The 22,000-year-old cave painting of an Atlantic salmon (Salmo salar) near the Vézère River in France is a reminder of our fascination with, and dependence on, Atlantic salmon throughout human history. Atlantic salmon belongs to the salmonid lineage which comprises 11 genera, with at least 70 species that exhibit a wide range of ecological adaptations and use a variety of marine and freshwater life history strategies 1 . Salmonids hold important positions as socially iconic species and economic resources within aquaculture, wild fisheries and recreational sport fisheries. Moreover, they serve as key indicator species of the health of North Atlantic and Pacific coastal and river ecosystems.All teleosts share at least three rounds of whole-genome duplication (WGD), 1R and 2R before the divergence of lamprey from the jawed vertebrates 2 , and a third teleost-specific WGD (Ts3R) at the base of the teleosts ~320 million years ago (Mya) [3][4][5] . Very little is known about the mechanisms of genomic and chromosomal reorganization after WGD in vertebrates because the 1R, 2R and Ts3R occurred so long ago that few clear signatures of post-WGD reorganization events remain. In contrast, a fourth WGD (the Ss4R salmonid-specific autotetraploidization event) occurred in the common ancestor of salmonids ~80 Mya after their divergence from Esociformes ~125 Mya 6-8 (Fig. 1), and the continued presence of multivalent pairing at meiosis and evidence of tetrasomic inheritance in salmonid species suggests that diploidy is not yet fully re-established 6,9,10 . Salmonids thus appear to provide an unprecedented opportunity for studying vertebrate genome evolution after an autotetraploid WGD 11,12 over a time period that is long enough to reveal long-term evolutionary patterns, but short enough to give a high-resolution picture of the process. In addition, they provide an excellent setting for contextualizing genome evolution with a dramatic post-WGD species radiation and intricate adaptations to a whole range of life history regimes.Here we present a high-quality reference genome assembly of the Atlantic salmon, and use it to describe major patterns characterizing the post-Ss4R salmonid genome evolution over the past 80 million years (Myr). Our results challenge the recent claim that rediploidization in salmonids has been a gradual process unlinked to significant genome rearrangements 13 . They also challenge current views about the relative importance of sub-and neofunctionalization in vertebrate genomes (reviewed in ref. 14), and the importance of dosage balance as a gene duplicate retention mechanism 15 . Genome characterizationThe Atlantic salmon reference genome assembly (GenBank: GCA_000233375.4) adds up to 2.97 gigabases (Gb) with aThe whole-genome duplication 80 million years ago of the common ancestor of salmonids (salmonid-specific fourth vertebrate whole-genome duplication, Ss4R) provides unique opportunities to learn about the evolutionary fate of a duplicated vertebrate genome in 70 extant lineages. Here we present a high...

In recent years, analogs of human insulin have been engineered with the aim of improving therapy for people with diabetes. To ensure that the safety profile of the human hormone is not compromised by the molecular modifications, the toxico-pharmacological properties of insulin analogs should be carefully monitored. In this study, we compared the insulin and IGF-I receptor binding properties and metabolic and mitogenic potencies of insulin aspart (B28Asp human insulin), insulin lispro (B28Lys,B29Pro human insulin), insulin glargine (A21Gly,B31Arg,B32Arg human insulin), insulin detemir (NN304) [B29Lys(-tetradecanoyl),desB30 human insulin], and reference insulin analogs. Receptor affinities were measured using purified human receptors, insulin receptor dissociation rates were determined using Chinese hamster ovary cells overexpressing the human insulin receptor, metabolic potencies were evaluated using primary mouse adipocytes, and mitogenic potencies were determined in human osteosarcoma cells. Metabolic potencies correlated well with insulin receptor affinities. Mitogenic potencies in general correlated better with IGF-I receptor affinities than with insulin receptor off-rates. The 2 rapid-acting insulin analogs aspart and lispro resembled human insulin on all parameters, except for a slightly elevated IGF-I receptor affinity of lispro. In contrast, the 2 long-acting insulin analogs, glargine and detemir, differed significantly from human insulin. The combination of the B31B32diArg and A21Gly substitutions provided insulin glargine with a 6-to 8-fold increased IGF-I receptor affinity and mitogenic potency compared with human insulin. The attachment of a fatty acid chain to LysB29 provided insulin detemir with reduced receptor affinities and metabolic and mitogenic potencies but did not change the balance between mitogenic and metabolic potencies. The safety implications of the increased growth-stimulating potential of insulin glargine are unclear. The reduced in vitro potency of insulin detemir might explain why this analog is not as effective on a molar basis as human insulin in humans.

Atlantic cod (Gadus morhua) is a large, cold-adapted teleost that sustains long-standing commercial fisheries and incipient aquaculture1,2. Here we present the genome sequence of Atlantic cod, showing evidence for complex thermal adaptations in its haemoglobin gene cluster and an unusual immune architecture compared to other sequenced vertebrates. The genome assembly was obtained exclusively by 454 sequencing of shotgun and paired-end libraries, and automated annotation identified 22,154 genes. The major histocompatibility complex (MHC) II is a conserved feature of the adaptive immune system of jawed vertebrates3,4, but we show that Atlantic cod has lost the genes for MHCII, CD4 and Ii that are essential for the function of this pathway. Nevertheless, Atlantic cod is not exceptionally susceptible to disease under natural conditions5. We find a highly expanded number of MHCI genes and a unique composition of its Toll-like receptor (TLR) families. This suggests how the Atlantic cod immune system has evolved compensatory mechanisms within both adaptive and innate immunity in the absence of MHCII. These observations affect fundamental assumptions about the evolution of the adaptive immune system and its components in vertebrates.

Signature databases are vital tools for identifying distant relationships in novel sequences and hence for inferring protein function. InterPro is an integrated documentation resource for protein families, domains and functional sites, which amalgamates the efforts of the PROSITE, PRINTS, Pfam and ProDom database projects. Each InterPro entry includes a functional description, annotation, literature references and links back to the relevant member database(s). Release 2.0 of InterPro (October 2000) contains over 3000 entries, representing families, domains, repeats and sites of post-translational modification encoded by a total of 6804 different regular expressions, profiles, fingerprints and Hidden Markov Models. Each InterPro entry lists all the matches against SWISS-PROT and TrEMBL (more than 1,000,000 hits from 462,500 proteins in SWISS-PROT and TrEMBL). The database is accessible for text- and sequence-based searches at http://www.ebi.ac.uk/interpro/. Questions can be emailed to interhelp@ebi.ac.uk.

PurposeBasal insulins with improved kinetic properties can potentially be produced using acylation by fatty acids that enable soluble, high-molecular weight complexes to form post-injection. A series of insulins, acylated at B29 with fatty acids via glutamic acid spacers, were examined to deduce the structural requirements.MethodsSelf-association, molecular masses and hexameric conformations of the insulins were studied using size exclusion chromatography monitored by UV or multi-angle light scattering and dynamic light scattering, and circular dichroism spectroscopy (CDS) in environments (changing phenol and zinc concentration) simulating a pharmaceutical formulation and changes following subcutaneous injection.ResultsWith depletion of phenol, insulin degludec and another fatty diacid–insulin analogue formed high molecular mass filament-like complexes, which disintegrated with depletion of zinc. CDS showed these analogues adopting stable T3R3 conformation in presence of phenol and zinc, changing to T6 with depletion of phenol. These findings suggest insulin degludec is dihexameric in pharmaceutical formulation becoming multihexameric after injection. The analogues showed weak dimeric association, indicating rapid release of monomers following hexamer disassembly.ConclusionsInsulins can be engineered that remain soluble but become highly self-associated after injection, slowly releasing monomers; this is critically dependent on the acylation moiety. One such analogue, insulin degludec, has therapeutic potential.

High throughput sequencing technology has great promise for biodiversity studies. However, an underlying assumption is that the primers used in these studies are universal for the prokaryotic or eukaryotic groups of interest. Full primer universality is difficult or impossible to achieve and studies using different primer sets make biodiversity comparisons problematic. The aim of this study was to design and optimize universal eukaryotic primers that could be used as a standard in future biodiversity studies. Using the alignment of all eukaryotic sequences from the publicly available SILVA database, we generated a full characterization of variable versus conserved regions in the 18S rRNA gene. All variable regions within this gene were analyzed and our results suggested that the V2, V4 and V9 regions were best suited for biodiversity assessments. Previously published universal eukaryotic primers as well as a number of self-designed primers were mapped to the alignment. Primer selection will depend on sequencing technology used, and this study focused on the 454 pyrosequencing GS FLX Titanium platform. The results generated a primer pair yielding theoretical matches to 80% of the eukaryotic and 0% of the prokaryotic sequences in the SILVA database. An empirical test of marine sediments using the AmpliconNoise pipeline for analysis of the high throughput sequencing data yielded amplification of sequences for 71% of all eukaryotic phyla with no isolation of prokaryotic sequences. To our knowledge this is the first characterization of the complete 18S rRNA gene using all eukaryotes present in the SILVA database, providing a robust test for universal eukaryotic primers. Since both in silico and empirical tests using high throughput sequencing retained high inclusion of eukaryotic phyla and exclusion of prokaryotes, we conclude that these primers are well suited for assessing eukaryote diversity, and can be used as a standard in biodiversity studies.

The protracted action of detemir is primarily achieved through slow absorption into blood. Dihexamerization and albumin binding of hexameric and dimeric detemir prolongs residence time at the injection depot. Some further retention of detemir occurs in the circulation where albumin binding causes buffering of insulin concentration. Insulin detemir provides a novel principle of protraction, enabling increased predictability of basal insulin.

Albumin is a multifunctional transport protein that binds a wide variety of endogenous substances and drugs. Insulins with affinity for albumin were engineered by acylation of the epsilon-amino group of LysB29 with saturated fatty acids containing 10-16 carbon atoms. The association constants for binding of the fatty acid acylated insulins to human albumin are in the order of 10(4)-10(5) M-1. The binding apparently involves both non-polar and ionic interactions with the protein. The acylated insulins bind at the long-chain fatty acid binding sites, but the binding affinity is lower than that of the free fatty acids and depends to a relatively small degree on the number of carbon atoms in the fatty acid chain. Differences in affinity of the acylated insulins for albumin are reflected in the relative timing of the blood-glucose-lowering effect after subcutaneous injection into rabbits. The acylated insulins provide a breakthrough in the search for soluble, prolonged-action insulin preparations for basal delivery of the hormone to the diabetic patient. We conclude that the biochemical concept of albumin binding can be applied to protract the effect of insulin, and suggest that derivatization with albumin-binding ligands could be generally applicable to prolong the action profile of peptide drugs.