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Additional file of Transcriptome sequencing and microarray development for the Manila clam, Ruditapes philippinarum: genomic tools for environmental monitoring

Additional file 2: GO terms associated to R. philippinarum transcripts represented in the microarray using "Generic GO slim" in Blast2GO software. Details about "Biological process", "Molecular function" and "Cellular component" GO terms. (DOC 158 KB)

Authors’ original file for figure 2

Authors’ original file for figure 3

SETDB1 is an essential H3K9 methyltransferase involved in silencing of retroviruses and gene regulation. We show here that its triple Tudor domain (3TD) specifically binds to doubly modified histone H3 containing K14 acetylation and K9 methylation. Crystal structures of 3TD in complex with H3K14ac/K9me peptides reveal that peptide binding and K14ac recognition occurs at the interface between Tudor domains (TD) TD2 and TD3. Structural and biochemical data demonstrate a pocket switch mechanism in histone code reading, because K9me1 or K9me2 is preferentially recognized by the aromatic cage of TD3, while K9me3 selectively binds to TD2. Mutations in the K14ac/K9me binding sites change the sub-nuclear localization of 3TD. ChIP-seq analyses show that SETDB1 is enriched at H3K9me3 regions and K9me3/K14ac is enriched at SETDB1 binding sites overlapping with LINE elements, suggesting that recruitment of the SETDB1 complex to K14ac/K9me regions has a role in silencing of active genomic regions., SETDB1 is a histone methyltransferase that generates H3K9me3 marks in euchromatic regions. Here the authors show that the triple Tudor domain (3TD) of SETDB1 binds histone H3 tails containing K14 acetylation combined with K9 methylation, and that the K9me–K14ac modification defines a novel chromatin state enriched at SETDB1 binding sites.

DNA methylation plays a critical role in the regulation and maintenance of cell-type specific transcriptional programs. Targeted epigenome editing is an emerging technology to specifically regulate cellular gene expression in order to modulate cell phenotypes or dissect the epigenetic mechanisms involved in their control. In this work, we employed a DNA methyltransferase Dnmt3a-Dnmt3L construct fused to the nuclease-inactivated dCas9 programmable targeting domain to introduce DNA methylation into the human genome specifically at the EpCAM, CXCR4 and TFRC gene promoters. We show that targeting of these loci with single gRNAs leads to efficient and widespread methylation of the promoters. Multiplexing of several guide RNAs does not increase the efficiency of methylation. Peaks of targeted methylation were observed around 25 bp upstream and 40 bp downstream of the PAM site, while 20-30 bp of the binding site itself are protected against methylation. Potent methylation is dependent on the multimerization of Dnmt3a/Dnmt3L complexes on the DNA. Furthermore, the introduced methylation causes transcriptional repression of the targeted genes. These new programmable epigenetic editors allow unprecedented control of the DNA methylation status in cells and will lead to further advances in the understanding of epigenetic signaling.

Interactions between co-existing microorganisms deeply affect the physiology of the involved organisms and, ultimately, the function of the ecosystem as a whole. Copiotrophic Alteromonas are marine gammaproteobacteria that thrive during the late stages of phytoplankton blooms in the marine environment and in laboratory co-cultures with cyanobacteria such as Trichodesmium. The response of this heterotroph to the sometimes rapid and transient changes in nutrient supply when the phototroph crashes is not well understood. Here, we isolated and sequenced the strain Alteromonas macleodii str. Te101 from a laboratory culture of Trichodesmium erythraeum IMS101, yielding a chromosome of 4.63 Mb and a single plasmid of 237 kb. Increasing salinities to ≥43 ppt inhibited the growth of Trichodesmium but stimulated growth of the associated Alteromonas. We characterized the transcriptomic responses of both microorganisms and identified the complement of active transcriptional start sites in Alteromonas at single-nucleotide resolution. In replicate cultures, a similar set of genes became activated in Alteromonas when growth rates of Trichodesmium declined and mortality was high. The parallel activation of fliA, rpoS and of flagellar assembly and growth-related genes indicated that Alteromonas might have increased cell motility, growth, and multiple biosynthetic activities. Genes with the highest expression in the data set were three small RNAs (Aln1a-c) that were identified as analogs of the small RNAs CsrB-C in E. coli or RsmX-Z in pathogenic bacteria. Together with the carbon storage protein A (CsrA) homolog Te101_05290, these RNAs likely control the expression of numerous genes in responding to changes in the environment., The ISME Journal, 12, ISSN:1751-7362, ISSN:1751-7370

Background: Sulfate-reducing bacteria (SRB) are key players of the carbon-and sulfur-cycles in the sediments of the world's oceans. Habitat relevant SRBs are often members of the Desulfosarcina-Desulfococcus clade belonging to the deltaproteobacterial family of Desulfobacteraceae. Despite this environmental recognition, their molecular (genome-based) physiology and their potential to contribute to organic carbon mineralization as well as to adapt to changing environmental conditions have been scarcely investigated. A metabolically versatile representative of this family is Desulfococcus multivorans that is able to completely oxidize (to CO2) a variety of organic acids, including fatty acids up to C-14, as well as aromatic compounds. Results: In this study the complete 4.46 Mbp and manually annotated genome of metabolically versatile Desulfococcus multivorans DSM 2059 is presented with particular emphasis on a proteomics-driven metabolic reconstruction. Proteomic profiling covered 17 substrate adaptation conditions (6 aromatic and 11 aliphatic compounds) and comprised 2D DIGE, shotgun proteomics and analysis of the membrane protein-enriched fractions. This comprehensive proteogenomic dataset allowed for reconstructing a metabolic network of degradation pathways and energy metabolism that consists of 170 proteins (154 detected; similar to 91 % coverage). Peripheral degradation routes feed via central benzoyl-CoA, (modified) beta-oxidation or methylmalonyl-CoA pathways into the Wood-Ljungdahl pathway for complete oxidation of acetyl-CoA to CO2. Dissimilatory sulfate reduction is fueled by a complex electron transfer network composed of cytoplasmic components (e.g., electron transfer flavoproteins) and diverse membrane redox complexes (Dsr, Qmo, Hmc, Tmc, Qrc, Nuo and Rnf). Overall, a high degree of substrate-specific formation of catabolic enzymes was observed, while most complexes involved in electron transfer appeared to be constitutively formed. Conclusions: A highly dynamic genome structure in combination with substrate-specifically formed catabolic subproteomes and a constitutive subproteome for energy metabolism and electron transfer appears to be a common trait of Desulfobacteraceae members.

Distribution of the 1,307 detected proteins by 2D DIGE, whole cell shotgun analysis and preparation of the membrane protein-enriched fraction of D. multivorans grown with 17 different substrates. Figure S2 Phylogenetic relationship of the class II benzoyl-CoA reductase catalytic subunit BamB and other uncharacterized aldehyde: ferredoxin oxidoreductases (AFOR) of selected Deltaproteobacteria. Figure S3 Scale model and chromosomal localization of transmembrane redox complex containing genes of D. multivorans. Table S1 Listing of locus tags of genes manually assigned to metabolic pathways and energy conservation as displayed in Figs. 2 and 3. (PDF 433 kb)

Bacteria of the class Dehalococcoidia (DEH) (phylum Chloroflexi) are widely distributed in the marine subsurface and are especially prevalent in deep marine sediments. Nevertheless, little is known about the specific distributions of DEH subgroups at different sites and depths. This study therefore specifically examined the distributions of DEH through depths of various marine sediment cores by quantitative PCR and pyrosequencing using newly designed DEH 16S rRNA gene targeting primers. Quantification of DEH showed populations may establish in shallow sediments (i.e. upper centimetres), although as low relative proportions of total Bacteria, yet often became more prevalent in deeper sediments. Pyrosequencing revealed pronounced diversity co-exists within single biogeochemical zones, and that clear and sometimes abrupt shifts in relative proportions of DEH subgroups occur with depth. These shifts indicate varying metabolic properties exist among DEH subgroups. The distributional changes in DEH subgroups with depth may be related to a combination of biogeochemical factors including the availability of electron acceptors such as sulfate, the composition of organic matter and depositional regimes. Collectively, the results suggest DEH exhibit wider metabolic and genomic diversity than previously recognized, and this contributes to their widespread occurrence in the marine subsurface.

BMC genomics 14, 793 (2013). doi:10.1186/1471-2164-14-793, Published by BioMed Central ; Berlin ; Heidelberg : Springer, London

Background Climate change will lead in the future to an occurrence of heat waves with a higher frequency and duration than observed today, which has the potential to cause severe damage to seedlings of temperate maize genotypes. In this study, we aimed to (I) assess phenotypic variation for heat tolerance of temperate European Flint and Dent maize inbred lines, (II) investigate the transcriptomic response of temperate maize to linearly increasing heat levels and, (III) identify genes associated with heat tolerance in a set of genotypes with contrasting heat tolerance behaviour. Results Strong phenotypic differences with respect to heat tolerance were observed between the examined maize inbred lines on a multi-trait level. We identified 607 heat responsive genes as well as 39 heat tolerance genes. Conclusion Our findings indicate that individual inbred lines developed different genetic mechanisms in response to heat stress. We applied a novel statistical approach enabling the integration of multiple genotypes and stress levels in the analysis of abiotic stress expression studies. Electronic supplementary material The online version of this article (doi:10.1186/s12864-015-1282-1) contains supplementary material, which is available to authorized users.

Background: DNA methylation and histone 3 lysine 9 (H3K9) methylation are considered as epigenetic marks that can be inherited through cell divisions. To explore the functional consequences and stability of these modifications, we employed targeted installment of DNA methylation and H3K9 methylation in the vascular endothelial growth factor A (VEGF-A) promoter using catalytic domains of DNA or H3K9 methyltransferases that are fused to a zinc finger protein which binds a site in the VEGF-A promoter. Results: Expression of the targeted DNA and H3K9 methyltransferases caused dense deposition of DNA methylation or H3K9 di- and trimethylation in the promoter of VEGF-A and downregulation of VEGF-A gene expression. We did not observe positive feedback between DNA methylation and H3K9 methylation. Upon loss of the targeted methyltransferases from the cells, the epigenetic marks, chromatin environment, and gene expression Levels returned to their original state, indicating that both methylation marks were not stably propagated after their installment. Conclusions: The clear anti-correlation between DNA or H3K9 methylation and gene expression suggests a direct role of these marks in transcriptional control. The lack of maintenance of the transiently induced silenced chromatin state suggests that the stability of epigenetic signaling is based on an epigenetic network consisting of several molecular marks. Therefore, for stable reprogramming, either multivalent deposition of functionally related epigenetic marks or longer-lasting trigger stimuli might be necessary.

In rocky shores, desiccation is triggered by daily tide changes, and experimental evidence suggests that local distribution of algal species across the intertidal rocky zone is related to their capacity to tolerate desiccation. In this context, the permanence of Pyropia columbina in the high intertidal rocky zone is explained by its exceptional physiological tolerance to desiccation. This study explored the metabolic pathways involved in tolerance to desiccation in the Chilean P. columbina, by characterizing its transcriptome under contrasting conditions of hydration. We obtained 1,410 ESTs from two subtracted cDNA libraries in naturally hydrated and desiccated fronds. Results indicate that transcriptome from both libraries contain transcripts from diverse metabolic pathways related to tolerance. Among the transcripts differentially expressed, 15% appears involved in protein synthesis, processing and degradation, 14.4% are related to photosynthesis and chloroplast, 13.1% to respiration and mitochondrial function (NADH dehydrogenase and cytochrome c oxidase proteins), 10.6% to cell wall metabolism, and 7.5% are involved in antioxidant activity, chaperone and defense factors (catalase, thioredoxin, heat shock proteins, cytochrome P450). Both libraries highlight the presence of genes/proteins never described before in algae. This information provides the first molecular work regarding desiccation tolerance in P. columbina, and helps, to some extent, explaining the classical patterns of ecological distribution described for algae across the intertidal zone. En zonas rocosas costeras, la desecación es gatillada por cambios diarios en los niveles de marea, y la evidencia experimental indica que la distribución de las algas en la zona intermareal está relacionada con su capacidad para tolerar la desecación. En este contexto, la presencia de Pyropia columbina en la zona alta del intermareal se explica por su excepcional tolerancia fisiológica a la desecación. Este estudio explora las vías metabólicas involucradas en la tolerancia a la desecación en P. columbina, a través de la caracterización de su transcriptoma bajo condiciones de hidratación contrastantes. Se obtuvo 1,410 ESTs provenientes de dos librerías de substracción de cDNA de frondas naturalmente hidratadas y desecadas. Los transcriptomas de ambas librerías contienen transcritos de diversas rutas metabólicas relacionadas a la tolerancia. Entre los transcritos expresados 15% están involucrados en la síntesis de proteínas, su procesamiento y degradación, 14,4% asociados a fotosíntesis y cloroplasto, 13,1% a respiración y función mitocondrial, 10,6% al metabolismo de la pared celular y 7,5% a la actividad antioxidante, proteínas chaperonas y factores de defensa (catalasa, tiorredoxina, proteínas de shock térmico, citocromo P450). En ambas librerías se destaca la presencia de genes/proteínas no descritos en algas. Esta información proporciona el primer trabajo molecular que estudia la tolerancia a desecación en P. columbina y sus resultados ayudan a explicar los patrones clásicos de distribución descritos para algas en la zona intermareal.

Skern-Mauritzen, Rasmus, Malde, Ketil, Besnier, Francois, Nilsen, Frank, Jonassen, Inge, Reinhardt, Richard, Koop, Ben, Dalvin, Sussie, Maehle, Stig, Kongshaug, Heidi, Glover, Kevin A. (2013): How does sequence variability affect de novo assembly quality? Journal of Natural History 47 (5-12): 901-910, DOI: 10.1080/00222933.2012.738833, URL: http://dx.doi.org/10.1080/00222933.2012.738833

Background Uncoupling proteins (UCP) are evolutionary conserved mitochondrial carriers that control energy metabolism and therefore play important roles in several physiological processes such as thermogenesis, regulation of reactive oxygen species (ROS), growth control, lipid metabolism and regulation of insulin secretion. Despite their importance in various physiological processes, their molecular function remains controversial. The evolution and phylogenetic distribution may assist to identify their general biological function and structure-function relationships. The exact number of uncoupling protein genes in the fish genome and their evolution is unresolved. Results Here we report the first characterisation of UCP gene family members in sea bass, Dicentrarchus labrax, and then retrace the evolution of the protein family in vertebrates. Four UCP genes that are shared by five other fish species were identified in sea bass genome. Phylogenetic reconstitution among vertebrate species and synteny analysis revealed that UCP1, UCP2 and UCP3 evolved from duplication events that occurred in the common ancestor of vertebrates, whereas the novel fourth UCP originated specifically in the teleost lineage. Functional divergence analysis among teleost species revealed specific amino acid positions that have been subjected to altered functional constraints after duplications. Conclusions This work provides the first unambiguous evidence for the presence of a fourth UCP gene in teleost fish genome and brings new insights into the evolutionary history of the gene family. Our results suggest functional divergence among paralogues which might result from long-term and differential selective pressures, and therefore, provide the indication that UCP genes may have diverse physiological functions in teleost fishes. Further experimental analysis of the critical amino acids identified here may provide valuable information on the physiological functions of UCP genes.

Periodontitis has been reported to be associated with coronary artery disease (CAD). Research is needed to determine if therapies that improve periodontal health also reduce systemic measures of inflammation associated with both diseases.The study registrar randomly assigned 128 eligible postmenopausal women with chronic periodontitis to a twice-daily regimen of subantimicrobial-dose-doxycycline (SDD) or placebo tablets for two years as an adjunct to periodontal maintenance therapy. Through a supplement to the main trial, in which they investigated alveolar bone and clinical attachment level changes, the authors assayed inflammatory mediators and lipid profiles in baseline, one-year and two-year serum samples. The authors analyzed the data by using generalized estimating equations.In the intent-to-treat analysis across two years, SDD treatment reduced median high-sensitivity C-reactive protein (hs-CRP) by 18 percent (primary outcome; P = .02) and reduced serum matrix metalloproteinase (MMP)-9 (92 kilodalton gelatinase; difference in mean scanning units, -28.44; P.001), with no significant effect on serum lipids. However, in women more than five years postmenopausal, SDD elevated the level of high-density lipoprotein (HDL) cholesterol (difference in means [milligrams per deciliter], 5.99; P = .01).A two-year SDD regimen in postmenopausal women significantly reduced the serum inflammatory biomarkers hs-CRP and MMP-9 and, among women more than five years postmenopausal, increased the HDL cholesterol level.SDD significantly reduced the systemic inflammatory biomarkers hs-CRP and MMP-9. More research is needed to determine whether SDD has a role in managing the care of patients at risk of developing CAD.

Microorganisms can degrade saturated hydrocarbons (alkanes) not only under oxic but also under anoxic conditions. Three denitrifying isolates (strains HxN1, OcN1, HdN1) able to grow under anoxic conditions by coupling alkane oxidation to CO(2) with NO(3) (-) reduction to N(2) were compared with respect to their alkane metabolism. Strains HxN1 and OcN1, which are both Betaproteobacteria, utilized n-alkanes from C(6) to C(8) and C(8) to C(12) respectively. Both activate alkanes anaerobically in a fumarate-dependent reaction yielding alkylsuccinates, as suggested by present and previous metabolite and gene analyses. However, strain HdN1 was unique in several respects. It belongs to the Gammaproteobacteria and was more versatile towards alkanes, utilizing the range from C(6) to C(30). Neither analysis of metabolites nor analysis of genes in the complete genome sequence of strain HdN1 hinted at fumarate-dependent alkane activation. Moreover, whereas strains HxN1 and OcN1 grew with alkanes and NO(3) (-), NO(2) (-) or N(2)O added to the medium, strain HdN1 oxidized alkanes only with NO(3) (-) or NO(2) (-) but not with added N(2)O; but N(2)O was readily used for growth with long-chain alcohols or fatty acids. Results suggest that NO(2) (-) or a subsequently formed nitrogen compound other than N(2)O is needed for alkane activation in strain HdN1. From an energetic point of view, nitrogen-oxygen species are generally rather strong oxidants. They may enable enzymatic mechanisms that are not possible under conditions of sulfate reduction or methanogenesis and thus allow a special mode of alkane activation.

Background: Gene function analysis of the obligate intracellular bacterium Chlamydia pneumoniae is hampered by the facts that this organism is inaccessible to genetic manipulations and not cultivable outside the host. The genomes of several strains have been sequenced; however, very little information is available on the gene structure and transcriptome of C. pneumoniae. Results: Using a differential RNA-sequencing approach with specific enrichment of primary transcripts, we defined the transcriptome of purified elementary bodies and reticulate bodies of C. pneumoniae strain CWL-029; 565 transcriptional start sites of annotated genes and novel transcripts were mapped. Analysis of adjacent genes for cotranscription revealed 246 polycistronic transcripts. In total, a distinct transcription start site or an affiliation to an operon could be assigned to 862 out of 1,074 annotated protein coding genes. Semi-quantitative analysis of mapped cDNA reads revealed significant differences for 288 genes in the RNA levels of genes isolated from elementary bodies and reticulate bodies. We have identified and in part confirmed 75 novel putative non-coding RNAs. The detailed map of transcription start sites at single nucleotide resolution allowed for the first time a comprehensive and saturating analysis of promoter consensus sequences in Chlamydia. Conclusions: The precise transcriptional landscape as a complement to the genome sequence will provide new insights into the organization, control and function of genes. Novel non-coding RNAs and identified common promoter motifs will help to understand gene regulation of this important human pathogen.

International audience

Next Generation Sequencing 2009 International Meeting, 1-3 Octubre 2009, Barcelona, Fish represent unique models to study vertebrate sex determination and differentiation, as well as gonad maturation for several reasons. First, fish are by far the most abundant type of vertebrates and exhibit all reproduction types known in vertebrates (gonochorism, hermaphroditism and unisexuality). Furthermore, the processes of sex determination, differentiation and gonad maturation are the result of complex genetic, environmental and social interactions. Thus, for example, temperature or population density can easily influence the course of sex differentiation, a phenomenon that represents one of the most dramatic examples of phenotypic plasticity. The gonads are then the only organ in vertebrates that starting from common undifferentiated rudiment can undergo two completely different developmental pathways, resulting in a testis or ovary, and the environmental conditions imposed by modern farming provide an excellent framework where to study these essential biological processes. We are applying next generation sequencing technologies, specifically 454 sequencing, to obtain a detailed overview of both the European sea bass (Dicentrarchus labrax) and turbot (Scophthalmus maximus) gonadal transcriptome under a variety of developmental and experimental conditions. In addition, oligo-based microarrays are being built enriched with genes expressed in the reproductive axis with the purpose of discovering new genes and signaling routes

Background: The facultatively anaerobic betaproteobacterium Castellaniella defragrans 65Phen utilizes acyclic, monocyclic and bicyclic monoterpenes as sole carbon source under oxic as well as anoxic conditions. A biotransformation pathway of the acyclic beta-myrcene required linalool dehydratase-isomerase as initial enzyme acting on the hydrocarbon. An in-frame deletion mutant did not use myrcene, but was able to grow on monocyclic monoterpenes. The genome sequence and a comparative proteome analysis together with a random transposon mutagenesis were conducted to identify genes involved in the monocyclic monoterpene metabolism. Metabolites accumulating in cultures of transposon and in-frame deletion mutants disclosed the degradation pathway. Results: Castellaniella defragrans 65Phen oxidizes the monocyclic monoterpene limonene at the primary methyl group forming perillyl alcohol. The genome of 3.95 Mb contained a 70 kb genome island coding for over 50 proteins involved in the monoterpene metabolism. This island showed higher homology to genes of another monoterpene-mineralizing betaproteobacterium, Thauera terpenica 58Eu(T), than to genomes of the family Alcaligenaceae, which harbors the genus Castellaniella. A collection of 72 transposon mutants unable to grow on limonene contained 17 inactivated genes, with 46 mutants located in the two genes ctmAB (cyclic terpene metabolism). CtmA and ctmB were annotated as FAD-dependent oxidoreductases and clustered together with ctmE, a 2Fe-2S ferredoxin gene, and ctmF, coding for a NADH: ferredoxin oxidoreductase. Transposon mutants of ctmA, B or E did not grow aerobically or anaerobically on limonene, but on perillyl alcohol. The next steps in the pathway are catalyzed by the geraniol dehydrogenase GeoA and the geranial dehydrogenase GeoB, yielding perillic acid. Two transposon mutants had inactivated genes of the monoterpene ring cleavage (mrc) pathway. 2-Methylcitrate synthase and 2-methylcitrate dehydratase were also essential for the monoterpene metabolism but not for growth on acetate. Conclusions: The genome of Castellaniella defragrans 65Phen is related to other genomes of Alcaligenaceae, but contains a genomic island with genes of the monoterpene metabolism. Castellaniella defragrans 65Phen degrades limonene via a limonene dehydrogenase and the oxidation of perillyl alcohol. The initial oxidation at the primary methyl group is independent of molecular oxygen.

Background Improving nutrient homeostasis is a major challenge of a sustainable maize cultivation, and cornerstone to ensure food supply for a growing world population. Although, iron constitutes an important nutrient, iron availability is limited. In this respect, iron deficiency associated chlorosis causes severe yield losses every year. Natural variation of the latter trait has yet not been addressed in maize and was therefore studied in the present analysis. Results In this study, we i) report about the contrasting chlorosis phenotypes of the inbreds B73 and Mo17 at 10 and 300 μM iron regime, ii) identified over 400 significantly regulated transcripts (FDR

Background Triosephosphate isomerase (TPI) is a central and conserved glycolytic enzyme. In humans, TPI is encoded by a single gene on 12p13, and associated with a rare genetic disorder, TPI deficiency. Reduced TPI activity can increase specific oxidant resistances of model organisms and TPI null-alleles have been hypothesized to promote a heterozygote advantage in man. However, comprehensive genetic information about the TPI1 locus is still lacking. Results Here, we sequenced the TPI1 locus in a sample of 357 German long-lived individuals (LLI) aged 95 to 110 years. We identified 17 different polymorphisms, of which 15 were rare and previously unknown. The two remaining SNPs occurred at much higher frequency and were tested for association with the longevity phenotype in larger samples of LLI (n = 1422) and younger controls (n = 967). Neither of the two markers showed a statistically significant difference in allele or genotype frequency between LLI and control subjects. Conclusion This study marks the TPI1 locus as extraordinarily conserved, even when analyzing intronic and non-coding regions of the gene. None of the identified sequence variations affected the amino acid composition of the TPI protein and hence, are unlikely to impact the catalytic activity of the enzyme. Thus, TPI variants occur less frequent than expected and inactive alleles are not enriched in German centenarians.

Low temperature X-ray structural analyses of monoclinic single crystals of S10 and S6 · Si10 (prepared from the components) show that the cyclic S10 molecule exhibits the same D2 conformation in both compounds with bond distances between 203.3 and 208.0 pm, bond angles (α) between 103 and 111°, and torsional angles (τ) between 73 and 124°. The S6 molecule (site symmetry Ci) in S6 · S10 is very similar to the one in pure S6 (dSS = 206.2 pm, α= 103°, τ = 74°). All intermolecular interactions are of van-der-Waals type. The Raman spectrum of S6 · S10 can be explained by a superposition of the S6 and S10 spectra.

Fifteen years of studies on Palladium! II) complexes of H ?0, NHL, and CI are summarized, including not only results from this laboratory but also from other laboratories, as have appeared in the chemical literature. More extensive report is given on several experiments not appearing elsewhere: the isomerism equilibria of Pd(NH 3 ) 2 Cl 2 and the ammonation kinetics of cis- Pd(NH 3 )gC1 2 . Tables are given for all presently known equilibrium a kinetic data involving these complexes, and estimates are also provided a number of reactions for which no data exist. Thirty-eight references are cited. supported in part by the Foundation Research Program of the Naval Postgraduate School with funds provided by the Chief of Naval Research http://archive.org/details/equilibriumkinet00rein N0001476WR60052 NA

The history of the construction of the stopped- flow apparatus at the Naval Postgraduate School is outlined and the design and characteristics of the present working model are given in constderable detail. Also provided are operating instructions and calibration procedures. Sixteen literature references are cited. Prepared for: Chief of Naval Research, Arlington, Virginia 22217, and Naval Postgraduate Schhool, Monterey, California 93940 http://archive.org/details/designconstructi00grah N0001476WR60052 NA

A petition to the President of the Executive Committee and members of Sigma XI by NPS members organized as a Club to establish a Chapter at the U.S. Naval Postgraduate School. Approved for public release; distribution is unlimited.

A Report to the Office of Naval Research on an investigation carried out under ONR Project Order No. NR051-350 http://archive.org/details/radiochemicalstu18rein NA NA

The article of record as published may be found at http://dx.doi.org/10.1021/ic50116a022 Upon treatment with excess aqueous HClO₄, Pd(NH₃)₄²⁺ is converted through four consecutive steps to Pd(OH₂)₄²⁺. Correlation of observed rate constants with actual mechanistic steps is based primarily on reconstruction of the absorption spectra of the intermediate species; it was thus found that the rate constants decrease steadily from one step to the next. At 50°, µ = 1, rate constants found are respectively 0.020, 0.008, 9.2 X 10ˉ³ and 2.2 X 10ˉ⁴ secˉ¹. Activation enthalpies range from 21 to 24 kcal/mol and activation entropies from +4 to -4 eu. Substituent effects are compared with those in chloride-containing complexes and with those for the reverse reactions. Office of Naval Research

The article of record as published may be found at http://dx.doi.org/10.1021/ic50048a013 Potentiometric and spectrophotometric studies have been made of the equilibria among several chloroammine complexes of Pd(II) in ammonium salt solutions at 25 and 30˚. For unit ionic strength at 25˚, equilibrium constants found potentiometrically for the substitution of one NH₃ by Clˉ are: in Pd(NH₃)₄²⁺, 6.1 X 10ˉ⁵; Pd(NH₃)₃CI⁺, 1.6 X 10ˉ⁴; Pd(NH₃)₂,CI₂, 1.9 X 10ˉ⁶. Aquation constants are, similarly: Pd(lNH₃)₃Cl⁺, 0.0010; Pd(NH₃)₂Cl₂, 0.0047. ΔH˚ for the substitution by Clˉ of each of the first two NH₃ molecules in Pd(NH₃)₄²⁺ is estimated as 10 kcal. Office of Naval Research

The article of record as published may be found at http://dx.doi.org/10.1021/ed043p382

The article of record as published may be found at http://dx.doi.org/10.1021/ic50091a014 Office of Naval Research

Solubilities have been determined at 25˚ for trans-Pd(NH₃)₂Cl₂ in solutions of NH₄NO₃, NH₄CI, and NaCl, in which media base hydrolysis is repressed. The data are interpreted in terms of the heterogeneous equilibrium involving the uncharged solute molecule and the homogeneous equilibrium for the first step of aquation, The concentration of neutral solute in the saturated solution is given as 9.5 X 10⁻⁴ M and the aquation equilibrium constant as 3.9 X 10⁻³. Office of Naval Research

Background DNA methylation and histone 3 lysine 9 (H3K9) methylation are considered as epigenetic marks that can be inherited through cell divisions. To explore the functional consequences and stability of these modifications, we employed targeted installment of DNA methylation and H3K9 methylation in the vascular endothelial growth factor A (VEGF-A) promoter using catalytic domains of DNA or H3K9 methyltransferases that are fused to a zinc finger protein which binds a site in the VEGF-A promoter. Results Expression of the targeted DNA and H3K9 methyltransferases caused dense deposition of DNA methylation or H3K9 di- and trimethylation in the promoter of VEGF-A and downregulation of VEGF-A gene expression. We did not observe positive feedback between DNA methylation and H3K9 methylation. Upon loss of the targeted methyltransferases from the cells, the epigenetic marks, chromatin environment, and gene expression levels returned to their original state, indicating that both methylation marks were not stably propagated after their installment. Conclusions The clear anti-correlation between DNA or H3K9 methylation and gene expression suggests a direct role of these marks in transcriptional control. The lack of maintenance of the transiently induced silenced chromatin state suggests that the stability of epigenetic signaling is based on an epigenetic network consisting of several molecular marks. Therefore, for stable reprogramming, either multivalent deposition of functionally related epigenetic marks or longer-lasting trigger stimuli might be necessary. Electronic supplementary material The online version of this article (doi:10.1186/s13072-015-0002-z) contains supplementary material, which is available to authorized users.

Understanding RNA processing and turnover requires knowledge of cleavages by major endoribonucleases within a living cell. We have employed TIER-seq (transiently inactivating an endoribonuclease followed by RNA-seq) to profile cleavage products of the essential endoribonuclease RNase E in Salmonella enterica. A dominating cleavage signature is the location of a uridine two nucleotides downstream in a single-stranded segment, which we rationalize structurally as a key recognition determinant that may favor RNase E catalysis. Our results suggest a prominent biogenesis pathway for bacterial regulatory small RNAs whereby RNase E acts together with the RNA chaperone Hfq to liberate stable 3′ fragments from various precursor RNAs. Recapitulating this process in vitro, Hfq guides RNase E cleavage of a representative small-RNA precursor for interaction with a mRNA target. In vivo, the processing is required for target regulation. Our findings reveal a general maturation mechanism for a major class of post-transcriptional regulators.

SETDB1 is an essential H3K9 methyltransferase involved in silencing of retroviruses and gene regulation. We show here that its triple Tudor domain (3TD) specifically binds to doubly modified histone H3 containing K14 acetylation and K9 methylation. Crystal structures of 3TD in complex with H3K14ac/K9me peptides reveal that peptide binding and K14ac recognition occurs at the interface between Tudor domains (TD) TD2 and TD3. Structural and biochemical data demonstrate a pocket switch mechanism in histone code reading, because K9me1 or K9me2 is preferentially recognized by the aromatic cage of TD3, while K9me3 selectively binds to TD2. Mutations in the K14ac/K9me binding sites change the subnuclear localization of 3TD. ChIP-seq analyses show that SETDB1 is enriched at H3K9me3 regions and K9me3/K14ac is enriched at SETDB1 binding sites overlapping with LINE elements, suggesting that recruitment of the SETDB1 complex to K14ac/K9me regions has a role in silencing of active genomic regions.

The characterization and chromosomal mapping of major histocompatibility complex (MHC)-containing BAC clones from platypus and the short-beaked echidna reveals new insights into the evolution of both the mammalian MHC and monotreme sex chromosomes., Background The monotremes, represented by the duck-billed platypus and the echidnas, are the most divergent species within mammals, featuring a flamboyant mix of reptilian, mammalian and specialized characteristics. To understand the evolution of the mammalian major histocompatibility complex (MHC), the analysis of the monotreme genome is vital. Results We characterized several MHC containing bacterial artificial chromosome clones from platypus (Ornithorhynchus anatinus) and the short-beaked echidna (Tachyglossus aculeatus) and mapped them onto chromosomes. We discovered that the MHC of monotremes is not contiguous and locates within pseudoautosomal regions of two pairs of their sex chromosomes. The analysis revealed an MHC core region with class I and class II genes on platypus and echidna X3/Y3. Echidna X4/Y4 and platypus Y4/X5 showed synteny to the human distal class III region and beyond. We discovered an intron-containing class I pseudogene on platypus Y4/X5 at a genomic location equivalent to the human HLA-B,C region, suggesting ancestral synteny of the monotreme MHC. Analysis of male meioses from platypus and echidna showed that MHC chromosomes occupy different positions in the meiotic chains of either species. Conclusion Molecular and cytogenetic analyses reveal new insights into the evolution of the mammalian MHC and the multiple sex chromosome system of monotremes. In addition, our data establish the first homology link between chicken microchromosomes and the smallest chromosomes in the monotreme karyotype. Our results further suggest that segments of the monotreme MHC that now reside on separate chromosomes must once have been syntenic and that the complex sex chromosome system of monotremes is dynamic and still evolving.

Helminth exposure, allergy and asthma each induce cellular responses in lymphoid and peripheral tissues that give rise to type-2 inflammation. Essential molecular mediators of this response are type-2 cytokines interleukin(IL)-4 and IL-13 derived from various subsets of immune cells. In lymphoid tissues, CD4+ Tfh cells make IL-4 to elicit IgE and high-affinity IgG1 production. In peripheral sites of infection, group 2 innate lymphoid (ILC2) cells make IL-13 and Th2 cells make both IL-13 and IL-4. Together, these cells mediate smooth muscle contraction, mucus production and recruitment of other innate effector cells, all of which are hallmarks of type-2 inflammation. As central mediators of type-2 inflammation, understanding the cell-specific expression and molecular regulation of type-2 cytokines in CD4+ T cells and ILC2 cells may lead to new therapies that ameliorate allergic disease and helminth infections. The AP-1 factor basic leucine zipper transcription factor ATF-like (BATF) has been identified as a pioneer factor in in vitro-generated Th17 cells. BATF facilitates chromatin remodeling at the IL-17 locus as well as loci of key Th17-associated lineage specifying factors. It has also been deemed essential to the generation of functional humoral immunity through the development of follicular helper T (Tfh) cells and germinal center B cells. However, the role of BATF in the development and function of other CD4+ T helper subsets and innate immune cells in vivo has remained unclear. I show here that mice deficient in BATF do not develop type-2 inflammation after exposure to the parasitic helminth Nippostongylus brasiliensis. Since type-2 cytokine expression by Th2 and ILC2 cells is essential for expedient helminth expulsion, I hypothesized that BATF likely has a role in the development and/or induction of cytokine expression in CD4+ Th2 cells and ILC2 cells. Consistent with this hypothesis, I found that BATF utilizes a novel mechanism to control Th2 cytokine expression in Th2 cells. Specifically, BATF promotes permissive epigenetic modifications to alter the chromatin landscape early during Th2 cell differentiation. In addition, my data show that BATF deficiency inhibits the activation of ILC2 cells, preventing ILC2-mediated helminth clearance. In addition to uncovering BATF-mediated regulations of type-2 inflammation, my work has revealed new insight into the role of a second bZIP transcription factor, cMaf, during type-2 immunity. As mentioned above, helminth exposure elicits IL-4 production by both CD4+ Tfh and Th2 cells. Although type-2 cytokine transcription has been well characterized in Th2 cells, Tfh cell-mediated IL-4 production has yet to be fully defined. Importantly, I show that IL-4 production by Tfh cells is sustained upon deletion of classical IL-4 regulatory factors signal transducer and activator of transcription 6 (STAT6) and STAT5 and is not dependent on high GATA-3 expression. In sum, Tfh-driven IL-4 production is induced independent of classical pathways in Th2 cells. Presently, the non-canonical transcription factors involved in IL-4 production by Tfh cells remain unclear. C-Maf works with BCL6, the master regulator of Tfh cells, to elicit Tfh formation. However, the precise role of c-Maf in Tfh cell fate and function remains unclear. So far, it has been shown that in Th2 cells, c-Maf binds to the IL-4 promoter and in Tfh cells, c-Maf binds to the CNS2 enhancer of the IL-4 locus to regulate IL-4 expression. Therefore, I hypothesized that c-Maf is important in non-canonical, GATA-3-independent IL-4 production by Tfh cells. Here, I show that Tfh cells lacking canonical Th2 pathways for IL-4 expression express high levels of c-Maf and IL-4 transcript. Deletion of c-Maf in CD4+ T cells resulted in normal induction of BCL6 expression. Thus the initial stages of Tfh cell generation were induced. However, cMaf-deficient CD4+ T cells did not express important molecules associated with Tfh cell migration. Immunohistochemistry also confirmed that c-Maf deficiency inhibited CD4+ T cell migration from the paracortex into the B cell follicle. These defects did not inhibit cMaf-deficient CD4+ T cells from making IL-4 transcript; however, IL-4 protein production was significantly impaired. Together, these results demonstrate that c-Maf is essential for Tfh cell-mediated immunity by promoting CD4+ T cell migration to the B cell follicles and the production of IL-4 protein in the germinal centers. Collectively, the objective of my thesis research is to define the roles of the bZIP transcription factors BATF and c-Maf in type-2 inflammation. My data demonstrate that BATF is essential for the differentiation and function of Tfh, Th2, and ILC2 cells during helminth infection. Additionally, I have shown that c-Maf is required for Tfh function and CD4+ T cell migration to the B cell follicle. Thus, BATF and c-Maf are central to the development of humoral and peripheral type-2 inflammatory responses against helminth infection. Given the wide spectrum of disorders associated with type-2 inflammation, the identification of factors relevant to the development and function of Th2-, ILC2- and Tfh-driven allergic pathologies is broadly relevant. A comprehensive characterization of core factors like BATF and c-Maf provide new avenues in which to explore novel therapies to modulate type-2 inflammatory responses.

Sequenzierung und Strukturen des Genoms von Pirellula sp. Stamm 1 stehen im Mittelpunkt der vorliegenden Arbeit. Bei dem Genom von Pirellula sp. Stamm 1 handelt es sich um das erste sequenzierte Genom aus dem tief abzweigenden Phylum Planctomycetales. Durch die Verwendung der whole genome shotgun Strategie gelang es auf hohem Qualitätsniveau die Sequenz des zirkulären 7´145´576 bp großen Genoms lückenlos zu bestimmen. Das notwendige fast achtfache Sequencing Coverage ließ sich durch einen hohen Grad der Automatisierung erreichen. Bei dem Genom von Pirellula sp. Stamm 1 handelt es sich um eines der größten bisher sequenzierten Bakteriengenome. Die im Genom auftretenden repetitive Elemente führten zunächst zu Problemen bei der Assemblierung der Einzelsequenzen. 62 identifizierte repetitive Elemente wurden in 13 Gruppen eingeteilt. Diese Gruppen beinhalten bisher unbekannte bakterielle Insertionssequenzen. Als weitere Besonderheiten dieses Genoms, die in dieser Arbeit hervorgehoben werden, sind die Verteilung der tRNAs und das nicht als klassische Einheit vorliegende rRNA-Operon zu nennen. Die tRNA-Verteilung zeigt außergewöhnliche lokale Konzentrationen, die in einem Fall ein Drittel der gesamten identifizierten tRNAs auf sich vereint. Der Abstand der 16S rRNA Untereinheit zu der 23S und der 5S rRNA Untereinheit von 467 kb bestätigt die bisherigen Erkenntnisse über das nicht als Einheit vorliegende rRNA-Operon im Phylum Planctomycetales auf deutliche Weise. Die vorliegende vollständige Sequenz von Pirellula sp. Stamm 1 stellt die Basis für weitere Analysen des Genoms dar.

This corrosion research has been conducted in two ways: microscopically and electrochemically . In the microscopic portion has been studied the mode and distribution of attack on metal coupons immersed in corrosive fluids. Here could be observed the effects of alloying, the corrosion product morphology, selective phase corrosion, and base metal microstructure . Both optical and scanning electron microscopy were used. In the electrochemical portion has been examined the effects of varying the corrosive electrolyte from synthetic seawater alone to one containing a marine fouling inhibitor, sodium hypochlorite. Here, corrosion rates were obtained for the specimens in the various corrosive media by a variety of methods. These ranged from rather simple controlled immersion tests to the more sophisticated electrochemical measurements such as linear polarization and potentiodynamic polarization. Chemical and x-ray analyses were performed on the corrosion products. The information gained from these two parts was integrated to provide predictions as to the suitability, probable rate and failure mode for each of the materials studied. http://archive.org/details/microgalvanicspe1094518797 Approved for public release; distribution is unlimited.

Cis -dichlorodiamminepalladium(II) was synthesized and identified by its unique infrared spectrum. The reaction of cis -dichlorodiamminepalladium(II) with ammonia in varying concentrations of sodium chloride was studied using stopped-flow techniques. An analysis of the following system was completed and discussed in the light of experimental rate constants. http://archive.org/details/stoppedflowkinet1094517254 Lieutenant Commander, United States Navy Approved for public release; distribution is unlimited.

The following substitution reactions involving the square complexes of palladium (II) in aqueous HC1 solution were studied by spectrophotometric and potentiometric methods... http://archive.org/details/substitutionreac109459648 Lieutenant Commander, Royal Canadian Navy

The need for a correlation between the amount of a substance added to a composite plastic material and its effect on the cubical coefficient of thermal expansion, or cubit expansivity, of the composite mixture is discussed, experimental values for the cubic expansivities of several composite plastics made from a thermoplastic resin, an inert salt filler, and various percentages of aluminum are reported. Four hypothetical correlations are tested, two of which are reported to give calculated values within 1% of those observed. Apparatus and procedures for obtaining these results are also described in detail. http://archive.org/details/theeffectoflumin1094526511 Lieutenant, United States Navy Approved for public release; distribution is unlimited.

Solutions of tetraamminepalladium (II) perchlorate were made up and analyzed. The following acid hydrolysis reactions of tetraamminepalladium (II) ion were studied by ultraviolet spectroscopy. First order rate constants were evaluated for each step of the reaction series at various temperatures. Activation parameters were determined. (Equations) Mechanisms for the consecutive reactions are proposed, and cis and trans effects are discussed. http://archive.org/details/thekineticsofsuc1094515789 Ensign, United States Naval Reserve Approved for public release; distribution is unlimited.