Synthetic protein scaffold-mediated manipulation of LOX-1 scavenger receptor in cell function, vascular physiology and disease

Binding and internalization of oxidized low-density lipoprotein (oxLDL) by immune cells within the arterial sub-intimal layer is a key step in the initiation and progression of atherosclerosis. Scavenger receptors (SRs) are a “super-family” of cell surface receptors expressed by immune cells which promote the removal of harmful non-self or altered-self targets, including oxidatively modified ligands such as oxLDL. Lectin-like oxidized LDL receptor-1 (LOX-1) is a member of the SR-E family of scavenger receptors. LOX-1 – oxLDL binding has been shown to exert significant pro-atherogenic effects. In this study, we used in vitro and in vivo models of atherosclerosis to explore the effectiveness of LOX-1 targeting in the inhibition of oxLDL binding and the amelioration of atherosclerosis and neointimal hyperplasia. LOX-1 was targeted using novel non-antibody artificial binding proteins, known as Affimers. Immunofluorescence studies using a tetracycline- inducible cell line expressing LOX-1 demonstrate the binding efficiency of fluorescent-labelled LOX-1 Affimers to LOX-1 in live cells. In same cell line, we co-incubated fluorescent-labelled oxLDL with increasing concentrations of LOX- 1 Affimers. The findings show that LOX-1 Affimers inhibit LOX-1 – oxLDL binding. ELISA studies were carried out to identify LOX-1 affimers with comparable affinity for both human and murine LOX-1. The most cross-reactive LOX-1 Affimer was taken forward in to a transgenic mouse model of diet-induced atherosclerosis. This model used APO-E null and APO-E/LOX-1 null mice to compare the effects of LOX-1 knockout with LOX-1 targeting using LOX-1 affimers. LOX-1 knockout resulted in a reduction in aortic and peripheral arterial atherosclerosis, and neointimal hyperplasia. LOX-1 targeting with affimers produced promising results. Treatment with LOX-1 affimers reduced atherosclerosis and NIH, but to a lesser extent compared with LOX-1 knockout. Treatment with LOX-1 affimer exerted additional metabolic benefits including protective effects against diet- induced obesity and insulin resistance. The results from these studies suggest that LOX-1 is an attractive target in the development of novel therapeutics in atherosclerosis. Targeted LOX-1 inhibition potentially offers protective benefits in associated metabolic disorders such as obesity and insulin resistance

Volumetric versus single slice measurements of core abdominal muscle for Sarcopenia

Objectives: We investigated whether total psoas muscle area (TPMA) was representative of the total psoas muscle volume (TPMV). Secondly, we assessed whether there was a relationship between the two commonly used single slice measurements of sarcopenia, TPMA and total abdominal muscle area (TAMA). Methods: Pre-operative CT imaging of 110 patients undergoing elective endovascular aneurysm repair were analysed by two trained independent observers. TPMA was measured at individual vertebral levels between the second lumbar vertebrae and sacrum. TPMV was also estimated between the second lumbar vertebrae and sacrum. TAMA was measured at the third lumbar vertebrae (L3). Observer differences were assessed using Bland-Altman plots. Associations between the different measures were assessed using linear regression and Pearson's correlation. Results: We found single slice measurements of the TPMA to be representative of the TPMV at individual levels between L2 to the sacrum. The strongest association was seen at L3 (adjusted regression coefficient 16.7, 95% CI 12.1 to 21.4, p < 0.001). There was no association between TPMA and TAMA (adjusted regression coefficient - 0.7, 95% CI - 4.1 to 2.8, p = 0.710). Conclusions: We demonstrate that measurements of the TPMA between L2 to the sacrum are representative of the TPMV, with the greatest association at the third lumbar vertebrae. There was no association between the TPMA and TAMA. Advances in Knowledge: We demonstrate that a single slice measurement of TPMA at L3 is representative of the muscle volume, contrary to previous criticism. Future sarcopenia studies can continue to measure TPMA which is representative of the TPMV

An overview of chromatin modifications

The last 15 years have witnessed tremendous progress in elucidating the roles of chromatin modifications in transcription regulation, DNA repair, replication, recombination, and other genomic processes. In this issue of Biopolymers, a series of reviews will summarize recent advances in our understanding of chromatin modifying enzymes and explore unresolved questions with respect to their regulation and functions in gene expression and other nuclear processes. © 2012 Wiley Periodicals, Inc. Biopolymers 99: 95–97, 2013.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/94507/1/22158_ftp.pd

“Affimer” synthetic protein scaffolds block oxidized LDL binding to the LOX-1 scavenger receptor and inhibit ERK1/2 activation

In multicellular organisms, a variety of lipid-protein particles control the systemic flow of triacylglycerides, cholesterol and fatty acids between cells in different tissues. The chemical modification by oxidation of these particles can trigger pathological responses, mediated by a group of membrane proteins termed scavenger receptors. The lectin-like oxidised low-density lipoprotein (LOX-1) scavenger receptor binds to oxidized low density lipoprotein (oxLDL) and mediates both signaling and trafficking outcomes. Here, we identified 5 synthetic proteins termed Affimers from a phage display library, each capable of binding recombinant LOX-1 extracellular (oxLDL-binding) domain with high specificity. These Affimers, based on a phytocystatin scaffold with loop regions of variable sequence, were able to bind to the plasma membrane of HEK293T cells exclusively when human LOX-1 was expressed. Binding and uptake of fluorescently-labelled oxLDL by the LOX-1-expressing cell model was inhibited with sub-nanomolar potency by all 5 Affimers. ERK1/2 activation, stimulated by oxLDL binding to LOX-1, was also significantly inhibited (p<0.01) by pre-incubation with LOX-1-specific Affimers, but these Affimers had no direct agonistic effect. Molecular modeling indicated that the LOX-1-specific Affimers bound predominantly via their variable loop regions to the surface of the LOX-1 lectin-like domain that contains a distinctive arrangement of arginine residues previously implicated in oxLDL binding, involving interactions with both subunits of the native, stable scavenger receptor homodimer. These data provide a new class of synthetic tools to probe and potentially modulate the oxLDL/LOX-1 interaction that plays an important role in vascular disease

Receding ice drove parallel expansions in Southern Ocean penguins

International audienceClimate shifts are key drivers of ecosystem change. Despite the critical importance of Antarctica and the Southern Ocean for global climate, the extent of climate-driven ecological change in this region remains controversial. In particular, the biological effects of changing sea ice conditions are poorly understood. We hypothesize that rapid postglacial reductions in sea ice drove biological shifts across multiple widespread Southern Ocean species. We test for demographic shifts driven by climate events over recent millennia by analyzing population genomic datasets spanning 3 penguin genera ( Eudyptes , Pygoscelis , and Aptenodytes ). Demographic analyses for multiple species (macaroni/royal, eastern rockhopper, Adélie, gentoo, king, and emperor) currently inhabiting southern coastlines affected by heavy sea ice conditions during the Last Glacial Maximum (LGM) yielded genetic signatures of near-simultaneous population expansions associated with postglacial warming. Populations of the ice-adapted emperor penguin are inferred to have expanded slightly earlier than those of species requiring ice-free terrain. These concerted high-latitude expansion events contrast with relatively stable or declining demographic histories inferred for 4 penguin species (northern rockhopper, western rockhopper, Fiordland crested, and Snares crested) that apparently persisted throughout the LGM in ice-free habitats. Limited genetic structure detected in all ice-affected species across the vast Southern Ocean may reflect both rapid postglacial colonization of subantarctic and Antarctic shores, in addition to recent genetic exchange among populations. Together, these analyses highlight dramatic, ecosystem-wide responses to past Southern Ocean climate change and suggest potential for further shifts as warming continues

Shifting the limits in wheat research and breeding using a fully annotated reference genome

Introduction: Wheat (Triticum aestivum L.) is the most widely cultivated crop on Earth, contributing about a fifth of the total calories consumed by humans. Consequently, wheat yields and production affect the global economy, and failed harvests can lead to social unrest. Breeders continuously strive to develop improved varieties by fine-tuning genetically complex yield and end-use quality parameters while maintaining stable yields and adapting the crop to regionally specific biotic and abiotic stresses. Rationale: Breeding efforts are limited by insufficient knowledge and understanding of wheat biology and the molecular basis of central agronomic traits. To meet the demands of human population growth, there is an urgent need for wheat research and breeding to accelerate genetic gain as well as to increase and protect wheat yield and quality traits. In other plant and animal species, access to a fully annotated and ordered genome sequence, including regulatory sequences and genome-diversity information, has promoted the development of systematic and more time-efficient approaches for the selection and understanding of important traits. Wheat has lagged behind, primarily owing to the challenges of assembling a genome that is more than five times as large as the human genome, polyploid, and complex, containing more than 85% repetitive DNA. To provide a foundation for improvement through molecular breeding, in 2005, the International Wheat Genome Sequencing Consortium set out to deliver a high-quality annotated reference genome sequence of bread wheat. Results: An annotated reference sequence representing the hexaploid bread wheat genome in the form of 21 chromosome-like sequence assemblies has now been delivered, giving access to 107,891 high-confidence genes, including their genomic context of regulatory sequences. This assembly enabled the discovery of tissue- and developmental stage–related gene coexpression networks using a transcriptome atlas representing all stages of wheat development. The dynamics of change in complex gene families involved in environmental adaptation and end-use quality were revealed at subgenome resolution and contextualized to known agronomic single-gene or quantitative trait loci. Aspects of the future value of the annotated assembly for molecular breeding and research were exemplarily illustrated by resolving the genetic basis of a quantitative trait locus conferring resistance to abiotic stress and insect damage as well as by serving as the basis for genome editing of the flowering-time trait. Conclusion: This annotated reference sequence of wheat is a resource that can now drive disruptive innovation in wheat improvement, as this community resource establishes the foundation for accelerating wheat research and application through improved understanding of wheat biology and genomics-assisted breeding. Importantly, the bioinformatics capacity developed for model-organism genomes will facilitate a better understanding of the wheat genome as a result of the high-quality chromosome-based genome assembly. By necessity, breeders work with the genome at the whole chromosome level, as each new cross involves the modification of genome-wide gene networks that control the expression of complex traits such as yield. With the annotated and ordered reference genome sequence in place, researchers and breeders can now easily access sequence-level information to precisely define the necessary changes in the genomes for breeding programs. This will be realized through the implementation of new DNA marker platforms and targeted breeding technologies, including genome editing

Reducing the environmental impact of surgery on a global scale: systematic review and co-prioritization with healthcare workers in 132 countries

Abstract Background Healthcare cannot achieve net-zero carbon without addressing operating theatres. The aim of this study was to prioritize feasible interventions to reduce the environmental impact of operating theatres. Methods This study adopted a four-phase Delphi consensus co-prioritization methodology. In phase 1, a systematic review of published interventions and global consultation of perioperative healthcare professionals were used to longlist interventions. In phase 2, iterative thematic analysis consolidated comparable interventions into a shortlist. In phase 3, the shortlist was co-prioritized based on patient and clinician views on acceptability, feasibility, and safety. In phase 4, ranked lists of interventions were presented by their relevance to high-income countries and low–middle-income countries. Results In phase 1, 43 interventions were identified, which had low uptake in practice according to 3042 professionals globally. In phase 2, a shortlist of 15 intervention domains was generated. In phase 3, interventions were deemed acceptable for more than 90 per cent of patients except for reducing general anaesthesia (84 per cent) and re-sterilization of ‘single-use’ consumables (86 per cent). In phase 4, the top three shortlisted interventions for high-income countries were: introducing recycling; reducing use of anaesthetic gases; and appropriate clinical waste processing. In phase 4, the top three shortlisted interventions for low–middle-income countries were: introducing reusable surgical devices; reducing use of consumables; and reducing the use of general anaesthesia. Conclusion This is a step toward environmentally sustainable operating environments with actionable interventions applicable to both high– and low–middle–income countries