Definition of Mafa-A and -B haplotypes in pedigreed cynomolgus macaques (Macaca fascicularis)

The major histocompatibility complex (MHC) class I B gene/allelic repertoire was investigated in a pedigreed population of cynomolgus macaques of mixed Indonesian/Malaysian origin. The Mafa-B alleles detected in this cohort are mostly specific for a given geographic area, and only a small number of alleles appears to be shared with other populations. This suggests the fast evolution of Mafa-B alleles due to adaptation to new environments. In contrast to humans, the B locus in Old World monkeys displays extensive copy number variation. The Mafa-B and previously defined -A gene combinations segregate in families and thus allowed the definition of extended haplotypes. In many cases it was possible to assign a particular Mafa-I allele to one of these Mafa-A/B haplotypes as well. The presence of a large number of stable haplotypes in this cohort of animals, which was pedigreed for up to eight generations, looks promising for developing discriminative MHC typing tools that are less cumbersome. Furthermore, the discovery of 53 unreported Mafa-B sequences expands the lexicon of alleles significantly, and may help in understanding the complex organisation of the macaque B region

Structure restraints from heteronuclear pseudocontact shifts generated by lanthanide tags at two different sites

Pseudocontact shifts (PCS) encode long-range information on 3D structures of protein backbones and side-chains. The level of structural detail that can be obtained increases with the number of different sites tagged with a paramagnetic metal ion to generate PCSs. Here we show that PCSs from two different sites can suffice to determine the structure of polypeptide chains and their location and orientation relative to the magnetic susceptibility tensor χ, provided that PCSs are available for 1H as well as heteronuclear spins. In addition, PCSs from two different sites are shown to provide detailed structural information on the conformation of methyl group-bearing amino-acid side-chains. A previously published ensemble structure of ubiquitin is shown to explain the magnetic susceptibility and alignment tensors slightly better than structures that try to explain the experimental data by a single conformation, illustrating the potential of PCSs as a tool to investigate small conformational changes.Financial support by the Australian Research Council is gratefully acknowledged

Introducing energetic and capacitive energy efficiency measures in the sector of chilled and frozen food storage via an electronic platform

The freshness of agricultural products is kept well when chilled or frozen. Additionally, chilled and frozen food is en vogue due to convenience reasons. The growing demand for chilled and frozen food on a global scale has some negative ecological side effects, though. The GEMIT Institute for Business process Management and IT developed the idea to combine an electronic market place for idle cold storage capacities with a tool that helps coldstores increase their energy efficiency. It is ecologically worthwhile in a branch characterized by high CO2-emissions and energy consumption. A future marketplace does not only serve as a spot market for short term demand in storage, where it guarantees cheaper storage space in proximity and higher independency from common cold storage services. Additionally, the supplier of empty cold storage space generates new revenues and at the same time spends the maintenance energy more efficiently. Finally, the participating suppliers can benefit from a benchmarking tool and information about their own energy savings potentials. The current market situation is especially advantageous for an introduction of ECO-Store, with growing cold storage spaces in Germany and an increasing consumption of chilled food worldwide. Aspects that can impose a barrier to the project are considered as well. It was found out that companies might not want to disclose data or cooperate with possible competitors and operating providers of cold storage space fear a decline in prices. Reaching a critical mass of participants in order to assure the cost effectiveness of the investment and to increase the attractiveness itself is a crucial issue. Finally, resembling concepts already exist on the market. Yet, what it makes unique is the combination of the electronic market place and a tool aiming at reducing individual energy consumption

Introducing energetic and capacitive energy efficiency measures in the sector of chilled and frozen food storage via an electronic platform

The freshness of agricultural products is kept well when chilled or frozen. Additionally, chilled and frozen food is en vogue due to convenience reasons. The growing demand for chilled and frozen food on a global scale has some negative ecological side effects, though. The GEMIT Institute for Business process Management and IT developed the idea to combine an electronic market place for idle cold storage capacities with a tool that helps coldstores increase their energy efficiency. It is ecologically worthwhile in a branch characterized by high CO2-emissions and energy consumption. A future marketplace does not only serve as a spot market for short term demand in storage, where it guarantees cheaper storage space in proximity and higher independency from common cold storage services. Additionally, the supplier of empty cold storage space generates new revenues and at the same time spends the maintenance energy more efficiently. Finally, the participating suppliers can benefit from a benchmarking tool and information about their own energy savings potentials. The current market situation is especially advantageous for an introduction of ECO-Store, with growing cold storage spaces in Germany and an increasing consumption of chilled food worldwide. Aspects that can impose a barrier to the project are considered as well. It was found out that companies might not want to disclose data or cooperate with possible competitors and operating providers of cold storage space fear a decline in prices. Reaching a critical mass of participants in order to assure the cost effectiveness of the investment and to increase the attractiveness itself is a crucial issue. Finally, resembling concepts already exist on the market. Yet, what it makes unique is the combination of the electronic market place and a tool aiming at reducing individual energy consumption

Compound Evolutionary History of the Rhesus Macaque Mhc Class I B Region Revealed by Microsatellite Analysis and Localization of Retroviral Sequences

In humans, the single polymorphic B locus of the major histocompatibility complex is linked to the microsatellite MIB. In rhesus macaques, however, haplotypes are characterized by the presence of unique combinations of multiple B genes, which may display different levels of polymorphism. The aim of the study was to shed light on the evolutionary history of this highly complex region. First, the robustness of the microsatellite MIB-linked to almost half of the B genes in rhesus macaques (Mamu-B)–for accurate B haplotyping was studied. Based on the physical map of an established haplotype comprising 7 MIB loci, each located next to a certain Mamu-B gene, two MIB loci, MIB1 and MIB6, were investigated in a panel of MHC homozygous monkeys. MIB1 revealed a complex genotyping pattern, whereas MIB6 analysis resulted in the detection of one or no amplicon. Both patterns are specific for a given B haplotype, show Mendelian segregation, and even allow a more precise haplotype definition than do traditional typing methods. Second, a search was performed for retroelements that may have played a role in duplication processes as observed in the macaque B region. This resulted in the description of two types of duplicons. One basic unit comprises an expressed Mamu-B gene, adjacent to an HERV16 copy closely linked to MIB. The second type of duplicon comprises a Mamu-B (pseudo)gene, linked to a truncated HERV16 structure lacking its MIB segment. Such truncation seems to coincide with the loss of B gene transcription. Subsequent to the duplication processes, recombination between MIB and Mamu-B loci appears to have occurred, resulting in a hyperplastic B region. Thus, analysis of MIB in addition to B loci allows deciphering of the compound evolutionary history of the class I B region in Old World monkeys

Solution conformations of a linked construct of the Zika virus NS2B-NS3 protease

The Zika virus presents a serious risk for global health. Crystal structures of different constructs of the Zika virus NS2B-NS3 protease (NS2B-NS3pro) have been determined with the aim to provide a basis for rational drug discovery. In these structures, the C-terminal β-hairpin of NS2B, NS2Bc, was observed to be either disordered (open conformation) or bound to NS3pro complementing the substrate binding site (closed conformation). Enzymatically active constructs of flaviviral NS2B-NS3 proteases commonly used for inhibitor testing contain a covalent peptide linker between NS2B and NS3pro. Using a linked construct of Zika virus NS2B-NS3pro, we studied the location of NS2Bc relative to NS3pro in solution by pseudocontact shifts generated by a paramagnetic lanthanide tag attached to NS3pro. Both closed and open conformations were observed with different inhibitors. As the NS2B co-factor is involved in substrate binding of flaviviral NS2B-NS3 proteases, the destabilization of the closed conformation in the linked construct makes it an attractive tool to search for inhibitors that interfere with the formation of the enzymatically active, closed conformation.C.N. thanks the Alexander vonHumbolt Foundation for a Feodor-Lynen fellowship. Financialsupport by the Australian Research Council is gratefully acknowl-edged. C.K. acknowledges support by the Deutsche For-schungsgemeinschaft, grant KL 1356/3

Sequential NMR assignments of labile protons in DNA using two-dimensional nuclear-overhauser-enhancemnt spectroscopy with three jump-and-return pulse sequences

Two-dimensional nuclear Overhauser enhancement (NOESY) spectra of labile protons were recorded in H2O solutions of a protein and of a DNA duplex, using a modification of the standard NOESY experiment with all three 90° pulses replaced by jump-and-return sequences. For the protein as well as the DNA fragment the strategically important spectral regions could be recorded with good sensitivity and free of artifacts. Using this procedure, sequence-specific assignments were obtained for the imino protons, C2H of adenine, and C4NH2 of cytosine in a 23-base-pair DNA duplex which includes the 17-base-pair OR3 repressor binding site of bacteriophage λ. Based on comparison with previously published results on the isolated OR3 binding site, these data were used for a study of chain termination effects on the chemical shifts of imino proton resonances of DNA duplexes

RIDME distance measurements using Gd(iii) tags with a narrow central transition

Methods based on pulse electron paramagnetic resonance allow measurement of the electron-electron dipolar coupling between two spin labels. Here we compare the most popular technique, Double Electron-Electron Resonance (DEER or PELDOR), with the dead-time free 5-pulse Relaxation-Induced Dipolar Modulation Enhancement (RIDME) method for Gd(iii)-Gd(iii) distance measurements at W-band (94.9 GHz, ≈3.5 T) using Gd(iii) tags with a small zero field splitting (ZFS). Such tags are important because of their high EPR sensitivity arising from their narrow central transition. Two systems were investigated: (i) a rigid model compound with an inter-spin distance of 2.35 nm, and (ii) two mutants of a homodimeric protein, both labeled with a DOTA-based Gd(iii) chelate and characterized by an inter-spin distance of around 6 nm, one having a narrow distance distribution and the other a broad distribution. Measurements on the model compound show that RIDME is less sensitive to the complications arising from the failure of the weak coupling approximation which affect DEER measurements on systems characterized by short inter-spin distances between Gd(iii) tags having a narrow central transition. Measurements on the protein samples, which are characterized by a long inter-spin distance, emphasize the complications due to the appearance of harmonics of the dipolar interaction frequency in the RIDME traces for S > 1/2 spin systems, as well as enhanced uncertainties in the background subtraction. In both cases the sensitivity of RIDME was found to be significantly better than DEER. The effects of the experimental parameters on the RIDME trace are discussed.This research was supported by the Israeli Science Foundation (grant 334/14) and was made possible in part by the historic generosity of the Harold Perlman Family (D. G.). We also acknowledge the Australian Research Council for a Discovery grant to G. O. and B. G. (DP150100383) and a Future Fellowship to B. G. (FT130100838). D. G. holds the Erich Klieger professorial chair in Chemical Physics

The mosaic of KIR haplotypes in rhesus macaques

To further refine and improve biomedical research in rhesus macaques, it is necessary to increase our knowledge concerning both the degree of allelic variation (polymorphism) and diversity (gene copy number variation) in the killer cell immunoglobulin-like receptor (KIR) gene cluster. Pedigreed animals in particular should be studied, as segregation data will provide clues to the linkage of particular KIR genes/alleles segregating on a haplotype and to its gene content as well. A dual strategy allowed us to screen the presence and absence of genes and the corresponding transcripts, as well as to track differences in transcription levels. On the basis of this approach, 14 diverse KIR haplotypes have been described. These haplotypes consist of multiple inhibitory and activating Mamu-KIR genes, and any gene present on one haplotype may be absent on another. This suggests that the cost of accelerated evolution by recombination may be the loss of certain framework genes on a haplotype