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5-Hydroxymethylcytosine (hmC) was recently detected as the sixth base in mammalian tissue at so far controversial levels. The function of the modified base is currently unknown, but it is certain that the base is generated from 5-methylcytosine (mC). This fuels the hypothesis that it represents an intermediate of an active demethylation process, which could involve further oxidation of the hydroxymethyl group to a formyl or carboxyl group followed by either deformylation or decarboxylation. Here, we use an ultra-sensitive and accurate isotope based LC-MS method to precisely determine the levels of hmC in various mouse tissues and we searched for 5-formylcytosine (fC), 5-carboxylcytosine (caC), and 5-hydroxymethyluracil (hmU) as putative active demethylation intermediates. Our data suggest that an active oxidative mC demethylation pathway is unlikely to occur. Additionally, we show using HPLC-MS analysis and immunohistochemistry that hmC is present in all tissues and cell types with highest concentrations in neuronal cells of the CNS.

The JmjC histone demethylases (KDMs) are linked to tumour cell proliferation and are current cancer targets; however, very few highly selective inhibitors for these are available. Here we report cyclic peptide inhibitors of the KDM4A-C with selectivity over other KDMs/2OG oxygenases, including closely related KDM4D/E isoforms. Crystal structures and biochemical analyses of one of the inhibitors (CP2) with KDM4A reveals that CP2 binds differently to, but competes with, histone substrates in the active site. Substitution of the active site binding arginine of CP2 to N -ɛ-trimethyl-lysine or methylated arginine results in cyclic peptide substrates, indicating that KDM4s may act on non-histone substrates. Targeted modifications to CP2 based on crystallographic and mass spectrometry analyses results in variants with greater proteolytic robustness. Peptide dosing in cells manifests KDM4A target stabilization. Although further development is required to optimize cellular activity, the results reveal the feasibility of highly selective non-metal chelating, substrate-competitive inhibitors of the JmjC KDMs. JmjC histone demethylases (KDMs) are cancer targets due to their links to cell proliferation, but selective inhibition remains a challenge. Here the authors identify potent inhibitors of KDM4A-C—via in vitro selection from a vast library of cyclic peptides—that show selectivity over other KDMs.

Recently, the clinical proof of concept for the first ultra-long oral insulin was reported, showing efficacy and safety similar to subcutaneously administered insulin glargine. Here, we report the molecular engineering as well as biological and pharmacological properties of these insulin analogues. Molecules were designed to have ultra-long pharmacokinetic profile to minimize variability in plasma exposure. Elimination plasma half-life of ~20 h in dogs and ~70 h in man is achieved by a strong albumin binding, and by lowering the insulin receptor affinity 500-fold to slow down receptor mediated clearance. These insulin analogues still stimulate efficient glucose disposal in rats, pigs and dogs during constant intravenous infusion and euglycemic clamp conditions. The albumin binding facilitates initial high plasma exposure with a concomitant delay in distribution to peripheral tissues. This slow appearance in the periphery mediates an early transient hepato-centric insulin action and blunts hypoglycaemia in dogs in response to overdosing. Recently, the first orally-administered ultra-long acting insulin was shown to have clinical efficacy. Here, the authors report the molecular engineering, as well as the biological and pharmacological properties of these insulin analogues.

AspH is an endoplasmic reticulum (ER) membrane-anchored 2-oxoglutarate oxygenase whose C-terminal oxygenase and tetratricopeptide repeat (TPR) domains present in the ER lumen. AspH catalyses hydroxylation of asparaginyl- and aspartyl-residues in epidermal growth factor-like domains (EGFDs). Here we report crystal structures of human AspH, with and without substrate, that reveal substantial conformational changes of the oxygenase and TPR domains during substrate binding. Fe(II)-binding by AspH is unusual, employing only two Fe(II)-binding ligands (His679/His725). Most EGFD structures adopt an established fold with a conserved Cys1–3, 2–4, 5–6 disulfide bonding pattern; an unexpected Cys3–4 disulfide bonding pattern is observed in AspH-EGFD substrate complexes, the catalytic relevance of which is supported by studies involving stable cyclic peptide substrate analogues and by effects of Ca(II) ions on activity. The results have implications for EGFD disulfide pattern processing in the ER and will enable medicinal chemistry efforts targeting human 2OG oxygenases. AspH catalyses hydroxylation of asparagine and aspartate residues in epidermal growth factor-like domains (EGFDs). Here, the authors present crystal structures of AspH with and without substrates and show that AspH uses EFGD substrates with a non-canonical disulfide pattern.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Despite exploring a number of fruitful lines of inquiry, research on emigration from the German Reich between 1933 and 1945 has for the most part neglected business people and entrepreneurs even if a few US American studies in the 1940s highlighted the economic significance of the refugees who had recently arrived from Europe. Against this background and on the basis of 166 individual cases, this article focuses on German-Jewish entrepreneurs who fled to the refugee metropolis of New York City. While there were parallels in terms of timing of emigration between this group and general trends in the emigration movement, it proved possible for about half of the emigrant entrepreneurs to establish themselves in a similar economic sector to that they had left behind. Owing to availability of sources, the article examines in particular the two largest groups of emigrant entrepreneurs, bankers and publishers. Various examples allow depiction, first of all, of the difficulties faced by the migrants in their new country. These included in particular the shortage of financial means which had resulted from the plundering of assets by the National Socialist state as well as the lack of an established reputation in the new country. A further factor was the lack of experience in dealing with American industrial structures and business methods. The examples, however, also make clear those advantages which the refugees possessed as they undertook entrepreneurial activity in their new environment. One of the most important of these lay in the possibility of being able to draw upon kinship structures and emigrant networks. In addition, though, business areas which were further developed in Germany than in the US could also be exploited entrepreneurially, and many of the German refugees thus became «pioneers» in new areas in the US and bearers of entrepreneurial transfer processes.

Despite exploring a number of fruitful lines of inquiry, research on emigration from the German Reich between 1933 and 1945 has for the most part neglected business people and entrepreneurs even if a few US American studies in the 1940s highlighted the economic significance on the refugees who had recently arrived from Europe. Against his background and on the basis of 166 individual cases, this article focuses on German-Jewish entrepreneurs who fled to the refugee metropolis of New York City. While there were parallels in terms of timing of emigration between this group and general trends in the emigration movement, it proved possible for about half of the emigrant entrepreneurs to establish themselves in a similar economic sector to that they had left behind. Owing to availability of sources, the article examines in particular the two largest groups of emigrant entrepreneurs, bankers and publishers. Various examples allow depiction, first of all, of the difficulties faced by the migrants in their new country. These included in particular the shortage of financial means which had resulted from the plundering of assets by the National Socialist state as well as the lack of an established reputation in the new country. A further factor was the lack of experience in dealing with American industrial structures and business methods. The examples, however, also make clear those advantages which the refugees possessed as they undertook entrepreneurial activity in their new environment. One of the most important of these lay in the possibility of being able to draw upon kinship structures and emigrant networks.

YcfD from Escherichia coli is a homologue of the human ribosomal oxygenases NO66 and MINA53, which catalyse histidyl-hydroxylation of the 60S subunit and affect cellular proliferation (Ge et al., Nat Chem Biol 12:960–962, 2012). Bioinformatic analysis identified a potential homologue of ycfD in the thermophilic bacterium Rhodothermus marinus (ycfDRM). We describe studies on the characterization of ycfDRM, which is a functional 2OG oxygenase catalysing (2S,3R)-hydroxylation of the ribosomal protein uL16 at R82, and which is active at significantly higher temperatures than previously reported for any other 2OG oxygenase. Recombinant ycfDRM manifests high thermostability (Tm 84 °C) and activity at higher temperatures (Topt 55 °C) than ycfDEC (Tm 50.6 °C, Topt 40 °C). Mass spectrometric studies on purified R. marinus ribosomal proteins demonstrate a temperature-dependent variation in uL16 hydroxylation. Kinetic studies of oxygen dependence suggest that dioxygen availability can be a limiting factor for ycfDRM catalysis at high temperatures, consistent with incomplete uL16 hydroxylation observed in R. marinus cells. Overall, the results that extend the known range of ribosomal hydroxylation, reveal the potential for ycfD-catalysed hydroxylation to be regulated by temperature/dioxygen availability, and that thermophilic 2OG oxygenases are of interest from a biocatalytic perspective.

Despite exploring a number of fruitful lines of inquiry, research on emigration from the German Reich between 1933 and 1945 has for the most part neglected business people and entrepreneurs even if a few US American studies in the 1940s highlighted the economic significance of the refugees who had recently arrived from Europe. Against this background and on the basis of 166 individual cases, this article focuses on German-Jewish entrepreneurs who fled to the refugee metropolis of New York City. While there were parallels in terms of timing of emigration between this group and general trends in the emigration movement, it proved possible for about half of the emigrant entrepreneurs to establish themselves in a similar economic sector to that they had left behind. Owing to availability of sources, the article examines in particular the two largest groups of emigrant entrepreneurs, bankers and publishers. Various examples allow depiction, first of all, of the difficulties faced by the migrants in their new country. These included in particular the shortage of financial means which had resulted from the plundering of assets by the National Socialist state as well as the lack of an established reputation in the new country. A further factor was the lack of experience in dealing with American industrial structures and business methods. The examples, however, also make clear those advantages which the refugees possessed as they undertook entrepreneurial activity in their new environment. One of the most important of these lay in the possibility of being able to draw upon kinship structures and emigrant networks. In addition, though, business areas which were further developed in Germany than in the US could also be exploited entrepreneurially, and many of the German refugees thus became «pioneers» in new areas in the US and bearers of entrepreneurial transfer processes. Abstract printed by permission of the publisher

Biomimetic synthesis is an attempt to assemble natural products along biosynthetic lines without recourse to the full enzymatic machinery of nature. We exemplify this with a total synthesis of exiguamine A and the newly isolated natural product exiguamine B. The most noteworthy feature of this work is an oxidative endgame drawing from the complex chemistry of catecholamines, which allows for ready access to a new class of nanomolar indoleamine-2,3-dioxygenase inhibitors.