Mechanism for nitrogen isotope fractionation during ammonium assimilation by Escherichia coli K12

Author Posting. © The Author(s), 2013. This is the author's version of the work. It is posted here by permission of National Academy of Sciences for personal use, not for redistribution. The definitive version was published in Proceedings of the National Academy of Sciences of the United States of America 110 (2013): 8696-8701, doi:10.1073/pnas.1216683110.Organisms that use ammonium as the sole nitrogen source discriminate between [15N] and [14N] ammonium. This selectivity leaves an isotopic signature in their biomass that depends on the external concentration of ammonium. To dissect how differences in discrimination arise molecularly, we examined a wild-type (WT) strain of Escherichia coli K12 and mutant strains with lesions affecting ammonium-assimilatory proteins. We used isotope ratio mass spectrometry (MS) to assess the nitrogen isotopic composition of cell material when the strains were grown in batch culture at either high or low external concentrations of NH3 (achieved by controlling total NH4Cl and pH of the medium). At high NH3 (≥0.89 µM), discrimination against the heavy isotope by the WT strain (−19.2‰) can be accounted for by the equilibrium isotope effect for dissociation of NH4+ to NH3 + H+. NH3 equilibrates across the cytoplasmic membrane, and glutamine synthetase does not manifest an isotope effect in vivo. At low NH3 (≤0.18 µM), discrimination reflects an isotope effect for the NH4+ channel AmtB (−14.1‰). By making E. coli dependent on the low-affinity ammonium-assimilatory pathway, we determined that biosynthetic glutamate dehydrogenase has an inverse isotope effect in vivo (+8.8‰). Likewise, by making unmediated diffusion of NH3 across the cytoplasmic membrane rate-limiting for cell growth in a mutant strain lacking AmtB, we could deduce an in vivo isotope effect for transport of NH3 across the membrane (−10.9‰). The paper presents the raw data from which our conclusions were drawn and discusses the assumptions underlying them.This work was supported by NIH grant GM38361 to S.K. J.M.H. thanks WHOI for support as an emeritus scientist.2013-11-0

Myth and Symbol II: Symbolic phenomena in Ancient Greek culture.

Papers from the second and third international symposia on symbolism at The Norwegian institute at Athens, September 21-24, 2000 and September 19-22,200

Using Genomic Sequencing for Classical Genetics in E. coli K12

We here develop computational methods to facilitate use of 454 whole genome shotgun sequencing to identify mutations in Escherichia coli K12. We had Roche sequence eight related strains derived as spontaneous mutants in a background without a whole genome sequence. They provided difference tables based on assembling each genome to reference strain E. coli MG1655 (NC_000913). Due to the evolutionary distance to MG1655, these contained a large number of both false negatives and positives. By manual analysis of the dataset, we detected all the known mutations (24 at nine locations) and identified and genetically confirmed new mutations necessary and sufficient for the phenotypes we had selected in four strains. We then had Roche assemble contigs de novo, which we further assembled to full-length pseudomolecules based on synteny with MG1655. This hybrid method facilitated detection of insertion mutations and allowed annotation from MG1655. After removing one genome with less than the optimal 20- to 30-fold sequence coverage, we identified 544 putative polymorphisms that included all of the known and selected mutations apart from insertions. Finally, we detected seven new mutations in a total of only 41 candidates by comparing single genomes to composite data for the remaining six and using a ranking system to penalize homopolymer sequencing and misassembly errors. An additional benefit of the analysis is a table of differences between MG1655 and a physiologically robust E. coli wild-type strain NCM3722. Both projects were greatly facilitated by use of comparative genomics tools in the CoGe software package (http://genomevolution.org/)

The \u3cem\u3eChlamydomonas\u3c/em\u3e Genome Reveals the Evolution of Key Animal and Plant Functions

Chlamydomonas reinhardtii is a unicellular green alga whose lineage diverged from land plants over 1 billion years ago. It is a model system for studying chloroplast-based photosynthesis, as well as the structure, assembly, and function of eukaryotic flagella (cilia), which were inherited from the common ancestor of plants and animals, but lost in land plants. We sequenced the ∼120-megabase nuclear genome of Chlamydomonas and performed comparative phylogenomic analyses, identifying genes encoding uncharacterized proteins that are likely associated with the function and biogenesis of chloroplasts or eukaryotic flagella. Analyses of the Chlamydomonas genome advance our understanding of the ancestral eukaryotic cell, reveal previously unknown genes associated with photosynthetic and flagellar functions, and establish links between ciliopathy and the composition and function of flagella

Transcription Factors Bind Thousands of Active and Inactive Regions in the Drosophila Blastoderm

Identifying the genomic regions bound by sequence-specific regulatory factors is central both to deciphering the complex DNA cis-regulatory code that controls transcription in metazoans and to determining the range of genes that shape animal morphogenesis. We used whole-genome tiling arrays to map sequences bound in Drosophila melanogaster embryos by the six maternal and gap transcription factors that initiate anterior–posterior patterning. We find that these sequence-specific DNA binding proteins bind with quantitatively different specificities to highly overlapping sets of several thousand genomic regions in blastoderm embryos. Specific high- and moderate-affinity in vitro recognition sequences for each factor are enriched in bound regions. This enrichment, however, is not sufficient to explain the pattern of binding in vivo and varies in a context-dependent manner, demonstrating that higher-order rules must govern targeting of transcription factors. The more highly bound regions include all of the over 40 well-characterized enhancers known to respond to these factors as well as several hundred putative new cis-regulatory modules clustered near developmental regulators and other genes with patterned expression at this stage of embryogenesis. The new targets include most of the microRNAs (miRNAs) transcribed in the blastoderm, as well as all major zygotically transcribed dorsal–ventral patterning genes, whose expression we show to be quantitatively modulated by anterior–posterior factors. In addition to these highly bound regions, there are several thousand regions that are reproducibly bound at lower levels. However, these poorly bound regions are, collectively, far more distant from genes transcribed in the blastoderm than highly bound regions; are preferentially found in protein-coding sequences; and are less conserved than highly bound regions. Together these observations suggest that many of these poorly bound regions are not involved in early-embryonic transcriptional regulation, and a significant proportion may be nonfunctional. Surprisingly, for five of the six factors, their recognition sites are not unambiguously more constrained evolutionarily than the immediate flanking DNA, even in more highly bound and presumably functional regions, indicating that comparative DNA sequence analysis is limited in its ability to identify functional transcription factor targets

The seeds of divergence: the economy of French North America, 1688 to 1760

Generally, Canada has been ignored in the literature on the colonial origins of divergence with most of the attention going to the United States. Late nineteenth century estimates of income per capita show that Canada was relatively poorer than the United States and that within Canada, the French and Catholic population of Quebec was considerably poorer. Was this gap long standing? Some evidence has been advanced for earlier periods, but it is quite limited and not well-suited for comparison with other societies. This thesis aims to contribute both to Canadian economic history and to comparative work on inequality across nations during the early modern period. With the use of novel prices and wages from Quebec—which was then the largest settlement in Canada and under French rule—a price index, a series of real wages and a measurement of Gross Domestic Product (GDP) are constructed. They are used to shed light both on the course of economic development until the French were defeated by the British in 1760 and on standards of living in that colony relative to the mother country, France, as well as the American colonies. The work is divided into three components. The first component relates to the construction of a price index. The absence of such an index has been a thorn in the side of Canadian historians as it has limited the ability of historians to obtain real values of wages, output and living standards. This index shows that prices did not follow any trend and remained at a stable level. However, there were episodes of wide swings—mostly due to wars and the monetary experiment of playing card money. The creation of this index lays the foundation of the next component. The second component constructs a standardized real wage series in the form of welfare ratios (a consumption basket divided by nominal wage rate multiplied by length of work year) to compare Canada with France, England and Colonial America. Two measures are derived. The first relies on a “bare bones” definition of consumption with a large share of land-intensive goods. This measure indicates that Canada was poorer than England and Colonial America and not appreciably richer than France. However, this measure overestimates the relative position of Canada to the Old World because of the strong presence of land-intensive goods. A second measure is created using a “respectable” definition of consumption in which the basket includes a larger share of manufactured goods and capital-intensive goods. This second basket better reflects differences in living standards since the abundance of land in Canada (and Colonial America) made it easy to achieve bare subsistence, but the scarcity of capital and skilled labor made the consumption of luxuries and manufactured goods (clothing, lighting, imported goods) highly expensive. With this measure, the advantage of New France over France evaporates and turns slightly negative. In comparison with Britain and Colonial America, the gap widens appreciably. This element is the most important for future research. By showing a reversal because of a shift to a different type of basket, it shows that Old World and New World comparisons are very sensitive to how we measure the cost of living. Furthermore, there are no sustained improvements in living standards over the period regardless of the measure used. Gaps in living standards observed later in the nineteenth century existed as far back as the seventeenth century. In a wider American perspective that includes the Spanish colonies, Canada fares better. The third component computes a new series for Gross Domestic Product (GDP). This is to avoid problems associated with using real wages in the form of welfare ratios which assume a constant labor supply. This assumption is hard to defend in the case of Colonial Canada as there were many signs of increasing industriousness during the eighteenth and nineteenth centuries. The GDP series suggest no long-run trend in living standards (from 1688 to circa 1765). The long peace era of 1713 to 1740 was marked by modest economic growth which offset a steady decline that had started in 1688, but by 1760 (as a result of constant warfare) living standards had sunk below their 1688 levels. These developments are accompanied by observations that suggest that other indicators of living standard declined. The flat-lining of incomes is accompanied by substantial increases in the amount of time worked, rising mortality and rising infant mortality. In addition, comparisons of incomes with the American colonies confirm the results obtained with wages— Canada was considerably poorer. At the end, a long conclusion is provides an exploratory discussion of why Canada would have diverged early on. In structural terms, it is argued that the French colony was plagued by the problem of a small population which prohibited the existence of scale effects. In combination with the fact that it was dispersed throughout the territory, the small population of New France limited the scope for specialization and economies of scale. However, this problem was in part created, and in part aggravated, by institutional factors like seigneurial tenure. The colonial origins of French America’s divergence from the rest of North America are thus partly institutional

St. Pancras New Church

Exterior

Sulfonation of kraft lignon to water soluble value added products

Kraft lignin is water insoluble and has limited end-use applications. The main objective of this MSc studies was to rend kraft lignin water soluble, and various value-added products including dispersants and flocculants. In this work, softwood kraft lignin was supplied from FPInnovations from its pilot facilities in Thunder Bay, ON. It was then modified using BURL lab facilities of Lakehead University. In one alternative, phenolation and hydroxymethylation of kraft lignin were followed as pre-treatment processes to improve the reactivity of lignin. Unmodified kraft lignin, phenolated lignin and hydroxymethylated lignin were then sulfonated through 1) concentrated sulfuric acid and 2) sodium sulfite treatments. All lignin samples treated with sodium sulfite exhibited increased in charge density and solubility. Additionally, sulfuric acid treatment of phenolated lignin yielded soluble product (SAP) with a high charge density (e.g. 3 meq/g). However, sulfuric acid treatment of phenolated lignin was unsuccessful in producing lignin with desired charge density and solubility. The synthesized soluble sulfonated lignin samples (SS, SSH, SSP, and SAP) demonstrated a greater solubility than kraft lignin, but weaker solubility than commercial and industrial lignosulfonates. The application of sulfonated lignin samples were evaluated as dispersants in cement and kaolinite; adsorbent on kaolinite and calcium carbonate to produce modified fillers for composites and papermaking as well as flocculants for textile industry. The addition of the sulfonated lignin to cement did not increase the fluidity of the cement, but improved the fluidity of kaolinite to some extent. The adsorption of sulfonated lignins on calcium carbonate and kaolinite were greater than that of kraft lignin, which shows that modified lignin can have a high adsorption capacity to produce modified fillers. The samples, which were phenolated, exhibited a greater adsorption affinity than other samples on calcium carbonate. In this study, ethyl violet and basic blue solutions were used as model wastewater samples of textile industry. The results showed that, the sulfonated lignin samples were generally able to remove ethyl violet, but were unsuccessful in removing basic blue from solution

The Erechtheion at Athens : fragments of Athenian architecture and a few remains in Attica Megara and Epirus

by Henry William Inwoo