SNP discovery using next generation transcriptomic sequencing in Atlantic herring (Clupea harengus)

The introduction of Next Generation Sequencing (NGS) has revolutionised population genetics, providing studies of non-model species with unprecedented genomic coverage, allowing evolutionary biologists to address questions previously far beyond the reach of available resources. Furthermore, the simple mutation model of Single Nucleotide Polymorphisms (SNPs) permits cost-effective high-throughput genotyping in thousands of individuals simultaneously. Genomic resources are scarce for the Atlantic herring (Clupea harengus), a small pelagic species that sustains high revenue fisheries. This paper details the development of 578 SNPs using a combined NGS and high-throughput genotyping approach. Eight individuals covering the species distribution in the eastern Atlantic were bar-coded and multiplexed into a single cDNA library and sequenced using the 454 GS FLX platform. SNP discovery was performed by de novo sequence clustering and contig assembly, followed by the mapping of reads against consensus contig sequences. Selection of candidate SNPs for genotyping was conducted using an in silico approach. SNP validation and genotyping were performed simultaneously using an Illumina 1,536 GoldenGate assay. Although the conversion rate of candidate SNPs in the genotyping assay cannot be predicted in advance, this approach has the potential to maximise cost and time efficiencies by avoiding expensive and time-consuming laboratory stages of SNP validation. Additionally, the in silico approach leads to lower ascertainment bias in the resulting SNP panel as marker selection is based only on the ability to design primers and the predicted presence of intron-exon boundaries. Consequently SNPs with a wider spectrum of minor allele frequencies (MAFs) will be genotyped in the final panel. The genomic resources presented here represent a valuable multi-purpose resource for developing informative marker panels for population discrimination, microarray development and for population genomic studies in the wild

A robust linkage map of the porcine autosomes based on gene-associated SNPs

<p>Abstract</p> <p>Background</p> <p>Genetic linkage maps are necessary for mapping of mendelian traits and quantitative trait loci (QTLs). To identify the actual genes, which control these traits, a map based on gene-associated single nucleotide polymorphism (SNP) markers is highly valuable. In this study, the SNPs were genotyped in a large family material comprising more than 5,000 piglets derived from 12 Duroc boars crossed with 236 Danish Landrace/Danish Large White sows. The SNPs were identified in sequence alignments of 4,600 different amplicons obtained from the 12 boars and containing coding regions of genes derived from expressed sequence tags (ESTs) and genomic shotgun sequences.</p> <p>Results</p> <p>Linkage maps of all 18 porcine autosomes were constructed based on 456 gene-associated and six porcine EST-based SNPs. The total length of the averaged-sex whole porcine autosome was estimated to 1,711.8 cM resulting in an average SNP spacing of 3.94 cM. The female and male maps were estimated to 2,336.1 and 1,441.5 cM, respectively. The gene order was validated through comparisons to the cytogenetic and/or physical location of 203 genes, linkage to evenly spaced microsatellite markers as well as previously reported conserved synteny. A total of 330 previously unmapped genes and ESTs were mapped to the porcine autosome while ten genes were mapped to unexpected locations.</p> <p>Conclusion</p> <p>The linkage map presented here shows high accuracy in gene order. The pedigree family network as well as the large amount of meiotic events provide good reliability and make this map suitable for QTL and association studies. In addition, the linkage to the RH-map of microsatellites makes it suitable for comparison to other QTL studies.</p

Structural characterization and comparison of iridium, platinum and gold/palladium ultra-thin film coatings for STM of biomolecules

Scanning tunneling microscopy (STM) is capable of atomic resolution and is ideally suited for imaging surfaces with uniform work function. A biological sample on a conducting substrate in air does not meet this criteria and requires a conductive coating for stable and reproducible STM imaging. In this paper, the authors describe the STM and transmission electron microscopy (TEM) characterization of ultra-thin ion-beam sputtered films of iridium and cathode sputtered gold/palladium and platinum films on highly ordered pyrolytic graphite (HOPG) which were developed for use as biomolecule coatings. The goals were the development of metal coatings sufficiently thin and fine grained that 15--20 {angstrom} features of biological molecules could be resolved using STM, and the development of a substrate/coating system which would allow complementary TEM information to be obtained for films and biological molecules. The authors demonstrate in this paper that ion-beam sputtered iridium on highly ordered pyrolytic graphite (HOPG) has met both these goals. The ion-beam sputtered iridium produced a very fine grained (< 10 {angstrom}) continuous film at 5--6 {angstrom} thickness suitable for stable air STM imaging. In comparison, cathode sputtered platinum produced 16 {angstrom} grains with the thinnest continuous film at 15 {angstrom} thickness, and the sputtered gold/palladium produced 25 {angstrom} grains with the thinnest continuous film at 18 {angstrom} thickness

Barrier anodic coatings formed on 6061-T6 aluminum alloy in electrolytes containing different ethanol to water ratios

We have studied barrier anodic film formation on 6061-T6 aluminum alloy substrates as a function of electrolyte composition for five mixtures of ammonium tartrate dissolved in water and diluted with different amounts of ethanol. The effects of electrolyte temperatures within the range of 18/degree/C to 38/degree/C were explored. The results of this study indicate that the best dielectric coatings and the shortest processing times occur for the 100% water-ammonium tartrate electrolyte. The second best coatings and processing times occur in conjunction with the use of 98% ethanol, 2% water plus ammonium tartrate electrolyte. In general, visibly flawed coatings, scintillation events at cell voltages in excess of approximately 750-800 volts and/or abnormally long processing times occur in conjunction with the use of electrolyte mixtures containing 20%, 60%, and 90% water. We analysed samples of electrolyte as a function of usage, and evaluated the composition of the coatings using Fourier Transform Infrared Analysis to better understand the mechanisms which contribute to anodic coating growth that result in the observed variations in the dielectric properties. All of the coatings exhibited similar compositions except with regard to the amount of CO2 that was physisorbed in the coatings. The dielectrically inferior coatings that were typically produced by the electrolytes containing ethanol contain substantially more CO2 than the coatings grown in the 100% water-based electrolyte. These results strongly suggest that the ethanol in the electrolyte oxidizes and forms CO2 which is incorporated in the coatings and results in inferior dielectric properties. 8 refs., 7 figs

Single-cell analysis reveals individual spore responses to simulated space vacuum

Outer space is a challenging environment for all forms of life, and dormant spores of bacteria have been frequently used to study the survival of terrestrial life in a space journey. Previous work showed that outer space vacuum alone can kill bacterial spores. However, the responses and mechanisms of resistance of individual spores to space vacuum are unclear. Here, we examined spores’ molecular changes under simulated space vacuum (~10−5 Pa) using micro-Raman spectroscopy and found that this vacuum did not cause significant denaturation of spore protein. Then, live-cell microscopy was developed to investigate the temporal events during germination, outgrowth, and growth of individual Bacillus spores. The results showed that after exposure to simulated space vacuum for 10 days, viability of spores of two Bacillus species was reduced up to 35%, but all spores retained their large Ca2 +-dipicolinic acid depot. Some of the killed spores did not germinate, and the remaining germinated but did not proceed to vegetative growth. The vacuum treatment slowed spore germination, and changed average times of all major germination events. In addition, viable vacuum-treated spores exhibited much greater sensitivity than untreated spores to dry heat and hyperosmotic stress. Among spores’ resistance mechanisms to high vacuum, DNA-protective α/β−type small acid-soluble proteins, and non- homologous end joining and base excision repair of DNA played the most important roles, especially against multiple cycles of vacuum treatment. Overall, these results give new insight into individual spore’s responses to space vacuum and provide new techniques for microorganism analysis at the single-cell level

Restriction of HIV-1 Replication in Monocytes Is Abolished by Vpx of SIVsmmPBj

Background: Human primary monocytes are refractory to infection with the human immunodeficiency virus 1 (HIV-1) or transduction with HIV-1-derived vectors. In contrast, efficient single round transduction of monocytes is mediated by vectors derived from simian immunodeficiency virus of sooty mangabeys (SIVsmmPBj), depending on the presence of the viral accessory protein Vpx. Methods and Findings: Here we analyzed whether Vpx of SIVsmmPBj is sufficient for transduction of primary monocytes by HIV-1-derived vectors. To enable incorporation of PBj Vpx into HIV-1 vector particles, a HA-Vpr/Vpx fusion protein was generated. Supplementation of HIV-1 vector particles with this fusion protein was not sufficient to facilitate transduction of human monocytes. However, monocyte transduction with HIV-1-derived vectors was significantly enhanced after delivery of Vpx proteins by virus-like particles (VLPs) derived from SIVsmmPBj. Moreover, pre-incubation with Vpx-containing VLPs restored replication capacity of infectious HIV-1 in human monocytes. In monocytes of non-human primates, single-round transduction with HIV-1 vectors was enabled. Conclusion: Vpx enhances transduction of primary human and even non-human monocytes with HIV-1-derived vectors, only if delivered in the background of SIVsmmPBj-derived virus-like particles. Thus, for accurate Vpx function the presence of SIVsmmPBj capsid proteins might be required. Vpx is essential to overcome a block of early infection steps in primary monocytes

IL4I1 Is a Metabolic Immune Checkpoint that Activates the AHR and Promotes Tumor Progression

Immune checkpoint blockade therapy induces the AHR-activating enzyme IL4I1, which promotes tumor progression, through its effects on tumor cell motility and adaptive immunity

The first multi-model ensemble of regional climate simulations at kilometer-scale resolution, part I: evaluation of precipitation

Here we present the first multi-model ensemble of regional climate simulations at kilometer-scale horizontal grid spacing over a decade long period. A total of 23 simulations run with a horizontal grid spacing of ∼3 km, driven by ERA-Interim reanalysis, and performed by 22 European research groups are analysed. Six different regional climate models (RCMs) are represented in the ensemble. The simulations are compared against available high-resolution precipitation observations and coarse resolution (∼ 12 km) RCMs with parameterized convection. The model simulations and observations are compared with respect to mean precipitation, precipitation intensity and frequency, and heavy precipitation on daily and hourly timescales in different seasons. The results show that kilometer-scale models produce a more realistic representation of precipitation than the coarse resolution RCMs. The most significant improvements are found for heavy precipitation and precipitation frequency on both daily and hourly time scales in the summer season. In general, kilometer-scale models tend to produce more intense precipitation and reduced wet-hour frequency compared to coarse resolution models. On average, the multi-model mean shows a reduction of bias from ∼ −40% at 12 km to ∼ −3% at 3 km for heavy hourly precipitation in summer. Furthermore, the uncertainty ranges i.e. the variability between the models for wet hour frequency is reduced by half with the use of kilometer-scale models. Although differences between the model simulations at the kilometer-scale and observations still exist, it is evident that these simulations are superior to the coarse-resolution RCM simulations in the representing precipitation in the present-day climate, and thus offer a promising way forward for investigations of climate and climate change at local to regional scales

The first multi-model ensemble of regional climate simulations at kilometer-scale resolution. Part I: Evaluation of precipitation

Here we present the first multi-model ensemble of regional climate simulations at kilometer-scale horizontal grid spacing over a decade long period. A total of 23 simulations run with a horizontal grid spacing of ∼ 3 km, driven by ERA-Interim reanalysis, and performed by 22 European research groups are analysed. Six different regional climate models (RCMs) are represented in the ensemble. The simulations are compared against available high-resolution precipitation observations and coarse resolution (∼ 12 km) RCMs with parameterized convection. The model simulations and observations are compared with respect to mean precipitation, precipitation intensity and frequency, and heavy precipitation on daily and hourly timescales in different seasons. The results show that kilometer-scale models produce a more realistic representation of precipitation than the coarse resolution RCMs. The most significant improvements are found for heavy precipitation and precipitation frequency on both daily and hourly time scales in the summer season. In general, kilometer-scale models tend to produce more intense precipitation and reduced wet-hour frequency compared to coarse resolution models. On average, the multi-model mean shows a reduction of bias from ∼ −40 at 12 km to ∼ −3 at 3 km for heavy hourly precipitation in summer. Furthermore, the uncertainty ranges i.e. the variability between the models for wet hour frequency is reduced by half with the use of kilometer-scale models. Although differences between the model simulations at the kilometer-scale and observations still exist, it is evident that these simulations are superior to the coarse-resolution RCM simulations in the representing precipitation in the present-day climate, and thus offer a promising way forward for investigations of climate and climate change at local to regional scales. © 2021, The Author(s)

SAMHD1-Deficient CD14+ Cells from Individuals with Aicardi-Goutières Syndrome Are Highly Susceptible to HIV-1 Infection

Myeloid blood cells are largely resistant to infection with human immunodeficiency virus type 1 (HIV-1). Recently, it was reported that Vpx from HIV-2/SIVsm facilitates infection of these cells by counteracting the host restriction factor SAMHD1. Here, we independently confirmed that Vpx interacts with SAMHD1 and targets it for ubiquitin-mediated degradation. We found that Vpx-mediated SAMHD1 degradation rendered primary monocytes highly susceptible to HIV-1 infection; Vpx with a T17A mutation, defective for SAMHD1 binding and degradation, did not show this activity. Several single nucleotide polymorphisms in the SAMHD1 gene have been associated with Aicardi-Goutières syndrome (AGS), a very rare and severe autoimmune disease. Primary peripheral blood mononuclear cells (PBMC) from AGS patients homozygous for a nonsense mutation in SAMHD1 (R164X) lacked endogenous SAMHD1 expression and support HIV-1 replication in the absence of exogenous activation. Our results indicate that within PBMC from AGS patients, CD14+ cells were the subpopulation susceptible to HIV-1 infection, whereas cells from healthy donors did not support infection. The monocytic lineage of the infected SAMHD1 -/- cells, in conjunction with mostly undetectable levels of cytokines, chemokines and type I interferon measured prior to infection, indicate that aberrant cellular activation is not the cause for the observed phenotype. Taken together, we propose that SAMHD1 protects primary CD14+ monocytes from HIV-1 infection confirming SAMHD1 as a potent lentiviral restriction factor