovery of chemosynthetic symbiosis in the tubeworm Riftia pachyptila, how organisms maintain their unique host-symbiont associations at the cellular level is still largely unknown. Previous studies primarily focus on symbionts associated with host lineages living in hydrothermal vents. To understand physiological adaptations and evolution in these holobiont systems in markedly different habitats, we characterized four novel siboglinid-symbiont genomes spanning deep-sea seep and sedimented environments. Our comparative analyses suggest that all sampled siboglinid chemoautotrophic symbionts, except for frenulate symbionts, can use both rTCA and Calvin cycle for carbon fixation. We hypothesize that over evolutionary time siboglinids have been able to utilize different bacterial lineages allowing greater metabolic flexibility of carbon fixation (e.g., rTCA) enabling tubeworms to thrive in more reducing habitats, such as vents and seeps. Moreover, we show that sulfur metabolism and molecular mechanisms related to initial infection are remarkably conserved across chemoautotrophic symbionts in different habitats. Unexpectedly, we find that the ability to use hydrogen, as an additional energy source, is potentially more widespread than previously recognized. Our comparative genomic results help elucidate potential mechanisms used to allow chemosynthetically dependent holobionts adapt to, and evolve in, different environments.
17. Assembly of a reference transcriptome for the gymnosome pteropod Clione limacina and profiling responses to short-term CO2 exposure. Thabet Ali A, Maas Amy E, Saber Samy A, Tarrant Ann M. Mar Genomics 2017. X:X. X. PMID 28385518
The gymnosome (unshelled) pteropod Clione limacina is a pelagic predatory mollusc found in polar and sub-polar regions. It has been studied for its distinctive swimming behavior and as an obligate predator on the closely related thecosome (shelled) pteropods. As concern about ocean acidification increases, it becomes useful to compare the physiological responses of closely-related calcifying and non-calcifying species to acidification. The goals of this study were thus to generate a reference transcriptome for Clione limacina, to expose individuals to CO2 for a period of 3days, and to explore differential patterns of gene expression. Our Trinity assembly contained 300,994 transcripts of which ~26% could be annotated. In total, only 41 transcripts were differentially expressed following the CO2 treatment, consistent with a limited physiological response of this species to short-term CO2 exposure. The differentially expressed genes identified in our study were largely distinct from those identified in previous studies of thecosome pteropods, although some similar transcripts were identified, suggesting that comparison of these transcriptomes and responses may provide insight into differences in responses to ocean acidification among phylogenetically and functionally distinct molluscan lineages.
18. Discovery and evolution of novel hemerythrin genes in annelid worms. Costa-Paiva Elisa M, Whelan Nathan V, Waits Damien S, Santos Scott R, Schrago Carlos G, Halanych Kenneth M. BMC Evol. Biol. 2017. 17:1. 85. PMID 28330441
Despite extensive study on hemoglobins and hemocyanins, little is known about hemerythrin (Hr) evolutionary history. Four subgroups of Hrs have been documented, including: circulating Hr (cHr), myohemerythrin (myoHr), ovohemerythrin (ovoHr), and neurohemerythrin (nHr). Annelids have the greatest diversity of oxygen carrying proteins among animals and are the only phylum in which all Hr subgroups have been documented. To examine Hr diversity in annelids and to further understand evolution of Hrs, we employed approaches to survey annelid transcriptomes in silico.
19. Comparative transcriptome analysis reveals conserved branching morphogenesis related genes involved in chamber formation of catfish swimbladder. Yang Yujia, Fu Qiang, Liu Yang, Wang Xiaozhu, Dunham Rex, Liu Shikai, Bao Lisui, Zeng Qifan, Zhou Tao, Li Ning, Qin Zhenkui, Jiang Chen, Gao Dongya, Liu Zhanjiang. Physiol. Genomics 2017. X:X. physiolgenomics.00089.2017. PMID 29167198
The swimbladder is an internal gas-filled organ in teleosts. Its major function is to regulate buoyancy. Swimbladder exhibits great variations in size, shape, number of compartments or chambers among teleosts. However, the genomic control of swimbladder variations is unknown. Channel catfish (Ictalurus punctatus), blue catfish (Ictalurus furcatus), and their F1 hybrids of female channel catfish x male blue catfish (C×B hybrid catfish) provide a good model to investigate the swimbladder morphology, because channel catfish possess a single-chambered swimbladder whereas blue catfish possess a bi-chambered swimbladder; and C×B hybrid catfish possess a bi-chambered swimbladder but with significantly reduced posterior chamber. Here we determined the transcriptional profiles of swimbladder from channel catfish, blue catfish, and C×B hybrid catfish. We examined their transcriptomes at both the fingerling and adult stages. Through comparative transcriptome analysis, approximately 4,000 differentially expressed genes (DEGs) were identified. Among these DEGs, members of the Wnt signaling pathway (wnt1, wnt2, nfatc1, rac2), Hedgehog signaling pathway (shh), and growth factors (fgf10, igf-1) were identified. As these genes were known to be important for branching morphogenesis of mammalian lung and of mammary glands, their association with budding of posterior chamber primordium and progressive development of bi-chambered swimbladder in fish suggested that these branching morphogenesis related genes and their functions in branching are evolutionarily conserved across a broad spectrum of species.
20. Transcriptomic profiling analysis of tilapia (Oreochromis niloticus) following Streptococcus agalactiae challenge. Zhu Jiajie, Fu Qiang, Ao Qiuwei, Tan Yun, Luo Yongju, Jiang Hesheng, Li Chao, Gan Xi. Fish Shellfish Immunol. 2017. X:X. X. PMID 28111359
Innate immune system is the primary defense mechanism against pathogen infection in teleost, which are living in pathogen-rich aquatic environment. It has been long hypothesized that the disease resistance in teleost are strongly correlated to the activities of innate immune genes. Tilapia is an important economical fish around the world, especially in China, where the production accounts for nearly half of the global production. Recently, S. agalactiae has become one of the most serious bacterial diseases in southern China, resulted in high cumulative mortality and economic loss to tilapia industry. Therefore, we sought here to characterize the expression profiles of tilapia against S. agalactiae infection at whole transcriptome level by RNA-seq technology. A total of 2822 genes were revealed significantly expressed in tilapia spleen with a general trend of induction. Notably, most of the genes were rapidly the most induced at the early timepoint. The significantly changed genes highlighted the function of pathogen attachment and recognition, antioxidant/apoptosis, cytoskeletal rearrangement, and immune activation. Collectively, the induced expression patterns suggested the strong ability of tilapia to rapidly recognize the invasive bacteria, and activation of downstream immune signaling pathways to clear the bacteria and prevent the tissue damage and bacteria triggered cell apoptosis. Our results heighted important roles of novel candidate genes which were often missed in previous tilapia studies. Further studies are needed to characterize the molecular relationships between key immune genes and disease resistance, and to identify the candidate genes for molecular-assistant selection of disease-resistant broodstock and evaluation of disease prevention and treatment measures.
21. Altered methylation of specific DNA loci in the liver of Bhmt-null mice results in repression of Iqgap2 and F2rl2 and is associated with development of preneoplastic foci. Lupu Daniel S, Orozco Luz D, Wang Ying, Cullen John M, Pellegrini Matteo, Zeisel Steven H. FASEB J. 2017. X:X. X. PMID 28179424
Folate B12-dependent remethylation of homocysteine is important, but less is understood about the importance of the alternative betaine-dependent methylation pathway-catalyzed by betaine-homocysteine methyltransferase (BHMT)-for establishing and maintaining adequate DNA methylation across the genome. We studied C57Bl/6J Bhmt (betaine-homocysteine methyltransferase)-null mice at age 4, 12, 24, and 52 wk (N = 8) and observed elevation of S-adenosylhomocysteine concentrations and development of preneoplastic foci in the liver (increased placental glutathione S-transferase and cytokeratin 8-18 activity; starting at 12 wk). At 4 wk, we identified 63 differentially methylated CpGs (DMCs; false discovery rate < 5%) proximal to 81 genes (across 14 chromosomes), of which 18 were differentially expressed. Of these DMCs, 52% were located in one 15.5-Mb locus on chromosome 13, which encompassed the Bhmt gene and defined a potentially sensitive region with mostly decreased methylation. Analyzing Hybrid Mouse Diversity Panel data, which consisted of 100 inbred strains of mice, we identified 97 DMCs that were affected by Bhmt genetic variation in the same region, with 7 overlapping those found in Bhmt-null mice (P < 0.001). At all time points, we found a hypomethylated region mapping to Iqgap2 (IQ motif-containing GTPase activating protein 2) and F2rl2 (proteinase-activated receptor-3), 2 genes that were also silenced and underexpressed, respectively.-Lupu, D. S., Orozco, L. D., Wang, Y., Cullen, J. M., Pellegrini, M., Zeisel, S. H. Altered methylation of specific DNA loci in the liver of Bhmt-null mice results in repression of Iqgap2 and F2rl2 and is associated with development of preneoplastic foci.
22. Comparative transcriptomics of Entelegyne spiders (Araneae, Entelegynae), with emphasis on molecular evolution of orphan genes. Carlson David E, Hedin Marshal. PLoS ONE 2017. 12:4. e0174102. PMID 28379977
Next-generation sequencing technology is rapidly transforming the landscape of evolutionary biology, and has become a cost-effective and efficient means of collecting exome information for non-model organisms. Due to their taxonomic diversity, production of interesting venom and silk proteins, and the relative scarcity of existing genomic resources, spiders in particular are excellent targets for next-generation sequencing (NGS) methods. In this study, the transcriptomes of six entelegyne spider species from three genera (Cicurina travisae, C. vibora, Habronattus signatus, H. ustulatus, Nesticus bishopi, and N. cooperi) were sequenced and de novo assembled. Each assembly was assessed for quality and completeness and functionally annotated using gene ontology information. Approximately 100 transcripts with evidence of homology to venom proteins were discovered. After identifying more than 3,000 putatively orthologous genes across all six taxa, we used comparative analyses to identify 24 instances of positively selected genes. In addition, between ~ 550 and 1,100 unique orphan genes were found in each genus. These unique, uncharacterized genes exhibited elevated rates of amino acid substitution, potentially consistent with lineage-specific adaptive evolution. The data generated for this study represent a valuable resource for future phylogenetic and molecular evolutionary research, and our results provide new insight into the forces driving genome evolution in taxa that span the root of entelegyne spider phylogeny.
23. Twitchin Kinase Inhibits Muscle Activity. Matsunaga Yohei, Hwang Hyundoo, Franke Barbara, Williams Rhys, Penley McKenna, Qadota Hiroshi, Yi Hong, Morran Levi T, Lu Hang, Mayans Olga, Benian Guy M. Mol. Biol. Cell 2017. X:X. X. PMID 28428253
Muscle sarcomeres contain giant polypeptides composed of multiple immunoglobulin and fibronectin domains and one or two protein kinase domains. Although binding partners for a number of this family's kinase domains have been identified, the catalytic necessity of these kinase domains remains unknown. In addition, various members of this kinase family are suspected pseudokinases with no, or little, activity. Here, we address catalytic necessity for the first time using the prototypic invertebrate representative twitchin (UNC-22) from C. elegans In in vitro experiments, change of a conserved lysine (K) that is involved in ATP coordination to alanine (A) resulted in elimination of kinase activity without affecting the overall structure of the kinase domain. The same mutation, unc-22(sf21), was generated in the endogenous twitchin gene. The unc-22(sf21) worms have well-organized sarcomeres. However, unc-22(sf21) mutants move faster than wild-type, and by optogenetic experiments, contract more than wild-type. Wild-type nematodes exhibited greater competitive fitness than unc-22(sf21) mutants. Thus, the catalytic activity of twitchin kinase has a role in vivo, where it inhibits muscle activity, and is likely maintained by selection.
24. Detection of prostate cancer-specific transcripts in extracellular vesicles isolated from post-DRE urine. Pellegrini Kathryn L, Patil Dattatraya, Douglas Kristen J S, Lee Grace, Wehrmeyer Kathryn, Torlak Mersiha, Clark Jeremy, Cooper Colin S, Moreno Carlos S, Sanda Martin G. Prostate 2017. X:X. X. PMID 28419548
The measurement of gene expression in post-digital rectal examination (DRE) urine specimens provides a non-invasive method to determine a patient's risk of prostate cancer. Many currently available assays use whole urine or cell pellets for the analysis of prostate cancer-associated genes, although the use of extracellular vesicles (EVs) has also recently been of interest. We investigated the expression of prostate-, kidney-, and bladder-specific transcripts and known prostate cancer biomarkers in urine EVs.
25. Chromatin States in Mouse Sperm Correlate with Embryonic and Adult Regulatory Landscapes. Jung Yoon Hee, Sauria Michael E G, Lyu Xiaowen, Cheema Manjinder S, Ausio Juan, Taylor James, Corces Victor G. Cell Rep 2017. 18:6. 1366-1382. PMID 28178516
The mammalian sperm genome is thought to lack substantial information for the regulation of future expression after fertilization. Here, we show that most promoters in mouse sperm are flanked by well-positioned nucleosomes marked by active histone modifications. Analysis of these modifications suggests that many enhancers and super-enhancers functional in embryonic and adult tissues are already specified in sperm. The sperm genome is bound by CTCF and cohesin at sites that are also present in round spermatids and embryonic stem cells (ESCs). These sites mediate interactions that organize the sperm genome into domains and compartments that overlap extensively with those found in mESCs. These results suggest that sperm carry a rich source of regulatory information, encoded in part by its three-dimensional folding specified by CTCF and cohesin. This information may contribute to future expression during embryonic and adult life, suggesting mechanisms by which environmental effects on the paternal germline are transmitted transgenerationally.
26. Deletion of Type I glutamine synthetase deregulates nitrogen metabolism and increases ethanol production in Clostridium thermocellum. Rydzak Thomas, Garcia David, Stevenson David M, Sladek Margaret, Klingeman Dawn M, Holwerda Evert K, Amador-Noguez Daniel, Brown Steven D, Guss Adam M. Metab. Eng. 2017. X:X. X. PMID 28400329
Clostridium thermocellum rapidly deconstructs cellulose and ferments resulting hydrolysis products into ethanol and other products, and is thus a promising platform organism for the development of cellulosic biofuel production via consolidated bioprocessing. While recent metabolic engineering strategies have targeted eliminating canonical fermentation products (acetate, lactate, formate, and H2), C. thermocellum also secretes amino acids, which has limited ethanol yields in engineered strains to approximately 70% of the theoretical maximum. To investigate approaches to decrease amino acid secretion, we attempted to reduce ammonium assimilation by deleting the Type I glutamine synthetase (glnA) in an essentially wild type strain of C. thermocellum. Deletion of glnA reduced levels of secreted valine and total amino acids by 53% and 44% respectively, and increased ethanol yields by 53%. RNA-seq analysis revealed that genes encoding the RNF-complex were more highly expressed in ?glnA and may have a role in improving NADH-availability for ethanol production. While a significant up-regulation of genes involved in nitrogen assimilation and urea uptake suggested that deletion of glnA induces a nitrogen starvation response, metabolomic analysis showed an increase in intracellular glutamine levels indicative of nitrogen-rich conditions. We propose that deletion of glnA causes deregulation of nitrogen metabolism, leading to overexpression of nitrogen metabolism genes and, in turn, elevated glutamine levels. Here we demonstrate that perturbation of nitrogen assimilation is a promising strategy to redirect flux from the production of nitrogenous compounds toward biofuels in C. thermocellum.
27. Genetic adaptation of microbial populations present in high-intensity catfish production systems with therapeutic oxytetracycline treatment. Zeng Qifan, Tian Xiangli, Wang Luxin. Sci Rep 2017. 7:1. 17491. PMID 29235508 PMC ID PMC5727513
Microbial communities that are present in aquaculture production systems play significant roles in degrading organic matter, controlling diseases, and formation of antibiotic resistance. It is important to understand the diversity and abundance of microbial communities and their genetic adaptations associated with environmental physical and chemical changes. Here we collected water and sediment samples from a high-intensity catfish production system and its original water reservoir. The metagenomic analysis showed that Proteobacteria, Actinobacteria, Bacteroidetes, Cyanobacteria, and Firmicutes were the top five phyla identified from all samples. The aquaculture production system significantly changed the structure of aquatic microbial populations. Substantial changes were also observed in SNP patterns among four sample types. The gene-specific sweep was found to be more common than genome-wide sweep. The selective sweep analysis revealed that 21 antibiotic resistant (AR) genes were under selection, with most belonging to antibiotic efflux pathways. Over 200 AR gene gains and losses were determined by changes in gene frequencies. Most of the AR genes were characterized as ABC efflux pumps, RND efflux pumps, and tetracycline MFS efflux pumps. Results of this study suggested that aquaculture waste, especially waste containing therapeutic antibiotics, has a significant impact on microbial population structures and their genetic structures.
28. Cell-Based Systems Biology Analysis of Human AS03-Adjuvanted H5N1 Avian Influenza Vaccine Responses: A Phase I Randomized Controlled Trial. Howard Leigh M, Hoek Kristen L, Goll Johannes B, Samir Parimal, Galassie Allison, Allos Tara M, Niu Xinnan, Gordy Laura E, Creech C Buddy, Prasad Nripesh, Jensen Travis L, Hill Heather, Levy Shawn E, Joyce Sebastian, Link Andrew J et al. PLoS ONE 2017. 12:1. e0167488. PMID 28099485
Vaccine development for influenza A/H5N1 is an important public health priority, but H5N1 vaccines are less immunogenic than seasonal influenza vaccines. Adjuvant System 03 (AS03) markedly enhances immune responses to H5N1 vaccine antigens, but the underlying molecular mechanisms are incompletely understood.
29. More than just antibodies: Protective mechanisms of a mucosal vaccine against fish pathogen Flavobacterium columnare. Zhang Dongdong, Thongda Wilawan, Li Chao, Zhao Honggang, Beck Benjamin H, Mohammed Haitham, Arias Covadonga R, Peatman Eric. Fish Shellfish Immunol. 2017. 71:X. 160-170. PMID 28989091
A recently developed attenuated vaccine for Flavobacterium columnare has been demonstrated to provide superior protection for channel catfish, Ictalurus punctatus, against genetically diverse columnaris isolates. We were interested in examining the mechanisms of this protection by comparing transcriptional responses to F. columnare challenge in vaccinated and unvaccinated juvenile catfish. Accordingly, 58 day old fingerling catfish (28 days post-vaccination or unvaccinated control) were challenged with a highly virulent F. columnare isolate (BGSF-27) and gill tissues collected pre-challenge (0 h), and 1 h and 2 h post infection, time points previously demonstrated to be critical in early host-pathogen interactions. Following RNA-sequencing and transcriptome assembly, differential expression (DE) analysis within and between treatments revealed several patterns and pathways potentially underlying improved survival of vaccinated fish. Most striking was a pattern of dramatically higher basal expression of an array of neuropeptides (e.g. somatostatin), hormones, complement factors, and proteases at 0 h in vaccinated fish. Previous studies indicate these are likely the preformed mediators of neuroendocrine cells and/or eosinophilic granular (mast-like) cells within the fish gill. Following challenge, these elements fell to almost undetectable levels (>100-fold downregulated) by 1 h in vaccinated fish, suggesting their rapid release and/or cessation of synthesis following degranulation. Concomitantly, levels of pro-inflammatory cytokines (IL-1b, IL-8, IL-17) were induced in unvaccinated fish. In contrast, in vaccinated catfish, we observed widespread induction of genes needed for collagen deposition and tissue remodeling. Taken together, our results indicate an important component of vaccine protection in fish mucosal tissues may be the sensitization, proliferation and arming of resident secretory cells in the period between primary and secondary challenge.
30. Contrasting seasonal drivers of virus abundance and production in the North Pacific Ocean. Gainer P Jackson, Pound Helena L, Larkin Alyse A, LeCleir Gary R, DeBruyn Jennifer M, Zinser Erik R, Johnson Zackary I, Wilhelm Steven W. PLoS ONE 2017. 12:9. e0184371. PMID 28880951
The North Pacific Ocean (between approximately 0°N and 50°N) contains the largest continuous ecosystem on Earth. This region plays a vital role in the cycling of globally important nutrients as well as carbon. Although the microbial communities in this region have been assessed, the dynamics of viruses (abundances and production rates) remains understudied. To address this gap, scientific cruises during the winter and summer seasons (2013) covered the North Pacific basin to determine factors that may drive virus abundances and production rates. Along with information on virus particle abundance and production, we collected a spectrum of oceanographic metrics as well as information on microbial diversity. The data suggest that both biotic and abiotic factors affect the distribution of virus particles. Factors influencing virus dynamics did not vary greatly between seasons, although the abundance of viruses was almost an order of magnitude greater in the summer. When considered in the context of microbial community structure, our observations suggest that members of the bacterial phyla Proteobacteria, Planctomycetes, and Bacteroidetes were correlated to both virus abundances and virus production rates: these phyla have been shown to be enriched in particle associated communities. The findings suggest that environmental factors influence virus community functions (e.g., virion particle degradation) and that particle-associated communities may be important drivers of virus activity.
31. Elevated p53 Activities Restrict Differentiation Potential of MicroRNA-Deficient Pluripotent Stem Cells. Liu Zhong, Zhang Cheng, Skamagki Maria, Khodadadi-Jamayran Alireza, Zhang Wei, Kong Dexin, Chang Chia-Wei, Feng Jingyang, Han Xiaosi, Townes Tim M, Li Hu, Kim Kitai, Zhao Rui. Stem Cell Reports 2017. 9:5. 1604-1617. PMID 29141234 PMC ID PMC5688240
Pluripotent stem cells (PSCs) deficient for microRNAs (miRNAs), such as Dgcr8-/- or Dicer-/- embryonic stem cells (ESCs), contain no mature miRNA and cannot differentiate into somatic cells. How miRNA deficiency causes differentiation defects remains poorly understood. Here, we report that miR-302 is sufficient to enable neural differentiation of differentiation-incompetent Dgcr8-/- ESCs. Our data showed that miR-302 directly suppresses the tumor suppressor p53, which is modestly upregulated in Dgcr8-/- ESCs and serves as a barrier restricting neural differentiation. We demonstrated that direct inactivation of p53 by SV40 large T antigen, a short hairpin RNA against Trp53, or genetic ablation of Trp53 in Dgcr8-/- PSCs enables neural differentiation, while activation of p53 by the MDM2 inhibitor nutlin-3a in wild-type ESCs inhibits neural differentiation. Together, we demonstrate that a major function of miRNAs in neural differentiation is suppression of p53 and that modest activation of p53 blocks neural differentiation of miRNA-deficient PSCs.
32. Ecophysiological examination of the Lake Erie Microcystis bloom in 2014: linkages between biology and the water supply shutdown of Toledo, Ohio. Steffen Morgan Michelle, Davis Timothy W, McKay Robert Michael, Bullerjahn George S, Krausfeldt Lauren E, Stough Joshua M A, Neitzey Michelle L, Gilbert Naomi E, Boyer Gregory L, Johengen Thomas H, Gossiaux Duane C, Burtner Ashley M, Palladino Danna, Rowe Mark, Dick Gregory J et al. Environ. Sci. Technol. 2017. X:X. X. PMID 28535339
Annual cyanobacterial blooms dominated by Microcystis have occurred in western Lake Erie (USA/Canada) during summer months since 1995. The production of toxins by bloom-forming cyanobacteria can lead to drinking water crises, such as the one experienced by the city of Toledo in August of 2014, when the city was rendered without drinking water for > 2 days. It is important to understand the conditions and environmental cues that were driving this specific bloom to provide a scientific framework for management of future bloom events. To this end, samples were collected and metatranscriptomes generated coincident with the collection of environmental metrics for eight sites located in the western basin of Lake Erie, including a station proximal to the water intake for the city of Toledo. These data were used to generate a basin-wide ecophysiological fingerprint of Lake Erie Microcystis populations in August 2014 for comparison to previous bloom communities. Our observations and analyses indicate that, at the time of sample collection, Microcystis populations were under dual nitrogen (N) and phosphorus (P) stress, as genes involved in scavenging of these nutrients were being actively transcribed. Targeted analysis of urea transport and hydrolysis suggests a potentially important role for exogenous urea as a nitrogen source during the 2014 event. Finally, simulation data suggest a wind event caused microcystin-rich water from Maumee Bay to be transported east along the southern shoreline past the Toledo water intake. Coupled with a significant cyanophage infection, these results reveal that a combination of biological and environmental factors led to the disruption of the Toledo water supply. This scenario was not atypical of re-occurring Lake Erie blooms and thus may re-occur in the future.
33. Looking beyond the exome: a phenotype-first approach to molecular diagnostic resolution in rare and undiagnosed diseases. Pena Loren D M, Jiang Yong-Hui, Schoch Kelly, Spillmann Rebecca C, Walley Nicole, Stong Nicholas, Rapisardo Horn Sarah, Sullivan Jennifer A, McConkie-Rosell Allyn, Kansagra Sujay, Smith Edward C, El-Dairi Mays, Bellet Jane, Keels Martha Ann, Jasien Joan et al. Genet. Med. 2017. X:X. X. PMID 28914269
PurposeTo describe examples of missed pathogenic variants on whole-exome sequencing (WES) and the importance of deep phenotyping for further diagnostic testing.MethodsGuided by phenotypic information, three children with negative WES underwent targeted single-gene testing.ResultsIndividual 1 had a clinical diagnosis consistent with infantile systemic hyalinosis, although WES and a next-generation sequencing (NGS)-based ANTXR2 test were negative. Sanger sequencing of ANTXR2 revealed a homozygous single base pair insertion, previously missed by the WES variant caller software. Individual 2 had neurodevelopmental regression and cerebellar atrophy, with no diagnosis on WES. New clinical findings prompted Sanger sequencing and copy number testing of PLA2G6. A novel homozygous deletion of the noncoding exon 1 (not included in the WES capture kit) was detected, with extension into the promoter, confirming the clinical suspicion of infantile neuroaxonal dystrophy. Individual 3 had progressive ataxia, spasticity, and magnetic resonance image changes of vanishing white matter leukoencephalopathy. An NGS leukodystrophy gene panel and WES showed a heterozygous pathogenic variant in EIF2B5; no deletions/duplications were detected. Sanger sequencing of EIF2B5 showed a frameshift indel, probably missed owing to failure of alignment.ConclusionThese cases illustrate potential pitfalls of WES/NGS testing and the importance of phenotype-guided molecular testing in yielding diagnoses.Genetics in Medicine advance online publication, 14 September 2017; doi:10.1038/gim.2017.128.
34. Molecular prediction of lytic vs lysogenic states for Microcystis phage: Metatranscriptomic evidence of lysogeny during large bloom events. Stough Joshua M A, Tang Xiangming, Krausfeldt Lauren E, Steffen Morgan M, Gao Guang, Boyer Gregory L, Wilhelm Steven W. PLoS ONE 2017. 12:9. e0184146. PMID 28873456
Microcystis aeruginosa is a freshwater bloom-forming cyanobacterium capable of producing the potent hepatotoxin, microcystin. Despite increased interest in this organism, little is known about the viruses that infect it and drive nutrient mobilization and transfer of genetic material between organisms. The genomic complement of sequenced phage suggests these viruses are capable of integrating into the host genome, though this activity has not been observed in the laboratory. While analyzing RNA-sequence data obtained from Microcystis blooms in Lake Tai (Taihu, China), we observed that a series of lysogeny-associated genes were highly expressed when genes involved in lytic infection were down-regulated. This pattern was consistent, though not always statistically significant, across multiple spatial and temporally distinct samples. For example, samples from Lake Tai (2014) showed a predominance of lytic virus activity from late July through October, while genes associated with lysogeny were strongly expressed in the early months (June-July) and toward the end of bloom season (October). Analyses of whole phage genome expression shows that transcription patterns are shared across sampling locations and that genes consistently clustered by co-expression into lytic and lysogenic groups. Expression of lytic-cycle associated genes was positively correlated to total dissolved nitrogen, ammonium concentration, and salinity. Lysogeny-associated gene expression was positively correlated with pH and total dissolved phosphorous. Our results suggest that lysogeny may be prevalent in Microcystis blooms and support the hypothesis that environmental conditions drive switching between temperate and lytic life cycles during bloom proliferation.
35. Global role of the bacterial post-transcriptional regulator CsrA revealed by integrated transcriptomics. Potts Anastasia H, Vakulskas Christopher A, Pannuri Archana, Yakhnin Helen, Babitzke Paul, Romeo Tony. Nat Commun 2017. 8:1. 1596. PMID 29150605
CsrA is a post-transcriptional regulatory protein that is widely distributed among bacteria. This protein influences bacterial lifestyle decisions by binding to the 5' untranslated and/or early coding regions of mRNA targets, causing changes in translation initiation, RNA stability, and/or transcription elongation. Here, we assess the contribution of CsrA to gene expression in Escherichia coli on a global scale. UV crosslinking immunoprecipitation and sequencing (CLIP-seq) identify RNAs that interact directly with CsrA in vivo, while ribosome profiling and RNA-seq uncover the impact of CsrA on translation, RNA abundance, and RNA stability. This combination of approaches reveals unprecedented detail about the regulatory role of CsrA, including novel binding targets and physiological roles, such as in envelope function and iron homeostasis. Our findings highlight the integration of CsrA throughout the E. coli regulatory network, where it orchestrates vast effects on gene expression.
36. Epigenetic regulation of Plasmodium falciparum clonally variant gene expression during development in Anopheles gambiae. Gómez-Díaz Elena, Yerbanga Rakiswendé S, Lefèvre Thierry, Cohuet Anna, Rowley M Jordan, Ouedraogo Jean Bosco, Corces Victor G. Sci Rep 2017. 7:X. 40655. PMID 28091569
P. falciparum phenotypic plasticity is linked to the variant expression of clonal multigene families such as the var genes. We have examined changes in transcription and histone modifications that occur during sporogonic development of P. falciparum in the mosquito host. All var genes are silenced or transcribed at low levels in blood stages (gametocyte/ring) of the parasite in the human host. After infection of mosquitoes, a single var gene is selected for expression in the oocyst, and transcription of this gene increases dramatically in the sporozoite. The same PF3D7_1255200 var gene was activated in 4 different experimental infections. Transcription of this var gene during parasite development in the mosquito correlates with the presence of low levels of H3K9me3 at the binding site for the PF3D7_1466400 AP2 transcription factor. This chromatin state in the sporozoite also correlates with the expression of an antisense long non-coding RNA (lncRNA) that has previously been shown to promote var gene transcription during the intraerythrocytic cycle in vitro. Expression of both the sense protein-coding transcript and the antisense lncRNA increase dramatically in sporozoites. The findings suggest a complex process for the activation of a single particular var gene that involves AP2 transcription factors and lncRNAs.
37. Physical Exercise Modulates L-DOPA-Regulated Molecular Pathways in the MPTP Mouse Model of Parkinson's Disease. Klemann Cornelius J H M, Xicoy Helena, Poelmans Geert, Bloem Bas R, Martens Gerard J M, Visser Jasper E. Mol. Neurobiol. 2017. X:X. X. PMID 29019056
Parkinson's disease (PD) is characterized by the degeneration of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc), resulting in motor and non-motor dysfunction. Physical exercise improves these symptoms in PD patients. To explore the molecular mechanisms underlying the beneficial effects of physical exercise, we exposed 1-methyl-4-phenyl-1,2,3,6-tetrahydropyrimidine (MPTP)-treated mice to a four-week physical exercise regimen, and subsequently explored their motor performance and the transcriptome of multiple PD-linked brain areas. MPTP reduced the number of DA neurons in the SNpc, whereas physical exercise improved beam walking, rotarod performance, and motor behavior in the open field. Further, enrichment analyses of the RNA-sequencing data revealed that in the MPTP-treated mice physical exercise predominantly modulated signaling cascades that are regulated by the top upstream regulators L-DOPA, RICTOR, CREB1, or bicuculline/dalfampridine, associated with movement disorders, mitochondrial dysfunction, and epilepsy-related processes. To elucidate the molecular pathways underlying these cascades, we integrated the proteins encoded by the exercise-induced differentially expressed mRNAs for each of the upstream regulators into a molecular landscape, for multiple key brain areas. Most notable was the opposite effect of physical exercise compared to previously reported effects of L-DOPA on the expression of mRNAs in the SN and the ventromedial striatum that are involved in-among other processes-circadian rhythm and signaling involving DA, neuropeptides, and endocannabinoids. Altogether, our findings suggest that physical exercise can improve motor function in PD and may, at the same time, counteract L-DOPA-mediated molecular mechanisms. Further, we hypothesize that physical exercise has the potential to improve non-motor symptoms of PD, some of which may be the result of (chronic) L-DOPA use.
38. A structural variant in the 5'-flanking region of the TWIST2 gene affects melanocyte development in belted cattle. Awasthi Mishra Nivedita, Drögemüller Cord, Jagannathan Vidhya, Keller Irene, Wüthrich Daniel, Bruggmann Rémy, Beck Julia, Schütz Ekkehard, Brenig Bertram, Demmel Steffi, Moser Simon, Signer-Hasler Heidi, Pie?kowska-Schelling Aldona, Schelling Claude, Sande Marcos et al. PLoS ONE 2017. 12:6. e0180170. PMID 28658273
Belted cattle have a circular belt of unpigmented hair and skin around their midsection. The belt is inherited as a monogenic autosomal dominant trait. We mapped the causative variant to a 37 kb segment on bovine chromosome 3. Whole genome sequence data of 2 belted and 130 control cattle yielded only one private genetic variant in the critical interval in the two belted animals. The belt-associated variant was a copy number variant (CNV) involving the quadruplication of a 6 kb non-coding sequence located approximately 16 kb upstream of the TWIST2 gene. Increased copy numbers at this CNV were strongly associated with the belt phenotype in a cohort of 333 cases and 1322 controls. We hypothesized that the CNV causes aberrant expression of TWIST2 during neural crest development, which might negatively affect melanoblasts. Functional studies showed that ectopic expression of bovine TWIST2 in neural crest in transgenic zebrafish led to a decrease in melanocyte numbers. Our results thus implicate an unsuspected involvement of TWIST2 in regulating pigmentation and reveal a non-coding CNV underlying a captivating Mendelian character.
39. dCATCH-Seq: improved sequencing of large continuous genomic targets with double-hybridization. Zhang Yanfeng, Song Jun, Day Kenneth, Absher Devin. BMC Genomics 2017. 18:1. 811. PMID 29061109 PMC ID PMC5653981
Targeted sequencing is a powerful tool with broad application in both basic and translational sciences. Relatively low on-target rates for most current targeted sequencing studies influence the required coverage and data quality for subsequent applications.
40. Genome-wide DNA methylation measurements in prostate tissues uncovers novel prostate cancer diagnostic biomarkers and transcription factor binding patterns. Kirby Marie K, Ramaker Ryne C, Roberts Brian S, Lasseigne Brittany N, Gunther David S, Burwell Todd C, Davis Nicholas S, Gulzar Zulfiqar G, Absher Devin M, Cooper Sara J, Brooks James D, Myers Richard M. BMC Cancer 2017. 17:1. 273. PMID 28412973 PMC ID PMC5392915
Current diagnostic tools for prostate cancer lack specificity and sensitivity for detecting very early lesions. DNA methylation is a stable genomic modification that is detectable in peripheral patient fluids such as urine and blood plasma that could serve as a non-invasive diagnostic biomarker for prostate cancer.
41. Defining age- and lactocrine-sensitive elements of the neonatal porcine uterine microRNA-mRNA interactome†,‡. George Ashley F, Rahman Kathleen M, Camp Meredith E, Prasad Nripesh, Bartol Frank F, Bagnell Carol A. Biol. Reprod. 2017. 96:2. 327-340. PMID 28203709
42. Modulation of microRNA-mRNA Target Pairs by Human Papillomavirus 16 Oncoproteins. Harden Mallory E, Prasad Nripesh, Griffiths Anthony, Munger Karl. MBio 2017. 8:1. X. PMID 28049151
The E6 and E7 proteins are the major oncogenic drivers encoded by high-risk human papillomaviruses (HPVs). While many aspects of the transforming activities of these proteins have been extensively studied, there are fewer studies that have investigated how HPV E6/E7 expression affects the expression of cellular noncoding RNAs. The goal of our study was to investigate HPV16 E6/E7 modulation of cellular microRNA (miR) levels and to determine the potential consequences for cellular gene expression. We performed deep sequencing of small and large cellular RNAs in primary undifferentiated cultures of human foreskin keratinocytes (HFKs) with stable expression of HPV16 E6/E7 or a control vector. After integration of the two data sets, we identified 51 differentially expressed cellular miRs associated with the modulation of 1,456 potential target mRNAs in HPV16 E6/E7-expressing HFKs. We discovered that the degree of differential miR expression in HFKs expressing HPV16 E6/E7 was not necessarily predictive of the number of corresponding mRNA targets or the potential impact on gene expression. Additional analyses of the identified miR-mRNA pairs suggest modulation of specific biological activities and biochemical pathways. Overall, our study supports the model that perturbation of cellular miR expression by HPV16 E6/E7 importantly contributes to the rewiring of cellular regulatory circuits by the high-risk HPV E6 and E7 proteins that contribute to oncogenic transformation.
The Lone Star tick, Amblyomma americanum, transmits several bacterial pathogens including species of Anaplasma and Ehrlichia. Amblyomma americanum also hosts a number of non-pathogenic bacterial endosymbionts. Recent studies of other arthropod and insect vectors have documented that commensal microflora can influence transmission of vector-borne pathogens; however, little is known about tick microbiomes and their possible influence on tick-borne diseases. Our objective was to compare bacterial communities associated with A. americanum, comparing Anaplasma/Ehrlichia -infected and uninfected ticks. Field-collected questing specimens (n = 50) were used in the analyses, of which 17 were identified as Anaplasma/Ehrlichia infected based on PCR amplification and sequencing of groEL genes. Bacterial communities from each specimen were characterized using Illumina sequencing of 16S rRNA gene amplicon libraries. There was a broad range in diversity between samples, with inverse Simpson's Diversity indices ranging from 1.28-89.5. There were no statistical differences in the overall microbial community structure between PCR diagnosed Anaplasma/Ehrlichia-positive and negative ticks, but there were differences based on collection method (P < 0.05), collection site (P < 0.05), and sex (P < 0.1) suggesting that environmental factors may structure A. americanum microbiomes. Interestingly, there was not always agreement between Illumina sequencing and PCR diagnostics: Ehrlichia was identified in 16S rRNA gene libraries from three PCR-negative specimens; conversely, Ehrlichia was not found in libraries of six PCR-positive ticks. Illumina sequencing also helped identify co-infections, for example, one specimen had both Ehrlichia and Anaplasma. Other taxa of interest in these specimens included Coxiella, Borrelia, and Rickettsia. Identification of bacterial community differences between specimens of a single tick species from a single geographical site indicates that intra-species differences in microbiomes were not due solely to pathogen presence/absence, but may be also driven by vector life history factors, including environment, life stage, population structure, and host choice.
44. Clinical Sequencing Exploratory Research Consortium: Accelerating Evidence-Based Practice of Genomic Medicine. Green Robert C, Goddard Katrina A B, Jarvik Gail P, Amendola Laura M, Appelbaum Paul S, Berg Jonathan S, Bernhardt Barbara A, Biesecker Leslie G, Biswas Sawona, Blout Carrie L, Bowling Kevin M, Brothers Kyle B, Burke Wylie, Caga-Anan Charlisse F, Chinnaiyan Arul M et al. Am. J. Hum. Genet. 2016. X:X. None. PMID 27181682
Despite rapid technical progress and demonstrable effectiveness for some types of diagnosis and therapy, much remains to be learned about clinical genome and exome sequencing (CGES) and its role within the practice of medicine. The Clinical Sequencing Exploratory Research (CSER) consortium includes 18 extramural research projects, one National Human Genome Research Institute (NHGRI) intramural project, and a coordinating center funded by the NHGRI and National Cancer Institute. The consortium is exploring analytic and clinical validity and utility, as well as the ethical, legal, and social implications of sequencing via multidisciplinary approaches; it has thus far recruited 5,577 participants across a spectrum of symptomatic and healthy children and adults by utilizing both germline and cancer sequencing. The CSER consortium is analyzing data and creating publically available procedures and tools related to participant preferences and consent, variant classification, disclosure and management of primary and secondary findings, health outcomes, and integration with electronic health records. Future research directions will refine measures of clinical utility of CGES in both germline and somatic testing, evaluate the use of CGES for screening in healthy individuals, explore the penetrance of pathogenic variants through extensive phenotyping, reduce discordances in public databases of genes and variants, examine social and ethnic disparities in the provision of genomics services, explore regulatory issues, and estimate the value and downstream costs of sequencing. The CSER consortium has established a shared community of research sites by using diverse approaches to pursue the evidence-based development of best practices in genomic medicine.
45. LacI Transcriptional Regulatory Networks in Clostridium thermocellum DSM1313. Wilson Charlotte M, Klingeman Dawn M, Schlachter Caleb, Syed Mustafa H, Wu Chia-Wei, Guss Adam M, Brown Steven D. Appl. Environ. Microbiol. 2016. X:X. X. PMID 28003194
Organisms regulate gene expression in response to the environment to coordinate metabolic reactions. Clostridium thermocellum expresses enzymes for both lignocellulose solubilization and its fermentation to produce ethanol. One LacI regulator termed GlyR3 in C. thermocellum ATCC27405 was previously identified as a repressor of neighboring genes with repression relieved by laminaribiose (a ?-1,3 disaccharide). To better understand the three C. thermocellum LacI regulons, deletion mutants were constructed using the genetically tractable DSM1313 strain. DSM1313 lacI genes Clo1313_2023, Clo1313_0089, and Clo1313_0396 encode homologs of GlyR1, GlyR2 and GlyR3 from strain ATCC27405, respectively. Growth on cellobiose or pretreated switchgrass was unaffected by any of the gene deletions under pH controlled fermentations. Global gene expression patterns from time course analyses identified glycoside hydrolase genes encoding hemicellulases, including cellulosomal enzymes, that were highly up-regulated (5 to 100 fold) in the absence of each LacI regulator, suggesting these were repressed under wild type conditions and that relatively few genes were controlled by each regulator under the conditions tested. Clo1313_2022, encoding lichenase enzyme LicB, was derepressed in a ?glyR1 strain. Higher Clo1313_1398 expression, which encodes the Man5A mannanase, was observed in a ?glyR2 strain and ?-mannobiose was identified as a probable inducer for GlyR2 regulated genes. For the ?glyR3 strain, up regulation of the two genes adjacent to glyR3 in the celC-glyR3-licA operon was consistent with earlier studies. Electrophoretic mobility shift assays have confirmed LacI transcription factor binding to specific regions of gene promoters.
46. Performance of ACMG-AMP Variant-Interpretation Guidelines among Nine Laboratories in the Clinical Sequencing Exploratory Research Consortium. Amendola Laura M, Jarvik Gail P, Leo Michael C, McLaughlin Heather M, Akkari Yassmine, Amaral Michelle D, Berg Jonathan S, Biswas Sawona, Bowling Kevin M, Conlin Laura K, Cooper Greg M, Dorschner Michael O, Dulik Matthew C, Ghazani Arezou A, Ghosh Rajarshi et al. Am. J. Hum. Genet. 2016. X:X. None. PMID 27181684
Evaluating the pathogenicity of a variant is challenging given the plethora of types of genetic evidence that laboratories consider. Deciding how to weigh each type of evidence is difficult, and standards have been needed. In 2015, the American College of Medical Genetics and Genomics (ACMG) and the Association for Molecular Pathology (AMP) published guidelines for the assessment of variants in genes associated with Mendelian diseases. Nine molecular diagnostic laboratories involved in the Clinical Sequencing Exploratory Research (CSER) consortium piloted these guidelines on 99 variants spanning all categories (pathogenic, likely pathogenic, uncertain significance, likely benign, and benign). Nine variants were distributed to all laboratories, and the remaining 90 were evaluated by three laboratories. The laboratories classified each variant by using both the laboratory's own method and the ACMG-AMP criteria. The agreement between the two methods used within laboratories was high (K-alpha = 0.91) with 79% concordance. However, there was only 34% concordance for either classification system across laboratories. After consensus discussions and detailed review of the ACMG-AMP criteria, concordance increased to 71%. Causes of initial discordance in ACMG-AMP classifications were identified, and recommendations on clarification and increased specification of the ACMG-AMP criteria were made. In summary, although an initial pilot of the ACMG-AMP guidelines did not lead to increased concordance in variant interpretation, comparing variant interpretations to identify differences and having a common framework to facilitate resolution of those differences were beneficial for improving agreement, allowing iterative movement toward increased reporting consistency for variants in genes associated with monogenic disease.
47. The innate immune protein calprotectin promotes Pseudomonas aeruginosa and Staphylococcus aureus interaction. Wakeman Catherine A, Moore Jessica L, Noto Michael J, Zhang Yaofang, Singleton Marc D, Prentice Boone M, Gilston Benjamin A, Doster Ryan S, Gaddy Jennifer A, Chazin Walter J, Caprioli Richard M, Skaar Eric P. Nat Commun 2016. 7:X. 11951. PMID 27301800
Microorganisms form biofilms containing differentiated cell populations. To determine factors driving differentiation, we herein visualize protein and metal distributions within Pseudomonas aeruginosa biofilms using imaging mass spectrometry. These in vitro experiments reveal correlations between differential protein distribution and metal abundance. Notably, zinc- and manganese-depleted portions of the biofilm repress the production of anti-staphylococcal molecules. Exposure to calprotectin (a host protein known to sequester metal ions at infectious foci) recapitulates responses occurring within metal-deplete portions of the biofilm and promotes interaction between P. aeruginosa and Staphylococcus aureus. Consistent with these results, the presence of calprotectin promotes co-colonization of the murine lung, and polymicrobial communities are found to co-exist in calprotectin-enriched airspaces of a cystic fibrosis lung explant. These findings, which demonstrate that metal fluctuations are a driving force of microbial community structure, have clinical implications because of the frequent occurrence of P. aeruginosa and S. aureus co-infections.
48. The National Disease Research Interchange and Collaborators on: What Are the Major Hurdles to the Recovery of Human Tissue to Advance Research? VonDran Melissa, Thomas Jeffrey A, Freund Michelle P, Ritsick Maggie, Orr Maureen, Kaye Wendy E, Bakker Annette, Knight Pamela. Biopreserv Biobank 2016. X:X. None. PMID 27845557
49. Canonical microRNAs enable differentiation, protect against DNA damage, and promote cholesterol biosynthesis in neural stem cells. Liu Zhong, Zhang Cheng, Khodadadi-Jamayran Alireza, Dang Lam, Han Xiaosi, Kim Kitai, Li Hu, Zhao Rui. Stem Cells Dev. 2016. X:X. None. PMID 27762676
Neural stem cells (NSCs) have the capacity to differentiate into neurons, astrocytes, and oligodendrocytes, and therefore represent a promising donor tissue source for treating neurodegenerative diseases and repairing injuries of the nervous system. However, it remains unclear how canonical microRNAs (miRNAs), the subset of miRNAs requiring the Drosha-Dgcr8 microprocessor and the type III RNase Dicer for biogenesis, regulate NSCs. In this study, we established and characterized <i>Dgcr8</i><sup>-/-</sup> NSCs from conditionally <i>Dgcr8</i>-disrupted mouse embryonic brain. RNA-seq analysis demonstrated that disruption of <i>Dgcr8</i> in NSCs causes a complete loss of canonical miRNAs and an accumulation of pri-miRNAs. <i>Dgcr8</i><sup>-/-</sup> NSCs can be stably propagated in vitro but progress through the cell cycle at reduced rates. When induced for differentiation, <i>Dgcr8</i><sup>-/-</sup> NSCs failed to differentiate into neurons, astrocytes, or oligodendrocytes under permissive conditions. Compared to <i>Dgcr8</i><sup>+/-</sup> NSCs, <i>Dgcr8</i><sup>-/-</sup> NSCs exhibit significantly increased DNA damage. Comparative RNA-seq analysis and gene set enrichment analysis (GSEA) revealed that <i>Dgcr8</i><sup>-/-</sup> NSCs significantly downregulate genes associated with neuronal differentiation, cell cycle progression, DNA replication, protein translation, and DNA damage repair. Furthermore, we discovered that <i>Dgcr8</i><sup>-/-</sup> NSCs significantly downregulate genes responsible for cholesterol biosynthesis and demonstrated that <i>Dgcr8</i><sup>-/-</sup> NSCs contain lower levels of cholesterol. Together, our data demonstrate that canonical miRNAs play essential roles in enabling lineage specification, protecting DNA against damage, and promoting cholesterol biosynthesis in NSCs.
50. The SH3 domain of UNC-89 (obscurin) interacts with paramyosin, a coiled-coil protein, in C. elegans muscle. Qadota Hiroshi, Mayans Olga, Matsunaga Yohei, McMurry Jonathan L, Wilson Kristy J, Kwon Grace E, Stanford Rachel, Deehan Kevin, Tinley Tina L, Ngwa Verra M, Benian Guy M. Mol. Biol. Cell 2016. X:X. None. PMID 27009202
UNC-89 is a giant polypeptide located at the sarcomeric M-line ofC. elegansmuscle. The human homolog is obscurin. To understand how UNC-89 is localized and functions, we are identifying its binding partners. Screening a yeast 2-hybrid library revealed that UNC-89 interacts with paramyosin. Paramyosin is an invertebrate-specific coiled-coil dimer protein that is homologous to the rod portion of myosin heavy chains, and resides in thick filament cores. Minimally, this interaction requires UNC-89's SH3 domain and residues 294-376 of paramyosin, and has a KDof ?1.1 ?M. Inunc-89loss of function mutants that lack the SH3 domain, paramyosin is found in accumulations. When the SH3 domain is overexpressed, paramyosin is mis-localized. SH3 domains usually interact with a proline-rich consensus sequence, but the region of paramyosin that interacts with UNC-89's SH3 is ?-helical and lacks prolines. Homology modeling of UNC-89's SH3 suggests structural features that might be responsible for this interaction. The SH3-binding region of paramyosin contains a "skip residue" which is likely to locally unwind the coiled-coil and perhaps contributes to the binding specificity.
51. Genetic predisposition to high anxiety- and depression-like behavior coincides with diminished DNA methylation in the adult rat amygdala. McCoy Chelsea R, Jackson Nateka L, Day Jeremy, Clinton Sarah M. Behav. Brain Res. 2016. X:X. X. PMID 27965039
Understanding biological mechanisms that shape vulnerability to emotional dysfunction is critical for elucidating the neurobiology of psychiatric illnesses like anxiety and depression. To elucidate molecular and epigenetic alterations in the brain that contribute to individual differences in emotionality, our laboratory utilized a rodent model of temperamental differences. Rats bred for low response to novelty (Low Responders, LRs) are inhibited in novel situations and display high anxiety, helplessness, and diminished sociability compared to High Novelty Responder (HR) rats. Our current transcriptome profiling experiment identified widespread gene expression differences in the amygdala of adult HR/LR rats; we hypothesize that HR/LR gene expression and downstream behavioral differences stem from distinct epigenetic (specifically DNA methylation) patterning in the HR/LR brain. Although we found similar levels of DNA methyltransferase proteins in the adult HR/LR amygdala, next-generation sequencing analysis of the methylome revealed 793 differentially methylated genomic sites between the groups. Most of the differentially methylated sites were hypermethylated in HR versus LR, so we next tested the hypothesis that enhancing DNA methylation in LRs would improve their anxiety/depression-like phenotype. We found that increasing DNA methylation in LRs (via increased dietary methyl donor content) improved their anxiety-like behavior and decreased their typically high levels of Forced Swim Test (FST) immobility; however, dietary methyl donor depletion exacerbated LRs' high FST immobility. These data are generally consistent with findings in depressed patients showing that treatment with DNA methylation-promoting agents improves depressive symptoms, and highlight epigenetic mechanisms that may contribute to individual differences in risk for emotional dysfunction.
52. Developmental Transcriptomics of the Hawaiian Anchialine Shrimp Halocaridina rubra Holthuis, 1963 (Crustacea: Atyidae). Havird Justin C, Santos Scott R. Integr. Comp. Biol. 2016. X:X. None. PMID 27400978
Many crustacean species progress through a series of metamorphoses during the developmental transition from embryo to adult. The molecular genetic basis of this transition, however, is not well characterized for a large number of crustaceans. Here, we employ multiple RNA-Seq methodologies to identify differentially expressed genes (DEGs) between "early" (i.e., Z1 - Z2) as well as "late" (i.e., Z3 - Z4) larval and adult developmental stages of Halocaridina rubra Holthuis (1963), an atyid shrimp endemic to the environmentally variable anchialine ecosystem of the Hawaiian Islands. Given the differences in salinity tolerance (narrow vs. wide range), energy acquisition (maternal yolk-bearing vs. microphagous grazing), and behavior (positively phototactic vs. not) between larvae and adults, respectively, of this species, we hypothesized the recovery of numerous DEGs belonging to functional categories relating to these characteristics. Consistent with this and regardless of methodology, hundreds of DEGs were identified, including upregulation of opsins and other light/stimulus detection genes and downregulation of genes related to ion transport, digestion, and reproduction in larvae relative to adults. Furthermore, isoform-switching, which has been largely unexplored in crustacean development, appears to be pervasive between H. rubra larvae and adults, especially among structural and oxygen-transport genes. Finally, by comparing RNA-Seq methodologies, we provide recommendations for future crustacean transcriptomic studies, including a demonstration of the pitfalls associated with identifying DEGs from single replicate samples as well as the utility of leveraging "prepackaged" bioinformatics pipelines.
53. Connecting the Dots: Therapy-Induced Senescence and a Tumor-Suppressive Immune Microenvironment. Vilgelm Anna E, Johnson C Andrew, Prasad Nripesh, Yang Jinming, Chen Sheau-Chiann, Ayers Gregory D, Pawlikowski Jeff S, Raman Dayanidhi, Sosman Jeffrey A, Kelley Mark, Ecsedy Jeffrey A, Shyr Yu, Levy Shawn E, Richmond Ann. J. Natl. Cancer Inst. 2016. 108:6. None. PMID 26719346
Tumor cell senescence is a common outcome of anticancer therapy. Here we investigated how therapy-induced senescence (TIS) affects tumor-infiltrating leukocytes (TILs) and the efficacy of immunotherapy in melanoma.
54. A Molecular Genetic Basis Explaining Altered Bacterial Behavior in Space. Zea Luis, Prasad Nripesh, Levy Shawn E, Stodieck Louis, Jones Angela, Shrestha Shristi, Klaus David. PLoS ONE 2016. 11:11. e0164359. PMID 27806055
Bacteria behave differently in space, as indicated by reports of reduced lag phase, higher final cell counts, enhanced biofilm formation, increased virulence, and reduced susceptibility to antibiotics. These phenomena are theorized, at least in part, to result from reduced mass transport in the local extracellular environment, where movement of molecules consumed and excreted by the cell is limited to diffusion in the absence of gravity-dependent convection. However, to date neither empirical nor computational approaches have been able to provide sufficient evidence to confirm this explanation. Molecular genetic analysis findings, conducted as part of a recent spaceflight investigation, support the proposed model. This investigation indicated an overexpression of genes associated with starvation, the search for alternative energy sources, increased metabolism, enhanced acetate production, and other systematic responses to acidity-all of which can be associated with reduced extracellular mass transport.
55. Metabarcoding reveals environmental factors influencing spatio-temporal variation in pelagic micro-eukaryotes. Brannock Pamela M, Ortmann Alice C, Moss Anthony G, Halanych Kenneth M. Mol. Ecol. 2016. X:X. None. PMID 27238767
Marine environments harbor a vast diversity of micro-eukaryotic organisms (protists and other small eukaryotes) that play important roles in structuring marine ecosystems. However, micro-eukaryote diversity is not well understood. Likewise, knowledge is limited regarding micro-eukaryote spatial and seasonal distribution, especially over long temporal scales. Given the importance of this group for mobilizing energy from lower trophic levels near the base of the food chain to larger organisms, assessing community stability, diversity, and resilience is important to understand ecosystem health. Herein, we use a metabarcoding approach to examine pelagic micro-eukaryote communities over a 2.5-year time series. Bi-monthly surface sampling (July 2009 to December 2011) was conducted at four locations within Mobile Bay (Bay) and along the Alabama continental shelf (Shelf). Alpha-diversity only showed significant differences in Shelf sites, with the greatest differences observed between summer and winter. Beta-diversity showed significant differences in community composition in relation to season and. The Bay was dominated by diatoms, while the Shelf was characterized by dinoflagellates and copepods. The northern Gulf of Mexico is heavily influenced by the Mobile River Basin, which brings low-salinity nutrient-rich water mostly during winter and spring. Community composition was correlated with salinity, temperature, and dissolved silicate. However, species interactions (e.g. predation and parasitism) may also contribute to the observed variation, especially on the Shelf, which warrant further exploration. Metabarcoding revealed clear patterns in surface pelagic micro-eukaryote communities that were consistent over multiple years, demonstrating how these techniques could be greatly beneficial to ecological monitoring and management along temporal scales. This article is protected by copyright. All rights reserved.
56. Kidney Cell-Adapted Infectious Bronchitis Virus Arkansas Delmarva Poultry Industry Vaccine Confers Effective Protection Against Challenge. Ghetas A M, van Santen V L, Joiner K, Toro H. Avian Dis. 2016. 60:2. 418-23. PMID 27309281
We previously demonstrated that adaptation of an embryo-attenuated infectious bronchitis virus (IBV) Arkansas Delmarva Poultry Industry (ArkDPI)-derived vaccine to chicken embryo kidney (CEK) cells shifted the virus population towards homogeneity in spike (S) and nonstructural protein genes. Moreover, the typical Ark vaccine subpopulations emerging in chickens vaccinated with commercial Ark vaccines were not detected in chickens vaccinated with the CEK-adapted virus. In this study, chickens vaccinated with a low dose (1.6 × 10(3) EID50/bird, where EID50 is 50% embryo infectious dose) of CEK-adapted Ark vaccine at 5 days of age showed a significant reduction of IBV RNA in lachrymal fluids and decreased incidence of IBV RNA detection in tracheal swabs 5 days after challenge compared to unvaccinated challenged chickens. In a second experiment, 5-day-old chickens were vaccinated with 10(4) or 10(5) EID50/chicken of CEK-adapted Ark vaccine, and protection was compared to chickens vaccinated with 10(5) EID50/chicken of the commercial ArkDPI-derived vaccine from which the CEK-adapted virus originated. All vaccinated chicken groups showed a significant reduction of respiratory signs and viral load 5 days after Ark virulent challenge compared to unvaccinated challenged controls. No viral subpopulations different from the challenge virus were detected in chickens vaccinated with CEK-Ark after challenge. In contrast, IBV S1 sequences differing from the predominant population in the challenge virus were detected in several chickens vaccinated with the commercial Ark attenuated vaccine. From an applied perspective, the CEK-adapted IBV ArkDPI-derived vaccine is an improved and effective vaccine candidate with which to protect chickens against virulent Ark-type strains.
57. A survey of current practices for genomic sequencing test interpretation and reporting processes in US laboratories. O'Daniel Julianne M, McLaughlin Heather M, Amendola Laura M, Bale Sherri J, Berg Jonathan S, Bick David, Bowling Kevin M, Chao Elizabeth C, Chung Wendy K, Conlin Laura K, Cooper Gregory M, Das Soma, Deignan Joshua L, Dorschner Michael O, Evans James P et al. Genet. Med. 2016. X:X. X. PMID 27811861
While the diagnostic success of genomic sequencing expands, the complexity of this testing should not be overlooked. Numerous laboratory processes are required to support the identification, interpretation, and reporting of clinically significant variants. This study aimed to examine the workflow and reporting procedures among US laboratories to highlight shared practices and identify areas in need of standardization.
58. Neonatal maternal separation stress elicits lasting DNA methylation changes in the hippocampus of stress-reactive Wistar Kyoto rats. McCoy Chelsea R, Rana Samir, Stringfellow Sara Anne, Day Jeremy J, Michael Wyss J, Clinton Sarah M, Kerman Ilan A. Eur. J. Neurosci. 2016. X:X. None. PMID 27643783
Early-life stress (ELS) can alter neurodevelopment in variable ways, ranging from producing deleterious outcomes to stress resilience. While most ELS studies focus on its harmful effects, recent work by our lab and others shows that ELS elicits positive effects in certain individuals. We exposed Wistar-Kyoto (WKY) rats, known for a stress reactive, anxiety-/depression-like phenotype, to maternal separation (MS), a model of ELS. MS exposure elicited anxiolytic and antidepressant behavioral effects as well as improved cardiovascular function in adult WKY offspring. The present study interrogates an epigenetic mechanism (DNA methylation) that may confer the adaptive effects of MS in WKY offspring. We quantified global genome methylation levels in limbic brain regions of adult WKYs exposed to daily 180-min MS or neonatal handling from postnatal day 1-14. MS exposure triggered dramatic DNA hypermethylation specifically in the hippocampus. Next-generation sequencing methylome profiling revealed reduced methylation at intragenic sites within two key nodes of insulin signaling pathways: the insulin receptor and one of its major downstream targets, mitogen activated protein kinase kinase kinase 5 (Map3k5). We then tested the hypothesis that enhancing DNA methylation in WKY rats would elicit adaptive changes akin to the effects of MS. Dietary methyl donor supplementation improved WKY rats' anxiety/depression-like behaviors and also improved cardiovascular measures, similar to previous observations following MS. Overall these data suggest a potential molecular mechanism that mediates a predicted adaptive response whereby ELS induces DNA methylation changes in the brain that may contribute to successful stress coping and adaptive physiological changes in adulthood. This article is protected by copyright. All rights reserved.
59. Host Double Strand Break Repair Generates HIV-1 Strains Resistant to CRISPR/Cas9. Yoder Kristine E, Bundschuh Ralf. Sci Rep 2016. 6:X. 29530. PMID 27404981
CRISPR/Cas9 genome editing has been proposed as a therapeutic treatment for HIV-1 infection. CRISPR/Cas9 induced double strand breaks (DSBs) targeted to the integrated viral genome have been shown to decrease production of progeny virus. Unfortunately HIV-1 evolves rapidly and may readily produce CRISPR/Cas9 resistant strains. Here we used next-generation sequencing to characterize HIV-1 strains that developed resistance to six different CRISPR/Cas9 guide RNAs (gRNAs). Reverse transcriptase (RT) derived base substitution mutations were commonly found at sites encoding unpaired bases of RNA stem-loop structures. In addition to RT mutations, insertion and/or deletion (indel) mutations were common. Indels localized to the CRISPR/Cas9 cleavage site were major contributors to CRISPR gRNA resistance. While most indels at non-coding regions were a single base pair, 3 base pair indels were observed when a coding region of HIV-1 was targeted. The DSB repair event may preserve the HIV-1 reading frame, while destroying CRISPR gRNA homology. HIV-1 may be successfully edited by CRISPR/Cas9, but the virus remains competent for replication and resistant to further CRISPR/Cas9 targeting at that site. These observations strongly suggest that host DSB repair at CRISPR/Cas9 cleavage sites is a novel and important pathway that may contribute to HIV-1 therapeutic resistance.
60. Vibrio elicits targeted transcriptional responses from copepod hosts. Almada Amalia A, Tarrant Ann M. FEMS Microbiol. Ecol. 2016. X:X. None. PMID 27056917
Copepods are abundant crustaceans that harbor diverse bacterial communities, yet the nature of their interactions with microbiota are poorly understood. Here, we report thatVibrioelicits targeted transcriptional responses in the estuarine copepodEurytemora affinis We pre-treatedE. affiniswith an antibiotic-cocktail and exposed them to either a zooplankton specialist (Vibrio sp. F10 9ZB36) or a free-living species (V. ordalii 12B09) for 24 hours. We then identified via RNA-Seq a total of 78 genes that were differentially expressed followingVibrioexposure, including homologs of C-type lectins, chitin-binding proteins and saposins. The response differed between the twoVibriotreatments, with the greatest changes elicited upon inoculation withV. sp. F10 We suggest that these differentially regulated genes play important roles in cuticle integrity, the innate immune response, and general stress responses, and that their expression may enableE. affinisto recognize and regulate symbiotic vibrios. We further report thatV. sp. F10culturability is specifically altered upon colonization ofE. affinis These findings suggest that rather than acting as passive environmental vectors, copepods discriminately interact with vibrios, which may ultimately impact the abundance and activity of copepod-associated bacteria.
61. Assembly of the Complete Sitka Spruce Chloroplast Genome Using 10X Genomics' GemCode Sequencing Data. Coombe Lauren, Warren René L, Jackman Shaun D, Yang Chen, Vandervalk Benjamin P, Moore Richard A, Pleasance Stephen, Coope Robin J, Bohlmann Joerg, Holt Robert A, Jones Steven J M, Birol Inanc. PLoS ONE 2016. 11:9. e0163059. PMID 27632164 PMC ID PMC5025161
The linked read sequencing library preparation platform by 10X Genomics produces barcoded sequencing libraries, which are subsequently sequenced using the Illumina short read sequencing technology. In this new approach, long fragments of DNA are partitioned into separate micro-reactions, where the same index sequence is incorporated into each of the sequencing fragment inserts derived from a given long fragment. In this study, we exploited this property by using reads from index sequences associated with a large number of reads, to assemble the chloroplast genome of the Sitka spruce tree (Picea sitchensis). Here we report on the first Sitka spruce chloroplast genome assembled exclusively from P. sitchensis genomic libraries prepared using the 10X Genomics protocol. We show that the resulting 124,049 base pair long genome shares high sequence similarity with the related white spruce and Norway spruce chloroplast genomes, but diverges substantially from a previously published P. sitchensis- P. thunbergii chimeric genome. The use of reads from high-frequency indices enabled separation of the nuclear genome reads from that of the chloroplast, which resulted in the simplification of the de Bruijn graphs used at the various stages of assembly.
62. Dynamic DNA Methylation Regulates Levodopa-Induced Dyskinesia. Figge David A, Eskow Jaunarajs Karen L, Standaert David G. J. Neurosci. 2016. 36:24. 6514-24. PMID 27307239
Levodopa-induced dyskinesia (LID) is a persistent behavioral sensitization that develops after repeated levodopa (l-DOPA) exposure in Parkinson disease patients. LID is a consequence of sustained changes in the transcriptional behavior of striatal neurons following dopaminergic stimulation. In neurons, transcriptional regulation through dynamic DNA methylation has been shown pivotal to many long-term behavioral modifications; however, its role in LID has not yet been explored. Using a rodent model, we show LID development leads to the aberrant expression of DNA demethylating enzymes and locus-specific changes to DNA methylation at the promoter regions of genes aberrantly transcribed following l-DOPA treatment. Looking for dynamic DNA methylation in LID genome-wide, we used reduced representation bisulfite sequencing and found an extensive reorganization of the dorsal striatal methylome. LID development led to significant demethylation at many important regulatory areas of aberrantly transcribed genes. We used pharmacologic treatments that alter DNA methylation bidirectionally and found them able to modulate dyskinetic behaviors. Together, these findings demonstrate that l-DOPA induces widespread changes to striatal DNA methylation and that these modifications are required for the development and maintenance of LID.
63. Dpy30 is critical for maintaining the identity and function of adult hematopoietic stem cells. Yang Zhenhua, Shah Kushani, Khodadadi-Jamayran Alireza, Jiang Hao. J. Exp. Med. 2016. X:X. None. PMID 27647347
As the major histone H3K4 methyltransferases in mammals, the Set1/Mll complexes play important roles in animal development and are associated with many diseases, including hematological malignancies. However, the role of the H3K4 methylation activity of these complexes in fate determination of hematopoietic stem and progenitor cells (HSCs and HPCs) remains elusive. Here, we address this question by generating a conditional knockout mouse for Dpy30, which is a common core subunit of all Set1/Mll complexes and facilitates genome-wide H3K4 methylation in cells. Dpy30 loss in the adult hematopoietic system results in severe pancytopenia but striking accumulation of HSCs and early HPCs that are defective in multilineage reconstitution, suggesting a differentiation block. In mixed bone marrow chimeras, Dpy30-deficient HSCs cannot differentiate or efficiently up-regulate lineage-regulatory genes, and eventually fail to sustain for long term with significant loss of HSC signature gene expression. Our molecular analyses reveal that Dpy30 directly and preferentially controls H3K4 methylation and expression of many hematopoietic development-associated genes including several key transcriptional and chromatin regulators involved in HSC function. Collectively, our results establish a critical and selective role of Dpy30 and the H3K4 methylation activity of the Set1/Mll complexes for maintaining the identity and function of adult HSCs.
64. Reproductive isolation and introgression between sympatric Mimulus species. Kenney Amanda M, Sweigart Andrea L. Mol. Ecol. 2016. X:X. None. PMID 27038381
Incompletely isolated species provide an opportunity to investigate the genetic mechanisms and evolutionary forces that maintain distinct species in the face of ongoing gene flow. Here, we use field surveys and reduced representation sequencing to characterize patterns of reproductive isolation, admixture, and genomic divergence between populations of the outcrossing wildflower Mimulus guttatus and selfing M. nasutus. Focusing on a single site where these two species have come into secondary contact, we find that phenological isolation is strong, although incomplete, and is likely driven by divergence in response to photoperiod. In contrast to previous field studies, which have suggested that F1 -hybrid formation might be rare, we discover patterns of genomic variation consistent with ongoing introgression. Strikingly, admixed individuals vary continuously from highly admixed to nearly pure M. guttatus, demonstrating ongoing hybridization and asymmetric introgression from M. nasutus into M. guttatus. Patterns of admixture and divergence across the genome show levels of introgression are more variable than expected by chance. Some genomic regions show reduced introgression, including one region that overlaps a critical photoperiod QTL, whereas other regions show elevated levels of interspecific gene flow. In addition, we observe a genome-wide negative relationship between absolute divergence and the local recombination rate, potentially indicating natural selection against M. nasutus ancestry in M. guttatus genetic backgrounds. Together, our results suggest that Mimulus speciation is both ongoing and dynamic, and that a combination of divergence in phenology and mating system, as well as selection against interspecific alleles, likely maintains these sympatric species. This article is protected by copyright. All rights reserved.
65. Functional and Structural Succession of Soil Microbial Communities below Decomposing Human Cadavers. Cobaugh Kelly L, Schaeffer Sean M, DeBruyn Jennifer M. PLoS ONE 2015. 10:6. e0130201. PMID 26067226
The ecological succession of microbes during cadaver decomposition has garnered interest in both basic and applied research contexts (e.g. community assembly and dynamics; forensic indicator of time since death). Yet current understanding of microbial ecology during decomposition is almost entirely based on plant litter. We know very little about microbes recycling carcass-derived organic matter despite the unique decomposition processes. Our objective was to quantify the taxonomic and functional succession of microbial populations in soils below decomposing cadavers, testing the hypotheses that a) periods of increased activity during decomposition are associated with particular taxa; and b) human-associated taxa are introduced to soils, but do not persist outside their host. We collected soils from beneath four cadavers throughout decomposition, and analyzed soil chemistry, microbial activity and bacterial community structure. As expected, decomposition resulted in pulses of soil C and nutrients (particularly ammonia) and stimulated microbial activity. There was no change in total bacterial abundances, however we observed distinct changes in both function and community composition. During active decay (7 - 12 days postmortem), respiration and biomass production rates were high: the community was dominated by Proteobacteria (increased from 15.0 to 26.1% relative abundance) and Firmicutes (increased from 1.0 to 29.0%), with reduced Acidobacteria abundances (decreased from 30.4 to 9.8%). Once decay rates slowed (10 - 23 d postmortem), respiration was elevated, but biomass production rates dropped dramatically; this community with low growth efficiency was dominated by Firmicutes (increased to 50.9%) and other anaerobic taxa. Human-associated bacteria, including the obligately anaerobic Bacteroides, were detected at high concentrations in soil throughout decomposition, up to 198 d postmortem. Our results revealed the pattern of functional and compositional succession in soil microbial communities during decomposition of human-derived organic matter, provided insight into decomposition processes, and identified putative predictor populations for time since death estimation.
66. The transcriptomic and proteomic basis for the evolution of a novel venom phenotype within the Timber Rattlesnake (Crotalus horridus). Rokyta Darin R, Wray Kenneth P, McGivern James J, Margres Mark J. Toxicon 2015. X:X. None. PMID 25727380
The genetics underlying adaptive trait evolution describes the intersection between the probability that particular types of mutation are beneficial and the rates they arise. Snake venoms can vary in a directly meaningful manner through coding mutations and regulatory mutations. The amounts of different components determine venom efficacy, but point mutations in coding sequences can also change efficacy and function. The Timber Rattlesnake (Crotalus horridus) has populations that have evolved neurotoxic venom from the typical hemorrhagic rattlesnake venom present throughout most of its range. We identified only a handful of nonsynonymous differences in just five loci between animals with each venom type, and these differences affected lower-abundance toxins. Expression of at least 18 loci encoding hemorrhagic toxins was severely reduced in the production of neurotoxic venom. The entire phospholipase A2 toxin family was completely replaced in the neurotoxic venom, possibly through intergeneric hybridization. Venom paedomorphosis could, at best, explain only some of the loss of expression of hemorrhagic toxins. The number of potential mechanisms for altering venom composition and the patterns observed for C. horridus suggest that rapid venom evolution should occur primarily through changes in venom composition, rather than point mutations affecting coding sequences.
67. Non-integrating episomal plasmid-based reprogramming of human amniotic fluid stem cells into induced pluripotent stem cells in chemically defined conditions. Slamecka Jaroslav, Salimova Lilia, McClellan Steven, van Kelle Mathieu, Kehl Debora, Laurini Javier, Cinelli Paolo, Owen Laurie, Hoerstrup Simon P, Weber Benedikt. Cell Cycle 2015. X:X. 0. PMID 26654216
Amniotic fluid stem cells (AFSC) represent an attractive potential cell source for fetal and pediatric cell-based therapies. However, upgrading them to pluripotency confers refractoriness towards senescence, higher proliferation rate and unlimited differentiation potential. AFSC were observed to rapidly and efficiently reacquire pluripotency which together with their easy recovery makes them an attractive cell source for reprogramming. The reprogramming process as well as the resulting iPSC epigenome could potentially benefit from the unspecialized nature of AFSC. iPSC derived from AFSC also have potential in disease modeling, such as Down syndrome or ?-thalassemia. Previous experiments involving AFSC reprogramming have largely relied on integrative vector transgene delivery and undefined serum-containing, feeder-dependent culture. Here, we describe non-integrative oriP/EBNA-1 episomal plasmid-based reprogramming of AFSC into iPSC and culture in fully chemically defined xeno-free conditions represented by vitronectin coating and E8 medium, a system that we found uniquely suited for this purpose. The derived AF-iPSC lines uniformly expressed a set of pluripotency markers Oct3/4, Nanog, Sox2, SSEA-1, SSEA-4, TRA-1-60, TRA-1-81 in a pattern typical for human primed PSC. Additionally, the cells formed teratomas, and were deemed pluripotent by PluriTest, a global expression microarray-based in-silico pluripotency assay. However, we found that the PluriTest scores were borderline, indicating a unique pluripotent signature in the defined condition. In the light of potential future clinical translation of iPSC technology, non-integrating reprogramming and chemically defined culture are more acceptable.
68. Age and Nursing Affect the Neonatal Porcine Uterine Transcriptome. Rahman Kathleen M, Camp Meredith E, Prasad Nripesh, McNeel Anthony K, Levy Shawn E, Bartol Frank F, Bagnell Carol A. Biol. Reprod. 2015. X:X. None. PMID 26632611
The lactocrine hypothesis for maternal programming of neonatal development was proposed to describe a mechanism through which milk-borne bioactive factors, delivered from mother to nursing offspring, could affect development of tissues, including the uterus. Porcine uterine development, initiated before birth, is completed postnatally. However, age- and lactocrine-sensitive elements of the neonatal porcine uterine developmental program are undefined. Here, effects of age and nursing for 48 h from birth (postnatal day = [PND] 0) on the uterine transcriptome at PND 2 were identified using RNA sequencing (RNAseq). Uterine tissues were obtained from neonatal gilts (n = 4/group) within 1 h of birth and prior to feeding (PND 0), or 48 h after nursing ad libitum (PND 2N), or feeding a commercial milk replacer (PND 2R). RNAseq analysis revealed differentially expressed genes (DEGs) associated with both age (PND 2N vs PND 0; 3,283 DEGs) and nursing on PND 2 (PND 2N vs PND 2R; 896 DEGs). Expression of selected uterine genes was validated using quantitative real-time PCR. Bioinformatic analyses revealed multiple biological processes enriched in response to both age and nursing, including cell adhesion, morphogenesis and cell-cell signaling. Age-sensitive pathways also included estrogen receptor-? and hedgehog signaling cascades. Lactocrine-sensitive processes in nursed gilts included those involved in response to wounding, the plasminogen activator network and coagulation. Overall, RNAseq analysis revealed comprehensive age- and nursing-related transcriptomic differences in the neonatal porcine uterus and identified novel pathways and biological processes regulating uterine development.
69. Widespread Rearrangement of 3D Chromatin Organization Underlies Polycomb-Mediated Stress-Induced Silencing. Li Li, Lyu Xiaowen, Hou Chunhui, Takenaka Naomi, Nguyen Huy Q, Ong Chin-Tong, Cubeñas-Potts Caelin, Hu Ming, Lei Elissa P, Bosco Giovanni, Qin Zhaohui S, Corces Victor G. Mol. Cell 2015. X:X. None. PMID 25818644
Chromosomes of metazoan organisms are partitioned in the interphase nucleus into discrete topologically associating domains (TADs). Borders between TADs are formed in regions containing active genes and clusters of architectural protein binding sites. The transcription of most genes is repressed after temperature stress in Drosophila. Here we show that temperature stress induces relocalization of architectural proteins from TAD borders to inside TADs, and this is accompanied by a dramatic rearrangement in the 3D organization of the nucleus. TAD border strength declines, allowing for an increase in long-distance inter-TAD interactions. Similar but quantitatively weaker effects are observed upon inhibition of transcription or depletion of individual architectural proteins. Heat shock-induced inter-TAD interactions result in increased contacts among enhancers and promoters of silenced genes, which recruit Pc and form Pc bodies in the nucleolus. These results suggest that the TAD organization of metazoan genomes is plastic and can be reconfigured quickly.
70. Next-Generation Sequencing of Duplication CNVs Reveals that Most Are Tandem and Some Create Fusion Genes at Breakpoints. Newman Scott, Hermetz Karen E, Weckselblatt Brooke, Rudd M Katharine. Am. J. Hum. Genet. 2015. X:X. None. PMID 25640679
Interpreting the genomic and phenotypic consequences of copy-number variation (CNV) is essential to understanding the etiology of genetic disorders. Whereas deletion CNVs lead obviously to haploinsufficiency, duplications might cause disease through triplosensitivity, gene disruption, or gene fusion at breakpoints. The mutational spectrum of duplications has been studied at certain loci, and in some cases these copy-number gains are complex chromosome rearrangements involving triplications and/or inversions. However, the organization of clinically relevant duplications throughout the genome has yet to be investigated on a large scale. Here we fine-mapped 184 germline duplications (14.7 kb-25.3 Mb; median 532 kb) ascertained from individuals referred for diagnostic cytogenetics testing. We performed next-generation sequencing (NGS) and whole-genome sequencing (WGS) to sequence 130 breakpoints from 112 subjects with 119 CNVs and found that most (83%) were tandem duplications in direct orientation. The remainder were triplications embedded within duplications (8.4%), adjacent duplications (4.2%), insertional translocations (2.5%), or other complex rearrangements (1.7%). Moreover, we predicted six in-frame fusion genes at sequenced duplication breakpoints; four gene fusions were formed by tandem duplications, one by two interconnected duplications, and one by duplication inserted at another locus. These unique fusion genes could be related to clinical phenotypes and warrant further study. Although most duplications are positioned head-to-tail adjacent to the original locus, those that are inverted, triplicated, or inserted can disrupt or fuse genes in a manner that might not be predicted by conventional copy-number assays. Therefore, interpreting the genetic consequences of duplication CNVs requires breakpoint-level analysis.
71. The genome and methylome of a beetle with complex social behavior, Nicrophorus vespilloides (Coleoptera: Silphidae). Cunningham Christopher B, Ji Lexiang, Wiberg R Axel W, Shelton Jennifer, McKinney Elizabeth C, Parker Darren J, Meagher Richard B, Benowitz Kyle M, Roy-Zokan Eileen M, Ritchie Michael G, Brown Susan J, Schmitz Robert J, Moore Allen J. Genome Biol Evol 2015. X:X. None. PMID 26454014
Testing for conserved and novel mechanisms underlying phenotypic evolution requires a diversity of genomes available for comparison spanning multiple independent lineages. For example, complex social behavior in insects has been investigated primarily with eusocial lineages, nearly all of which are Hymenoptera. If conserved genomic influences on sociality do exist, we need data from a wider range of taxa that also vary in their levels of sociality. Here, we present the assembled and annotated genome of the subsocial beetle Nicrophorus vespilloides, a species long used to investigate evolutionary questions of complex social behavior. We used this genome to address two questions. First, do aspects of life history, such as using a carcass to breed, predict overlap in gene models more strongly than phylogeny? We found the overlap in gene models was similar between N. vespilloides and all other insect groups regardless of life history. Second, like other insects with highly developed social behavior but unlike other beetles, does N. vespilloides have DNA methylation? We found strong evidence for an active DNA methylation system. The distribution of methylation was similar to other insects with exons having the most methylated CpGs. Methylation status appears highly conserved; 85% of the methylated genes in N. vespilloides are also methylated in the hymentopteran Nasonia vitripennis. The addition of this genome adds a coleopteran resource to answer questions about the evolution and mechanistic basis of sociality and to address questions about the potential role of methylation in social behavior.
72. Enhanced stability and polyadenylation of select mRNAs support rapid thermogenesis in the brown fat of a hibernator. Grabek Katharine R, Diniz Behn Cecilia, Barsh Gregory S, Hesselberth Jay R, Martin Sandra L. Elife 2015. 4:X. None. PMID 25626169
During hibernation, animals cycle between torpor and arousal. These cycles involve dramatic but poorly understood mechanisms of dynamic physiological regulation at the level of gene expression. Each cycle, Brown Adipose Tissue (BAT) drives periodic arousal from torpor by generating essential heat. We applied digital transcriptome analysis to precisely timed samples to identify molecular pathways that underlie the intense activity cycles of hibernator BAT. A cohort of transcripts increased during torpor, paradoxical because transcription effectively ceases at these low temperatures. We show that this increase occurs not by elevated transcription but rather by enhanced stabilization associated with maintenance and/or extension of long poly(A) tails. Mathematical modeling further supports a temperature-sensitive mechanism to protect a subset of transcripts from ongoing bulk degradation instead of increased transcription. This subset was enriched in a C-rich motif and genes required for BAT activation, suggesting a model and mechanism to prioritize translation of key proteins for thermogenesis.
73. CTCF-dependent co-localization of canonical Smad signaling factors at architectural protein binding sites in D. melanogaster. Van Bortle Kevin, Peterson Aidan J, Takenaka Naomi, O'Connor Michael B, Corces Victor G. Cell Cycle 2015. X:X. 0. PMID 26125535
The transforming growth factor beta (TGF-?) and bone morphogenic protein (BMP) pathways transduce extracellular signals into tissue-specific transcriptional responses. During this process, signaling effector Smad proteins translocate into the nucleus to direct changes in transcription, but how and where they localize to DNA remain important questions. We have mapped Drosophila TGF-? signaling factors Mad, dSmad2, Medea, and Schnurri genome-wide in Kc cells and find that numerous sites for these factors overlap with the architectural protein CTCF. Depletion of CTCF by RNAi results in the disappearance of a subset of Smad sites, suggesting Smad proteins localize to CTCF binding sites in a CTCF-dependent manner. Sensitive Smad binding sites are enriched at low occupancy CTCF peaks within topological domains, rather than at the physical domain boundaries where CTCF may function as an insulator. In response to Decapentaplegic, CTCF binding is not significantly altered, whereas Mad, Medea, and Schnurri are redirected from CTCF to non-CTCF binding sites. These results suggest that CTCF participates in the recruitment of Smad proteins to a subset of genomic sites and in the redistribution of these proteins in response to BMP signaling.
74. Phylogenetic utility, and variability in structure and content, of complete mitochondrial genomes among genetic lineages of the Hawaiian anchialine shrimp Halocaridina rubra Holthuis 1963 (Atyidae:Decapoda). Justice Joshua L, Weese David A, Santos Scott Ross. Mitochondrial DNA 2015. X:X. 1-9. PMID 26061341
The Atyidae are caridean shrimp possessing hair-like setae on their claws and are important contributors to ecological services in tropical and temperate fresh and brackish water ecosystems. Complete mitochondrial genomes have only been reported from five of the 449 species in the family, thus limiting understanding of mitochondrial genome evolution and the phylogenetic utility of complete mitochondrial sequences in the Atyidae. Here, comparative analyses of complete mitochondrial genomes from eight genetic lineages of Halocaridina rubra, an atyid endemic to the anchialine ecosystem of the Hawaiian Archipelago, are presented. Although gene number, order, and orientation were syntenic among genomes, three regions were identified and further quantified where conservation was substantially lower: (1) high length and sequence variability in the tRNA-Lys and tRNA-Asp intergenic region; (2) a 317-bp insertion between the NAD6 and CytB genes confined to a single lineage and representing a partial duplication of CytB; and (3) the putative control region. Phylogenetic analyses utilizing complete mitochondrial sequences provided new insights into relationships among the H. rubra genetic lineages, with the topology of one clade correlating to the geologic sequence of the islands. However, deeper nodes in the phylogeny lacked bootstrap support. Overall, our results from H. rubra suggest intra-specific mitochondrial genomic diversity could be underestimated across the Metazoa since the vast majority of complete genomes are from just a single individual of a species.
75. Mitogenomics reveals phylogeny and repeated motifs in control regions of the deep-sea family Siboglinidae (Annelida). Li Yuanning, Kocot Kevin M, Schander Christoffer, Santos Scott R, Thornhill Daniel J, Halanych Kenneth M. Mol. Phylogenet. Evol. 2015. X:X. None. PMID 25721539
Deep-sea tubeworms in the annelid family Siboglinidae have drawn considerable interest regarding their ecology and evolutionary biology. As adults, they lack a digestive tract and rely on endosymbionts for nutrition. Moreover, they are important members of chemosynthetic environments including hydrothermal vents, cold seeps, muddy sediments, and whale bones. Evolution and diversification of siboglinids has been associated with host-symbiont relationships and reducing habitats. Despite their importance, the taxonomy and phylogenetics of this clade are debated due to conflicting results. In this study, 10 complete and 2 partial mitochondrial genomes and one transcriptome were sequenced and analyzed to address siboglinid evolution. Notably, repeated nucleotide motifs were found in control regions of these mt genomes, which may explain previous challenges of sequencing siboglinid mt genomes. Phylogenetic analyses of amino acid and nucleotide datasets were conducted in order to infer evolutionary history. Both analyses generally had strong nodal support and suggest Osedax is most closely related to the Vestimentifera+Sclerolinum clade, rather than Frenulata, as recently reported. These results imply Osedax, the only siboglinid lineage with heterotrophic endosymbionts, evolved from a lineage utilizing chemoautotrophic symbionts.
76. A gene-expression screen identifies a non-toxic sumoylation inhibitor that Mimics SUMO-less human LRH-1 in liver. Suzawa Miyuki, Miranda Diego A, Ramos Karmela A, Ang Kenny K-H, Faivre Emily J, Wilson Christopher G, Caboni Laura, Arkin Michelle R, Kim Yeong-Sang, Fletterick Robert J, Diaz Aaron, Schneekloth John S, Ingraham Holly A. Elife 2015. 4:X. None. PMID 26653140
SUMO-modification of nuclear proteins has profound effects on gene expression. However, non-toxic chemical tools that modulate sumoylation in cells are lacking. Here, to identify small molecule sumoylation inhibitors we developed a cell-based screen that focused on the well-sumoylated substrate, human Liver Receptor Homolog-1 (hLRH-1, NR5A2). Our primary gene-expression screen assayed two SUMO-sensitive transcripts, APOC3 and MUC1 that are upregulated by SUMO-less hLRH-1 or by siUBC9 knockdown, respectively. A polyphenol, tannic acid (TA) emerged as a potent sumoylation inhibitor in vitro (IC50 = 12.8 µM) and in cells. TA also increased hLRH-1 occupancy on SUMO-sensitive transcripts. Most significantly, when tested in humanized mouse primary hepatocytes, TA inhibits hLRH-1 sumoylation and induces SUMO-sensitive genes, thereby recapitulating the effects of expressing SUMO-less hLRH-1 in mouse liver. Our findings underscore the benefits of phenotypic screening for targeting post-translational modifications, and illustrate the potential utility of TA for probing the cellular consequences of sumoylation.
77. Trancriptomic profiling revealed the signatures of acute immune response in Tilapia (Oreochromis niloticus) following Streptococcus iniae challenge. Zhu Jiajie, Li Chao, Ao Qiuwei, Tan Yun, Luo Yongju, Guo Yafen, Lan Ganqiu, Jiang Hesheng, Gan Xi. Fish Shellfish Immunol. 2015. X:X. None. PMID 26117728
Streptococcus iniae is the most significant bacterial disease of tilapia throughout the world, and commonly leads to tremendous economic losses. In contrast to other important fish species, our knowledge about the molecular mechanisms of tilapia in response to bacterial infection is still limited. Here, therefore, we utilized RNA-seq to first profiling of host responses in tilapia spleen following S.iniae infection at transcriptome level. A total of 223 million reads were obtained and assembled into 192,884 contigs with average length 844 bp. Gene expression analysis between control and infected samples at 5 h, 50 h, and 7 d revealed 1,475 differentially expressed genes. In particular, the differentially expressed gene set was dramatically induced as early as 5 h, and rapidly declined to basal levels at 50 h. Enrichment and pathway analysis of the differentially expressed genes revealed the centrality of the pathogen attachment and recognition, cytoskeletal rearrangement and immune activation/inflammation in the pathogen entry and host inflammatory responses. Understanding of these responses can highlight mechanisms of tilapia host defense, and expand our knowledge of teleost immunology. Our findings will set a foundation of valuable biomarkers for future individual, strain, and family-level studies to evaluate immune effect of vaccine and individual response in host defense mechanisms to S.iniae infection, to select disease resistant families and strains.
78. Methyl-CpG Binding Protein 2 Regulates Microglia and Macrophage Gene Expression in Response to Inflammatory Stimuli. Cronk James C, Derecki Noël C, Ji Emily, Xu Yang, Lampano Aaron E, Smirnov Igor, Baker Wendy, Norris Geoffrey T, Marin Ioana, Coddington Nathan, Wolf Yochai, Turner Stephen D, Aderem Alan, Klibanov Alexander L, Harris Tajie H et al. Immunity 2015. 42:4. 679-91. PMID 25902482 PMC ID PMC4407145
Mutations in MECP2, encoding the epigenetic regulator methyl-CpG-binding protein 2, are the predominant cause of Rett syndrome, a disease characterized by both neurological symptoms and systemic abnormalities. Microglial dysfunction is thought to contribute to disease pathogenesis, and here we found microglia become activated and subsequently lost with disease progression in Mecp2-null mice. Mecp2 was found to be expressed in peripheral macrophage and monocyte populations, several of which also became depleted in Mecp2-null mice. RNA-seq revealed increased expression of glucocorticoid- and hypoxia-induced transcripts in Mecp2-deficient microglia and peritoneal macrophages. Furthermore, Mecp2 was found to regulate inflammatory gene transcription in response to TNF stimulation. Postnatal re-expression of Mecp2 using Cx3cr1(creER) increased the lifespan of otherwise Mecp2-null mice. These data suggest that Mecp2 regulates microglia and macrophage responsiveness to environmental stimuli to promote homeostasis. Dysfunction of tissue-resident macrophages might contribute to the systemic pathologies observed in Rett syndrome.
79. Non-adaptive plasticity potentiates rapid adaptive evolution of gene expression in nature. Ghalambor Cameron K, Hoke Kim L, Ruell Emily W, Fischer Eva K, Reznick David N, Hughes Kimberly A. Nature 2015. X:X. None. PMID 26331546
Phenotypic plasticity is the capacity for an individual genotype to produce different phenotypes in response to environmental variation. Most traits are plastic, but the degree to which plasticity is adaptive or non-adaptive depends on whether environmentally induced phenotypes are closer or further away from the local optimum. Existing theories make conflicting predictions about whether plasticity constrains or facilitates adaptive evolution. Debate persists because few empirical studies have tested the relationship between initial plasticity and subsequent adaptive evolution in natural populations. Here we show that the direction of plasticity in gene expression is generally opposite to the direction of adaptive evolution. We experimentally transplanted Trinidadian guppies (Poecilia reticulata) adapted to living with cichlid predators to cichlid-free streams, and tested for evolutionary divergence in brain gene expression patterns after three to four generations. We find 135 transcripts that evolved parallel changes in expression within the replicated introduction populations. These changes are in the same direction exhibited in a native cichlid-free population, suggesting rapid adaptive evolution. We find 89% of these transcripts exhibited non-adaptive plastic changes in expression when the source population was reared in the absence of predators, as they are in the opposite direction to the evolved changes. By contrast, the remaining transcripts exhibiting adaptive plasticity show reduced population divergence. Furthermore, the most plastic transcripts in the source population evolved reduced plasticity in the introduction populations, suggesting strong selection against non-adaptive plasticity. These results support models predicting that adaptive plasticity constrains evolution, whereas non-adaptive plasticity potentiates evolution by increasing the strength of directional selection. The role of non-adaptive plasticity in evolution has received relatively little attention; however, our results suggest that it may be an important mechanism that predicts evolutionary responses to new environments.
80. Meiofaunal community analysis by high-throughput sequencing: Comparison of extraction, quality filtering, and clustering methods. Brannock Pamela M, Halanych Kenneth M. Mar Genomics 2015. X:X. None. PMID 26001512
Using molecular tools to examine community composition of meiofauna, animals 45?m to 1mm in size living between sediment grains in aquatic environments, is relatively new in comparison to bacterial and archaeal microbial studies. Although high-throughput molecular approaches are starting to be applied to these ccommunities, effectiveness of different approaches for nucleic acid extraction from meiofauna is poorly known and bioinformatic pipelines vary between studies. Given this situation, there is a need for protocols to be developed that promote consistency in sample collection and processing, sequence quality filtering, and Operational Taxonomic Unit (OTU) clustering methods. Herein, we assess different approaches used for DNA extraction (DNA extracted directly from sediment versus elutriated material retained on a 45?m sieve) as well as how different quality filtering methods of sequences and OTU clustering algorithms impact genetic assessment of meiofauna community composition. DNA extracted directly from sediment resulted in higher presence of non-metazoan eukaryotic taxa; in contrast, an elutriation (resuspension with decanting) approach increased meiofauna abundance and enriched metazoan OTUs. In regards to bioinformatics analyses, the number of overall OTUs varied by clustering algorithm, primarily due to the applied method of sequence quality filtering. However, alpha and beta diversity analyses showed similar trends regardless of bioinformatics pipeline utilized. Based on our results, we recommend studies of meiofauna communities first elutriate samples prior to DNA extraction and include multiple biological replicates to account for variation in community-level composition. The quality filtering method should be carefully considered as this step accounted for large discrepancy in the number of OTUs inferred.
81. Serotonin Receptor Agonist 5-Nonyloxytryptamine Alters the Kinetics of Reovirus Cell Entry. Mainou Bernardo A, Ashbrook Alison W, Smith Everett Clinton, Dorset Daniel C, Denison Mark R, Dermody Terence S. J. Virol. 2015. 89:17. 8701-12. PMID 26109733 PMC ID PMC4524060
Mammalian orthoreoviruses (reoviruses) are nonenveloped double-stranded RNA viruses that infect most mammalian species, including humans. Reovirus binds to cell surface glycans, junctional adhesion molecule A (JAM-A), and the Nogo-1 receptor (depending on the cell type) and enters cells by receptor-mediated endocytosis. Within the endocytic compartment, reovirus undergoes stepwise disassembly, which is followed by release of the transcriptionally active viral core into the cytoplasm. In a small-molecule screen to identify host mediators of reovirus infection, we found that treatment of cells with 5-nonyloxytryptamine (5-NT), a prototype serotonin receptor agonist, diminished reovirus cytotoxicity. 5-NT also blocked reovirus infection. In contrast, treatment of cells with methiothepin mesylate, a serotonin antagonist, enhanced infection by reovirus. 5-NT did not alter cell surface expression of JAM-A or attachment of reovirus to cells. However, 5-NT altered the distribution of early endosomes with a concomitant impairment of reovirus transit to late endosomes and a delay in reovirus disassembly. Consistent with an inhibition of viral disassembly, 5-NT treatment did not alter infection by in vitro-generated infectious subvirion particles, which bind to JAM-A but bypass a requirement for proteolytic uncoating in endosomes to infect cells. We also found that treatment of cells with 5-NT decreased the infectivity of alphavirus chikungunya virus and coronavirus mouse hepatitis virus. These data suggest that serotonin receptor signaling influences cellular activities that regulate entry of diverse virus families and provides a new, potentially broad-spectrum target for antiviral drug development.
82. A Cell-Based Systems Biology Assessment of Human Blood to Monitor Immune Responses after Influenza Vaccination. Hoek Kristen L, Samir Parimal, Howard Leigh M, Niu Xinnan, Prasad Nripesh, Galassie Allison, Liu Qi, Allos Tara M, Floyd Kyle A, Guo Yan, Shyr Yu, Levy Shawn E, Joyce Sebastian, Edwards Kathryn M, Link Andrew J. PLoS ONE 2015. 10:2. e0118528. PMID 25706537
Systems biology is an approach to comprehensively study complex interactions within a biological system. Most published systems vaccinology studies have utilized whole blood or peripheral blood mononuclear cells (PBMC) to monitor the immune response after vaccination. Because human blood is comprised of multiple hematopoietic cell types, the potential for masking responses of under-represented cell populations is increased when analyzing whole blood or PBMC. To investigate the contribution of individual cell types to the immune response after vaccination, we established a rapid and efficient method to purify human T and B cells, natural killer (NK) cells, myeloid dendritic cells (mDC), monocytes, and neutrophils from fresh venous blood. Purified cells were fractionated and processed in a single day. RNA-Seq and quantitative shotgun proteomics were performed to determine expression profiles for each cell type prior to and after inactivated seasonal influenza vaccination. Our results show that transcriptomic and proteomic profiles generated from purified immune cells differ significantly from PBMC. Differential expression analysis for each immune cell type also shows unique transcriptomic and proteomic expression profiles as well as changing biological networks at early time points after vaccination. This cell type-specific information provides a more comprehensive approach to monitor vaccine responses.
83. An Integrated Approach for Analyzing Clinical Genomic Variant Data from Next-Generation Sequencing. Crowgey Erin L, Stabley Deborah L, Chen Chuming, Huang Hongzhan, Robbins Katherine M, Polson Shawn W, Sol-Church Katia, Wu Cathy H. J Biomol Tech 2015. X:X. None. PMID 25649353 PMC ID PMC4310222
Next-generation sequencing (NGS) technologies provide the potential for developing high-throughput and low-cost platforms for clinical diagnostics. A limiting factor to clinical applications of genomic NGS is downstream bioinformatics analysis for data interpretation. We have developed an integrated approach for end-to-end clinical NGS data analysis from variant detection to functional profiling. Robust bioinformatics pipelines were implemented for genome alignment, single nucleotide polymorphism (SNP), small insertion/deletion (InDel), and copy number variation (CNV) detection of whole exome sequencing (WES) data from the Illumina platform. Quality-control metrics were analyzed at each step of the pipeline by use of a validated training dataset to ensure data integrity for clinical applications. We annotate the variants with data regarding the disease population and variant impact. Custom algorithms were developed to filter variants based on criteria, such as quality of variant, inheritance pattern, and impact of variant on protein function. The developed clinical variant pipeline links the identified rare variants to Integrated Genome Viewer for visualization in a genomic context and to the Protein Information Resource's iProXpress for rich protein and disease information. With the application of our system of annotations, prioritizations, inheritance filters, and functional profiling and analysis, we have created a unique methodology for downstream variant filtering that empowers clinicians and researchers to interpret more effectively the relevance of genomic alterations within a rare genetic disease.
84. Transcriptomic profiling of differential responses to drought in two freshwater mussel species, the giant floater Pyganodon grandis and the pondhorn Uniomerus tetralasmus. Luo Yupeng, Li Chao, Landis Andrew Gascho, Wang Guiling, Stoeckel James, Peatman Eric. PLoS ONE 2014. 9:2. e89481. PMID 24586812 PMC ID PMC3934898
The southeastern US has experienced recurrent drought during recent decades. Increasing demand for water, as precipitation decreases, exacerbates stress on the aquatic biota of the Southeast: a global hotspot for freshwater mussel, crayfish, and fish diversity. Freshwater unionid mussels are ideal candidates to study linkages between ecophysiological and behavioral responses to drought. Previous work on co-occurring mussel species suggests a coupling of physiology and behavior along a gradient ranging from intolerant species such as Pyganodon grandis (giant floater) that track receding waters and rarely burrow in the substrates to tolerant species such as Uniomerus tetralasmus (pondhorn) that rarely track receding waters, but readily burrow into the drying sediments. We utilized a next-generation sequencing-based RNA-Seq approach to examine heat/desiccation-induced transcriptomic profiles of these two species in order to identify linkages between patterns of gene expression, physiology and behavior. Sequencing produced over 425 million 100 bp reads. Using the de novo assembly package Trinity, we assembled the short reads into 321,250 contigs from giant floater (average length 835 bp) and 385,735 contigs from pondhorn (average length 929 bp). BLAST-based annotation and gene expression analysis revealed 2,832 differentially expressed genes in giant floater and 2,758 differentially expressed genes in pondhorn. Trancriptomic responses included changes in molecular chaperones, oxidative stress profiles, cell cycling, energy metabolism, immunity, and cytoskeletal rearrangements. Comparative analyses between species indicated significantly higher induction of molecular chaperones and cytoskeletal elements in the intolerant P. grandis as well as important differences in genes regulating apoptosis and immunity.
85. Comparative transcriptome analysis reveals the genetic basis of skin color variation in common carp. Jiang Yanliang, Zhang Songhao, Xu Jian, Feng Jianxin, Mahboob Shahid, Al-Ghanim Khalid A, Sun Xiaowen, Xu Peng. PLoS ONE 2014. 9:9. e108200. PMID 25255374 PMC ID PMC4177847
The common carp is an important aquaculture species that is widely distributed across the world. During the long history of carp domestication, numerous carp strains with diverse skin colors have been established. Skin color is used as a visual criterion to determine the market value of carp. However, the genetic basis of common carp skin color has not been extensively studied.
86. Insights into the epigenomic landscape of the human malaria vector Anopheles gambiae. Gómez-Díaz Elena, Rivero Ana, Chandre Fabrice, Corces Victor G. Front Genet 2014. 5:X. 277. PMID 25177345 PMC ID PMC4133732
The epigenome of the human malaria vector Anopheles gambiae was characterized in midgut cells by mapping the distribution and levels of two post-translational histone modifications, H3K27ac and H3K27me3. These histone profiles were then correlated with levels of gene expression obtained by RNA-seq. Analysis of the transcriptome of A. gambiae midguts and salivary glands led to the discovery of 13,898 new transcripts not present in the most recent genome assembly. A subset of these transcripts is differentially expressed between midgut and salivary glands. The enrichment profiles of H3K27ac and H3K27me3 are mutually exclusive and associate with high and low levels of transcription, respectively. This distribution agrees with previous findings in Drosophila showing association of these two histone modifications with either active or inactive transcriptional states, including Polycomb-associated domains in silenced genes. This study provides a mosquito epigenomics platform for future comparative studies in other mosquito species, opening future investigations into the role of epigenetic processes in vector-borne systems of medical and economic importance.
87. Identification and Analysis of Genome-Wide SNPs Provide Insight into Signatures of Selection and Domestication in Channel Catfish (Ictalurus punctatus). Sun Luyang, Liu Shikai, Wang Ruijia, Jiang Yanliang, Zhang Yu, Zhang Jiaren, Bao Lisui, Kaltenboeck Ludmilla, Dunham Rex, Waldbieser Geoff, Liu Zhanjiang. PLoS ONE 2014. 9:10. e109666. PMID 25313648 PMC ID PMC4196944
Domestication and selection for important performance traits can impact the genome, which is most often reflected by reduced heterozygosity in and surrounding genes related to traits affected by selection. In this study, analysis of the genomic impact caused by domestication and artificial selection was conducted by investigating the signatures of selection using single nucleotide polymorphisms (SNPs) in channel catfish (Ictalurus punctatus). A total of 8.4 million candidate SNPs were identified by using next generation sequencing. On average, the channel catfish genome harbors one SNP per 116 bp. Approximately 6.6 million, 5.3 million, 4.9 million, 7.1 million and 6.7 million SNPs were detected in the Marion, Thompson, USDA103, Hatchery strain, and wild population, respectively. The allele frequencies of 407,861 SNPs differed significantly between the domestic and wild populations. With these SNPs, 23 genomic regions with putative selective sweeps were identified that included 11 genes. Although the function for the majority of the genes remain unknown in catfish, several genes with known function related to aquaculture performance traits were included in the regions with selective sweeps. These included hypoxia-inducible factor 1?· HIF?? ¨ and the transporter gene ATP-binding cassette sub-family B member 5 (ABCB5). HIF1?· is important for response to hypoxia and tolerance to low oxygen levels is a critical aquaculture trait. The large numbers of SNPs identified from this study are valuable for the development of high-density SNP arrays for genetic and genomic studies of performance traits in catfish.
88. Targeted Sequencing of Large Genomic Regions with CATCH-Seq. Day Kenneth, Song Jun, Absher Devin. PLoS ONE 2014. 9:10. e111756. PMID 25357200
Current target enrichment systems for large-scale next-generation sequencing typically require synthetic oligonucleotides used as capture reagents to isolate sequences of interest. The majority of target enrichment reagents are focused on gene coding regions or promoters en masse. Here we introduce development of a customizable targeted capture system using biotinylated RNA probe baits transcribed from sheared bacterial artificial chromosome clone templates that enables capture of large, contiguous blocks of the genome for sequencing applications. This clone adapted template capture hybridization sequencing (CATCH-Seq) procedure can be used to capture both coding and non-coding regions of a gene, and resolve the boundaries of copy number variations within a genomic target site. Furthermore, libraries constructed with methylated adapters prior to solution hybridization also enable targeted bisulfite sequencing. We applied CATCH-Seq to diverse targets ranging in size from 125 kb to 3.5 Mb. Our approach provides a simple and cost effective alternative to other capture platforms because of template-based, enzymatic probe synthesis and the lack of oligonucleotide design costs. Given its similarity in procedure, CATCH-Seq can also be performed in parallel with commercial systems.
89. Phenotypic and genomic plasticity of alternative male reproductive tactics in sailfin mollies. Fraser Bonnie A, Janowitz Ilana, Thairu Margaret, Travis Joseph, Hughes Kimberly A. Proc. Biol. Sci. 2014. 281:1781. 20132310. PMID 24573842 PMC ID PMC3953829
A major goal of modern evolutionary biology is to understand the causes and consequences of phenotypic plasticity, the ability of a single genotype to produce multiple phenotypes in response to variable environments. While ecological and quantitative genetic studies have evaluated models of the evolution of adaptive plasticity, some long-standing questions about plasticity require more mechanistic approaches. Here, we address two of those questions: does plasticity facilitate adaptive evolution? And do physiological costs place limits on plasticity? We examine these questions by comparing genetically and plastically regulated behavioural variation in sailfin mollies (Poecilia latipinna), which exhibit striking variation in plasticity for male mating behaviour. In this species, some genotypes respond plastically to a change in the social environment by switching between primarily courting and primarily sneaking behaviour. In contrast, other genotypes have fixed mating strategies (either courting or sneaking) and do not display plasticity. We found that genetic and plastic variation in behaviour were accompanied by partially, but not completely overlapping changes in brain gene expression, in partial support of models that predict that plasticity can facilitate adaptive evolution. We also found that behavioural plasticity was accompanied by broader and more robust changes in brain gene expression, suggesting a substantial physiological cost to plasticity. We also observed that sneaking behaviour, but not courting, was associated with upregulation of genes involved in learning and memory, suggesting that sneaking is more cognitively demanding than courtship.
90. Nutrients drive transcriptional changes that maintain metabolic homeostasis but alter genome architecture in Microcystis. Steffen Morgan M, Dearth Stephen P, Dill Brian D, Li Zhou, Larsen Kristen M, Campagna Shawn R, Wilhelm Steven W. ISME J 2014. 8:10. 2080-92. PMID 24858783 PMC ID PMC4184021
The cyanobacterium Microcystis aeruginosa is a globally distributed bloom-forming organism that degrades freshwater systems around the world. Factors that drive its dispersion, diversification and success remain, however, poorly understood. To develop insight into cellular-level responses to nutrient drivers of eutrophication, RNA sequencing was coupled to a comprehensive metabolomics survey of M. aeruginosa sp. NIES 843 grown in various nutrient-reduced conditions. Transcriptomes were generated for cultures grown in nutrient-replete (with nitrate as the nitrogen (N) source), nitrogen-reduced (with nitrate, urea or ammonium acting as the N sources) and phosphate-reduced conditions. Extensive expression differences (up to 696 genes for urea-grown cells) relative to the control treatment were observed, demonstrating that the chemical variant of nitrogen available to cells affected transcriptional activity. Of particular note, a high number of transposase genes (up to 81) were significantly and reproducibly up-regulated relative to the control when grown on urea. Conversely, phosphorus (P) reduction resulted in a significant cessation in transcription of transposase genes, indicating that variation in nutrient chemistry may influence transcription of transposases and may impact the highly mosaic genomic architecture of M. aeruginosa. Corresponding metabolomes showed comparably few differences between treatments, suggesting broad changes to gene transcription are required to maintain metabolic homeostasis under nutrient reduction. The combined observations provide novel and extensive insight into the complex cellular interactions that take place in this important bloom-forming organism during variable nutrient conditions and highlight a potential unknown molecular mechanism that may drive Microcystis blooms and evolution.
91. An expressed retrogene of the master embryonic stem cell gene POU5F1 is associated with prostate cancer susceptibility. Breyer Joan P, Dorset Daniel C, Clark Travis A, Bradley Kevin M, Wahlfors Tiina A, McReynolds Kate M, Maynard William H, Chang Sam S, Cookson Michael S, Smith Joseph A, Schleutker Johanna, Dupont William D, Smith Jeffrey R. Am. J. Hum. Genet. 2014. 94:3. 395-404. PMID 24581739 PMC ID PMC3951923
Genetic association studies of prostate and other cancers have identified a major risk locus at chromosome 8q24. Several independent risk variants at this locus alter transcriptional regulatory elements, but an affected gene and mechanism for cancer predisposition have remained elusive. The retrogene POU5F1B within the locus has a preserved open reading frame encoding a homolog of the master embryonic stem cell transcription factor Oct4. We find that 8q24 risk alleles are expression quantitative trait loci correlated with reduced expression of POU5F1B in prostate tissue and that predicted deleterious POU5F1B missense variants are also associated with risk of transformation. POU5F1 is known to be self-regulated by the encoded Oct4 transcription factor. We further observe that POU5F1 expression is directly correlated with POU5F1B expression. Our results suggest that a pathway critical to self-renewal of embryonic stem cells may also have a role in the origin of cancer.
92. Repurposed Transcriptomic Data Facilitate Discovery of Innate Immunity Toll-Like Receptor (TLR) Genes Across Lophotrochozoa. Halanych Kenneth M, Kocot Kevin M. Biol. Bull. 2014. 227:2. 201-9. PMID 25411377
The growing volume of genomic data from across life represents opportunities for deriving valuable biological information from data that were initially collected for another purpose. Here, we use transcriptomes collected for phylogenomic studies to search for toll-like receptor (TLR) genes in poorly sampled lophotrochozoan clades (Annelida, Mollusca, Brachiopoda, Phoronida, and Entoprocta) and one ecdysozoan clade (Priapulida). TLR genes are involved in innate immunity across animals by recognizing potential microbial infection. They have an extracellular leucine-rich repeat (LRR) domain connected to a transmembrane domain and an intracellular toll/interleukin-1 receptor (TIR) domain. Consequently, these genes are important in initiating a signaling pathway to trigger defense. We found at least one TLR ortholog in all but two taxa examined, suggesting that a broad array of lophotrochozoans may have innate immune systems similar to those observed in vertebrates and arthropods. Comparison to the SMART database confirmed the presence of both the LRR and the TIR protein motifs characteristic of TLR genes. Because we looked at only one transcriptome per species, discovery of TLR genes was limited for most taxa. However, several TRL-like genes that vary in the number and placement of LRR domains were found in phoronids. Additionally, several contigs contained LRR domains but lacked TIR domains, suggesting they were not TLRs. Many of these LRR-containing contigs had other domains (e.g., immunoglobin) and are likely involved in innate immunity.
93. High-throughput sequencing characterizes intertidal meiofaunal communities in northern gulf of Mexico (dauphin island and mobile bay, alabama). Brannock Pamela M, Waits Damien S, Sharma Jyotsna, Halanych Kenneth M. Biol. Bull. 2014. 227:2. 161-74. PMID 25411374
Meiofauna are important components of food webs and for nutrient exchange between the benthos and water column. Recent studies have focused on these communities in the Gulf of Mexico due to potential impacts of the Deepwater Horizon Oil Spill (DWHOS). In particular, intertidal meiofaunal communities from Mobile Bay and Dauphin Island, Alabama, were previously shown to shift from predominately metazoan taxa prior to DWHOS to a fungal-dominated community after the spill. However, knowledge of variability within these communities remains unknown. Herein, we used Illumina high-throughput amplicon sequencing to examine variation throughout a year for the same locations for which the organismal shift was noted. Sediment samples were collected bi-monthly for a year (July 2011-July 2012) from which the meiofaunal community was examined by sequencing the eukaryotic hypervariable V9 region of the 18S rRNA gene. Results showed that the presence of fungal taxa was limited within these communities, suggesting that previously reported acute impacts of the DWHOS on meiofauna were apparently short term. However, these meiofaunal communities show shifts in proportions of metazoan taxa compared to pre-spill samples. Whether this change is due to prolonged impacts of the spill or variation in community composition is unclear. Taxonomic variation within and between sampled locations throughout the study was observed, suggesting potential yearly variation in communities. Continued sampling over a longer timeframe will provide a more complete understanding of seasonality and variation within these communities. Such a baseline is required to assess future anthropogenic impacts.
94. Nutritional impacts on gene expression in the surface mucosa of blue catfish (Ictalurus furcatus). Li Chao, Beck Benjamin H, Peatman Eric. Dev. Comp. Immunol. 2014. 44:1. 226-34. PMID 24378224
Short-term feed deprivation is a common occurrence in both wild and farmed fish species, due to reproductive processes, seasonal variations in temperature, or in response to a disease outbreak. Fasting can have dramatic physiological and biological consequences for fish, including impacts on mucosal immunity which can, in turn, change host susceptibility to pathogens. Culture and selection of blue catfish (Ictalurus furcatus) has gained importance as the production of a channel catfish×blue catfish (Ictalurus punctatus×I. furcatus) hybrid has increased in the Southeast US. Following a recent examination of fasting-induced impacts on mucosal immunity in channel catfish, here we utilized Illumina-based RNA-seq expression profiling to compare changes in blue catfish gill and skin after a brief (7 day) period of fasting. Transcriptome sequencing and de novo assembly of over 194 million 100 base-pair transcript reads was followed by differential expression analysis. Fasting altered a total of 530 genes in the surface mucosa, including genes regulating the immune response, energy metabolism, mucus production, cellular cytoskeletal structure, cell proliferation, and antioxidant responses. In particular, fasting perturbed arginine synthesis and metabolism pathways in a manner likely altering macrophage activation states and immune readiness. Our findings highlight key mediators of the critical interaction between nutrition and immunity at points of pathogen adherence and entry.
95. Characterization of the Merkel Cell Carcinoma miRNome. Ning Matthew S, Kim Annette S, Prasad Nripesh, Levy Shawn E, Zhang Huiqiu, Andl Thomas. J Skin Cancer 2014. 2014:X. 289548. PMID 24627810 PMC ID PMC3929981
MicroRNAs have been implicated in various skin cancers, including melanoma, squamous cell carcinoma, and basal cell carcinoma; however, the expression of microRNAs and their role in Merkel cell carcinoma (MCC) have yet to be explored in depth. To identify microRNAs specific to MCC (MCC-miRs), next-generation sequencing (NGS) of small RNA libraries was performed on different tissue samples including MCCs, other cutaneous tumors, and normal skin. Comparison of the profiles identified several microRNAs upregulated and downregulated in MCC. For validation, their expression was measured via qRT-PCR in a larger group of MCC and in a comparison group of non-MCC cutaneous tumors and normal skin. Eight microRNAs were upregulated in MCC: miR-502-3p, miR-9, miR-7, miR-340, miR-182, miR-190b, miR-873, and miR-183. Three microRNAs were downregulated: miR-3170, miR-125b, and miR-374c. Many of these MCC-miRs, the miR-183/182/96a cistron in particular, have connections to tumorigenic pathways implicated in MCC pathogenesis. In situ hybridization confirmed that the highly expressed MCC-miR, miR-182, is localized within tumor cells. Furthermore, NGS and qRT-PCR reveal that several of these MCC-miRs are highly expressed in the patient-derived MCC cell line, MS-1. These data indicate that we have identified a set of MCC-miRs with important implications for MCC research.
96. Islet microenvironment, modulated by vascular endothelial growth factor-A signaling, promotes ? cell regeneration. Brissova Marcela, Aamodt Kristie, Brahmachary Priyanka, Prasad Nripesh, Hong Ji-Young, Dai Chunhua, Mellati Mahnaz, Shostak Alena, Poffenberger Greg, Aramandla Radhika, Levy Shawn E, Powers Alvin C. Cell Metab. 2014. 19:3. 498-511. PMID 24561261 PMC ID PMC4012856
Pancreatic islet endocrine cell and endothelial cell (EC) interactions mediated by vascular endothelial growth factor-A (VEGF-A) signaling are important for islet differentiation and the formation of highly vascularized islets. To dissect how VEGF-A signaling modulates intra-islet vasculature, islet microenvironment, and ? cell mass, we transiently increased VEGF-A production by ? cells. VEGF-A induction dramatically increased the number of intra-islet ECs but led to ? cell loss. After withdrawal of the VEGF-A stimulus, ? cell mass, function, and islet structure normalized as a result of a robust, but transient, burst in proliferation of pre-existing ? cells. Bone marrow-derived macrophages (M?s) recruited to the site of ? cell injury were crucial for the ? cell proliferation, which was independent of pancreatic location and circulating factors such as glucose. Identification of the signals responsible for the proliferation of adult, terminally differentiated ? cells will improve strategies aimed at ? cell regeneration and expansion.
97. Optimizing Transcriptome Assemblies for Eleusine indica Leaf and Seedling by Combining Multiple Assemblies from Three De Novo Assemblers Chen Shu, McElroy Scott J, Peatman Eric. Plant Genome 2014. 8:1. X. PMID None
Due to rapid advances in sequencing technology, increasing amounts of genomic and transcriptomic data are available for plant species, presenting enormous challenges for biocomputing analysis. A crucial first step for a successful transcriptomics-based study is the building of a high-quality assembly. Here, we utilized three different de novo assemblers (Trinity, Velvet, and CLC) and the EvidentialGene pipeline tr2aacds to assemble two optimized transcript sets for the notorious weed species, Eleusine indica. Two RNA sequencing (RNA-seq) datasets from leaf and aboveground seedlings were processed using three assemblers, which resulted in 20 assemblies for each dataset. The contig numbers and N50 values of each assembly were compared to study the effect of read number, k-mer size, and in silico normalization on assembly output. The 20 assemblies were then processed through the tr2aacds pipeline to remove redundant transcripts and to select the transcript set with the best coding potential. Each assembly contributed a considerable proportion to the final transcript combination with the exception of the CLC-k14. Thus each assembler and parameter set did assemble better contigs for certain transcripts. The redundancy, total contig number, N50, fully assembled contig number, and transcripts related to target-site herbicide resistance were evaluated for the EvidentialGene and Trinity assemblies. Comparing the EvidentialGene set with the Trinity assembly revealed improved quality and reduced redundancy in both leaf and seedling EvidentialGene sets. The optimized transcriptome references will be useful for studying herbicide resistance in E. indica and the evolutionary process in the three allotetraploid E. indica offspring.
98. Consensus Genotyper for Exome Sequencing (CGES): improving the quality of exome variant genotypes. Trubetskoy Vassily, Rodriguez Alex, Dave Uptal, Campbell Nicholas, Crawford Emily L, Cook Edwin H, Sutcliffe James S, Foster Ian, Madduri Ravi, Cox Nancy J, Davis Lea K. Bioinformatics 2014. X:X. None. PMID 25270638
The development of cost-effective next-generation sequencing methods has spurred the development of high-throughput bioinformatics tools for detection of sequence variation. With many disparate variant-calling algorithms available, investigators must ask, 'Which method is best for my data?' Machine learning research has shown that so-called ensemble methods that combine the output of multiple models can dramatically improve classifier performance. Here we describe a novel variant-calling approach based on an ensemble of variant-calling algorithms, which we term the Consensus Genotyper for Exome Sequencing (CGES). CGES uses a two-stage voting scheme among four algorithm implementations. While our ensemble method can accept variants generated by any variant-calling algorithm, we used GATK2.8, SAMtools, FreeBayes and Atlas-SNP2 in building CGES because of their performance, widespread adoption and diverse but complementary algorithms.
99. Development and evaluation of the first high-throughput SNP array for common carp (Cyprinus carpio). Xu Jian, Zhao Zixia, Zhang Xiaofeng, Zheng Xianhu, Li Jiongtang, Jiang Yanliang, Kuang Youyi, Zhang Yan, Feng Jianxin, Li Chuangju, Yu Juhua, Li Qiang, Zhu Yuanyuan, Liu Yuanyuan, Xu Peng et al. BMC Genomics 2014. 15:X. 307. PMID 24762296
A large number of single nucleotide polymorphisms (SNPs) have been identified in common carp (Cyprinus carpio) but, as yet, no high-throughput genotyping platform is available for this species. C. carpio is an important aquaculture species that accounts for nearly 14% of freshwater aquaculture production worldwide. We have developed an array for C. carpio with 250,000 SNPs and evaluated its performance using samples from various strains of C. carpio.
100. A novel IFITM5 mutation in severe atypical osteogenesis imperfecta type VI impairs osteoblast production of pigment epithelium?derived factor. Farber Charles R, Reich Adi, Barnes Aileen M, Becerra Patricia, Rauch Frank, Cabral Wayne A, Bae Alison, Quinlan Aaron, Glorieux Francis H, Clemens Thomas L, Marini Joan C. J. Bone Miner. Res. 2014. 29:6. 1402-11. PMID 24519609
Osteogenesis imperfecta (OI) types V and VI are caused, respectively, by a unique dominant mutation in IFITM5, encoding BRIL, a transmembrane ifitm-like protein most strongly expressed in the skeletal system, and recessive null mutations in SERPINF1, encoding pigment epithelium-derived factor (PEDF). We identified a 25-year-old woman with severe OI whose dermal fibroblasts and cultured osteoblasts displayed minimal secretion of PEDF, but whose serum PEDF level was in the normal range. SERPINF1 sequences were normal despite bone histomorphometry consistent with type VI OI and elevated childhood serum alkaline phosphatase. We performed exome sequencing on the proband, both parents, and an unaffected sibling. IFITM5 emerged as the candidate gene from bioinformatics analysis, and was corroborated by membership in a murine bone co-expression network module containing all currently known OI genes. The de novo IFITM5 mutation was confirmed in one allele of the proband, resulting in a p.S40L substitution in the intracellular domain of BRIL but was absent in unaffected family members. IFITM5 expression was normal in proband fibroblasts and osteoblasts, and BRIL protein level was similar to control in differentiated proband osteoblasts on Western blot and in permeabilized mutant osteoblasts by microscopy. In contrast, SERPINF1 expression was decreased in proband osteoblasts; PEDF was barely detectable in conditioned media of proband cells. Expression and secretion of type I collagen was similarly decreased in proband osteoblasts; the expression pattern of several osteoblast markers largely overlapped reported values from cells with a primary PEDF defect. In contrast, osteoblasts from a typical case of type V OI, with an activating mutation at the 5'-terminus of BRIL, have increased SERPINF1 expression and PEDF secretion during osteoblast differentiation. Together, these data suggest that BRIL and PEDF have a relationship that connects the genes for types V and VI OI and their roles in bone mineralization.
101. Progressive increase in mtDNA 3243A>G heteroplasmy causes abrupt transcriptional reprogramming. Picard Martin, Zhang Jiangwen, Hancock Saege, Derbeneva Olga, Golhar Ryan, Golik Pawel, O'Hearn Sean, Levy Shawn, Potluri Prasanth, Lvova Maria, Davila Antonio, Lin Chun Shi, Perin Juan Carlos, Rappaport Eric F, Hakonarson Hakon et al. Proc. Natl. Acad. Sci. U.S.A. 2014. 111:38. E4033-42. PMID 25192935 PMC ID PMC4183335
Variation in the intracellular percentage of normal and mutant mitochondrial DNAs (mtDNA) (heteroplasmy) can be associated with phenotypic heterogeneity in mtDNA diseases. Individuals that inherit the common disease-causing mtDNA tRNA(Leu(UUR)) 3243A>G mutation and harbor ?10-30% 3243G mutant mtDNAs manifest diabetes and occasionally autism; individuals with ?50-90% mutant mtDNAs manifest encephalomyopathies; and individuals with ?90-100% mutant mtDNAs face perinatal lethality. To determine the basis of these abrupt phenotypic changes, we generated somatic cell cybrids harboring increasing levels of the 3243G mutant and analyzed the associated cellular phenotypes and nuclear DNA (nDNA) and mtDNA transcriptional profiles by RNA sequencing. Small increases in mutant mtDNAs caused relatively modest defects in oxidative capacity but resulted in sharp transitions in cellular phenotype and gene expression. Cybrids harboring 20-30% 3243G mtDNAs had reduced mtDNA mRNA levels, rounded mitochondria, and small cell size. Cybrids with 50-90% 3243G mtDNAs manifest induction of glycolytic genes, mitochondrial elongation, increased mtDNA mRNA levels, and alterations in expression of signal transduction, epigenomic regulatory, and neurodegenerative disease-associated genes. Finally, cybrids with 100% 3243G experienced reduced mtDNA transcripts, rounded mitochondria, and concomitant changes in nuclear gene expression. Thus, striking phase changes occurred in nDNA and mtDNA gene expression in response to the modest changes of the mtDNA 3243G mutant levels. Hence, a major factor in the phenotypic variation in heteroplasmic mtDNA mutations is the limited number of states that the nucleus can acquire in response to progressive changes in mitochondrial retrograde signaling.
102. Comparative transcriptomics of maturity-associated color change in Hawaiian spiders. Yim Kristina M, Brewer Michael S, Miller Craig T, Gillespie Rosemary G. J. Hered. 2014. 105 Sup:X. 771-81. PMID 25149253
Discontinuous variation within individuals is increasingly recognized as playing a role in diversification and ecological speciation. This study is part of an effort to investigate the molecular genetic underpinnings of adaptive radiation in Hawaiian spiders (genus Tetragnatha). This radiation is found throughout the Hawaiian Islands, showing a common pattern of evolutionary progression from older to younger islands. Moreover, the species are characterized by repeated evolution of similar ecomorphs that can be recognized on the basis of color--Green, Maroon, Large Brown, and Small Brown. However, 2 species (including T. kauaiensis from Kauai) are polyphenic, changing from 1 ecomorph (Green) to another (Maroon) at a specific developmental period. The current study focuses on the age-associated color change in the early stages of the radiation to determine whether this ancestral flexibility in phenotype may have translated into diversification of more derived taxa. We conducted a comparative analysis of transcriptome data (expressed genes) from the Maroon morph of T. kauaiensis and T. perreirai (Oahu), which exhibits a single fixed ecomorph (Maroon). Over 70 million sequence reads were generated using Illumina sequencing of messenger RNA. Using reciprocal best hit BLAST searches, 9027 orthologous genes were identified, of which 32 showed signatures of positive selection between the 2 taxa and may be involved in the loss of the ancestral developmental polyphenism and associated switch to separate monophenic ecomorphs. These results provide critical groundwork that will allow us to advance our understanding of the genomic elements associated with adaptive radiations.
103. First report of resistance to acetolactate-synthase-inhibiting herbicides in yellow nutsedge (Cyperus esculentus): confirmation and characterization. Tehranchian Parsa, Norsworthy Jason K, Nandula Vijay, McElroy Scott, Chen Shu, Scott Robert C. Pest Manag. Sci. 2014. X:X. None. PMID 25307777
Yellow nutsedge is one of the most problematic sedges in Arkansas rice, requiring the frequent use of halosulfuron (sulfonylurea) for its control. In the summer of 2012, halosulfuron at 53 g ha(-1) (labeled field rate) failed to control yellow nutsedge. The level of resistance to halosulfuron was determined in the putative resistant biotype, and its cross-resistance to other acetolactate synthase (ALS) inhibitors from four different herbicide families. ALS enzyme assays and analysis of the ALS gene were used to ascertain the resistance mechanism.
104. Utilizing next-generation sequencing to study homeologous polymorphisms and herbicide-resistance-endowing mutations in Poa annua acetolactate synthase genes. Chen Shu, McElroy J Scott, Flessner Michael L, Dane Fenny. Pest Manag. Sci. 2014. X:X. None. PMID 25180862
Detection of single nucleotide polymorphisms (SNPs) related to herbicide resistance in non-model polyploid weed species is fraught with difficulty owing to the gene duplication and lack of reference sequences. Our research seeks to overcome these obstacles by Illumina HiSeq read mapping, SNP calling and allele frequency determinations. Our focus is on the acetolactate synthase (ALS) gene, the target site of ALS-inhibiting herbicides, in Poa annua, an allotetraploid weed species originating from two diploid parents, P. supina and P. infirma.
105. Transcriptome annotation and marker discovery in white bass (Morone chrysops) and striped bass (Morone saxatilis). Li Chao, Beck Benjamin H, Fuller S Adam, Peatman Eric. Anim. Genet. 2014. 45:6. 885-7. PMID 25160849
Striped bass (Morone saxatilis) and white bass (Morone chrysops) are the parental species of the hybrid striped bass, a major U.S. aquaculture species. Currently, genomic resources for striped bass, white bass, and their hybrid lag behind those of other aquaculture species. Current resources consist of a medium-density genetic linkage map and a well-annotated ovarian transcriptome. A well-annotated transcriptome from across striped bass and white bass tissues is needed to advance both broad-based RNA-seq studies of gene expression as well as aid in more targeted studies of important genes and pathways critical for reproductive physiology and immunity. Here, we carried out Illumina-based transcriptome sequencing and annotation in both species utilizing the trinity and trinotate packages. The assembled Moronid reference transcriptomes and identified SSRs and SNPs should advance ongoing studies of reproduction, physiology, and immunology in these species and provide markers for broodstock management and selection.
106. Effects of transgenic sterilization constructs and their repressor compounds on hatch, developmental rate and early survival of electroporated channel catfish embryos and fry. Su Baofeng, Shang Mei, Li Chao, Perera Dayan A, Pinkert Carl A, Irwin Michael H, Peatman Eric, Grewe Peter, Patil Jawahar G, Dunham Rex A. Transgenic Res. 2014. X:X. None. PMID 25367204
Channel catfish (Ictalurus punctatus) embryos were electroporated with sterilization constructs targeting primordial germ cell proteins or with buffer. Some embryos then were treated with repressor compounds, cadmium chloride, copper sulfate, sodium chloride or doxycycline, to prevent expression of the transgene constructs. Promoters included channel catfish nanos and vasa, salmon transferrin (TF), modified yeast Saccharomyces cerevisiae copper transport protein (MCTR) and zebrafish racemase (RM). Knock-down systems were the Tet-off (nanos and vasa constructs), MCTR, RM and TF systems. Knock-down genes included shRNAi targeting 5' nanos (N1), 3' nanos (N2) or dead end (DND), or double-stranded nanos RNA (dsRNA) for overexpression of nanos mRNA. These constructs previously were demonstrated to knock down nanos, vasa and dead end, with the repressors having variable success. Exogenous DNA affected percentage hatch (% hatch), as all 14 constructs, except for the TF dsRNA, TF N1 (T), RM DND (C), vasa DND (C), vasa N1 (C) and vasa N2 (C), had lower % hatch than the control electroporated with buffer. The MCTR and RM DND (T) constructs resulted in delayed hatch, and the vasa and nanos constructs had minimal effects on time of hatch (P < 0.05). Cadmium chloride appeared to counteract the slow development caused by the TF constructs in two TF treatments (P < 0.05). The 4 ppt sodium chloride treatment for the RM system decreased % hatch (P < 0.05) and slowed development. In the case of nanos constructs, doxycycline greatly delayed hatch (P < 0.05). Adverse effects of the transgenes and repressors continued for several treatments for the first 6 days after hatch, but only in a few treatments during the next 10 days. Repressors and gene expression impacted the yield of putative transgenic channel catfish fry, and need to be considered and accounted for in the hatchery phase of producing transgenically sterilized catfish fry and their fertile counterparts. This fry output should be considered to ensure that sufficient numbers of transgenic fish are produced for future applications and for defining repressor systems that are the most successful.
107. Discovery and validation of gene-linked diagnostic SNP markers for assessing hybridization between Largemouth bass (Micropterus salmoides) and Florida bass (M. floridanus). Li Chao, Gowan Spencer, Anil Ammu, Beck Benjamin H, Thongda Wilawan, Kucuktas Huseyin, Kaltenboeck Ludmilla, Peatman Eric. Mol Ecol Resour 2014. X:X. None. PMID 25047482
Efforts to improve recreational fisheries have included widespread stocking of Micropterus floridanus outside its native range of peninsular Florida. Hybridization of Florida bass (M. floridanus) with largemouth bass (Micropterus salmoides) has now dramatically expanded beyond a naturally occurring intergrade zone in the southeast U.S. In recent years, there has been growing interest in protecting the genetic integrity of native basses and assessing the impact and nature of M. salmoides/M. floridanus introgression from the standpoint of hatchery and sport-fishery managers, fish biologists, ecologists and evolutionary biologists. Here, we conducted RNA-seq-based sequencing of the transcriptomes of M. salmoides, M. floridanus and their F1 hybrid and identified a set of 3674 SNP markers with fixed-allelic differences from 2112 unique genes. We then developed a subset of 25 of these markers into a single diagnostic multiplex assay and validated its capacity for assessing integrity and hybridization in hatchery and wild populations of largemouth and Florida bass. The availability of this resource, high-quality transcriptomes and a large set of gene-linked SNPs, should greatly facilitate functional and population genomics studies in these key species and allow the identification of traits and processes under selection during introgressive hybridization.
108. Multi-platform assessment of transcriptome profiling using RNA-seq in the ABRF next-generation sequencing study. Li Sheng, Tighe Scott W, Nicolet Charles M, Grove Deborah, Levy Shawn, Farmerie William, Viale Agnes, Wright Chris, Schweitzer Peter A, Gao Yuan, Kim Dewey, Boland Joe, Hicks Belynda, Kim Ryan, Chhangawala Sagar et al. Nat. Biotechnol. 2014. 32:9. 915-25. PMID 25150835 PMC ID PMC4167418
High-throughput RNA sequencing (RNA-seq) greatly expands the potential for genomics discoveries, but the wide variety of platforms, protocols and performance capabilitites has created the need for comprehensive reference data. Here we describe the Association of Biomolecular Resource Facilities next-generation sequencing (ABRF-NGS) study on RNA-seq. We carried out replicate experiments across 15 laboratory sites using reference RNA standards to test four protocols (poly-A-selected, ribo-depleted, size-selected and degraded) on five sequencing platforms (Illumina HiSeq, Life Technologies PGM and Proton, Pacific Biosciences RS and Roche 454). The results show high intraplatform (Spearman rank R > 0.86) and inter-platform (R > 0.83) concordance for expression measures across the deep-count platforms, but highly variable efficiency and cost for splice junction and variant detection between all platforms. For intact RNA, gene expression profiles from rRNA-depletion and poly-A enrichment are similar. In addition, rRNA depletion enables effective analysis of degraded RNA samples. This study provides a broad foundation for cross-platform standardization, evaluation and improvement of RNA-seq.
109. Diversity and genomic insights into the uncultured Chloroflexi from the human microbiota. Campbell Alisha G, Schwientek Patrick, Vishnivetskaya Tatiana, Woyke Tanja, Levy Shawn, Beall Clifford J, Griffen Ann, Leys Eugene, Podar Mircea. Environ. Microbiol. 2014. 16:9. 2635-43. PMID 24738594 PMC ID PMC4149597
Many microbial phyla that are widely distributed in open environments have few or no representatives within animal-associated microbiota. Among them, the Chloroflexi comprises taxonomically and physiologically diverse lineages adapted to a wide range of aquatic and terrestrial habitats. A distinct group of uncultured chloroflexi related to free-living anaerobic Anaerolineae inhabits the mammalian gastrointestinal tract and includes low-abundance human oral bacteria that appear to proliferate in periodontitis. Using a single-cell genomics approach, we obtained the first draft genomic reconstruction for these organisms and compared their inferred metabolic potential with free-living chloroflexi. Genomic data suggest that oral chloroflexi are anaerobic heterotrophs, encoding abundant carbohydrate transport and metabolism functionalities, similar to those seen in environmental Anaerolineae isolates. The presence of genes for a unique phosphotransferase system and N-acetylglucosamine metabolism suggests an important ecological niche for oral chloroflexi in scavenging material from lysed bacterial cells and the human tissue. The inferred ability to produce sialic acid for cell membrane decoration may enable them to evade the host defence system and colonize the subgingival space. As with other low abundance but persistent members of the microbiota, discerning community and host factors that influence the proliferation of oral chloroflexi may help understand the emergence of oral pathogens and the microbiota dynamics in health and disease states.
110. Whole-exome resequencing distinguishes cystic kidney diseases from phenocopies in renal ciliopathies. Gee Heon Yung, Otto Edgar A, Hurd Toby W, Ashraf Shazia, Chaki Moumita, Cluckey Andrew, Vega-Warner Virginia, Saisawat Pawaree, Diaz Katrina A, Fang Humphrey, Kohl Stefan, Allen Susan J, Airik Rannar, Zhou Weibin, Ramaswami Gokul et al. Kidney Int. 2014. 85:4. 880-7. PMID 24257694 PMC ID PMC3972265
Rare single-gene disorders cause chronic disease. However, half of the 6000 recessive single gene causes of disease are still unknown. Because recessive disease genes can illuminate, at least in part, disease pathomechanism, their identification offers direct opportunities for improved clinical management and potentially treatment. Rare diseases comprise the majority of chronic kidney disease (CKD) in children but are notoriously difficult to diagnose. Whole-exome resequencing facilitates identification of recessive disease genes. However, its utility is impeded by the large number of genetic variants detected. We here overcome this limitation by combining homozygosity mapping with whole-exome resequencing in 10 sib pairs with a nephronophthisis-related ciliopathy, which represents the most frequent genetic cause of CKD in the first three decades of life. In 7 of 10 sibships with a histologic or ultrasonographic diagnosis of nephronophthisis-related ciliopathy, we detect the causative gene. In six sibships, we identify mutations of known nephronophthisis-related ciliopathy genes, while in two additional sibships we found mutations in the known CKD-causing genes SLC4A1 and AGXT as phenocopies of nephronophthisis-related ciliopathy. Thus, whole-exome resequencing establishes an efficient, noninvasive approach towards early detection and causation-based diagnosis of rare kidney diseases. This approach can be extended to other rare recessive disorders, thereby providing accurate diagnosis and facilitating the study of disease mechanisms.
111. ALS-Resistant Smallflower Umbrella Sedge (Cyperus difformis) in Arkansas Rice: Physiological and Molecular Basis of Resistance Tehranchian Parsa, Riar Dilpreet S, Norsworthy Jason K, Nandula Vijay, McElroy Scott, Chen Shu, Scott Robert C. Weed Science 2014. X:X. X. PMID NOTINPMED000001
Smallflower umbrella sedge is a problematic weed in direct-seeded rice in the midsouthern U.S. It recently has evolved resistance to the acetolactate synthase (ALS)-inhibiting herbicide halosulfuron in Arkansas rice. Studies were conducted to (1) determine if the resistant biotype is cross resistant to other ALS-inhibiting herbicides, (2) evaluate alternative herbicide control options, and (3) determine the mechanism of resistance. Whole plant bioassay revealed that halosulfuron-resistant plants were not controlled by bispyribac-sodium, imazamox, and penoxsulam at the labeled field rate of each herbicide. The level of resistance to these herbicides, based on the lethal dose needed to kill 50% of plants (LD50) was >15 fold compared to a susceptible biotype. Both biotypes were controlled >96% with bentazon and propanil and <23% with quinclorac, thiobencarb, and 2,4-D. Hence, effective control measures exist; albeit, the number of herbicide options appear limited. Based on in vitro ALS enzyme assays, altered target-site is the mechanism of resistance to halosulfuron and imazamox. Massively parallel sequencing using the Illumina HiSeq detected an amino acid substitution of Pro197-to-His in the resistant biotype that is consistent with ALS-inhibiting herbicide resistance in other weed species.
112. The genomic landscape of mantle cell lymphoma is related to the epigenetically determined chromatin state of normal B cells. Zhang Jenny, Jima Dereje, Moffitt Andrea B, Liu Qingquan, Czader Magdalena, Hsi Eric D, Fedoriw Yuri, Dunphy Cherie H, Richards Kristy L, Gill Javed I, Sun Zhen, Love Cassandra, Scotland Paula, Lock Eric, Levy Shawn et al. Blood 2014. 123:19. 2988-96. PMID 24682267 PMC ID PMC4014841
In this study, we define the genetic landscape of mantle cell lymphoma (MCL) through exome sequencing of 56 cases of MCL. We identified recurrent mutations in ATM, CCND1, MLL2, and TP53. We further identified a number of novel genes recurrently mutated in patients with MCL including RB1, WHSC1, POT1, and SMARCA4. We noted that MCLs have a distinct mutational profile compared with lymphomas from other B-cell stages. The ENCODE project has defined the chromatin structure of many cell types. However, a similar characterization of primary human mature B cells has been lacking. We defined, for the first time, the chromatin structure of primary human naïve, germinal center, and memory B cells through chromatin immunoprecipitation and sequencing for H3K4me1, H3K4me3, H3Ac, H3K36me3, H3K27me3, and PolII. We found that somatic mutations that occur more frequently in either MCLs or Burkitt lymphomas were associated with open chromatin in their respective B cells of origin, naïve B cells, and germinal center B cells. Our work thus elucidates the landscape of gene-coding mutations in MCL and the critical interplay between epigenetic alterations associated with B-cell differentiation and the acquisition of somatic mutations in cancer.
113. Monoallelic and biallelic mutations in MAB21L2 cause a spectrum of major eye malformations. Rainger Joe, Pehlivan Davut, Johansson Stefan, Bengani Hemant, Sanchez-Pulido Luis, Williamson Kathleen A, Ture Mehmet, Barker Heather, Rosendahl Karen, Spranger Jürgen, Horn Denise, Meynert Alison, Floyd James A B, Prescott Trine, Anderson Carl A et al. Am. J. Hum. Genet. 2014. 94:6. 915-23. PMID 24906020 PMC ID PMC4121478
We identified four different missense mutations in the single-exon gene MAB21L2 in eight individuals with bilateral eye malformations from five unrelated families via three independent exome sequencing projects. Three mutational events altered the same amino acid (Arg51), and two were identical de novo mutations (c.151C>T [p.Arg51Cys]) in unrelated children with bilateral anophthalmia, intellectual disability, and rhizomelic skeletal dysplasia. c.152G>A (p.Arg51His) segregated with autosomal-dominant bilateral colobomatous microphthalmia in a large multiplex family. The fourth heterozygous mutation (c.145G>A [p.Glu49Lys]) affected an amino acid within two residues of Arg51 in an adult male with bilateral colobomata. In a fifth family, a homozygous mutation (c.740G>A [p.Arg247Gln]) altering a different region of the protein was identified in two male siblings with bilateral retinal colobomata. In mouse embryos, Mab21l2 showed strong expression in the developing eye, pharyngeal arches, and limb bud. As predicted by structural homology, wild-type MAB21L2 bound single-stranded RNA, whereas this activity was lost in all altered forms of the protein. MAB21L2 had no detectable nucleotidyltransferase activity in vitro, and its function remains unknown. Induced expression of wild-type MAB21L2 in human embryonic kidney 293 cells increased phospho-ERK (pERK1/2) signaling. Compared to the wild-type and p.Arg247Gln proteins, the proteins with the Glu49 and Arg51 variants had increased stability. Abnormal persistence of pERK1/2 signaling in MAB21L2-expressing cells during development is a plausible pathogenic mechanism for the heterozygous mutations. The phenotype associated with the homozygous mutation might be a consequence of complete loss of MAB21L2 RNA binding, although the cellular function of this interaction remains unknown.
114. SNP discovery in wild and domesticated populations of blue catfish, Ictalurus furcatus, using genotyping-by-sequencing and subsequent SNP validation. Li Chao, Waldbieser Geoff, Bosworth Brian, Beck Benjamin H, Thongda Wilawan, Peatman Eric. Mol Ecol Resour 2014. 14:6. 1261-70. PMID 24797164
Blue catfish, Ictalurus furcatus, are valued in the United States as a trophy fishery for their capacity to reach large sizes, sometimes exceeding 45 kg. Additionally, blue catfish × channel catfish (I. punctatus) hybrid food fish production has recently increased the demand for blue catfish broodstock. However, there has been little study of the genetic impacts and interaction of farmed, introduced and stocked populations of blue catfish. We utilized genotyping-by-sequencing (GBS) to capture and genotype SNP markers on 190 individuals from five wild and domesticated populations (Mississippi River, Missouri, D&B, Rio Grande and Texas). Stringent filtering of SNP-calling parameters resulted in 4275 SNP loci represented across all five populations. Population genetics and structure analyses revealed potential shared ancestry and admixture between populations. We utilized the Sequenom MassARRAY to validate two multiplex panels of SNPs selected from the GBS data. Selection criteria included SNPs shared between populations, SNPs specific to populations, number of reads per individual and number of individuals genotyped by GBS. Putative SNPs were validated in the discovery population and in two additional populations not used in the GBS analysis. A total of 64 SNPs were genotyped successfully in 191 individuals from nine populations. Our results should guide the development of highly informative, flexible genotyping multiplexes for blue catfish from the larger GBS SNP set as well as provide an example of a rapid, low-cost approach to generate and genotype informative marker loci in aquatic species with minimal previous genetic information.
115. Vascular endothelial growth factor coordinates islet innervation via vascular scaffolding. Reinert Rachel B, Cai Qing, Hong Ji-Young, Plank Jennifer L, Aamodt Kristie, Prasad Nripesh, Aramandla Radhika, Dai Chunhua, Levy Shawn E, Pozzi Ambra, Labosky Patricia A, Wright Christopher V E, Brissova Marcela, Powers Alvin C. Development 2014. 141:7. 1480-91. PMID 24574008 PMC ID PMC3957372
Neurovascular alignment is a common anatomical feature of organs, but the mechanisms leading to this arrangement are incompletely understood. Here, we show that vascular endothelial growth factor (VEGF) signaling profoundly affects both vascularization and innervation of the pancreatic islet. In mature islets, nerves are closely associated with capillaries, but the islet vascularization process during embryonic organogenesis significantly precedes islet innervation. Although a simple neuronal meshwork interconnects the developing islet clusters as they begin to form at E14.5, the substantial ingrowth of nerve fibers into islets occurs postnatally, when islet vascularization is already complete. Using genetic mouse models, we demonstrate that VEGF regulates islet innervation indirectly through its effects on intra-islet endothelial cells. Our data indicate that formation of a VEGF-directed, intra-islet vascular plexus is required for development of islet innervation, and that VEGF-induced islet hypervascularization leads to increased nerve fiber ingrowth. Transcriptome analysis of hypervascularized islets revealed an increased expression of extracellular matrix components and axon guidance molecules, with these transcripts being enriched in the islet-derived endothelial cell population. We propose a mechanism for coordinated neurovascular development within pancreatic islets, in which endocrine cell-derived VEGF directs the patterning of intra-islet capillaries during embryogenesis, forming a scaffold for the postnatal ingrowth of essential autonomic nerve fibers.
116. Intragenic motifs regulate the transcriptional complexity of Pkhd1/PKHD1. Boddu Ravindra, Yang Chaozhe, O'Connor Amber K, Hendrickson Robert Curtis, Boone Braden, Cui Xiangqin, Garcia-Gonzalez Miguel, Igarashi Peter, Onuchic Luiz F, Germino Gregory G, Guay-Woodford Lisa M. J. Mol. Med. 2014. 92:10. 1045-56. PMID 24984783 PMC ID PMC4197071
Autosomal recessive polycystic kidney disease (ARPKD) results from mutations in the human PKHD1 gene. Both this gene, and its mouse ortholog, Pkhd1, are primarily expressed in renal and biliary ductal structures. The mouse protein product, fibrocystin/polyductin complex (FPC), is a 445-kDa protein encoded by a 67-exon transcript that spans >500 kb of genomic DNA. In the current study, we observed multiple alternatively spliced Pkhd1 transcripts that varied in size and exon composition in embryonic mouse kidney, liver, and placenta samples, as well as among adult mouse pancreas, brain, heart, lung, testes, liver, and kidney. Using reverse transcription PCR and RNASeq, we identified 22 novel Pkhd1 kidney transcripts with unique exon junctions. Various mechanisms of alternative splicing were observed, including exon skipping, use of alternate acceptor/donor splice sites, and inclusion of novel exons. Bioinformatic analyses identified, and exon-trapping minigene experiments validated, consensus binding sites for serine/arginine-rich proteins that modulate alternative splicing. Using site-directed mutagenesis, we examined the functional importance of selected splice enhancers. In addition, we demonstrated that many of the novel transcripts were polysome bound, thus likely translated. Finally, we determined that the human PKHD1 R760H missense variant alters a splice enhancer motif that disrupts exon splicing in vitro and is predicted to truncate the protein. Taken together, these data provide evidence of the complex transcriptional regulation of Pkhd1/PKHD1 and identified motifs that regulate its splicing. Our studies indicate that Pkhd1/PKHD1 transcription is modulated, in part by intragenic factors, suggesting that aberrant PKHD1 splicing represents an unappreciated pathogenic mechanism in ARPKD.
117. Insulator function and topological domain border strength scale with architectural protein occupancy. Van Bortle Kevin, Nichols Michael H, Li Li, Ong Chin-Tong, Takenaka Naomi, Qin Zhaohui S, Corces Victor G. Genome Biol. 2014. 15:6. R82. PMID 24981874
Chromosome conformation capture studies suggest that eukaryotic genomes are organized into structures called topologically associating domains. The borders of these domains are highly enriched for architectural proteins with characterized roles in insulator function. However, a majority of architectural protein binding sites localize within topological domains, suggesting sites associated with domain borders represent a functionally different subclass of these regulatory elements. How topologically associating domains are established and what differentiates border-associated from non-border architectural protein binding sites remain unanswered questions.
118. Large inverted duplications in the human genome form via a fold-back mechanism. Hermetz Karen E, Newman Scott, Conneely Karen N, Martin Christa L, Ballif Blake C, Shaffer Lisa G, Cody Jannine D, Rudd M Katharine. PLoS Genet. 2014. 10:1. e1004139. PMID 24497845 PMC ID PMC3907307
Inverted duplications are a common type of copy number variation (CNV) in germline and somatic genomes. Large duplications that include many genes can lead to both neurodevelopmental phenotypes in children and gene amplifications in tumors. There are several models for inverted duplication formation, most of which include a dicentric chromosome intermediate followed by breakage-fusion-bridge (BFB) cycles, but the mechanisms that give rise to the inverted dicentric chromosome in most inverted duplications remain unknown. Here we have combined high-resolution array CGH, custom sequence capture, next-generation sequencing, and long-range PCR to analyze the breakpoints of 50 nonrecurrent inverted duplications in patients with intellectual disability, autism, and congenital anomalies. For half of the rearrangements in our study, we sequenced at least one breakpoint junction. Sequence analysis of breakpoint junctions reveals a normal-copy disomic spacer between inverted and non-inverted copies of the duplication. Further, short inverted sequences are present at the boundary of the disomic spacer and the inverted duplication. These data support a mechanism of inverted duplication formation whereby a chromosome with a double-strand break intrastrand pairs with itself to form a "fold-back" intermediate that, after DNA replication, produces a dicentric inverted chromosome with a disomic spacer corresponding to the site of the fold-back loop. This process can lead to inverted duplications adjacent to terminal deletions, inverted duplications juxtaposed to translocations, and inverted duplication ring chromosomes.
119. Patterns of variation influencing antipsychotic treatment outcomes in South African first-episode schizophrenia patients. Drogemöller Britt I, Niehaus Dana J H, Chiliza Bonginkosi, van der Merwe Lize, Asmal Laila, Malhotra Anil K, Wright Galen E B, Emsley Robin, Warnich Louise. Pharmacogenomics 2014. 15:2. 189-99. PMID 24444409
Many antipsychotic pharmacogenetics studies have been performed examining candidate genes or known variation; however, our understanding of the genetic factors involved in antipsychotic pharmacogenetic traits remains limited.
120. The maxillary palp of Aedes aegypti, a model of multisensory integration. Bohbot Jonathan D, Sparks Jackson T, Dickens Joseph C. Insect Biochem. Mol. Biol. 2014. 48:X. 29-39. PMID 24613607
Female yellow-fever mosquitoes, Aedes aegypti, are obligate blood-feeders and vectors of the pathogens that cause dengue fever, yellow fever and Chikungunya. This feeding behavior concludes a series of multisensory events guiding the mosquito to its host from a distance. The antennae and maxillary palps play a major role in host detection and other sensory-mediated behaviors. Compared to the antennae, the maxillary palps are a relatively simple organ and thus an attractive model for exploration of the neuromolecular networks underlying chemo- and mechanosensation. In this study, we surveyed the expressed genetic components and examined their potential involvement with these sensory modalities. Using Illumina sequencing, we identified the transcriptome of the maxillary palps of physiologically mature female Ae. aegypti. Genes expressed in the maxillary palps included those involved in sensory reception, signal transduction and neuromodulation. In addition to previously reported chemosensory genes, we identified candidate transcripts potentially involved in mechanosensation and thermosensation. This survey lays the groundwork to explore sensory networks in an insect appendage. The identification of genes involved in thermosensation provides prospective molecular targets for the development of chemicals aimed at disrupting the behavior of this medically important insect.
121. Mutations in EMP2 cause childhood-onset nephrotic syndrome. Gee Heon Yung, Ashraf Shazia, Wan Xiaoyang, Vega-Warner Virginia, Esteve-Rudd Julian, Lovric Svjetlana, Fang Humphrey, Hurd Toby W, Sadowski Carolin E, Allen Susan J, Otto Edgar A, Korkmaz Emine, Washburn Joseph, Levy Shawn, Williams David S et al. Am. J. Hum. Genet. 2014. 94:6. 884-90. PMID 24814193 PMC ID PMC4121470
Nephrotic syndrome (NS) is a genetically heterogeneous group of diseases that are divided into steroid-sensitive NS (SSNS) and steroid-resistant NS (SRNS). SRNS inevitably leads to end-stage kidney disease, and no curative treatment is available. To date, mutations in more than 24 genes have been described in Mendelian forms of SRNS; however, no Mendelian form of SSNS has been described. To identify a genetic form of SSNS, we performed homozygosity mapping, whole-exome sequencing, and multiplex PCR followed by next-generation sequencing. We thereby detected biallelic mutations in EMP2 (epithelial membrane protein 2) in four individuals from three unrelated families affected by SRNS or SSNS. We showed that EMP2 exclusively localized to glomeruli in the kidney. Knockdown of emp2 in zebrafish resulted in pericardial effusion, supporting the pathogenic role of mutated EMP2 in human NS. At the cellular level, we showed that knockdown of EMP2 in podocytes and endothelial cells resulted in an increased amount of CAVEOLIN-1 and decreased cell proliferation. Our data therefore identify EMP2 mutations as causing a recessive Mendelian form of SSNS.
122. Novel SACS mutations identified by whole exome sequencing in a norwegian family with autosomal recessive spastic ataxia of Charlevoix-Saguenay. Tzoulis Charalampos, Johansson Stefan, Haukanes Bjørn Ivar, Boman Helge, Knappskog Per Morten, Bindoff Laurence A. PLoS ONE 2013. 8:6. e66145. PMID 23785480 PMC ID PMC3681964
We employed whole exome sequencing to investigate three Norwegian siblings with an autosomal recessive spastic ataxia and epilepsy. All patients were compound heterozygous (c.13352T>C, p.Leu4451Pro; c.6890T>G, p.Leu2297Trp) for mutations in the SACS gene establishing the diagnosis of autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS). The clinical features shown by our patients were typical of this disorder with the exception of epilepsy, which is a rare manifestation. This is the first report of ARSACS in Scandinavian patients and our findings expand the genetic and clinical spectrum of this rare disorder. Moreover, we show that exome sequencing is a powerful and cost-effective tool for the diagnosis of genetically heterogeneous disorders such as the hereditary ataxias.
123. Patient-specific iPSC-derived photoreceptor precursor cells as a means to investigate retinitis pigmentosa. Tucker Budd A, Mullins Robert F, Streb Luan M, Anfinson Kristin, Eyestone Mari E, Kaalberg Emily, Riker Megan J, Drack Arlene V, Braun Terry A, Stone Edwin M. Elife 2013. 2:X. e00824. PMID 23991284 PMC ID PMC3755341
Next-generation and Sanger sequencing were combined to identify disease-causing USH2A mutations in an adult patient with autosomal recessive RP. Induced pluripotent stem cells (iPSCs), generated from the patient's keratinocytes, were differentiated into multi-layer eyecup-like structures with features of human retinal precursor cells. The inner layer of the eyecups contained photoreceptor precursor cells that expressed photoreceptor markers and exhibited axonemes and basal bodies characteristic of outer segments. Analysis of the USH2A transcripts of these cells revealed that one of the patient's mutations causes exonification of intron 40, a translation frameshift and a premature stop codon. Western blotting revealed upregulation of GRP78 and GRP94, suggesting that the patient's other USH2A variant (Arg4192His) causes disease through protein misfolding and ER stress. Transplantation into 4-day-old immunodeficient Crb1 (-/-) mice resulted in the formation of morphologically and immunohistochemically recognizable photoreceptor cells, suggesting that the mutations in this patient act via post-developmental photoreceptor degeneration. DOI:http://dx.doi.org/10.7554/eLife.00824.001.
124. Short-term feed deprivation alters immune status of surface mucosa in channel catfish (Ictalurus punctatus). Liu Lisa, Li Chao, Su Baofeng, Beck Benjamin H, Peatman Eric. PLoS ONE 2013. 8:9. e74581. PMID 24023952 PMC ID PMC3762756
Short-term feed deprivation (or fasting) is a common occurrence in aquacultured fish species whether due to season, production strategies, or disease. In channel catfish (Ictalurus punctatus) fasting impacts susceptibility to several bacterial pathogens including Flavobacterium columnare, the causative agent of columnaris disease. As columnaris gains entry through the gills and skin of fish, we examined here changes in transcriptional regulation induced in these surface mucosal tissues due to short-term (7 day) fasting. RNA-seq expression analysis revealed a total of 1,545 genes perturbed by fasting. Fasting significantly altered expression of critical innate immune factors in a manner consistent with lower immune fitness as well as dysregulating key genes involved in energy metabolism and cell cycling/proliferation. Downregulation of innate immune actors such as iNOS2b, Lysozyme C, and peptidoglycan recognition protein 6 is predicted to impact the delicate recognition/tolerance balance for commensal and pathogenic bacteria on the skin and gill. The highlighted expression profiles reveal potential mechanistic similarities between gut and surface mucosa and underscore the complex interrelationships between nutrition, mucosal integrity, and immunity in teleost fish.
125. Male-biased genes in catfish as revealed by RNA-Seq analysis of the testis transcriptome. Sun Fanyue, Liu Shikai, Gao Xiaoyu, Jiang Yanliang, Perera Dayan, Wang Xiuli, Li Chao, Sun Luyang, Zhang Jiaren, Kaltenboeck Ludmilla, Dunham Rex, Liu Zhanjiang. PLoS ONE 2013. 8:7. e68452. PMID 23874634 PMC ID PMC3709890
Catfish has a male-heterogametic (XY) sex determination system, but genes involved in gonadogenesis, spermatogenesis, testicular determination, and sex determination are poorly understood. As a first step of understanding the transcriptome of the testis, here, we conducted RNA-Seq analysis using high throughput Illumina sequencing.
126. Generation of physical map contig-specific sequences useful for whole genome sequence scaffolding. Jiang Yanliang, Ninwichian Parichart, Liu Shikai, Zhang Jiaren, Kucuktas Huseyin, Sun Fanyue, Kaltenboeck Ludmilla, Sun Luyang, Bao Lisui, Liu Zhanjiang. PLoS ONE 2013. 8:10. e78872. PMID 24205335 PMC ID PMC3811975
Along with the rapid advances of the nextgen sequencing technologies, more and more species are added to the list of organisms whose whole genomes are sequenced. However, the assembled draft genome of many organisms consists of numerous small contigs, due to the short length of the reads generated by nextgen sequencing platforms. In order to improve the assembly and bring the genome contigs together, more genome resources are needed. In this study, we developed a strategy to generate a valuable genome resource, physical map contig-specific sequences, which are randomly distributed genome sequences in each physical contig. Two-dimensional tagging method was used to create specific tags for 1,824 physical contigs, in which the cost was dramatically reduced. A total of 94,111,841 100-bp reads and 315,277 assembled contigs are identified containing physical map contig-specific tags. The physical map contig-specific sequences along with the currently available BAC end sequences were then used to anchor the catfish draft genome contigs. A total of 156,457 genome contigs (~79% of whole genome sequencing assembly) were anchored and grouped into 1,824 pools, in which 16,680 unique genes were annotated. The physical map contig-specific sequences are valuable resources to link physical map, genetic linkage map and draft whole genome sequences, consequently have the capability to improve the whole genome sequences assembly and scaffolding, and improve the genome-wide comparative analysis as well. The strategy developed in this study could also be adopted in other species whose whole genome assembly is still facing a challenge.
127. Functional Development of the Octenol Response in Aedes aegypti. Bohbot Jonathan D, Durand Nicolas F, Vinyard Bryan T, Dickens Joseph C. Front Physiol 2013. 4:X. 39. PMID 23471139 PMC ID PMC3590643
Attraction of female Aedes aegypti mosquitoes to 1-octen-3-ol (octenol), CO2, lactic acid, or ammonia emitted by vertebrate hosts is not only contingent on the presence of odorants in the environment, but is also influenced by the insect's physiological state. For anautogenous mosquito species, like A. aegypti, newly emerged adult females neither respond to host odors nor engage in blood-feeding; the bases for these behaviors are poorly understood. Here we investigated detection of two components of an attractant blend emitted by vertebrate hosts, octenol, and CO2, by female A. aegypti mosquitoes using electrophysiological, behavioral, and molecular approaches. An increase in sensitivity of octenol olfactory receptor neurons (ORNs) was correlated with an increase in odorant receptor gene (Or) expression and octenol-mediated attractive behavior from day 1 to day 6 post-emergence. While the sensitivity of octenol ORNs was maintained through day 10, behavioral responses to octenol decreased as did the ability of females to discriminate between octenol and octenol?+?CO2. Our results show differing age-related roles for the peripheral receptors for octenol and higher order neural processing in the behavior of female mosquitoes.
128. Transcriptome sequencing and analysis of wild Amur Ide (Leuciscus waleckii) inhabiting an extreme alkaline-saline lake reveals insights into stress adaptation. Xu Jian, Ji Peifeng, Wang Baosen, Zhao Lan, Wang Jian, Zhao Zixia, Zhang Yan, Li Jiongtang, Xu Peng, Sun Xiaowen. PLoS ONE 2013. 8:4. e59703. PMID 23573207 PMC ID PMC3613414
Amur ide (Leuciscus waleckii) is an economically and ecologically important species in Northern Asia. The Dali Nor population inhabiting Dali Nor Lake, a typical saline-alkaline lake in Inner Mongolia, is well-known for its adaptation to extremely high alkalinity. Genome information is needed for conservation and aquaculture purposes, as well as to gain further understanding into the genetics of stress tolerance. The objective of the study is to sequence the transcriptome and obtain a well-assembled transcriptome of Amur ide.
129. The genesis of an exceptionally lethal venom in the timber rattlesnake (Crotalus horridus) revealed through comparative venom-gland transcriptomics. Rokyta Darin R, Wray Kenneth P, Margres Mark J. BMC Genomics 2013. 14:X. 394. PMID 23758969 PMC ID PMC3701607
Snake venoms generally show sequence and quantitative variation within and between species, but some rattlesnakes have undergone exceptionally rapid, dramatic shifts in the composition, lethality, and pharmacological effects of their venoms. Such shifts have occurred within species, most notably in Mojave (Crotalus scutulatus), South American (C. durissus), and timber (C. horridus) rattlesnakes, resulting in some populations with extremely potent, neurotoxic venoms without the hemorrhagic effects typical of rattlesnake bites.
130. MRI characterisation of adult onset alpha-methylacyl-coA racemase deficiency diagnosed by exome sequencing. Haugarvoll Kristoffer, Johansson Stefan, Tzoulis Charalampos, Haukanes Bjørn Ivar, Bredrup Cecilie, Neckelmann Gesche, Boman Helge, Knappskog Per Morten, Bindoff Laurence A. Orphanet J Rare Dis 2013. 8:X. 1. PMID 23286897 PMC ID PMC3567975
Correct diagnosis is pivotal to understand and treat neurological disease. Herein, we report the diagnostic work-up utilizing exome sequencing and the characterization of clinical features and brain MRI in two siblings with a complex, adult-onset phenotype; including peripheral neuropathy, epilepsy, relapsing encephalopathy, bilateral thalamic lesions, type 2 diabetes mellitus, cataract, pigmentary retinopathy and tremor.
131. Exome sequencing reveals FAM20c mutations associated with fibroblast growth factor 23-related hypophosphatemia, dental anomalies, and ectopic calcification. Rafaelsen Silje Hjorth, Raeder Helge, Fagerheim Anne Kristine, Knappskog Per, Carpenter Thomas O, Johansson Stefan, Bjerknes Robert. J. Bone Miner. Res. 2013. 28:6. 1378-85. PMID 23325605
Fibroblast growth factor 23 (FGF23) plays a crucial role in renal phosphate regulation, exemplified by the causal role of PHEX and DMP1 mutations in X-linked hypophosphatemic rickets and autosomal recessive rickets type 1, respectively. Using whole exome sequencing we identified compound heterozygous mutations in family with sequence similarity 20, member C (FAM20C) in two siblings referred for hypophosphatemia and severe dental demineralization disease. FAM20C mutations were not found in other undiagnosed probands of a national Norwegian population of familial hypophosphatemia. Our results demonstrate that mutations in FAM20C provide a putative new mechanism in human subjects leading to dysregulated FGF23 levels, hypophosphatemia, hyperphosphaturia, dental anomalies, intracerebral calcifications and osteosclerosis of the long bones in the absence of rickets.
132. Malignant transformation of colonic epithelial cells by a colon-derived long noncoding RNA. Franklin Jeffrey L, Rankin Carl R, Levy Shawn, Snoddy Jay R, Zhang Bing, Washington Mary Kay, Thomson J Michael, Whitehead Robert H, Coffey Robert J. Biochem. Biophys. Res. Commun. 2013. 440:1. 99-104. PMID 24045012 PMC ID PMC3875348
Recent progress has been made in the identification of protein-coding genes and miRNAs that are expressed in and alter the behavior of colonic epithelia. However, the role of long non-coding RNAs (lncRNAs) in colonic homeostasis is just beginning to be explored. By gene expression profiling of post-mitotic, differentiated tops and proliferative, progenitor-compartment bottoms of microdissected adult mouse colonic crypts, we identified several lncRNAs more highly expressed in crypt bottoms. One identified lncRNA, designated non-coding Nras functional RNA (ncNRFR), resides within the Nras locus but appears to be independent of the Nras coding transcript. Stable overexpression of ncNRFR in non-transformed, conditionally immortalized mouse colonocytes results in malignant transformation, as determined by growth in soft agar and formation of highly invasive tumors in nude mice. Moreover, ncNRFR appears to inhibit the function of the tumor suppressor let-7. These results suggest precise regulation of ncNRFR is necessary for proper cell growth in the colonic crypt, and its misregulation results in neoplastic transformation.
133. Pitx1 broadly associates with limb enhancers and is enriched on hindlimb cis-regulatory elements. Infante Carlos R, Park Sungdae, Mihala Alexandra G, Kingsley David M, Menke Douglas B. Dev. Biol. 2013. 374:1. 234-44. PMID 23201014 PMC ID PMC3640454
Extensive functional analyses have demonstrated that the pituitary homeodomain transcription factor Pitx1 plays a critical role in specifying hindlimb morphology in vertebrates. However, much less is known regarding the target genes and cis-regulatory elements through which Pitx1 acts. Earlier studies suggested that the hindlimb transcription factors Tbx4, HoxC10, and HoxC11 might be transcriptional targets of Pitx1, but definitive evidence for direct regulatory interactions has been lacking. Using ChIP-Seq on embryonic mouse hindlimbs, we have pinpointed the genome-wide location of Pitx1 binding sites during mouse hindlimb development and identified potential gene targets for Pitx1. We determined that Pitx1 binding is significantly enriched near genes involved in limb morphogenesis, including Tbx4, HoxC10, and HoxC11. Notably, Pitx1 is bound to the previously identified HLEA and HLEB hindlimb enhancers of the Tbx4 gene and to a newly identified Tbx2 hindlimb enhancer. Moreover, Pitx1 binding is significantly enriched on hindlimb relative to forelimb-specific cis-regulatory features that are differentially marked by H3K27ac. However, our analysis revealed that Pitx1 also strongly associates with many functionally verified limb enhancers that exhibit similar levels of activity in the embryonic mesenchyme of forelimbs and hindlimbs. We speculate that Pitx1 influences hindlimb morphology both through the activation of hindlimb-specific enhancers as well as through the hindlimb-specific modulation of enhancers that are active in both sets of limbs.
134. A subset of Drosophila Myc sites remain associated with mitotic chromosomes colocalized with insulator proteins. Yang Jingping, Sung Elizabeth, Donlin-Asp Paul G, Corces Victor G. Nat Commun 2013. 4:X. 1464. PMID 23403565 PMC ID PMC3573855
Myc has been characterized as a transcription factor that activates expression of genes involved in pluripotency and cancer, and as a component of the replication complex. Here we find that Myc is present at promoters and enhancers of Drosophila melanogaster genes during interphase. Myc colocalizes with Orc2, which is part of the prereplication complex, during G1. As is the case in mammals, Myc associates preferentially with paused genes, suggesting that it may also be involved in the release of RNA polymerase II from the promoter-proximal pausing in Drosophila. Interestingly, about 40% of Myc sites present in interphase persists during mitosis. None of the Myc mitotic sites correspond to enhancers, and only some correspond to promoters. The rest of the mitotic Myc sites overlap with binding sites for multiple insulator proteins that are also maintained in mitosis. These results suggest alternative mechanisms to explain the role of Myc in pluripotency and cancer.
135. Distinct isoforms of the Drosophila Brd4 homologue are present at enhancers, promoters and insulator sites. Kellner Wendy A, Van Bortle Kevin, Li Li, Ramos Edward, Takenaka Naomi, Corces Victor G. Nucleic Acids Res. 2013. 41:20. 9274-83. PMID 23945939 PMC ID PMC3814382
Brd4 is a double bromodomain protein that has been shown to interact with acetylated histones to regulate transcription by recruiting Positive Transcription Elongation Factor b to the promoter region. Brd4 is also involved in gene bookmarking during mitosis and is a therapeutic target for the treatment of acute myeloid leukemia. The Drosophila melanogaster Brd4 homologue is called Fs(1)h and, like its vertebrate counterpart, encodes different isoforms. We have used ChIP-seq to examine the genome-wide distribution of Fs(1)h isoforms. We are able to distinguish the Fs(1)h-L and Fs(1)h-S binding profiles and discriminate between the genomic locations of the two isoforms. Fs(1)h-S is present at enhancers and promoters and its amount parallels transcription levels. Correlations between the distribution of Fs(1)h-S and various forms of acetylated histones H3 and H4 suggest a preference for binding to H3K9acS10ph. Surprisingly, Fs(1)h-L is located at sites in the genome where multiple insulator proteins are also present. The results suggest that Fs(1)h-S may be responsible for the classical role assigned to this protein, whereas Fs(1)h-L may have a new and unexpected role in chromatin architecture by working in conjunction with insulator proteins to mediate intra- or inter-chromosome interactions.
136. Congenital myopathy is caused by mutation of HACD1. Muhammad Emad, Reish Orit, Ohno Yusuke, Scheetz Todd, Deluca Adam, Searby Charles, Regev Miriam, Benyamini Lilach, Fellig Yakov, Kihara Akio, Sheffield Val C, Parvari Ruti. Hum. Mol. Genet. 2013. 22:25. 5229-36. PMID 23933735 PMC ID PMC3842179
Congenital myopathies are heterogeneous inherited diseases of muscle characterized by a range of distinctive histologic abnormalities. We have studied a consanguineous family with congenital myopathy. Genome-wide linkage analysis and whole-exome sequencing identified a homozygous non-sense mutation in 3-hydroxyacyl-CoA dehydratase 1 (HACD1) in affected individuals. The mutation results in non-sense mediated decay of the HACD1 mRNA to 31% of control levels in patient muscle and completely abrogates the enzymatic activity of dehydration of 3-hydroxyacyl-CoA, the third step in the elongation of very long-chain fatty acids (VLCFAs). We describe clinical findings correlated with a deleterious mutation in a gene not previously known to be associated with congenital myopathy in humans. We suggest that the mutation in the HACD1 gene causes a reduction in the synthesis of VLCFAs, which are components of membrane lipids and participants in physiological processes, leading to congenital myopathy. These data indicate that HACD1 is necessary for muscle function.
137. Gene expression changes leading extreme alkaline tolerance in Amur ide (Leuciscus waleckii) inhabiting soda lake. Xu Jian, Li Qiang, Xu Liming, Wang Shaolin, Jiang Yanliang, Zhao Zixia, Zhang Yan, Li Jiongtang, Dong Chuanju, Xu Peng, Sun Xiaowen. BMC Genomics 2013. 14:X. 682. PMID 24094069 PMC ID PMC3852516
Amur ide (Leuciscus waleckii) is an economically and ecologically important cyprinid species in Northern Asia. The Dali Nor population living in the soda lake Dali Nor can adapt the extremely high alkalinity, providing us a valuable material to understand the adaptation mechanism against extreme environmental stress in teleost.
138. Non-exomic and synonymous variants in ABCA4 are an important cause of Stargardt disease. Braun Terry A, Mullins Robert F, Wagner Alex H, Andorf Jeaneen L, Johnston Rebecca M, Bakall Benjamin B, Deluca Adam P, Fishman Gerald A, Lam Byron L, Weleber Richard G, Cideciyan Artur V, Jacobson Samuel G, Sheffield Val C, Tucker Budd A, Stone Edwin M. Hum. Mol. Genet. 2013. 22:25. 5136-45. PMID 23918662 PMC ID PMC3842174
Mutations in ABCA4 cause Stargardt disease and other blinding autosomal recessive retinal disorders. However, sequencing of the complete coding sequence in patients with clinical features of Stargardt disease sometimes fails to detect one or both mutations. For example, among 208 individuals with clear clinical evidence of ABCA4 disease ascertained at a single institution, 28 had only one disease-causing allele identified in the exons and splice junctions of the primary retinal transcript of the gene. Haplotype analysis of these 28 probands revealed 3 haplotypes shared among ten families, suggesting that 18 of the 28 missing alleles were rare enough to be present only once in the cohort. We hypothesized that mutations near rare alternate splice junctions in ABCA4 might cause disease by increasing the probability of mis-splicing at these sites. Next-generation sequencing of RNA extracted from human donor eyes revealed more than a dozen alternate exons that are occasionally incorporated into the ABCA4 transcript in normal human retina. We sequenced the genomic DNA containing 15 of these minor exons in the 28 one-allele subjects and observed five instances of two different variations in the splice signals of exon 36.1 that were not present in normal individuals (P < 10(-6)). Analysis of RNA obtained from the keratinocytes of patients with these mutations revealed the predicted alternate transcript. This study illustrates the utility of RNA sequence analysis of human donor tissue and patient-derived cell lines to identify mutations that would be undetectable by exome sequencing.
139. Bulk segregant RNA-seq reveals expression and positional candidate genes and allele-specific expression for disease resistance against enteric septicemia of catfish. Wang Ruijia, Sun Luyang, Bao Lisui, Zhang Jiaren, Jiang Yanliang, Yao Jun, Song Lin, Feng Jianbin, Liu Shikai, Liu Zhanjiang. BMC Genomics 2013. 14:X. 929. PMID 24373586 PMC ID PMC3890627
The application of RNA-seq has accelerated gene expression profiling and identification of gene-associated SNPs in many species. However, the integrated studies of gene expression along with SNP mapping have been lacking. Coupling of RNA-seq with bulked segregant analysis (BSA) should allow correlation of expression patterns and associated SNPs with the phenotypes.
140. Whole-transcriptome shotgun sequencing (RNA-seq) screen reveals upregulation of cellobiose and motility operons of Lactobacillus ruminis L5 during growth on tetrasaccharides derived from barley ?-glucan. Lawley Blair, Sims Ian M, Tannock Gerald W. Appl. Environ. Microbiol. 2013. 79:18. 5661-9. PMID 23851085 PMC ID PMC3754151
Lactobacillus ruminis is an inhabitant of human bowels and bovine rumens. None of 10 isolates (three from bovine rumen, seven from human feces) of L. ruminis that were tested could utilize barley ?-glucan for growth. Seven of the strains of L. ruminis were, however, able to utilize tetrasaccharides (3-O-?-cellotriosyl-d-glucose [LDP4] or 4-O-?-laminaribiosyl-d-cellobiose [CDP4]) present in ?-glucan hydrolysates for growth. The tetrasaccharides were generated by the use of lichenase or cellulase, respectively. To learn more about the utilization of tetrasaccharides by L. ruminis, whole-transcriptome shotgun sequencing (RNA-seq) was tested as a transcriptional screen to detect altered gene expression when an autochthonous human strain (L5) was grown in medium containing CDP4. RNA-seq results were confirmed and extended by reverse transcription-quantitative PCR assays of selected genes in two upregulated operons when cells were grown as batch cultures in medium containing either CDP4 or LDP4. The cellobiose utilization operon had increased transcription, particularly in early growth phase, whereas the chemotaxis/motility operon was upregulated in late growth phase. Phenotypic changes were seen in relation to upregulation of chemotaxis/flagellar operons: flagella were rarely seen by electron microscopy on glucose-grown cells but cells cultured in tetrasaccharide medium were commonly flagellated. Chemotactic movement toward tetrasaccharides was demonstrated in capillary cultures. L. ruminis utilized 3-O-?-cellotriosyl-d-glucose released by ?-glucan hydrolysis due to bowel commensal Coprococcus sp., indicating that cross feeding of tetrasaccharide between bacteria could occur. Therefore, the RNA-seq screen and subsequent experiments had utility in revealing foraging attributes of gut commensal Lactobacillus ruminis.
141. CYP1B1, MYOC, and LTBP2 mutations in primary congenital glaucoma patients in the United States. Lim Sing-Hui, Tran-Viet Khanh-Nhat, Yanovitch Tammy L, Freedman Sharon F, Klemm Thomas, Call Whitney, Powell Caldwell, Ravichandran Ajay, Metlapally Ravikanth, Nading Erica B, Rozen Steve, Young Terri L. Am. J. Ophthalmol. 2013. 155:3. 508-517.e5. PMID 23218701 PMC ID PMC3736560
To screen primary congenital glaucoma patients in the United States for sequence variants within the CYP1B1, LTBP2, and MYOC genes using Sanger and whole exome sequencing.
142. Reovirus cell entry requires functional microtubules. Mainou Bernardo A, Zamora Paula F, Ashbrook Alison W, Dorset Daniel C, Kim Kwang S, Dermody Terence S. MBio 2013. 4:4. None. PMID 23820395 PMC ID PMC3705452
Mammalian reovirus binds to cell-surface glycans and junctional adhesion molecule A and enters cells by receptor-mediated endocytosis in a process dependent on ?1 integrin. Within the endocytic compartment, reovirus undergoes stepwise disassembly, allowing release of the transcriptionally active viral core into the cytoplasm. To identify cellular mediators of reovirus infectivity, we screened a library of small-molecule inhibitors for the capacity to block virus-induced cytotoxicity. In this screen, reovirus-induced cell killing was dampened by several compounds known to impair microtubule dynamics. Microtubule inhibitors were assessed for blockade of various stages of the reovirus life cycle. While these drugs did not alter reovirus cell attachment or internalization, microtubule inhibitors diminished viral disassembly kinetics with a concomitant decrease in infectivity. Reovirus virions colocalize with microtubules and microtubule motor dynein 1 during cell entry, and depolymerization of microtubules results in intracellular aggregation of viral particles. These data indicate that functional microtubules are required for proper sorting of reovirus virions following internalization and point to a new drug target for pathogens that use the endocytic pathway to invade host cells.
143. Poly(ADP-ribosyl)ation regulates insulator function and intrachromosomal interactions in Drosophila. Ong Chin-Tong, Van Bortle Kevin, Ramos Edward, Corces Victor G. Cell 2013. 155:1. 148-59. PMID 24055367 PMC ID PMC3816015
Insulators mediate inter- and intrachromosomal contacts to regulate enhancer-promoter interactions and establish chromosome domains. The mechanisms by which insulator activity can be regulated to orchestrate changes in the function and three-dimensional arrangement of the genome remain elusive. Here, we demonstrate that Drosophila insulator proteins are poly(ADP-ribosyl)ated and that mutation of the poly(ADP-ribose) polymerase (Parp) gene impairs their function. This modification is not essential for DNA occupancy of insulator DNA-binding proteins dCTCF and Su(Hw). However, poly(ADP-ribosyl)ation of K566 in CP190 promotes protein-protein interactions with other insulator proteins, association with the nuclear lamina, and insulator activity in vivo. Consistent with these findings, the nuclear clustering of CP190 complexes is disrupted in Parp mutant cells. Importantly, poly(ADP-ribosyl)ation facilitates intrachromosomal interactions between insulator sites measured by 4C. These data suggest that the role of insulators in organizing the three-dimensional architecture of the genome may be modulated by poly(ADP-ribosyl)ation.
144. Architectural protein subclasses shape 3D organization of genomes during lineage commitment. Phillips-Cremins Jennifer E, Sauria Michael E G, Sanyal Amartya, Gerasimova Tatiana I, Lajoie Bryan R, Bell Joshua S K, Ong Chin-Tong, Hookway Tracy A, Guo Changying, Sun Yuhua, Bland Michael J, Wagstaff William, Dalton Stephen, McDevitt Todd C, Sen Ranjan et al. Cell 2013. 153:6. 1281-95. PMID 23706625 PMC ID PMC3712340
Understanding the topological configurations of chromatin may reveal valuable insights into how the genome and epigenome act in concert to control cell fate during development. Here, we generate high-resolution architecture maps across seven genomic loci in embryonic stem cells and neural progenitor cells. We observe a hierarchy of 3D interactions that undergo marked reorganization at the submegabase scale during differentiation. Distinct combinations of CCCTC-binding factor (CTCF), Mediator, and cohesin show widespread enrichment in chromatin interactions at different length scales. CTCF/cohesin anchor long-range constitutive interactions that might form the topological basis for invariant subdomains. Conversely, Mediator/cohesin bridge short-range enhancer-promoter interactions within and between larger subdomains. Knockdown of Smc1 or Med12 in embryonic stem cells results in disruption of spatial architecture and downregulation of genes found in cohesin-mediated interactions. We conclude that cell-type-specific chromatin organization occurs at the submegabase scale and that architectural proteins shape the genome in hierarchical length scales.
145. RNA-Seq analysis reveals genes associated with resistance to Taura syndrome virus (TSV) in the Pacific white shrimp Litopenaeus vannamei. Sookruksawong Suchonma, Sun Fanyue, Liu Zhanjiang, Tassanakajon Anchalee. Dev. Comp. Immunol. 2013. 41:4. 523-33. PMID 23921257
Outbreak of Taura syndrome virus (TSV) is one of the major pathogens of the Pacific white shrimp Litopenaeus vannamei. Although selective breeding for improvement of TSV resistance in L. vannamei has been successfully developed and has led to a great benefit to the shrimp farming industry worldwide. The molecular mechanisms underlying the viral resistance in shrimp remain largely unknown. In the present study, we conducted the first transcriptomic profiling of host responses in hemolymph and hemocytes in order to identify the differentially expressed genes associated with resistance to TSV in L. vannamei. High-throughput RNA-Seq was employed, obtaining 193.6 and 171.2 million high-quality Illumina reads from TSV-resistant and susceptible L. vannamei lines respectively. A total of 61,937 contigs were generated with an average length of 546.26 bp. BLASTX-based gene annotation (E-value < 10(-5)) allowed the identification of 12,398 unique proteins against the NCBI non-redundant NR database. In addition, comparison of digital gene expression between resistant and susceptible strains revealed 1374 significantly differentially expressed contigs (representing 697 unigenes). Gene pathway analysis of the differentially expressed gene set highlighted several putative genes involved in the immune response activity including (1) pathogen/antigen recognition including immune regulator, adhesive protein and signal transducer; (2) coagulation; (3) proPO pathway cascade; (4) antioxidation; and (5) protease. The expression patterns of 22 differentially expressed genes involving immune response were validated by quantitative real-time RT-PCR (average correlation coefficients 0.94, p-value < 0.001). Our results provide valuable information on gene functions associated with resistance to TSV in L. vannamei.
146. RNA-Seq reveals expression signatures of genes involved in oxygen transport, protein synthesis, folding, and degradation in response to heat stress in catfish. Liu Shikai, Wang Xiuli, Sun Fanyue, Zhang Jiaren, Feng Jianbin, Liu Hong, Rajendran K V, Sun Luyang, Zhang Yu, Jiang Yanliang, Peatman Eric, Kaltenboeck Ludmilla, Kucuktas Huseyin, Liu Zhanjiang. Physiol. Genomics 2013. 45:12. 462-76. PMID 23632418
Temperature is one of the most prominent abiotic factors affecting ectotherms. Most fish species, as ectotherms, have extraordinary ability to deal with a wide range of temperature changes. While the molecular mechanism underlying temperature adaptation has long been of interest, it is still largely unexplored with fish. Understanding of the fundamental mechanisms conferring tolerance to temperature fluctuations is a topic of increasing interest as temperature may continue to rise as a result of global climate change. Catfish have a wide natural habitat and possess great plasticity in dealing with environmental variations in temperature. However, no studies have been conducted at the transcriptomic level to determine heat stress-induced gene expression. In the present study, we conducted an RNA-Seq analysis to identify heat stress-induced genes in catfish at the transcriptome level. Expression analysis identified a total of 2,260 differentially expressed genes with a cutoff of twofold change. qRT-PCR validation suggested the high reliability of the RNA-Seq results. Gene ontology, enrichment, and pathway analyses were conducted to gain insight into physiological and gene pathways. Specifically, genes involved in oxygen transport, protein folding and degradation, and metabolic process were highly induced, while general protein synthesis was dramatically repressed in response to the lethal temperature stress. This is the first RNA-Seq-based expression study in catfish in response to heat stress. The candidate genes identified should be valuable for further targeted studies on heat tolerance, thereby assisting the development of heat-tolerant catfish lines for aquaculture.
147. Dynamic changes in the genomic localization of DNA replication-related element binding factor during the cell cycle. Gurudatta B V, Yang Jingping, Van Bortle Kevin, Donlin-Asp Paul G, Corces Victor G. Cell Cycle 2013. 12:10. 1605-15. PMID 23624840 PMC ID PMC3680540
DREF was first characterized for its role in the regulation of transcription of genes encoding proteins involved in DNA replication and found to interact with sequences similar to the DNA recognition motif of the BEAF-32 insulator protein. Insulators are DNA-protein complexes that mediate intra- and inter-chromosome interactions. Several DNA-binding insulator proteins have been described in Drosophila, including BEAF-32, dCTCF and Su(Hw). Here we find that DREF and BEAF-32 co-localize at the same genomic sites, but their enrichment shows an inverse correlation. Furthermore, DREF co-localizes in the genome with other insulator proteins, suggesting that the function of this protein may require components of Drosophila insulators. This is supported by the finding that mutations in insulator proteins modulate DREF-induced cell proliferation. DREF persists bound to chromatin during mitosis at a subset of sites where it also co-localizes with dCTCF, BEAF-32 and CP190. These sites are highly enriched for sites where Orc2 and Mcm2 are present during interphase and at the borders of topological domains of chromosomes defined by Hi-C. The results suggest that DREF and insulator proteins may help maintain chromosome organization during the cell cycle and mark a subset of genomic sites for the assembly of pre-replication complexes and gene bookmarking during the M/G1 transition.
148. Variants of anterior segment dysgenesis and cerebral involvement in a large family with a novel COL4A1 mutation. Rødahl Eyvind, Knappskog Per M, Majewski Jacek, Johansson Stefan, Telstad Wenche, Kråkenes Jostein, Boman Helge. Am. J. Ophthalmol. 2013. 155:5. 946-53. PMID 23394911
To investigate the diverse ocular manifestations and identify the causative mutation in a large family with autosomal dominant anterior segment dysgenesis accompanied in some individuals by cerebral vascular disease.
149. Differential DNA methylation with age displays both common and dynamic features across human tissues that are influenced by CpG landscape. Day Kenneth, Waite Lindsay L, Thalacker-Mercer Anna, West Andrew, Bamman Marcas M, Brooks James D, Myers Richard M, Absher Devin. Genome Biol. 2013. 14:9. R102. PMID 24034465 PMC ID PMC4053985
DNA methylation is an epigenetic modification that changes with age in human tissues, although the mechanisms and specificity of this process are still poorly understood. We compared CpG methylation changes with age across 283 human blood, brain, kidney, and skeletal muscle samples using methylation arrays to identify tissue-specific age effects.
150. SHORT syndrome with partial lipodystrophy due to impaired phosphatidylinositol 3 kinase signaling. Chudasama Kishan Kumar, Winnay Jonathon, Johansson Stefan, Claudi Tor, König Rainer, Haldorsen Ingfrid, Johansson Bente, Woo Ju Rang, Aarskog Dagfinn, Sagen Jørn V, Kahn C Ronald, Molven Anders, Njølstad Pål Rasmus. Am. J. Hum. Genet. 2013. 93:1. 150-7. PMID 23810379 PMC ID PMC3710758
The phosphatidylinositol 3 kinase (PI3K) pathway regulates fundamental cellular processes such as metabolism, proliferation, and survival. A central component in this pathway is the p85? regulatory subunit, encoded by PIK3R1. Using whole-exome sequencing, we identified a heterozygous PIK3R1 mutation (c.1945C>T [p.Arg649Trp]) in two unrelated families affected by partial lipodystrophy, low body mass index, short stature, progeroid face, and Rieger anomaly (SHORT syndrome). This mutation led to impaired interaction between p85? and IRS-1 and reduced AKT-mediated insulin signaling in fibroblasts from affected subjects and in reconstituted Pik3r1-knockout preadipocytes. Normal PI3K activity is critical for adipose differentiation and insulin signaling; the mutated PIK3R1 therefore provides a unique link among lipodystrophy, growth, and insulin signaling.
151. Basal polarization of the mucosal compartment in Flavobacterium columnare susceptible and resistant channel catfish (Ictalurus punctatus). Peatman Eric, Li Chao, Peterson Brian C, Straus David L, Farmer Bradley D, Beck Benjamin H. Mol. Immunol. 2013. 56:4. 317-27. PMID 23895942
The freshwater bacterial pathogen, Flavobacterium columnare, infects a variety of ornamental and farmed fish species worldwide through mucosal attachment points on the gill and skin. While previous studies have demonstrated a chemotactic response of F. columnare to fish mucus, little is known about how host gill mucosal molecular and cellular constituents may impact rates of adhesion, tissue invasion, and ultimately, mortality. Here, we describe the use of RNA-seq to profile gill expression differences between channel catfish (Ictalurus punctatus) differing in their susceptibility to F. columnare both basally (before infection) and at three early timepoints post-infection (1 h, 2 h, and 8 h). After sequencing and de novo assembly of over 350 million 100 base-pair transcript reads, between group comparisons revealed 1714 unique genes differentially expressed greater than 1.5-fold at one or more timepoints. In the large dataset, we focused our analysis on basal differential expression between resistant and susceptible catfish as these genes could potentially reveal genetic and/or environmental factors linked with differential rates of infection. A number of critical innate immune components including iNOS2b, lysozyme C, IL-8, and TNF-alpha were constitutively higher in resistant catfish gill, while susceptible fish showed high expression levels of secreted mucin forms, a rhamnose-binding lectin previously linked to susceptibility, and mucosal immune factors such as CD103 and IL-17. Taken together, the immune and mucin profiles obtained by RNA-seq suggest a basal polarization in the gill mucosa, with susceptible fish possessing a putative mucosecretory, toleragenic phenotype which may predispose them to F. columnare infection.
152. ZMYND10 is mutated in primary ciliary dyskinesia and interacts with LRRC6. Zariwala Maimoona A, Gee Heon Yung, Kurkowiak Ma?gorzata, Al-Mutairi Dalal A, Leigh Margaret W, Hurd Toby W, Hjeij Rim, Dell Sharon D, Chaki Moumita, Dougherty Gerard W, Adan Mohamed, Spear Philip C, Esteve-Rudd Julian, Loges Niki T, Rosenfeld Margaret et al. Am. J. Hum. Genet. 2013. 93:2. 336-45. PMID 23891469 PMC ID PMC3738827
Defects of motile cilia cause primary ciliary dyskinesia (PCD), characterized by recurrent respiratory infections and male infertility. Using whole-exome resequencing and high-throughput mutation analysis, we identified recessive biallelic mutations in ZMYND10 in 14 families and mutations in the recently identified LRRC6 in 13 families. We show that ZMYND10 and LRRC6 interact and that certain ZMYND10 and LRRC6 mutations abrogate the interaction between the LRRC6 CS domain and the ZMYND10 C-terminal domain. Additionally, ZMYND10 and LRRC6 colocalize with the centriole markers SAS6 and PCM1. Mutations in ZMYND10 result in the absence of the axonemal protein components DNAH5 and DNALI1 from respiratory cilia. Animal models support the association between ZMYND10 and human PCD, given that zmynd10 knockdown in zebrafish caused ciliary paralysis leading to cystic kidneys and otolith defects and that knockdown in Xenopus interfered with ciliogenesis. Our findings suggest that a cytoplasmic protein complex containing ZMYND10 and LRRC6 is necessary for motile ciliary function.
153. The venom-gland transcriptome of the eastern coral snake (Micrurus fulvius) reveals high venom complexity in the intragenomic evolution of venoms. Margres Mark J, Aronow Karalyn, Loyacano Jacob, Rokyta Darin R. BMC Genomics 2013. 14:X. 531. PMID 23915248 PMC ID PMC3750283
Snake venom is shaped by the ecology and evolution of venomous species, and signals of positive selection in toxins have been consistently documented, reflecting the role of venoms as an ecologically critical phenotype. New World coral snakes (Elapidae) are represented by three genera and over 120 species and subspecies that are capable of causing significant human morbidity and mortality, yet coral-snake venom composition is poorly understood in comparison to that of Old World elapids. High-throughput sequencing is capable of identifying thousands of loci, while providing characterizations of expression patterns and the molecular evolutionary forces acting within the venom gland.
154. Type 1 and type 2 strains of Mycoplasma pneumoniae form different biofilms. Simmons Warren L, Daubenspeck James M, Osborne John D, Balish Mitchell F, Waites Ken B, Dybvig Kevin. Microbiology (Reading, Engl.) 2013. 159:Pt 4. 737-47. PMID 23412845 PMC ID PMC4036059
Several mycoplasma species have been shown to form biofilms that confer resistance to antimicrobials and which may affect the host immune system, thus making treatment and eradication of the pathogens difficult. The present study shows that the biofilms formed by two strains of the human pathogen Mycoplasma pneumoniae differ quantitatively and qualitatively. Compared with strain UAB PO1, strain M129 grows well but forms biofilms that are less robust, with towers that are less smooth at the margins. A polysaccharide containing N-acetylglucosamine is secreted by M129 into the culture medium but found in tight association with the cells of UAB PO1. The polysaccharide may have a role in biofilm formation, contributing to differences in virulence, chronicity and treatment outcome between strains of M. pneumoniae. The UAB PO1 genome was found to be that of a type 2 strain of M. pneumoniae, whereas M129 is type 1. Examination of other M. pneumoniae isolates suggests that the robustness of the biofilm correlates with the strain type.
155. Gene signature distinguishes patients with chronic ulcerative colitis harboring remote neoplastic lesions. Pekow Joel, Dougherty Urszula, Huang Yong, Gometz Edward, Nathanson Jeff, Cohen Greg, Levy Shawn, Kocherginsky Masha, Venu Nanda, Westerhoff Maria, Hart John, Noffsinger Amy E, Hanauer Stephen B, Hurst Roger D, Fichera Alessandro et al. Inflamm. Bowel Dis. 2013. 19:3. 461-70. PMID 23388545 PMC ID PMC3836269
Individuals with ulcerative colitis (UC) are at increased risk for colorectal cancer. The standard method of surveillance for neoplasia in UC by colonoscopy is invasive and can miss flat lesions. We sought to identify a gene expression signature in nondysplastic mucosa without active inflammation that could serve as a marker for remote neoplastic lesions.
156. ARHGDIA mutations cause nephrotic syndrome via defective RHO GTPase signaling. Gee Heon Yung, Saisawat Pawaree, Ashraf Shazia, Hurd Toby W, Vega-Warner Virginia, Fang Humphrey, Beck Bodo B, Gribouval Olivier, Zhou Weibin, Diaz Katrina A, Natarajan Sivakumar, Wiggins Roger C, Lovric Svjetlana, Chernin Gil, Schoeb Dominik S et al. J. Clin. Invest. 2013. 123:8. 3243-53. PMID 23867502 PMC ID PMC3726174
Nephrotic syndrome (NS) is divided into steroid-sensitive (SSNS) and -resistant (SRNS) variants. SRNS causes end-stage kidney disease, which cannot be cured. While the disease mechanisms of NS are not well understood, genetic mapping studies suggest a multitude of unknown single-gene causes. We combined homozygosity mapping with whole-exome resequencing and identified an ARHGDIA mutation that causes SRNS. We demonstrated that ARHGDIA is in a complex with RHO GTPases and is prominently expressed in podocytes of rat glomeruli. ARHGDIA mutations (R120X and G173V) from individuals with SRNS abrogated interaction with RHO GTPases and increased active GTP-bound RAC1 and CDC42, but not RHOA, indicating that RAC1 and CDC42 are more relevant to the pathogenesis of this SRNS variant than RHOA. Moreover, the mutations enhanced migration of cultured human podocytes; however, enhanced migration was reversed by treatment with RAC1 inhibitors. The nephrotic phenotype was recapitulated in arhgdia-deficient zebrafish. RAC1 inhibitors were partially effective in ameliorating arhgdia-associated defects. These findings identify a single-gene cause of NS and reveal that RHO GTPase signaling is a pathogenic mediator of SRNS.
157. Significance of expression of ITGA5 and its splice variants in acute myeloid leukemia: a report from the Children's Oncology Group. Walter Roland B, Laszlo George S, Alonzo Todd A, Gerbing Robert B, Levy Shawn, Fitzgibbon Matthew P, Gudgeon Chelsea J, Ries Rhonda E, Harrington Kimberly H, Raimondi Susana C, Hirsch Betsy A, Gamis Alan S, W McIntosh Martin, Meshinchi Soheil. Am. J. Hematol. 2013. 88:8. 694-702. PMID 23686445 PMC ID PMC3757130
Acute myeloid leukemia (AML) encompasses a heterogeneous group of diseases, and novel biomarkers for risk refinement and stratification are needed to optimize patient care. To identify novel risk factors, we performed transcriptome sequencing on 68 diagnostic AML samples and identified 2 transcript variants (-E2 and -E2/3) of the ?-subunit (ITGA5) of the very late antigen-5 integrin. We then quantified expression of ITGA5 and these splice variants in specimens from participants of the AAML03P1 trial. We found no association between ITGA5 expression and clinical outcome. In contrast, patients with the highest relative expression (Q4) of the -E2/3 ITGA5 splice variant less likely had low-risk disease than Q1-3 patients (21% vs. 38%, P = 0.027). Q4 patients had worse response to chemotherapy with a higher proportion having persistent minimal residual disease (50% vs. 23%, P = 0.003) and inferior overall survival (at 5 years: 48% vs. 67%, P = 0.015); the latter association was limited to low-risk patients (Q4 vs. Q1-3: 56% vs. 85%, P = 0.043) and was not seen in standard-risk (51% vs. 60%, P = 0.340) or high-risk (33% vs. 38%, P = 0.952) patients. Our exploratory studies indicate that transcriptome sequencing is useful for biomarker discovery, as exemplified by the identification of ITGA5 -E2/3 splice variant as potential novel adverse prognostic marker for low-risk AML that, if confirmed, could serve to further risk-stratify this patient subset.
158. ADCK4 mutations promote steroid-resistant nephrotic syndrome through CoQ10 biosynthesis disruption. Ashraf Shazia, Gee Heon Yung, Woerner Stephanie, Xie Letian X, Vega-Warner Virginia, Lovric Svjetlana, Fang Humphrey, Song Xuewen, Cattran Daniel C, Avila-Casado Carmen, Paterson Andrew D, Nitschké Patrick, Bole-Feysot Christine, Cochat Pierre, Esteve-Rudd Julian et al. J. Clin. Invest. 2013. 123:12. 5179-89. PMID 24270420 PMC ID PMC3859425
Identification of single-gene causes of steroid-resistant nephrotic syndrome (SRNS) has furthered the understanding of the pathogenesis of this disease. Here, using a combination of homozygosity mapping and whole human exome resequencing, we identified mutations in the aarF domain containing kinase 4 (ADCK4) gene in 15 individuals with SRNS from 8 unrelated families. ADCK4 was highly similar to ADCK3, which has been shown to participate in coenzyme Q10 (CoQ10) biosynthesis. Mutations in ADCK4 resulted in reduced CoQ10 levels and reduced mitochondrial respiratory enzyme activity in cells isolated from individuals with SRNS and transformed lymphoblasts. Knockdown of adck4 in zebrafish and Drosophila recapitulated nephrotic syndrome-associated phenotypes. Furthermore, ADCK4 was expressed in glomerular podocytes and partially localized to podocyte mitochondria and foot processes in rat kidneys and cultured human podocytes. In human podocytes, ADCK4 interacted with members of the CoQ10 biosynthesis pathway, including COQ6, which has been linked with SRNS and COQ7. Knockdown of ADCK4 in podocytes resulted in decreased migration, which was reversed by CoQ10 addition. Interestingly, a patient with SRNS with a homozygous ADCK4 frameshift mutation had partial remission following CoQ10 treatment. These data indicate that individuals with SRNS with mutations in ADCK4 or other genes that participate in CoQ10 biosynthesis may be treatable with CoQ10.
159. Whole exome sequencing identifies a mutation for a novel form of corneal intraepithelial dyskeratosis. Soler Vincent José, Tran-Viet Khanh-Nhat, Galiacy Stéphane D, Limviphuvadh Vachiranee, Klemm Thomas Patrick, St Germain Elizabeth, Fournié Pierre R, Guillaud Céline, Maurer-Stroh Sebastian, Hawthorne Felicia, Suarez Cyrielle, Kantelip Bernadette, Afshari Natalie A, Creveaux Isabelle, Luo Xiaoyan et al. J. Med. Genet. 2013. 50:4. 246-54. PMID 23349227 PMC ID PMC4115150
Corneal intraepithelial dyskeratosis is an extremely rare condition. The classical form, affecting Native American Haliwa-Saponi tribe members, is called hereditary benign intraepithelial dyskeratosis (HBID). Herein, we present a new form of corneal intraepithelial dyskeratosis for which we identified the causative gene by using deep sequencing technology.
160. A neurodegeneration-specific gene-expression signature of acutely isolated microglia from an amyotrophic lateral sclerosis mouse model. Chiu Isaac M, Morimoto Emiko T A, Goodarzi Hani, Liao Jennifer T, O'Keeffe Sean, Phatnani Hemali P, Muratet Michael, Carroll Michael C, Levy Shawn, Tavazoie Saeed, Myers Richard M, Maniatis Tom. Cell Rep 2013. 4:2. 385-401. PMID 23850290
Microglia are resident immune cells of the CNS that are activated by infection, neuronal injury, and inflammation. Here, we utilize flow cytometry and deep RNA sequencing of acutely isolated spinal cord microglia to define their activation in vivo. Analysis of resting microglia identified 29 genes that distinguish microglia from other CNS cells and peripheral macrophages/monocytes. We then analyzed molecular changes in microglia during neurodegenerative disease activation using the SOD1(G93A) mouse model of amyotrophic lateral sclerosis (ALS). We found that SOD1(G93A) microglia are not derived from infiltrating monocytes, and that both potentially neuroprotective and toxic factors, including Alzheimer's disease genes, are concurrently upregulated. Mutant microglia differed from SOD1(WT), lipopolysaccharide-activated microglia, and M1/M2 macrophages, defining an ALS-specific phenotype. Concurrent messenger RNA/fluorescence-activated cell sorting analysis revealed posttranscriptional regulation of microglia surface receptors and T cell-associated changes in the transcriptome. These results provide insights into microglia biology and establish a resource for future studies of neuroinflammation.
161. Genetic heterogeneity of diffuse large B-cell lymphoma. Zhang Jenny, Grubor Vladimir, Love Cassandra L, Banerjee Anjishnu, Richards Kristy L, Mieczkowski Piotr A, Dunphy Cherie, Choi William, Au Wing Yan, Srivastava Gopesh, Lugar Patricia L, Rizzieri David A, Lagoo Anand S, Bernal-Mizrachi Leon, Mann Karen P et al. Proc. Natl. Acad. Sci. U.S.A. 2013. 110:4. 1398-403. PMID 23292937 PMC ID PMC3557051
Diffuse large B-cell lymphoma (DLBCL) is the most common form of lymphoma in adults. The disease exhibits a striking heterogeneity in gene expression profiles and clinical outcomes, but its genetic causes remain to be fully defined. Through whole genome and exome sequencing, we characterized the genetic diversity of DLBCL. In all, we sequenced 73 DLBCL primary tumors (34 with matched normal DNA). Separately, we sequenced the exomes of 21 DLBCL cell lines. We identified 322 DLBCL cancer genes that were recurrently mutated in primary DLBCLs. We identified recurrent mutations implicating a number of known and not previously identified genes and pathways in DLBCL including those related to chromatin modification (ARID1A and MEF2B), NF-?B (CARD11 and TNFAIP3), PI3 kinase (PIK3CD, PIK3R1, and MTOR), B-cell lineage (IRF8, POU2F2, and GNA13), and WNT signaling (WIF1). We also experimentally validated a mutation in PIK3CD, a gene not previously implicated in lymphomas. The patterns of mutation demonstrated a classic long tail distribution with substantial variation of mutated genes from patient to patient and also between published studies. Thus, our study reveals the tremendous genetic heterogeneity that underlies lymphomas and highlights the need for personalized medicine approaches to treating these patients.
162. RNA-seq analysis of mucosal immune responses reveals signatures of intestinal barrier disruption and pathogen entry following Edwardsiella ictaluri infection in channel catfish, Ictalurus punctatus. Li Chao, Zhang Yu, Wang Ruijia, Lu Jianguo, Nandi Samiran, Mohanty Sriprakash, Terhune Jeffery, Liu Zhanjiang, Peatman Eric. Fish Shellfish Immunol. 2012. 32:5. 816-27. PMID 22366064
The mucosal surfaces of fish (gill, skin, gastrointestinal tract) are important sites of bacterial exposure and host defense mechanisms. In mammalian systems, the intestinal epithelium is well characterized as both a selectively permeable barrier regulated by junctional proteins and as a primary site of infection for a number of enteric pathogens including viruses, bacteria, and parasites. The causative bacterium of enteric septicemia of catfish, Edwardsiella ictaluri, is believed to gain entry through the intestinal epithelium, with previous research using a rat intestinal epithelial cell line (IEC-6) indicating actin polymerization and receptor-mediated endocytosis as potential mechanisms of uptake. Here, we utilized high-throughput RNA-seq to characterize the role of the intestinal epithelial barrier following E. ictaluri challenge. A total of 197.6 million reads were obtained and assembled into 176,481 contigs with an average length of 893.7 bp and N50 of 1676 bp. The assembled contigs contained 14,457 known unigenes, including 2719 genes not previously identified in other catfish transcriptome studies. Comparison of digital gene expression between challenged and control samples revealed 1633 differentially expressed genes at 3 h, 24 h, and 3 day following exposure. Gene pathway analysis of the differentially expressed gene set indicated the centrality of actin cytoskeletal polymerization/remodelling and junctional regulation in pathogen entry and subsequent inflammatory responses. The expression patterns of fifteen differentially expressed genes related to intestinal epithelial barrier dysfunction were validated by quantitative real-time RT-PCR (average correlation coeff. 0.92, p < 0.001). Our results set a foundation for future studies comparing mechanisms of pathogen entry and mucosal immunity across several important catfish pathogens including E. ictaluri, Edwardsiellatarda, Flavobacterium columnare, and virulent atypical Aeromonas hydrophila. Understanding of molecular mechanisms of pathogen entry during infection will provide insight into strategies for selection of resistant catfish brood stocks against various diseases.
163. Steps to ensure accuracy in genotype and SNP calling from Illumina sequencing data. Liu Qi, Guo Yan, Li Jiang, Long Jirong, Zhang Bing, Shyr Yu. BMC Genomics 2012. 13 Supp:X. S8. PMID 23281772 PMC ID PMC3535703
Accurate calling of SNPs and genotypes from next-generation sequencing data is an essential prerequisite for most human genetics studies. A number of computational steps are required or recommended when translating the raw sequencing data into the final calls. However, whether each step does contribute to the performance of variant calling and how it affects the accuracy still remain unclear, making it difficult to select and arrange appropriate steps to derive high quality variants from different sequencing data. In this study, we made a systematic assessment of the relative contribution of each step to the accuracy of variant calling from Illumina DNA sequencing data.
164. The use of next generation sequencing technology to study the effect of radiation therapy on mitochondrial DNA mutation. Guo Yan, Cai Qiuyin, Samuels David C, Ye Fei, Long Jirong, Li Chung-I, Winther Jeanette F, Tawn E Janet, Stovall Marilyn, Lähteenmäki Päivi, Malila Nea, Levy Shawn, Shaffer Christian, Shyr Yu, Shu Xiao-Ou et al. Mutat. Res. 2012. 744:2. 154-60. PMID 22387842 PMC ID PMC3354959
The human mitochondrial genome has an exclusively maternal mode of inheritance. Mitochondrial DNA (mtDNA) is particularly vulnerable to environmental insults due in part to an underdeveloped DNA repair system, limited to base excision and homologous recombination repair. Radiation exposure to the ovaries may cause mtDNA mutations in oocytes, which may in turn be transmitted to offspring. We hypothesized that the children of female cancer survivors who received radiation therapy may have an increased rate of mtDNA heteroplasmy mutations, which conceivably could increase their risk of developing cancer and other diseases. We evaluated 44 DNA blood samples from 17 Danish and 1 Finnish families (18 mothers and 26 children). All mothers had been treated for cancer as children and radiation doses to their ovaries were determined based on medical records and computational models. DNA samples were sequenced for the entire mitochondrial genome using the Illumina GAII system. Mother's age at sample collection was positively correlated with mtDNA heteroplasmy mutations. There was evidence of heteroplasmy inheritance in that 9 of the 18 families had at least one child who inherited at least one heteroplasmy site from his or her mother. No significant difference in single nucleotide polymorphisms between mother and offspring, however, was observed. Radiation therapy dose to ovaries also was not significantly associated with the heteroplasmy mutation rate among mothers and children. No evidence was found that radiotherapy for pediatric cancer is associated with the mitochondrial genome mutation rate in female cancer survivors and their children.
165. A high-throughput fluorescence polarization anisotropy assay for the 70N domain of replication protein A. Souza-Fagundes Elaine M, Frank Andreas O, Feldkamp Michael D, Dorset Daniel C, Chazin Walter J, Rossanese Olivia W, Olejniczak Edward T, Fesik Stephen W. Anal. Biochem. 2012. 421:2. 742-9. PMID 22197419 PMC ID PMC3274598
Replication protein A (RPA) interacts with multiple checkpoint proteins and promotes signaling through the ATR kinase, a key regulator of checkpoint pathways in the mammalian response to DNA damage. In cancer cells, increased DNA repair activity contributes to resistance to chemotherapy. Therefore, small molecules that block binding of checkpoint proteins to RPA may inhibit the DNA damage response and, thus, sensitize cancer cells to DNA-damaging agents. Here we report on the development of a homogeneous, high-throughput fluorescence polarization assay for identifying compounds that block the critical protein-protein interaction site in the basic cleft of the 70N domain of RPA (RPA70N). A fluorescein isothiocyanate (FITC)-labeled peptide derived from the ATR cofactor, ATRIP, was used as a probe in the binding assay. The ability of the assay to accurately detect relevant ligands was confirmed using peptides derived from ATRIP, RAD9, MRE11, and p53. The assay was validated for use in high-throughput screening using the Spectrum collection of 2000 compounds. The FPA assay was performed with a Z' factor of ? 0.76 in a 384-well format and identified several compounds capable of inhibiting the RPA70N binding interface.
166. De novo gene mutations highlight patterns of genetic and neural complexity in schizophrenia. Xu Bin, Ionita-Laza Iuliana, Roos J Louw, Boone Braden, Woodrick Scarlet, Sun Yan, Levy Shawn, Gogos Joseph A, Karayiorgou Maria. Nat. Genet. 2012. 44:12. 1365-9. PMID 23042115 PMC ID PMC3556813
To evaluate evidence for de novo etiologies in schizophrenia, we sequenced at high coverage the exomes of families recruited from two populations with distinct demographic structures and history. We sequenced a total of 795 exomes from 231 parent-proband trios enriched for sporadic schizophrenia cases, as well as 34 unaffected trios. We observed in cases an excess of de novo nonsynonymous single-nucleotide variants as well as a higher prevalence of gene-disruptive de novo mutations relative to controls. We found four genes (LAMA2, DPYD, TRRAP and VPS39) affected by recurrent de novo events within or across the two populations, which is unlikely to have occurred by chance. We show that de novo mutations affect genes with diverse functions and developmental profiles, but we also find a substantial contribution of mutations in genes with higher expression in early fetal life. Our results help define the genomic and neural architecture of schizophrenia.
167. Patterns and rates of exonic de novo mutations in autism spectrum disorders. Neale Benjamin M, Kou Yan, Liu Li, Ma'ayan Avi, Samocha Kaitlin E, Sabo Aniko, Lin Chiao-Feng, Stevens Christine, Wang Li-San, Makarov Vladimir, Polak Paz, Yoon Seungtai, Maguire Jared, Crawford Emily L, Campbell Nicholas G et al. Nature 2012. 485:7397. 242-5. PMID 22495311 PMC ID PMC3613847
Autism spectrum disorders (ASD) are believed to have genetic and environmental origins, yet in only a modest fraction of individuals can specific causes be identified. To identify further genetic risk factors, here we assess the role of de novo mutations in ASD by sequencing the exomes of ASD cases and their parents (n = 175 trios). Fewer than half of the cases (46.3%) carry a missense or nonsense de novo variant, and the overall rate of mutation is only modestly higher than the expected rate. In contrast, the proteins encoded by genes that harboured de novo missense or nonsense mutations showed a higher degree of connectivity among themselves and to previous ASD genes as indexed by protein-protein interaction screens. The small increase in the rate of de novo events, when taken together with the protein interaction results, are consistent with an important but limited role for de novo point mutations in ASD, similar to that documented for de novo copy number variants. Genetic models incorporating these data indicate that most of the observed de novo events are unconnected to ASD; those that do confer risk are distributed across many genes and are incompletely penetrant (that is, not necessarily sufficient for disease). Our results support polygenic models in which spontaneous coding mutations in any of a large number of genes increases risk by 5- to 20-fold. Despite the challenge posed by such models, results from de novo events and a large parallel case-control study provide strong evidence in favour of CHD8 and KATNAL2 as genuine autism risk factors.
168. Exome sequencing and genetic testing for MODY. Johansson Stefan, Irgens Henrik, Chudasama Kishan K, Molnes Janne, Aerts Jan, Roque Francisco S, Jonassen Inge, Levy Shawn, Lima Kari, Knappskog Per M, Bell Graeme I, Molven Anders, Njølstad Pål R. PLoS ONE 2012. 7:5. e38050. PMID 22662265 PMC ID PMC3360646
Genetic testing for monogenic diabetes is important for patient care. Given the extensive genetic and clinical heterogeneity of diabetes, exome sequencing might provide additional diagnostic potential when standard Sanger sequencing-based diagnostics is inconclusive.
169. Exome capture reveals ZNF423 and CEP164 mutations, linking renal ciliopathies to DNA damage response signaling. Chaki Moumita, Airik Rannar, Ghosh Amiya K, Giles Rachel H, Chen Rui, Slaats Gisela G, Wang Hui, Hurd Toby W, Zhou Weibin, Cluckey Andrew, Gee Heon Yung, Ramaswami Gokul, Hong Chen-Jei, Hamilton Bruce A, Cervenka Igor et al. Cell 2012. 150:3. 533-48. PMID 22863007 PMC ID PMC3433835
Nephronophthisis-related ciliopathies (NPHP-RC) are degenerative recessive diseases that affect kidney, retina, and brain. Genetic defects in NPHP gene products that localize to cilia and centrosomes defined them as "ciliopathies." However, disease mechanisms remain poorly understood. Here, we identify by whole-exome resequencing, mutations of MRE11, ZNF423, and CEP164 as causing NPHP-RC. All three genes function within the DNA damage response (DDR) pathway. We demonstrate that, upon induced DNA damage, the NPHP-RC proteins ZNF423, CEP164, and NPHP10 colocalize to nuclear foci positive for TIP60, known to activate ATM at sites of DNA damage. We show that knockdown of CEP164 or ZNF423 causes sensitivity to DNA damaging agents and that cep164 knockdown in zebrafish results in dysregulated DDR and an NPHP-RC phenotype. Our findings link degenerative diseases of the kidney and retina, disorders of increasing prevalence, to mechanisms of DDR.
170. Increased protein-coding mutations in the mitochondrial genome of African American women with preeclampsia. Ding David, Scott Nicole M, Thompson Emma E, Chaiworapongsa Tinnakorn, Torres Raul, Billstrand Christine, Murray Kathleen, Dexheimer Phillip J, Ismail Mahmoud, Kay Helen, Levy Shawn, Romero Roberto, Lindheimer Marshall D, Nicolae Dan L, Ober Carole. Reprod Sci 2012. 19:12. 1343-51. PMID 22902742 PMC ID PMC4046444
Preeclampsia occurs more frequently in women of African ancestry. The cause of this hypertensive complication is unclear, but placental oxidative stress may play a role. Because mitochondria are the major sites of oxidative phosphorylation, we hypothesized that placentas of preeclamptic pregnancies harbor mitochondrial DNA (mtDNA) mutations. Next-generation sequencing of placental mtDNA in African American preeclamptics (N = 30) and controls (N = 38) from Chicago revealed significant excesses in preeclamptics of nonsynonymous substitutions in protein-coding genes and mitochondrially encoded nicotinamide adenine dinucleotide dehydrogenase 5 gene and an increase in the substitution rate (P = .0001). Moreover, 88% of preeclamptics and 53% of controls carried at least one nonsynonymous substitution (P = .005; odds ratio [OR] = 6.36, 95% confidence interval [CI]: 1.5-39.1). These results were not replicated in a sample of African American preeclamptics (N = 162) and controls (N = 171) from Detroit. Differences in study design and heterogeneity may account for this lack of replication. Nonsynonymous substitutions in mtDNA may be risk factors for preeclampsia in some African American women, but additional studies are required to establish this relationship.
171. The BEAF-32 insulator coordinates genome organization and function during the evolution of Drosophila species. Yang Jingping, Ramos Edward, Corces Victor G. Genome Res. 2012. 22:11. 2199-207. PMID 22895281 PMC ID PMC3483549
Understanding the relationship between genome organization and expression is central to understanding genome function. Closely apposed genes in a head-to-head orientation share the same upstream region and are likely to be coregulated. Here we identify the Drosophila BEAF-32 insulator as a cis regulatory element separating close head-to-head genes with different transcription regulation modes. We then compare the binding landscapes of the BEAF-32 insulator protein in four different Drosophila genomes and highlight the evolutionarily conserved presence of this protein between close adjacent genes. We find that changes in binding of BEAF-32 to sites in the genome of different Drosophila species correlate with alterations in genome organization caused by DNA rearrangements or genome size expansion. The cross-talk between BEAF-32 genomic distribution and genome organization contributes to new gene-expression profiles, which in turn translate into specific and distinct phenotypes. The results suggest a mechanism for the establishment of differences in transcription patterns during evolution.
172. The effect of strand bias in Illumina short-read sequencing data. Guo Yan, Li Jiang, Li Chung-I, Long Jirong, Samuels David C, Shyr Yu. BMC Genomics 2012. 13:X. 666. PMID 23176052 PMC ID PMC3532123
When using Illumina high throughput short read data, sometimes the genotype inferred from the positive strand and negative strand are significantly different, with one homozygous and the other heterozygous. This phenomenon is known as strand bias. In this study, we used Illumina short-read sequencing data to evaluate the effect of strand bias on genotyping quality, and to explore the possible causes of strand bias.
173. Mutations in the colony stimulating factor 1 receptor (CSF1R) gene cause hereditary diffuse leukoencephalopathy with spheroids. Rademakers Rosa, Baker Matt, Nicholson Alexandra M, Rutherford Nicola J, Finch NiCole, Soto-Ortolaza Alexandra, Lash Jennifer, Wider Christian, Wojtas Aleksandra, DeJesus-Hernandez Mariely, Adamson Jennifer, Kouri Naomi, Sundal Christina, Shuster Elizabeth A, Aasly Jan et al. Nat. Genet. 2012. 44:2. 200-5. PMID 22197934 PMC ID PMC3267847
Hereditary diffuse leukoencephalopathy with spheroids (HDLS) is an autosomal-dominant central nervous system white-matter disease with variable clinical presentations, including personality and behavioral changes, dementia, depression, parkinsonism, seizures and other phenotypes. We combined genome-wide linkage analysis with exome sequencing and identified 14 different mutations affecting the tyrosine kinase domain of the colony stimulating factor 1 receptor (encoded by CSF1R) in 14 families with HDLS. In one kindred, we confirmed the de novo occurrence of the mutation. Follow-up sequencing identified an additional CSF1R mutation in an individual diagnosed with corticobasal syndrome. In vitro, CSF-1 stimulation resulted in rapid autophosphorylation of selected tyrosine residues in the kinase domain of wild-type but not mutant CSF1R, suggesting that HDLS may result from partial loss of CSF1R function. As CSF1R is a crucial mediator of microglial proliferation and differentiation in the brain, our findings suggest an important role for microglial dysfunction in HDLS pathogenesis.
174. Genome-wide SNP discovery from transcriptome of four common carp strains. Xu Jian, Ji Peifeng, Zhao Zixia, Zhang Yan, Feng Jianxin, Wang Jian, Li Jiongtang, Zhang Xiaofeng, Zhao Lan, Liu Guangzan, Xu Peng, Sun Xiaowen. PLoS ONE 2012. 7:10. e48140. PMID 23110192 PMC ID PMC3482183
Single nucleotide polymorphisms (SNPs) have been used as genetic marker for genome-wide association studies in many species. Gene-associated SNPs could offer sufficient coverage in trait related research and further more could themselves be causative SNPs for traits. Common carp (Cyprinus carpio) is one of the most important aquaculture species in the world accounting for nearly 14% of freshwater aquaculture production. There are various strains of common carp with different economic traits, however, the genetic mechanism underlying the different traits have not been elucidated yet. In this project, we identified a large number of gene-associated SNPs from four strains of common carp using next-generation sequencing.
175. The genetic landscape of mutations in Burkitt lymphoma. Love Cassandra, Sun Zhen, Jima Dereje, Li Guojie, Zhang Jenny, Miles Rodney, Richards Kristy L, Dunphy Cherie H, Choi William W L, Srivastava Gopesh, Lugar Patricia L, Rizzieri David A, Lagoo Anand S, Bernal-Mizrachi Leon, Mann Karen P et al. Nat. Genet. 2012. 44:12. 1321-5. PMID 23143597 PMC ID PMC3674561
Burkitt lymphoma is characterized by deregulation of MYC, but the contribution of other genetic mutations to the disease is largely unknown. Here, we describe the first completely sequenced genome from a Burkitt lymphoma tumor and germline DNA from the same affected individual. We further sequenced the exomes of 59 Burkitt lymphoma tumors and compared them to sequenced exomes from 94 diffuse large B-cell lymphoma (DLBCL) tumors. We identified 70 genes that were recurrently mutated in Burkitt lymphomas, including ID3, GNA13, RET, PIK3R1 and the SWI/SNF genes ARID1A and SMARCA4. Our data implicate a number of genes in cancer for the first time, including CCT6B, SALL3, FTCD and PC. ID3 mutations occurred in 34% of Burkitt lymphomas and not in DLBCLs. We show experimentally that ID3 mutations promote cell cycle progression and proliferation. Our work thus elucidates commonly occurring gene-coding mutations in Burkitt lymphoma and implicates ID3 as a new tumor suppressor gene.
176. The pan-ErbB negative regulator Lrig1 is an intestinal stem cell marker that functions as a tumor suppressor. Powell Anne E, Wang Yang, Li Yina, Poulin Emily J, Means Anna L, Washington Mary K, Higginbotham James N, Juchheim Alwin, Prasad Nripesh, Levy Shawn E, Guo Yan, Shyr Yu, Aronow Bruce J, Haigis Kevin M, Franklin Jeffrey L et al. Cell 2012. 149:1. 146-58. PMID 22464327 PMC ID PMC3563328
Lineage mapping has identified both proliferative and quiescent intestinal stem cells, but the molecular circuitry controlling stem cell quiescence is incompletely understood. By lineage mapping, we show Lrig1, a pan-ErbB inhibitor, marks predominately noncycling, long-lived stem cells that are located at the crypt base and that, upon injury, proliferate and divide to replenish damaged crypts. Transcriptome profiling of Lrig1(+) colonic stem cells differs markedly from the profiling of highly proliferative, Lgr5(+) colonic stem cells; genes upregulated in the Lrig1(+) population include those involved in cell cycle repression and response to oxidative damage. Loss of Apc in Lrig1(+) cells leads to intestinal adenomas, and genetic ablation of Lrig1 results in heightened ErbB1-3 expression and duodenal adenomas. These results shed light on the relationship between proliferative and quiescent intestinal stem cells and support a model in which intestinal stem cell quiescence is maintained by calibrated ErbB signaling with loss of a negative regulator predisposing to neoplasia.
177. Effects of short-term exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin on microRNA expression in zebrafish embryos. Jenny Matthew J, Aluru Neelakanteswar, Hahn Mark E. Toxicol. Appl. Pharmacol. 2012. 264:2. 262-73. PMID 22921993 PMC ID PMC3471217
Although many drugs and environmental chemicals are teratogenic, the mechanisms by which most toxicants disrupt embryonic development are not well understood. MicroRNAs, single-stranded RNA molecules of ~22 nt that regulate protein expression by inhibiting mRNA translation and promoting mRNA sequestration or degradation, are important regulators of a variety of cellular processes including embryonic development and cellular differentiation. Recent studies have demonstrated that exposure to xenobiotics can alter microRNA expression and contribute to the mechanisms by which environmental chemicals disrupt embryonic development. In this study we tested the hypothesis that developmental exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a well-known teratogen, alters microRNA expression during zebrafish development. We exposed zebrafish embryos to DMSO (0.1%) or TCDD (5nM) for 1h at 30hours post fertilization (hpf) and measured microRNA expression using several methods at 36 and 60hpf. TCDD caused strong induction of CYP1A at 36hpf (62-fold) and 60hpf (135-fold) as determined by real-time RT-PCR, verifying the effectiveness of the exposure. MicroRNA expression profiles were determined using microarrays (Agilent and Exiqon), next-generation sequencing (SOLiD), and real-time RT-PCR. The two microarray platforms yielded results that were similar but not identical; both showed significant changes in expression of miR-451, 23a, 23b, 24 and 27e at 60hpf. Multiple analyses were performed on the SOLiD sequences yielding a total of 16 microRNAs as differentially expressed by TCDD in zebrafish embryos. However, miR-27e was the only microRNA to be identified as differentially expressed by all three methods (both microarrays, SOLiD sequencing, and real-time RT-PCR). These results suggest that TCDD exposure causes modest changes in expression of microRNAs, including some (miR-451, 23a, 23b, 24 and 27e) that are critical for hematopoiesis and cardiovascular development.
178. Drosophila CTCF tandemly aligns with other insulator proteins at the borders of H3K27me3 domains. Van Bortle Kevin, Ramos Edward, Takenaka Naomi, Yang Jingping, Wahi Jessica E, Corces Victor G. Genome Res. 2012. 22:11. 2176-87. PMID 22722341 PMC ID PMC3483547
Several multiprotein DNA complexes capable of insulator activity have been identified in Drosophila melanogaster, yet only CTCF, a highly conserved zinc finger protein, and the transcription factor TFIIIC have been shown to function in mammals. CTCF is involved in diverse nuclear activities, and recent studies suggest that the proteins with which it associates and the DNA sequences that it targets may underlie these various roles. Here we show that the Drosophila homolog of CTCF (dCTCF) aligns in the genome with other Drosophila insulator proteins such as Suppressor of Hairy wing [SU(HW)] and Boundary Element Associated Factor of 32 kDa (BEAF-32) at the borders of H3K27me3 domains, which are also enriched for associated insulator proteins and additional cofactors. RNAi depletion of dCTCF and combinatorial knockdown of gene expression for other Drosophila insulator proteins leads to a reduction in H3K27me3 levels within repressed domains, suggesting that insulators are important for the maintenance of appropriate repressive chromatin structure in Polycomb (Pc) domains. These results shed new insights into the roles of insulators in chromatin domain organization and support recent models suggesting that insulators underlie interactions important for Pc-mediated repression. We reveal an important relationship between dCTCF and other Drosophila insulator proteins and speculate that vertebrate CTCF may also align with other nuclear proteins to accomplish similar functions.
179. Gene density, transcription, and insulators contribute to the partition of the Drosophila genome into physical domains. Hou Chunhui, Li Li, Qin Zhaohui S, Corces Victor G. Mol. Cell 2012. 48:3. 471-84. PMID 23041285 PMC ID PMC3496039
The mechanisms responsible for the establishment of physical domains in metazoan chromosomes are poorly understood. Here we find that physical domains in Drosophila chromosomes are demarcated at regions of active transcription and high gene density that are enriched for transcription factors and specific combinations of insulator proteins. Physical domains contain different types of chromatin defined by the presence of specific proteins and epigenetic marks, with active chromatin preferentially located at the borders and silenced chromatin in the interior. Domain boundaries participate in long-range interactions that may contribute to the clustering of regions of active or silenced chromatin in the nucleus. Analysis of transgenes suggests that chromatin is more accessible and permissive to transcription at the borders than inside domains, independent of the presence of active or silencing histone modifications. These results suggest that the higher-order physical organization of chromatin may impose an additional level of regulation over classical epigenetic marks.
180. ANKRD7 and CYTL1 are novel risk genes for alcohol drinking behavior. Chen Xiang-ding, Xiong Dong-hai, Yang Tie-lin, Pei Yu-fang, Guo Yan-fang, Li Jian, Yang Fang, Pan Feng, Tan Li-jun, Yan Han, Liu Xiao-gang, Lei Shu-feng, Li Xi, Ning Ling-ling, Zhu Xue-zhen et al. Chin. Med. J. 2012. 125:6. 1127-34. PMID 22613542 PMC ID PMC4174677
Alcohol dependence (AD) is a complex disorder characterized by impaired control over drinking. It is determined by both genetic and environmental factors. The recent approach of genome-wide association study (GWAS) is a powerful tool for identifying complex disease-associated susceptibility alleles, however, a few GWASs have been conducted for AD, and their results are largely inconsistent. The present study aimed to screen the loci associated with alcohol-related phenotypes using GWAS technology.
181. Transcriptomic signatures of attachment, NF-?B suppression and IFN stimulation in the catfish gill following columnaris bacterial infection. Sun Fanyue, Peatman Eric, Li Chao, Liu Shikai, Jiang Yanliang, Zhou Zunchun, Liu Zhanjiang. Dev. Comp. Immunol. 2012. 38:1. 169-80. PMID 22669032
Outbreaks of columnaris disease (Flavobacterium columnare) are common in wild and cultured freshwater fish worldwide. Disease occurrences, particularly those caused by virulent genomovar II isolates, in aquaculture species such as channel catfish can be devastating. In contrast to other important aquaculture pathogens, little is known about host immune responses to columnaris. Adhesion of F. columnare to gill tissue has been correlated in some previous studies to virulence and host susceptibility. Here, therefore, we conducted the first transcriptomic profiling of host responses to columnaris following an experimental challenge. We utilized Illumina-based RNA-seq expression profiling to examine transcript profiles at three timepoints (4h, 24h, and 48h) in catfish gill after bath immersion infection. Enrichment and pathway analyses of the differentially expressed genes revealed several central signatures following infection. These included the dramatic upregulation of a rhamnose-binding lectin, with putative roles in bacterial attachment and aggregation, suppression of NF-?B signalling via I?Bs, BCL-3, TAX1BP1, and olfactomedin 4, and strong induction of IFN-inducible responses including iNOS2b, IFI44, and VHSV genes. Fifteen differentially expressed genes with varying expression profiles by RNA-seq, were validated by QPCR (correlation coefficients 0.85-0.94, p-value <0.001). Our results highlight several putative immune pathways and individual candidate genes deserving of further investigation in the context of development of therapeutic regimens and laying the foundation for selection of resistant catfish lines against columnaris.
182. Genome-wide phosphoacetylation of histone H3 at Drosophila enhancers and promoters. Kellner Wendy A, Ramos Edward, Van Bortle Kevin, Takenaka Naomi, Corces Victor G. Genome Res. 2012. 22:6. 1081-8. PMID 22508764 PMC ID PMC3371715
Transcription regulation is mediated by enhancers that bind sequence-specific transcription factors, which in turn interact with the promoters of the genes they control. Here, we show that the JIL-1 kinase is present at both enhancers and promoters of ecdysone-induced Drosophila genes, where it phosphorylates the Ser10 and Ser28 residues of histone H3. JIL-1 is also required for CREB binding protein (CBP)-mediated acetylation of Lys27, a well-characterized mark of active enhancers. The presence of these proteins at enhancers and promoters of ecdysone-induced genes results in the establishment of the H3K9acS10ph and H3K27acS28ph marks at both regulatory sequences. These modifications are necessary for the recruitment of 14-3-3, a scaffolding protein capable of facilitating interactions between two simultaneously bound proteins. Chromatin conformation capture assays indicate that interaction between the enhancer and the promoter is dependent on the presence of JIL-1, 14-3-3, and CBP. Genome-wide analyses extend these conclusions to most Drosophila genes, showing that the presence of JIL-1, H3K9acS10ph, and H3K27acS28ph is a general feature of enhancers and promoters in this organism.
183. Phylogenomic resolution of paleozoic divergences in harvestmen (Arachnida, Opiliones) via analysis of next-generation transcriptome data. Hedin Marshal, Starrett James, Akhter Sajia, Schönhofer Axel L, Shultz Jeffrey W. PLoS ONE 2012. 7:8. e42888. PMID 22936998 PMC ID PMC3427324
Next-generation sequencing technologies are rapidly transforming molecular systematic studies of non-model animal taxa. The arachnid order Opiliones (commonly known as "harvestmen") includes more than 6,400 described species placed into four well-supported lineages (suborders). Fossil plus molecular clock evidence indicates that these lineages were diverging in the late Silurian to mid-Carboniferous, with some fossil harvestmen representing the earliest known land animals. Perhaps because of this ancient divergence, phylogenetic resolution of subordinal interrelationships within Opiliones has been difficult. We present the first phylogenomics analysis for harvestmen, derived from comparative RNA-Seq data for eight species representing all suborders. Over 30 gigabases of original Illumina short-read data were used in de novo assemblies, resulting in 50-80,000 transcripts per taxon. Transcripts were compared to published scorpion and tick genomics data, and a stringent filtering process was used to identify over 350 putatively single-copy, orthologous protein-coding genes shared among taxa. Phylogenetic analyses using various partitioning strategies, data coding schemes, and analytical methods overwhelmingly support the "classical" hypothesis of Opiliones relationships, including the higher-level clades Palpatores and Phalangida. Relaxed molecular clock analyses using multiple alternative fossil calibration strategies corroborate ancient divergences within Opiliones that are possibly deeper than the recorded fossil record indicates. The assembled data matrices, comprising genes that are conserved, highly expressed, and varying in length and phylogenetic informativeness, represent an important resource for future molecular systematic studies of Opiliones and other arachnid groups.
Boundary Element Associated Factor-32 (BEAF-32) is an insulator protein predominantly found near gene promoters and thought to play a role in gene expression. We find that mutations in BEAF-32 are lethal, show loss of epithelial morphology in imaginal discs and cause neoplastic growth defects. To investigate the molecular mechanisms underlying this phenotype, we carried out a genome-wide analysis of BEAF-32 localization in wing imaginal disc cells. Mutation of BEAF-32 results in miss-regulation of 3850 genes by at least 1.5-fold, 794 of which are bound by this protein in wing imaginal cells. Up-regulated genes encode proteins involved in cell polarity, cell proliferation and cell differentiation. Among the down-regulated genes are those encoding components of the wingless pathway, which is required for cell differentiation. Miss-regulation of these genes explains the unregulated cell growth and neoplastic phenotypes observed in imaginal tissues of BEAF-32 mutants.
185. Familial diarrhea syndrome caused by an activating GUCY2C mutation. Fiskerstrand Torunn, Arshad Najla, Haukanes Bjørn Ivar, Tronstad Rune Rose, Pham Khanh Do-Cong, Johansson Stefan, Håvik Bjarte, Tønder Siv L, Levy Shawn E, Brackman Damien, Boman Helge, Biswas Kabir Hassan, Apold Jaran, Hovdenak Nils, Visweswariah Sandhya S et al. N. Engl. J. Med. 2012. 366:17. 1586-95. PMID 22436048
Familial diarrhea disorders are, in most cases, severe and caused by recessive mutations. We describe the cause of a novel dominant disease in 32 members of a Norwegian family. The affected members have chronic diarrhea that is of early onset, is relatively mild, and is associated with increased susceptibility to inflammatory bowel disease, small-bowel obstruction, and esophagitis.
186. The venom-gland transcriptome of the eastern diamondback rattlesnake (Crotalus adamanteus). Rokyta Darin R, Lemmon Alan R, Margres Mark J, Aronow Karalyn. BMC Genomics 2012. 13:X. 312. PMID 23025625 PMC ID PMC3472243
Snake venoms have significant impacts on human populations through the morbidity and mortality associated with snakebites and as sources of drugs, drug leads, and physiological research tools. Genes expressed by venom-gland tissue, including those encoding toxic proteins, have therefore been sequenced but only with relatively sparse coverage resulting from the low-throughput sequencing approaches available. High-throughput approaches based on 454 pyrosequencing have recently been applied to the study of snake venoms to give the most complete characterizations to date of the genes expressed in active venom glands, but such approaches are costly and still provide a far-from-complete characterization of the genes expressed during venom production.
187. Efficient assembly and annotation of the transcriptome of catfish by RNA-Seq analysis of a doubled haploid homozygote. Liu Shikai, Zhang Yu, Zhou Zunchun, Waldbieser Geoff, Sun Fanyue, Lu Jianguo, Zhang Jiaren, Jiang Yanliang, Zhang Hao, Wang Xiuli, Rajendran K V, Khoo Lester, Kucuktas Huseyin, Peatman Eric, Liu Zhanjiang. BMC Genomics 2012. 13:X. 595. PMID 23127152 PMC ID PMC3582483
Upon the completion of whole genome sequencing, thorough genome annotation that associates genome sequences with biological meanings is essential. Genome annotation depends on the availability of transcript information as well as orthology information. In teleost fish, genome annotation is seriously hindered by genome duplication. Because of gene duplications, one cannot establish orthologies simply by homology comparisons. Rather intense phylogenetic analysis or structural analysis of orthologies is required for the identification of genes. To conduct phylogenetic analysis and orthology analysis, full-length transcripts are essential. Generation of large numbers of full-length transcripts using traditional transcript sequencing is very difficult and extremely costly.
188. RAF265 inhibits the growth of advanced human melanoma tumors. Su Yingjun, Vilgelm Anna E, Kelley Mark C, Hawkins Oriana E, Liu Yan, Boyd Kelli L, Kantrow Sara, Splittgerber Ryan C, Short Sarah P, Sobolik Tammy, Zaja-Milatovic Snjezana, Dahlman Kimberly Brown, Amiri Katayoun I, Jiang Aixiang, Lu Pengcheng et al. Clin. Cancer Res. 2012. 18:8. 2184-98. PMID 22351689 PMC ID PMC3724517
The purpose of this preclinical study was to determine the effectiveness of RAF265, a multikinase inhibitor, for treatment of human metastatic melanoma and to characterize traits associated with drug response.
189. Exome sequencing generates high quality data in non-target regions. Guo Yan, Long Jirong, He Jing, Li Chung-I, Cai Qiuyin, Shu Xiao-Ou, Zheng Wei, Li Chun. BMC Genomics 2012. 13:X. 194. PMID 22607156 PMC ID PMC3416685
Exome sequencing using next-generation sequencing technologies is a cost efficient approach to selectively sequencing coding regions of human genome for detection of disease variants. A significant amount of DNA fragments from the capture process fall outside target regions, and sequence data for positions outside target regions have been mostly ignored after alignment.
190. Exome analysis of a family with pleiotropic congenital heart disease. Arrington Cammon B, Bleyl Steven B, Matsunami Norisada, Bonnell Gabriel D, Otterud Brith E M, Nielsen Douglas C, Stevens Jeffrey, Levy Shawn, Leppert Mark F, Bowles Neil E. Circ Cardiovasc Genet 2012. 5:2. 175-82. PMID 22337856 PMC ID PMC3329568
A number of single gene defects have been identified in patients with isolated or nonsyndromic congenital heart defects (CHDs). However, due to significant genetic heterogeneity, candidate gene approaches have had limited success in finding high-risk alleles in most cases. The purpose of this study was to use exome sequencing to identify high-risk gene variants in a family with highly penetrant pleiotropic CHD.
191. FAN1 mutations cause karyomegalic interstitial nephritis, linking chronic kidney failure to defective DNA damage repair. Zhou Weibin, Otto Edgar A, Cluckey Andrew, Airik Rannar, Hurd Toby W, Chaki Moumita, Diaz Katrina, Lach Francis P, Bennett Geoffrey R, Gee Heon Yung, Ghosh Amiya K, Natarajan Sivakumar, Thongthip Supawat, Veturi Uma, Allen Susan J et al. Nat. Genet. 2012. 44:8. 910-5. PMID 22772369 PMC ID PMC3412140
Chronic kidney disease (CKD) represents a major health burden. Its central feature of renal fibrosis is not well understood. By exome sequencing, we identified mutations in FAN1 as a cause of karyomegalic interstitial nephritis (KIN), a disorder that serves as a model for renal fibrosis. Renal histology in KIN is indistinguishable from that of nephronophthisis, except for the presence of karyomegaly. The FAN1 protein has nuclease activity and acts in DNA interstrand cross-link (ICL) repair within the Fanconi anemia DNA damage response (DDR) pathway. We show that cells from individuals with FAN1 mutations have sensitivity to the ICL-inducing agent mitomycin C but do not exhibit chromosome breakage or cell cycle arrest after diepoxybutane treatment, unlike cells from individuals with Fanconi anemia. We complemented ICL sensitivity with wild-type FAN1 but not with cDNA having mutations found in individuals with KIN. Depletion of fan1 in zebrafish caused increased DDR, apoptosis and kidney cysts. Our findings implicate susceptibility to environmental genotoxins and inadequate DNA repair as novel mechanisms contributing to renal fibrosis and CKD.
192. Generation of genome-scale gene-associated SNPs in catfish for the construction of a high-density SNP array. Liu Shikai, Zhou Zunchun, Lu Jianguo, Sun Fanyue, Wang Shaolin, Liu Hong, Jiang Yanliang, Kucuktas Huseyin, Kaltenboeck Ludmilla, Peatman Eric, Liu Zhanjiang. BMC Genomics 2011. 12:X. 53. PMID 21255432 PMC ID PMC3033819
Single nucleotide polymorphisms (SNPs) have become the marker of choice for genome-wide association studies. In order to provide the best genome coverage for the analysis of performance and production traits, a large number of relatively evenly distributed SNPs are needed. Gene-associated SNPs may fulfill these requirements of large numbers and genome wide distribution. In addition, gene-associated SNPs could themselves be causative SNPs for traits. The objective of this project was to identify large numbers of gene-associated SNPs using high-throughput next generation sequencing.
193. Streptomyces scopuliridis sp. nov., a bacteriocin-producing soil streptomycete. Farris M Heath, Duffy Carol, Findlay Robert H, Olson Julie B. Int. J. Syst. Evol. Microbiol. 2011. 61:Pt 9. 2112-6. PMID 20870885 PMC ID PMC3352161
Actinomycete strain RB72(T) was isolated from woodland bluff soil in northern Alabama, USA, and shown to produce a broad spectrum bacteriocin. Based on morphological and chemotaxonomic characteristics, the strain was determined to belong to the genus Streptomyces. Phylogenetic analysis of the near-complete 16S rRNA gene sequence indicated that it differed from those of the described streptomycetes available in public databases. The distinctive white aerial hyphae and lack of sporulation suggest a deficiency in the whi pathway of the organism. A combination of substrate utilization patterns, morphological and chemotaxonomic characteristics and DNA-DNA hybridization results supported the affiliation of strain RB72(T) to the genus Streptomyces and enabled the genotypic and phenotypic differentiation of strain RB72(T) from closely related reference strains. Strain RB72(T) therefore represents a novel species of the genus Streptomyces, for which the name Streptomyces scopuliridis sp. nov. is proposed. The type strain is RB72(T) (?=?DSM 41917(T) ?=?NRRL B-24574(T)).
194. Genome-wide association study identifies breast cancer risk variant at 10q21.2: results from the Asia Breast Cancer Consortium. Cai Qiuyin, Long Jirong, Lu Wei, Qu Shimian, Wen Wanqing, Kang Daehee, Lee Ji-Young, Chen Kexin, Shen Hongbing, Shen Chen-Yang, Sung Hyuna, Matsuo Keitaro, Haiman Christopher A, Khoo Ui Soon, Ren Zefang et al. Hum. Mol. Genet. 2011. 20:24. 4991-9. PMID 21908515 PMC ID PMC3221542
Although approximately 20 common genetic susceptibility loci have been identified for breast cancer risk through genome-wide association studies (GWASs), genetic risk variants reported to date explain only a small fraction of heritability for this common cancer. We conducted a four-stage GWAS including 17 153 cases and 16 943 controls among East-Asian women to search for new genetic risk factors for breast cancer. After analyzing 684 457 SNPs in 2062 cases and 2066 controls (Stage I), we selected for replication among 5969 Chinese women (4146 cases and 1823 controls) the top 49 SNPs that had neither been reported previously nor were in strong linkage disequilibrium with reported SNPs (Stage II). Three SNPs were further evaluated in up to 13 152 Chinese and Japanese women (6436 cases and 6716 controls) (Stage III). Finally, two SNPs were evaluated in 10 847 Korean women (4509 cases and 6338 controls) (Stage IV). SNP rs10822013 on chromosome 10q21.2, located in the zinc finger protein 365 (ZNF365) gene, showed a consistent association with breast cancer risk in all four stages with a combined per-risk allele odds ratio of 1.10 (95% CI: 1.07-1.14) (P-value for trend = 5.87 × 10(-9)). In vitro electrophoretic mobility shift assays demonstrated the potential functional significance of rs10822013. Our results strongly implicate rs10822013 at 10q21.2 as a genetic risk variant for breast cancer among East-Asian women.
195. Exome sequencing and analysis of induced pluripotent stem cells identify the cilia-related gene male germ cell-associated kinase (MAK) as a cause of retinitis pigmentosa. Tucker Budd A, Scheetz Todd E, Mullins Robert F, DeLuca Adam P, Hoffmann Jeremy M, Johnston Rebecca M, Jacobson Samuel G, Sheffield Val C, Stone Edwin M. Proc. Natl. Acad. Sci. U.S.A. 2011. 108:34. E569-76. PMID 21825139 PMC ID PMC3161526
Retinitis pigmentosa (RP) is a genetically heterogeneous heritable disease characterized by apoptotic death of photoreceptor cells. We used exome sequencing to identify a homozygous Alu insertion in exon 9 of male germ cell-associated kinase (MAK) as the cause of disease in an isolated individual with RP. Screening of 1,798 unrelated RP patients identified 20 additional probands homozygous for this insertion (1.2%). All 21 affected probands are of Jewish ancestry. MAK encodes a kinase involved in the regulation of photoreceptor-connecting cilium length. Immunohistochemistry of human donor tissue revealed that MAK is expressed in the inner segments, cell bodies, and axons of rod and cone photoreceptors. Several isoforms of MAK that result from alternative splicing were identified. Induced pluripotent stem cells were derived from the skin of the proband and a patient with non-MAK-associated RP (RP control). In the RP control individual, we found that a transcript lacking exon 9 was predominant in undifferentiated cells, whereas a transcript bearing exon 9 and a previously unrecognized exon 12 predominated in cells that were differentiated into retinal precursors. However, in the proband with the Alu insertion, the developmental switch to the MAK transcript bearing exons 9 and 12 did not occur. In addition to showing the use of induced pluripotent stem cells to efficiently evaluate the pathogenicity of specific mutations in relatively inaccessible tissues like retina, this study reveals algorithmic and molecular obstacles to the discovery of pathogenic insertions and suggests specific changes in strategy that can be implemented to more fully harness the power of sequencing technologies.
196. Novel human genetic variants associated with extrapulmonary tuberculosis: a pilot genome wide association study. Oki Noffisat O, Motsinger-Reif Alison A, Antas Paulo Rz, Levy Shawn, Holland Steven M, Sterling Timothy R. BMC Res Notes 2011. 4:X. 28. PMID 21281516 PMC ID PMC3041678
Approximately 5-10% of persons infected with M. tuberculosis develop tuberculosis, but the factors associated with disease progression are incompletely understood. Both linkage and association studies have identified human genetic variants associated with susceptibility to pulmonary tuberculosis, but few genetic studies have evaluated extrapulmonary disease. Because extrapulmonary and pulmonary tuberculosis likely have different underlying pathophysiology, identification of genetic mutations associated with extrapulmonary disease is important.
197. A pilot study for channel catfish whole genome sequencing and de novo assembly. Jiang Yanliang, Lu Jianguo, Peatman Eric, Kucuktas Huseyin, Liu Shikai, Wang Shaolin, Sun Fanyue, Liu Zhanjiang. BMC Genomics 2011. 12:X. 629. PMID 22192763 PMC ID PMC3266365
Recent advances in next-generation sequencing technologies have drastically increased throughput and significantly reduced sequencing costs. However, the average read lengths in next-generation sequencing technologies are short as compared with that of traditional Sanger sequencing. The short sequence reads pose great challenges for de novo sequence assembly. As a pilot project for whole genome sequencing of the catfish genome, here we attempt to determine the proper sequence coverage, the proper software for assembly, and various parameters used for the assembly of a BAC physical map contig spanning approximately a million of base pairs.
198. Characterizing the impact of smoking and lung cancer on the airway transcriptome using RNA-Seq. Beane Jennifer, Vick Jessica, Schembri Frank, Anderlind Christina, Gower Adam, Campbell Joshua, Luo Lingqi, Zhang Xiao Hui, Xiao Ji, Alekseyev Yuriy O, Wang Shenglong, Levy Shawn, Massion Pierre P, Lenburg Marc, Spira Avrum. Cancer Prev Res (Phila) 2011. 4:6. 803-17. PMID 21636547 PMC ID PMC3694393
Cigarette smoke creates a molecular field of injury in epithelial cells that line the respiratory tract. We hypothesized that transcriptome sequencing (RNA-Seq) will enhance our understanding of the field of molecular injury in response to tobacco smoke exposure and lung cancer pathogenesis by identifying gene expression differences not interrogated or accurately measured by microarrays. We sequenced the high-molecular-weight fraction of total RNA (>200 nt) from pooled bronchial airway epithelial cell brushings (n = 3 patients per pool) obtained during bronchoscopy from healthy never smoker (NS) and current smoker (S) volunteers and smokers with (C) and without (NC) lung cancer undergoing lung nodule resection surgery. RNA-Seq libraries were prepared using 2 distinct approaches, one capable of capturing non-polyadenylated RNA (the prototype NuGEN Ovation RNA-Seq protocol) and the other designed to measure only polyadenylated RNA (the standard Illumina mRNA-Seq protocol) followed by sequencing generating approximately 29 million 36 nt reads per pool and approximately 22 million 75 nt paired-end reads per pool, respectively. The NuGEN protocol captured additional transcripts not detected by the Illumina protocol at the expense of reduced coverage of polyadenylated transcripts, while longer read lengths and a paired-end sequencing strategy significantly improved the number of reads that could be aligned to the genome. The aligned reads derived from the two complementary protocols were used to define the compendium of genes expressed in the airway epithelium (n = 20,573 genes). Pathways related to the metabolism of xenobiotics by cytochrome P450, retinol metabolism, and oxidoreductase activity were enriched among genes differentially expressed in smokers, whereas chemokine signaling pathways, cytokine-cytokine receptor interactions, and cell adhesion molecules were enriched among genes differentially expressed in smokers with lung cancer. There was a significant correlation between the RNA-Seq gene expression data and Affymetrix microarray data generated from the same samples (P < 0.001); however, the RNA-Seq data detected additional smoking- and cancer-related transcripts whose expression was were either not interrogated by or was not found to be significantly altered when using microarrays, including smoking-related changes in the inflammatory genes S100A8 and S100A9 and cancer-related changes in MUC5AC and secretoglobin (SCGB3A1). Quantitative real-time PCR confirmed differential expression of select genes and non-coding RNAs within individual samples. These results demonstrate that transcriptome sequencing has the potential to provide new insights into the biology of the airway field of injury associated with smoking and lung cancer. The measurement of both coding and non-coding transcripts by RNA-Seq has the potential to help elucidate mechanisms of response to tobacco smoke and to identify additional biomarkers of lung cancer risk and novel targets for chemoprevention.
199. Exome sequencing supports a de novo mutational paradigm for schizophrenia. Xu Bin, Roos J Louw, Dexheimer Phillip, Boone Braden, Plummer Brooks, Levy Shawn, Gogos Joseph A, Karayiorgou Maria. Nat. Genet. 2011. 43:9. 864-8. PMID 21822266 PMC ID PMC3196550
Despite its high heritability, a large fraction of individuals with schizophrenia do not have a family history of the disease (sporadic cases). Here we examined the possibility that rare de novo protein-altering mutations contribute to the genetic component of schizophrenia by sequencing the exomes of 53 sporadic cases, 22 unaffected controls and their parents. We identified 40 de novo mutations in 27 cases affecting 40 genes, including a potentially disruptive mutation in DGCR2, a gene located in the schizophrenia-predisposing 22q11.2 microdeletion region. A comparison to rare inherited variants indicated that the identified de novo mutations show a large excess of non-synonymous changes in schizophrenia cases, as well as a greater potential to affect protein structure and function. Our analyses suggest a major role for de novo mutations in schizophrenia as well as a large mutational target, which together provide a plausible explanation for the high global incidence and persistence of the disease.
200. Transcriptome profiling of chemosensory appendages in the malaria vector Anopheles gambiae reveals tissue- and sex-specific signatures of odor coding. Pitts R Jason, Rinker David C, Jones Patrick L, Rokas Antonis, Zwiebel Laurence J. BMC Genomics 2011. 12:X. 271. PMID 21619637 PMC ID PMC3126782
Chemosensory signal transduction guides the behavior of many insects, including Anopheles gambiae, the major vector for human malaria in sub-Saharan Africa. To better understand the molecular basis of mosquito chemosensation we have used whole transcriptome RNA sequencing (RNA-seq) to compare transcript expression profiles between the two major chemosensory tissues, the antennae and maxillary palps, of adult female and male An. gambiae.
201. Regulation of chromatin organization and inducible gene expression by a Drosophila insulator. Wood Ashley M, Van Bortle Kevin, Ramos Edward, Takenaka Naomi, Rohrbaugh Margaret, Jones Brian C, Jones Keith C, Corces Victor G. Mol. Cell 2011. 44:1. 29-38. PMID 21981916 PMC ID PMC3190163
Insulators are multiprotein-DNA complexes thought to affect gene expression by mediating inter- and intrachromosomal interactions. Drosophila insulators contain specific DNA-binding proteins plus common components, such as CP190, that facilitate these interactions. Here, we examine changes in the distribution of Drosophila insulator proteins during the heat-shock and ecdysone responses. We find that CP190 recruitment to insulator sites is the main regulatable step in controlling insulator function during heat shock. In contrast, both CP190 and DNA-binding protein recruitment are regulated during the ecdysone response. CP190 is necessary to stabilize specific chromatin loops and for proper activation of transcription of genes regulated by this hormone. These findings suggest that cells may regulate recruitment of insulator proteins to DNA to activate insulator activity at specific sites and create distinct patterns of nuclear organization that are necessary to achieve proper gene expression in response to different stimuli.
202. Complete bacteriophage transfer in a bacterial endosymbiont (Wolbachia) determined by targeted genome capture. Kent Bethany N, Salichos Leonidas, Gibbons John G, Rokas Antonis, Newton Irene L G, Clark Michael E, Bordenstein Seth R. Genome Biol Evol 2011. 3:X. 209-18. PMID 21292630 PMC ID PMC3068000
Bacteriophage flux can cause the majority of genetic diversity in free-living bacteria. This tenet of bacterial genome evolution generally does not extend to obligate intracellular bacteria owing to their reduced contact with other microbes and a predominance of gene deletion over gene transfer. However, recent studies suggest intracellular coinfections in the same host can facilitate exchange of mobile elements between obligate intracellular bacteria-a means by which these bacteria can partially mitigate the reductive forces of the intracellular lifestyle. To test whether bacteriophages transfer as single genes or larger regions between coinfections, we sequenced the genome of the obligate intracellular Wolbachia strain wVitB from the parasitic wasp Nasonia vitripennis and compared it against the prophage sequences of the divergent wVitA coinfection. We applied, for the first time, a targeted sequence capture array to specifically trap the symbiont's DNA from a heterogeneous mixture of eukaryotic, bacterial, and viral DNA. The tiled array successfully captured the genome with 98.3% efficiency. Examination of the genome sequence revealed the largest transfer of bacteriophage and flanking genes (52.2 kb) to date between two obligate intracellular coinfections. The mobile element transfer occurred in the recent evolutionary past based on the 99.9% average nucleotide identity of the phage sequences between the two strains. In addition to discovering an evolutionary recent and large-scale horizontal phage transfer between coinfecting obligate intracellular bacteria, we demonstrate that "targeted genome capture" can enrich target DNA to alleviate the problem of isolating symbiotic microbes that are difficult to culture or purify from the conglomerate of organisms inside eukaryotes.
203. A genome-wide association analysis implicates SOX6 as a candidate gene for wrist bone mass. Tan LiJun, Liu Rong, Lei ShuFeng, Pan Rong, Yang TieLin, Yan Han, Pei YuFang, Yang Fang, Zhang Feng, Pan Feng, Zhang YinPin, Hu HongGang, Levy Shawn, Deng HongWen. Sci China Life Sci 2010. 53:9. 1065-72. PMID 21104366
Osteoporosis is a highly heritable common bone disease leading to fractures that severely impair the life quality of patients. Wrist fractures caused by osteoporosis are largely due to the scarcity of wrist bone mass. Here we report the results of a genome-wide association study (GWAS) of wrist bone mineral density (BMD). We examined ?500000 SNP markers in 1000 unrelated homogeneous Caucasian subjects and found a novel allelic association with wrist BMD at rs11023787 in the SOX6 (SRY (sex determining region Y)-box 6) gene (P=9.00×10(-5)). Subjects carrying the C allele of rs11023787 in SOX6 had significantly higher mean wrist BMD values than those with the T allele (0.485:0.462 g cm(-2) for C allele vs. T allele carriers). For validation, we performed SOX6 association for BMD in an independent Chinese sample and found that SNP rs11023787 was significantly associated with wrist BMD in the Chinese sample (P=6.41×10(-3)). Meta-analyses of the GWAS scan and the replication studies yielded P-values of 5.20×10(-6) for rs11023787. Results of this study, together with the functional relevance of SOX6 in cartilage formation, support the SOX6 gene as an important gene for BMD variation.
204. Candidate exome capture identifies mutation of SDCCAG8 as the cause of a retinal-renal ciliopathy. Otto Edgar A, Hurd Toby W, Airik Rannar, Chaki Moumita, Zhou Weibin, Stoetzel Corinne, Patil Suresh B, Levy Shawn, Ghosh Amiya K, Murga-Zamalloa Carlos A, van Reeuwijk Jeroen, Letteboer Stef J F, Sang Liyun, Giles Rachel H, Liu Qin et al. Nat. Genet. 2010. 42:10. 840-50. PMID 20835237 PMC ID PMC2947620
Nephronophthisis-related ciliopathies (NPHP-RC) are recessive disorders that feature dysplasia or degeneration occurring preferentially in the kidney, retina and cerebellum. Here we combined homozygosity mapping with candidate gene analysis by performing 'ciliopathy candidate exome capture' followed by massively parallel sequencing. We identified 12 different truncating mutations of SDCCAG8 (serologically defined colon cancer antigen 8, also known as CCCAP) in 10 families affected by NPHP-RC. We show that SDCCAG8 is localized at both centrioles and interacts directly with OFD1 (oral-facial-digital syndrome 1), which is associated with NPHP-RC. Depletion of sdccag8 causes kidney cysts and a body axis defect in zebrafish and induces cell polarity defects in three-dimensional renal cell cultures. This work identifies loss of SDCCAG8 function as a cause of a retinal-renal ciliopathy and validates exome capture analysis for broadly heterogeneous single-gene disorders.
205. Polymorphisms in IL-1beta, vitamin D receptor Fok1, and Toll-like receptor 2 are associated with extrapulmonary tuberculosis. Motsinger-Reif Alison A, Antas Paulo R Z, Oki Noffisat O, Levy Shawn, Holland Steven M, Sterling Timothy R. BMC Med. Genet. 2010. 11:X. 37. PMID 20196868 PMC ID PMC2837863
Human genetic variants may affect tuberculosis susceptibility, but the immunologic correlates of the genetic variants identified are often unclear.
206. Genome-wide association study of homocysteine levels in Filipinos provides evidence for CPS1 in women and a stronger MTHFR effect in young adults. Lange Leslie A, Croteau-Chonka Damien C, Marvelle Amanda F, Qin Li, Gaulton Kyle J, Kuzawa Christopher W, McDade Thomas W, Wang Yunfei, Li Yun, Levy Shawn, Borja Judith B, Lange Ethan M, Adair Linda S, Mohlke Karen L. Hum. Mol. Genet. 2010. 19:10. 2050-8. PMID 20154341 PMC ID PMC2860887
Plasma homocysteine (Hcy) level is associated with cardiovascular disease and may play an etiologic role in vascular damage, a precursor for atherosclerosis. We performed a genome-wide association study for Hcy in 1786 unrelated Filipino women from the Cebu Longitudinal Health and Nutrition Survey (CLHNS). The most strongly associated single-nucleotide polymorphism (SNP) (rs7422339, P = 4.7 x 10(-13)) encodes Thr1405Asn in the gene CPS1 and explained 3.0% of variation in the Hcy level. The widely studied MTHFR C677T SNP (rs1801133) was also highly significant (P = 8.7 x 10(-10)) and explained 1.6% of the trait variation. We also genotyped these two SNPs in 1679 CLHNS young adult offspring. The MTHFR C677T SNP was strongly associated with Hcy (P = 1.9 x 10(-26)) and explained approximately 5.1% of the variation in the offspring. In contrast, the CPS1 variant was significant only in females (P = 0.11 in all; P = 0.0087 in females). Combined analysis of all samples confirmed that the MTHFR variant was more strongly associated with Hcy in the offspring (interaction P = 1.2 x 10(-5)). Furthermore, although there was evidence for a positive synergistic effect between the CPS1 and MTHFR SNPs in the offspring (interaction P = 0.0046), there was no significant evidence for an interaction in the mothers (P = 0.55). These data confirm a recent finding that CPS1 is a locus influencing Hcy levels in women and suggest that genetic effects on Hcy may differ across developmental stages.
207. Genome-wide association study identifies a new breast cancer susceptibility locus at 6q25.1. Zheng Wei, Long Jirong, Gao Yu-Tang, Li Chun, Zheng Ying, Xiang Yong-Bin, Wen Wanqing, Levy Shawn, Deming Sandra L, Haines Jonathan L, Gu Kai, Fair Alecia Malin, Cai Qiuyin, Lu Wei, Shu Xiao-Ou. Nat. Genet. 2009. 41:3. 324-8. PMID 19219042 PMC ID PMC2754845
We carried out a genome-wide association study among Chinese women to identify risk variants for breast cancer. After analyzing 607,728 SNPs in 1,505 cases and 1,522 controls, we selected 29 SNPs for a fast-track replication in an independent set of 1,554 cases and 1,576 controls. We further investigated four replicated loci in a third set of samples comprising 3,472 cases and 900 controls. SNP rs2046210 at 6q25.1, located upstream of the gene encoding estrogen receptor alpha (ESR1), showed strong and consistent association with breast cancer across all three stages. Adjusted odds ratio (95% CI) were 1.36 (1.24-1.49) and 1.59 (1.40-1.82), respectively, for genotypes A/G and A/A versus G/G (P for trend 2.0 x 10(-15)) in the pooled analysis of samples from all three stages. We also found a similar, albeit weaker, association in an independent study comprising 1,591 cases and 1,466 controls of European ancestry (P(trend) = 0.01). These results strongly implicate 6q25.1 as a susceptibility locus for breast cancer.
208. Whole genome distribution and ethnic differentiation of copy number variation in Caucasian and Asian populations. Li Jian, Yang Tielin, Wang Liang, Yan Han, Zhang Yinping, Guo Yan, Pan Feng, Zhang Zhixin, Peng Yumei, Zhou Qi, He Lina, Zhu Xuezhen, Deng Hongyi, Levy Shawn, Papasian Christopher J et al. PLoS ONE 2009. 4:11. e7958. PMID 19956714 PMC ID PMC2776354
Although copy number variation (CNV) has recently received much attention as a form of structure variation within the human genome, knowledge is still inadequate on fundamental CNV characteristics such as occurrence rate, genomic distribution and ethnic differentiation. In the present study, we used the Affymetrix GeneChip(R) Mapping 500K Array to discover and characterize CNVs in the human genome and to study ethnic differences of CNVs between Caucasians and Asians. Three thousand and nineteen CNVs, including 2381 CNVs in autosomes and 638 CNVs in X chromosome, from 985 Caucasian and 692 Asian individuals were identified, with a mean length of 296 kb. Among these CNVs, 190 had frequencies greater than 1% in at least one ethnic group, and 109 showed significant ethnic differences in frequencies (p<0.01). After merging overlapping CNVs, 1135 copy number variation regions (CNVRs), covering approximately 439 Mb (14.3%) of the human genome, were obtained. Our findings of ethnic differentiation of CNVs, along with the newly constructed CNV genomic map, extend our knowledge on the structural variation in the human genome and may furnish a basis for understanding the genomic differentiation of complex traits across ethnic groups.