A peptide mimic of the chemotaxis inhibitory protein of Staphylococcus aureus: towards the development of novel anti-inflammatory compounds

Complement factor C5a is one of the most powerful pro-inflammatory agents involved in recruitment of leukocytes, activation of phagocytes and other inflammatory responses. C5a triggers inflammatory responses by binding to its G-protein-coupled C5a-receptor (C5aR). Excessive or erroneous activation of the C5aR has been implicated in numerous inflammatory diseases. The C5aR is therefore a key target in the development of specific anti-inflammatory compounds. A very potent natural inhibitor of the C5aR is the 121-residue chemotaxis inhibitory protein of Staphylococcus aureus (CHIPS). Although CHIPS effectively blocks C5aR activation by binding tightly to its extra-cellular N terminus, it is not suitable as a potential anti-inflammatory drug due to its immunogenic properties. As a first step in the development of an improved CHIPS mimic, we designed and synthesized a substantially shorter 50-residue adapted peptide, designated CHOPS. This peptide included all residues important for receptor binding as based on the recent structure of CHIPS in complex with the C5aR N terminus. Using isothermal titration calorimetry we demonstrate that CHOPS has micromolar affinity for a model peptide comprising residues 7–28 of the C5aR N terminus including two O-sulfated tyrosine residues at positions 11 and 14. CD and NMR spectroscopy showed that CHOPS is unstructured free in solution. Upon addition of the doubly sulfated model peptide, however, the NMR and CD spectra reveal the formation of structural elements in CHOPS reminiscent of native CHIPS

Integrating global energy and climate governance: The changing role of the International Energy Agency

Despite the long-recognized interlinkages between global energy consumption and climate change, there has historically been only limited policy interaction, let alone integration, between the two fields. This compartmentalization is mirrored in scholarship, where much research has focused on the fragmentation of, respectively, global energy and global climate governance, but only little has been said about how these fields might be integrated. Our analysis of the International Energy Agency’s (IEA) changing activities in recent years shows that governance integration – both within global energy governance and between global energy and climate governance – is now happening. The IEA has broadened its portfolio to embrace the full spectrum of energy issues, including renewable energy and climate change; it has built and is expanding key partnerships with both the UN climate convention and the International Renewable Energy Agency (IRENA); and it has become an authoritative advocate for the inter-related goals of a low-carbon transition and climate change mitigation. We show that these developments are not the result of a top-down plan, but have rather emerged through the Agency’s various efforts to pursue its energy-centric mandate in a fast-changing global policy environment

Opportunities for improving animal welfare in rodent models of epilepsy and seizures

Animal models of epilepsy and seizures, mostly involving mice and rats, are used to understand the pathophysiology of the different forms of epilepsy and their comorbidities, to identify biomarkers, and to discover new antiepileptic drugs and treatments for comorbidities. Such models represent an important area for application of the 3Rs (replacement, reduction and refinement of animal use). This report provides background information and recommendations aimed at minimising pain, suffering and distress in rodent models of epilepsy and seizures in order to improve animal welfare and optimise the quality of studies in this area. The report includes practical guidance on principles of choosing a model, induction procedures, in vivo recordings, perioperative care, welfare assessment, humane endpoints, social housing, environmental enrichment, reporting of studies and data sharing. In addition, some model-specific welfare considerations are discussed, and data gaps and areas for further research are identified. The guidance is based upon a systematic review of the scientific literature, survey of the international epilepsy research community, consultation with veterinarians and animal care and welfare officers, and the expert opinion and practical experience of the members of a Working Group convened by the United Kingdom's National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs)

Evaluation of experimental genetic management in reintroduced bighorn sheep

Positive demographic responses have been reported in several species where the immigration or supplementation of genetically distinct individuals into wild populations has resulted in a genetic rescue effect. However, rarely have researchers incorporated what could be considerable risk of outbreeding depression into planning for genetic management programs. We assess the genetic effects of an experiment in genetic management involving replicate populations of California bighorn sheep (Ovis canadensis californiana) in Oregon, USA, which previously experienced poor productivity and numerical declines. In the experiment, two declining populations were supplemented with ewes from a more genetically diverse population of California bighorn sheep in Nevada. We incorporated analysis of genetic samples representing both experimental populations prior to supplementation, samples from the supplemented individuals, and samples collected from both experimental populations approximately one generation after supplementation. We used genetic analyses to assess the integration of supplemented and resident populations by identifying interpopulation hybrids. Further, we incorporated demographic simulations to assess the risk of outbreeding depression as a result of the experimental augmentation. Finally, we used data from microsatellites and mitochondrial sequences to determine if genetic management increased genetic diversity in the experimental populations. Our analyses demonstrated the success of genetic management by documenting interpopulation hybrids, identifying no evidence for outbreeding depression as a result of contact between the genetically distinct supplemented and resident populations, and by identifying increased population-level metrics of genetic diversity in postsupplementation populations compared with presupplementation levels

Highly Differentiated, Resting Gn-Specific Memory CD8+ T Cells Persist Years after Infection by Andes Hantavirus

In man, infection with South American Andes virus (ANDV) causes hantavirus cardiopulmonary syndrome (HCPS). HCPS due to ANDV is endemic in Southern Chile and much of Argentina and increasing numbers of cases are reported all over South America. A case-fatality rate of about 36% together with the absence of successful antiviral therapies urge the development of a vaccine. Although T-cell responses were shown to be critically involved in immunity to hantaviruses in mouse models, no data are available on the magnitude, specificity and longevity of ANDV-specific memory T-cell responses in patients. Using sets of overlapping peptides in IFN-γ ELISPOT assays, we herein show in 78 Chilean convalescent patients that Gn-derived epitopes were immunodominant as compared to those from the N- and Gc-proteins. Furthermore, while the relative contribution of the N-specific response significantly declined over time, Gn-specific responses remained readily detectable ex vivo up to 13 years after the acute infection. Tetramer analysis further showed that up to 16.8% of all circulating CD3+CD8+ T cells were specific for the single HLA-B*3501-restricted epitope Gn465–473 years after the acute infection. Remarkably, Gn465–473–specific cells readily secreted IFN-γ, granzyme B and TNF-α but not IL-2 upon stimulation and showed a ‘revertant’ CD45RA+CD27−CD28−CCR7−CD127− effector memory phenotype, thereby resembling a phenotype seen in other latent virus infections. Most intriguingly, titers of neutralizing antibodies increased over time in 10/17 individuals months to years after the acute infection and independently of whether they were residents of endemic areas or not. Thus, our data suggest intrinsic, latent antigenic stimulation of Gn-specific T-cells. However, it remains a major task for future studies to proof this hypothesis by determination of viral antigen in convalescent patients. Furthermore, it remains to be seen whether Gn-specific T cells are critical for viral control and protective immunity. If so, Gn-derived immunodominant epitopes could be of high value for future ANDV vaccines

A New Pathway for Protein Haptenation by beta-Lactams

"This is the peer reviewed version of the following article: Pérez-Ruíz, Raúl, Emilio Lence, Inmaculada Andreu, Daniel Limones-Herrero, Concepción González-Bello, Miguel A. Miranda, and M. Consuelo Jiménez. 2017. A New Pathway for Protein Haptenation by β-Lactams. Chemistry - A European Journal 23 (56). Wiley: 13986 94. doi:10.1002/chem.201702643, which has been published in final form at https://doi.org/10.1002/chem.201702643. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving."[EN] The covalent binding of beta-lactams to proteins upon photochemical activation has been demonstrated by using an integrated approach that combines photochemical, proteomic and computational studies, selecting human serum albumin (HSA) as a target protein and ezetimibe (1) as a probe. The results have revealed a novel protein haptenation pathway for this family of drugs that is an alternative to the known nucleophilic ring opening of beta-lactams by the free amino group of lysine residues. Thus, photochemical ring splitting of the beta-lactam ring, following a formal retro-Staudinger reaction, gives a highly reactive ketene intermediate that is trapped by the neighbouring lysine residues, leading to an amide adduct. For the investigated 1/HSA system, covalent modification of residues Lys414 and Lys525, which are located in sub-domains IIIA and IIIB, respectively, occurs. The observed photobinding may constitute the key step in the sequence of events leading to photoallergy. Docking and molecular dynamics simulation studies provide an insight into the molecular basis of the selectivity of 1 for these HSA sub-domains and the covalent modification mechanism. Computational studies also reveal positive cooperative binding of sub-domain IIIB that explains the experimentally observed modification of Lys414, which is located in a barely accessible pocket (sub-domain IIIA).Financial support from Ministerio de Economia, Industria y Competitividad (CTQ2013-47872-C2-1-P, CTQ2016-78875-P, SAF2013-42899-R, SAF2016-75638-R), Instituto de Salud Carlos III (RD12/0013/0009 and RD16/0006/0030), Generalitat Valenciana (PROMETEOII/2013/005), Xunta de Galicia (Centro singular de investigacion de Galicia accreditation 2016-2019, ED431G/09) and European Union (European Regional Development Fund -ERDF) is gratefully acknowledged. E.L. thanks the Xunta de Galicia for a postdoctoral fellowship. We are grateful to the Centro de Supercomputacion de Galicia (CESGA) for use of the Finis Terrae II supercomputer. The proteomic analysis was performed in the proteomics facility of SCSIE University of Valencia that belongs to ProteoRed PRB2-ISCIII and is supported by grant PT13/0001, of the PE I+D+i 2013-2016, funded by ISCIII and FEDER.Pérez-Ruiz, R.; Lence, E.; Andreu Ros, MI.; Limones Herrero, D.; González-Bello, C.; Miranda Alonso, MÁ.; Jiménez Molero, MC. (2017). A New Pathway for Protein Haptenation by beta-Lactams. Chemistry - A European Journal. 23(56):13986-13994. https://doi.org/10.1002/chem.201702643S13986139942356Van Boeckel, T. P., Gandra, S., Ashok, A., Caudron, Q., Grenfell, B. T., Levin, S. A., & Laxminarayan, R. (2014). Global antibiotic consumption 2000 to 2010: an analysis of national pharmaceutical sales data. The Lancet Infectious Diseases, 14(8), 742-750. doi:10.1016/s1473-3099(14)70780-7Elander, R. P. (2003). Industrial production of β-lactam antibiotics. 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Impaired Release of Antimicrobial Peptides into Nasal Fluid of Hyper-IgE and CVID Patients

Patients with primary immunodeficiency (PID) often suffer from frequent respiratory tract infections. Despite standard treatment with IgG-substitution and antibiotics many patients do not improve significantly. Therefore, we hypothesized that additional immune deficits may be present among these patients.To investigate if PID patients exhibit impaired production of antimicrobial peptides (AMPs) in nasal fluid and a possible link between AMP-expression and Th17-cells.Nasal fluid, nasopharyngeal swabs and peripheral blood mononuclear cells (PBMCs) were collected from patients and healthy controls. AMP levels were measured in nasal fluid by Western blotting. Nasal swabs were cultured for bacteria. PBMCs were stimulated with antigen and the supernatants were assessed for IL-17A release by ELISA.In healthy controls and most patients, AMP levels in nasal fluid were increased in response to pathogenic bacteria. However, this increase was absent in patients with common variable immunodeficiency (CVID) and Hyper-IgE syndrome (HIES), despite the presence of pathogenic bacteria. Furthermore, stimulation of PBMCs revealed that both HIES and CVID patients exhibited an impaired production of IL-17A.CVID and HIES patients appear to have a dysregulated AMP response to pathogenic bacteria in the upper respiratory tract, which could be linked to an aberrant Th17 cell response

Amino Acid Similarity Accounts for T Cell Cross-Reactivity and for “Holes” in the T Cell Repertoire

Background: Cytotoxic T cell (CTL) cross-reactivity is believed to play a pivotal role in generating immune responses but the extent and mechanisms of CTL cross-reactivity remain largely unknown. Several studies suggest that CTL clones can recognize highly diverse peptides, some sharing no obvious sequence identity. The emerging realization in the field is that T cell receptors (TcR) recognize multiple distinct ligands. Principal Findings: First, we analyzed peptide scans of the HIV epitope SLFNTVATL (SFL9) and found that TCR specificity is position dependent and that biochemically similar amino acid substitutions do not drastically affect recognition. Inspired by this, we developed a general model of TCR peptide recognition using amino acid similarity matrices and found that such a model was able to predict the cross-reactivity of a diverse set of CTL epitopes. With this model, we were able to demonstrate that seemingly distinct T cell epitopes, i.e., ones with low sequence identity, are in fact more biochemically similar than expected. Additionally, an analysis of HIV immunogenicity data with our model showed that CTLs have the tendency to respond mostly to peptides that do not resemble self-antigens. Conclusions: T cell cross-reactivity can thus, to an extent greater than earlier appreciated, be explained by amino acid similarity. The results presented in this paper will help resolving some of the long-lasting discussions in the field of T cel

On this and other worlds: Voices from Amazonia

This edited volume offers a collection of twelve interlinear texts reflecting the vast linguistic diversity of Amazonia as well as the rich verbal arts and oral literature traditions of Amazonian peoples. Contributions to the volume come from a variety of geographic regions and represent the Carib, Jê, Tupi, East Tukano, Nadahup, and Pano language families, as well as three linguistic isolates. The selected texts exemplify a variety of narrative styles recounting the origins of constellations, crops, and sacred cemeteries, and of travel to worlds beyond death. We hear tales of tricksters and of encounters between humans and other beings, learn of battles between enemies, and gain insight into history and the indigenous perspective of creation, cordiality and confrontation. The contributions to this volume are the result of research efforts conducted since 2000, and as such, exemplify rapidly expanding investment and interest in documenting native Amazonian voices. They moreover demonstrate the collaborative efforts of linguists, anthropologists, and indigenous leaders, storytellers, and researchers to study and preserve Amazonian languages and cultures. Each chapter offers complete interlinear analysis as well as ample commentary on both linguistic and cultural aspects, appealing to a wide audience, including linguists, historians, anthropologists, and other social scientists. This collection is the first of its type, constituting a significant contribution to focused study of Amazonian linguistic diversity and a relevant addition to our broader knowledge of Amerindian languages and cosmologies