A large nested association mapping population for breeding and quantitative trait locus mapping in Ethiopian durum wheat

The Ethiopian plateau hosts thousands of durum wheat (Triticum turgidum subsp. durum) farmer varieties (FV) with high adaptability and breeding potential. To harness their unique allelic diversity, we produced a large nested association mapping (NAM) population intercrossing fifty Ethiopian FVs with an international elite durum wheat variety (Asassa). The Ethiopian NAM population (EtNAM) is composed of fifty interconnected bi‐parental families, totalling 6280 recombinant inbred lines (RILs) that represent both a powerful quantitative trait loci (QTL) mapping tool, and a large pre‐breeding panel. Here, we discuss the molecular and phenotypic diversity of the EtNAM founder lines, then we use an array featuring 13 000 single nucleotide polymorphisms (SNPs) to characterize a subset of 1200 EtNAM RILs from 12 families. Finally, we test the usefulness of the population by mapping phenology traits and plant height using a genome wide association (GWA) approach. EtNAM RILs showed high allelic variation and a genetic makeup combining genetic diversity from Ethiopian FVs with the international durum wheat allele pool. EtNAM SNP data were projected on the fully sequenced AB genome of wild emmer wheat, and were used to estimate pairwise linkage disequilibrium (LD) measures that reported an LD decay distance of 7.4 Mb on average, and balanced founder contributions across EtNAM families. GWA analyses identified 11 genomic loci individually affecting up to 3 days in flowering time and more than 1.6 cm in height. We argue that the EtNAM is a powerful tool to support the production of new durum wheat varieties targeting local and global agriculture.     

The metallo-beta-lactamase GOB is a mono-Zn(II) enzyme with a novel active site

Metallo-beta-lactamases (MbetaLs) are zinc-dependent enzymes able to hydrolyze and inactivate most beta-lactam antibiotics. The large diversity of active site structures and metal content among MbetaLs from different sources has limited the design of a pan-MbetaL inhibitor. Here we report the biochemical and biophysical characterization of a novel MbetaL, GOB-18, from a clinical isolate of a Gram-negative opportunistic pathogen, Elizabethkingia meningoseptica. Different spectroscopic techniques, three-dimensional modeling, and mutagenesis experiments, reveal that the Zn(II) ion is bound to Asp120, His121, His263, and a solvent molecule, i.e. in the canonical Zn2 site of dinuclear MbetaLs. Contrasting all other related MbetaLs, GOB-18 is fully active against a broad range of beta-lactam substrates using a single Zn(II) ion in this site. These data further enlarge the structural diversity of MbetaLs.     

Tick tock,tick tock: Mouse culture and tissue aging captured by an epigenetic clock

Aging is associated with dramatic changes to DNA methylation (DNAm), although the causes and consequences of such alterations are unknown. Our ability to experimentally uncover mechanisms of epigenetic aging will be greatly enhanced by our ability to study and manipulate these changes using in vitro models. However, it remains unclear whether the changes elicited by cells in culture can serve as a model of what is observed in aging tissues in vivo. To test this, we serially passaged mouse embryonic fibroblasts (MEFs) and assessed changes in DNAm at each time point via reduced representation bisulfite sequencing. By developing a measure that tracked cellular aging in vitro, we tested whether it tracked physiological aging in various mouse tissues and whether anti‐aging interventions modulate this measure. Our measure, termed CultureAGE, was shown to strongly increase with age when examined in multiple tissues (liver, lung, kidney, blood, and adipose). As a control, we confirmed that the measure was not a marker of cellular senescence, suggesting that it reflects a distinct yet progressive cellular aging phenomena that can be induced in vitro. Furthermore, we demonstrated slower epigenetic aging in animals undergoing caloric restriction and a resetting of our measure in lung and kidney fibroblasts when re‐programmed to iPSCs. Enrichment and clustering analysis implicated EED and Polycomb group (PcG) factors as potentially important chromatin regulators in translational culture aging phenotypes. Overall, this study supports the concept that physiologically relevant aging changes can be induced in vitro and used to uncover mechanistic insights into epigenetic aging.     

Defective dendritic cell migration and activation of adaptive immunity in PI3Kgamma-deficient mice

Gene-targeted mice were used to evaluate the role of the gamma isoform of phosphoinositide 3-kinase (PI3Kgamma) in dendritic cell (DC) migration and induction of specific T-cell-mediated immune responses. DC obtained from PI3Kgamma-/- mice showed a reduced ability to respond to chemokines in vitro and ex vivo and to travel to draining lymph nodes under inflammatory conditions. PI3Kgamma-/- mice had a selective defect in the number of skin Langerhans cells and in lymph node CD8alpha- DC. Furthermore, PI3Kgamma-/- mice showed a defective capacity to mount contact hypersensitivity and delayed-type hypersensitivity reactions. This defect was directly related to the reduced ability of antigen-loaded DC to migrate from the periphery to draining lymph nodes. Thus, PI3Kgamma plays a nonredundant role in DC trafficking and in the activation of specific immunity. Therefore, PI3Kgamma may be considered a new target to control exaggerated immune reactions.     

Amplification of trial-to-trial response variability by neurons in visual cortex

The visual cortex responds to repeated presentations of the same stimulus with high variability. Because the firing mechanism is remarkably noiseless, the source of this variability is thought to lie in the membrane potential fluctuations that result from summated synaptic input. Here this hypothesis is tested through measurements of membrane potential during visual stimulation. Surprisingly, trial-to-trial variability of membrane potential is found to be low. The ratio of variance to mean is much lower for membrane potential than for firing rate. The high variability of firing rate is explained by the threshold present in the function that converts inputs into firing rates. Given an input with small, constant noise, this function produces a firing rate with a large variance that grows with the mean. This model is validated on responses recorded both intracellularly and extracellularly. In neurons of visual cortex, thus, a simple deterministic mechanism amplifies the low variability of summated synaptic inputs into the large variability of firing rate. The computational advantages provided by this amplification are not known.     

Stopped-flow kinetic investigations of the activation of soybean lipoxygenase-1 and the influence of inhibitors on the allosteric site

Herein, we report on the role of the allosteric site in the activation mechanism of soybean lipoxygenase-1 utilizing stopped-flow inhibition kinetic studies. The K(D) for the activation was determined to be 25.9 +/- 2.3 microM and the rate constant for the oxidation of the iron cofactor, k(2), to be 182 +/- 4 s(-1). Two inhibitors were employed in this study, (Z)-9-octadecenyl sulfate (OS) and (Z)-9-palmitoleyl sulfate (PS), of which OS is an allosteric inhibitor of the turnover process, while PS is a linear mixed inhibitor with a K(i) of 13.7 +/- 1.3 microM for the catalytic site and a K(i)' of 140 +/- 9 microM for the allosteric site. It was found that OS does not inhibit the activation of soybean lipoxygenase-1, while PS acts as a competitive inhibitor versus the product, 13-hydroperoxy-9,11-(Z,E)-octadecadienoic acid, with a K(i) of 17.5 +/- 3.8 microM. These results suggest that OS binds to an allosteric site that is separate from the catalytic iron site. We further observed that the allosteric site binding selectivity is sensitive to inhibitor length as seen by its preference for OS over that of PS, which is two carbons longer than PS.