Peptidomic Analysis of Skin Secretions of the Caribbean Frogs Leptodactylus insularum and Leptodactylus nesiotus (Leptodactylidae) Identifies an Ocellatin with Broad Spectrum Antimicrobial Activity

International audienceOcellatins are peptides produced in the skins of frogs belonging to the genus Leptodactylus that generally display weak antimicrobial activity against Gram-negative bacteria only. Peptidomic analysis of norepinephrine-stimulated skin secretions from Leptodactylus insularum Barbour 1906 and Leptodactylus nesiotus Heyer 1994, collected in the Icacos Peninsula, Trinidad, led to the purification and structural characterization of five ocellatin-related peptides from L. insularum (ocellatin-1I together with its (1-16) fragment, ocellatin-2I and its (1-16) fragment, and ocellatin-3I) and four ocellatins from L. nesiotus (ocellatin-1N,-2N,-3N, and-4N). While ocellatins-1I,-2I, and-1N showed a typically low antimicrobial potency against Gram-negative bacteria, ocellatin-3N (GIFDVLKNLAKGVITSLAS.NH 2) was active against an antibiotic-resistant strain of Klebsiella pneumoniae and reference strains of Escherichia coli, K. pneumoniae, Pseudomonas aeruginosa, and Salmonella typhimurium (minimum inhibitory concentrations (MICs) in the range 31.25-62.5 µM), and was the only peptide active against Gram-positive Staphylococcus aureus (MIC = 31.25 µM) and Enterococcus faecium (MIC = 62.5 µM). The therapeutic potential of ocellatin-3N is limited by its moderate hemolytic activity (LC 50 = 98 µM) against mouse erythrocytes. The peptide represents a template for the design of long-acting, non-toxic, and broad-spectrum antimicrobial agents for targeting multidrug-resistant pathogens

Ion mobility mass spectrometry uncovers the impact of the patterning of oppositely charged residues on the conformational distributions of intrinsically disordered proteins

The global dimensions and amplitudes of conformational fluctuations of intrinsically disordered proteins are governed, in part, by the linear segregation versus clustering of oppositely charged residues within the primary sequence. Ion mobility-mass spectrometry (IM-MS) affords unique advantages for probing the conformational consequences of the linear patterning of oppositely charged residues because it measures and separates proteins electrosprayed from solution on the basis of charge and shape. Here, we use IM-MS to measure the conformational consequences of charge patterning on the C-terminal intrinsically disordered region (p27 IDR) of the cell cycle inhibitory protein p27Kip1. We report the range of charge states and accompanying collisional cross section distributions for wild-type p27 IDR and two variants with identical amino acid compositions, κ14 and κ56, distinguished by the extent of linear mixing versus segregation of oppositely charged residues. Wild-type p27 IDR (κ31) and κ14, where the oppositely charged residues are more evenly distributed, exhibit a broad distribution of charge states. This is concordant with high degrees of conformational heterogeneity in solution. By contrast, κ56 with linear segregation of oppositely charged residues leads to limited conformational heterogeneity and a narrow distribution of charged states. Gas-phase molecular dynamics simulations demonstrate that the interplay between chain solvation and intrachain interactions (self-solvation) leads to conformational distributions that are modulated by salt concentration, with the wild-type sequence showing the most sensitivity to changes in salt concentration. These results suggest that the charge patterning within the wild-type p27 IDR may be optimized to sample both highly solvated and self-solvated conformational states

Peptidomic analysis of the host-defense peptides in skin secretions of the Amazon River frog Lithobates palmipes (Ranidae)

International audienc

An integrated analysis and comparison of serum, saliva and sebum for COVID-19 metabolomics.

The majority of metabolomics studies to date have utilised blood serum or plasma, biofluids that do not necessarily address the full range of patient pathologies. Here, correlations between serum metabolites, salivary metabolites and sebum lipids are studied for the first time. 83 COVID-19 positive and negative hospitalised participants provided blood serum alongside saliva and sebum samples for analysis by liquid chromatography mass spectrometry. Widespread alterations to serum-sebum lipid relationships were observed in COVID-19 positive participants versus negative controls. There was also a marked correlation between sebum lipids and the immunostimulatory hormone dehydroepiandrosterone sulphate in the COVID-19 positive cohort. The biofluids analysed herein were also compared in terms of their ability to differentiate COVID-19 positive participants from controls; serum performed best by multivariate analysis (sensitivity and specificity of 0.97), with the dominant changes in triglyceride and bile acid levels, concordant with other studies identifying dyslipidemia as a hallmark of COVID-19 infection. Sebum performed well (sensitivity 0.92; specificity 0.84), with saliva performing worst (sensitivity 0.78; specificity 0.83). These findings show that alterations to skin lipid profiles coincide with dyslipidaemia in serum. The work also signposts the potential for integrated biofluid analyses to provide insight into the whole-body atlas of pathophysiological conditions

Purpurogallin-A heme binding component of oak galls

Recently, it has been shown that Purpurogallin (PPG), an orange benztropolone constituent of oak galls and its derivative, CU-CPT22, can compete with the binding of the specific lipoprotein ligand to toll-like receptors (TLRs), which are type I transmembrane proteins. These recognize pathogen-derived macromolecules that play a key role in the innate immune system. This system provides an attractive target for the treatment of various immune disorders. Notably, PPG also interacts with various metals and its mode of action against HIV in vitro may involve inhibition of metal containing integrases. In the current study, an optimised synthesis of PPG is presented together with its gas phase behaviour (probed by mass spectrometry) as well as its redox behaviour with porphyrins such as heme. This interaction may also explain its effects at metal containing integrases within HIV in vitro as well as its action during processing of iron complexes within Plasmodia. This compound could serve as a novel prototype for the synthesis of novel redox active antimalarials

Positioning discourse on homophobia in schools: what have lesbian and gay families got to say?

This paper reports findings from a study in England, which investigated the experiences of lesbian and gay parents in relation to homophobia in primary and secondary schools. The study was part of a larger European Union project investigating the impact of family and school alliances against homophobic and transphobic bullying in schools across six nation states. Qualitative in-depth semi-structured interviews with seven lesbian and gay parents from five families were conducted to explore their unique experience and perspectives on these issues. Discourse analysis was used to facilitate understanding of how lesbian and gay families negotiated the outsider/insider and public/private spheres of the school and communities of which they were a part. Parents identified a number of strategies to address their experiences of homophobia within schools. The findings have implications for how social work recognises and promotes diversity and equality when working with lesbian, gay, bisexual and transgender families, as social workers have a powerful role in supporting families. This involves recognising the strengths of lesbian, gay, bisexual and transgender families in their assessments

Positioning discourse on homophobia in schools: What have lesbian and gay families got to say?

This paper reports findings from a study in England, which investigated the experiences of lesbian and gay parents in relation to homophobia in primary and secondary schools. The study was part of a larger European Union project investigating the impact of family and school alliances against homophobic and transphobic bullying in schools across six nation states. Qualitative in-depth semi-structured interviews with seven lesbian and gay parents from five families were conducted to explore their unique experience and perspectives on these issues. Discourse analysis was used to facilitate understanding of how lesbian and gay families negotiated the outsider/insider and public/private spheres of the school and communities of which they were a part. Parents identified a number of strategies to address their experiences of homophobia within schools. The findings have implications for how social work recognises and promotes diversity and equality when working with lesbian, gay, bisexual and transgender families, as social workers have a powerful role in supporting families. This involves recognising the strengths of lesbian, gay, bisexual and transgender families in their assessments

Novel allosteric mechanism of p53 activation by small molecules for targeted anticancer therapy

Given the immense significance of p53 restoration for anti-cancer therapy and that p53-activating molecules are in clinical trials, elucidation of the mechanisms of action of p53-activating molecules is of the utmost importance. Here we report a discovery of a novel allosteric modulation of p53 by small molecules, which is an unexpected turn in the p53 story. We identified a structural element involved in allosteric regulation of p53, whose targeting by small molecules RITA, PpIX and licofelone blocks the binding of two p53 inhibitors, MDM2 and MDMX, thereby restoring p53 function. Deletion and mutation analysis followed by molecular modeling and its thorough validation, identified the key p53 residues S33 and S37 targeted by RITA and PpIX. We propose that the binding of small molecules to the identified site in p53 induces a conformational trap preventing p53 from the interaction with MDM2 and MDMX. These results point to a high potential of allosteric activators as targeted drugs. Our study provides a basis for the development of therapeutics with a novel mechanism of action, thus extending the p53 pharmacopeia

Partial Deletion of Chromosome 8 β-defensin Cluster Confers Sperm Dysfunction and Infertility in Male Mice

β-defensin peptides are a family of antimicrobial peptides present at mucosal surfaces, with the main site of expression under normal conditions in the male reproductive tract. Although they kill microbes in vitro and interact with immune cells, the precise role of these genes in vivo remains uncertain. We show here that homozygous deletion of a cluster of nine β-defensin genes (DefbΔ9) in the mouse results in male sterility. The sperm derived from the mutants have reduced motility and increased fragility. Epididymal sperm isolated from the cauda should require capacitation to induce the acrosome reaction but sperm from the mutants demonstrate precocious capacitation and increased spontaneous acrosome reaction compared to wild-types but have reduced ability to bind the zona pellucida of oocytes. Ultrastructural examination reveals a defect in microtubule structure of the axoneme with increased disintegration in mutant derived sperm present in the epididymis cauda region, but not in caput region or testes. Consistent with premature acrosome reaction, sperm from mutant animals have significantly increased intracellular calcium content. Thus we demonstrate in vivo that β-defensins are essential for successful sperm maturation, and their disruption leads to alteration in intracellular calcium, inappropriate spontaneous acrosome reaction and profound male infertility

Post-assembly modification of kinetically metastable Fe(II)2L3 triple helicates.

We report the covalent post-assembly modification of kinetically metastable amine-bearing Fe(II)2L3 triple helicates via acylation and azidation. Covalent modification of the metastable helicates prevented their reorganization to the thermodynamically favored Fe(II)4L4 tetrahedral cages, thus trapping the system at the non-equilibrium helicate structure. This functionalization strategy also conveniently provides access to a higher-order tris(porphyrinatoruthenium)-helicate complex that would be difficult to prepare by de novo ligand synthesis.This work was supported by the UK Engineering and Physical Sciences Research Council (EPSRC). D.A.R. acknowledges the Gates Cambridge Trust for Ph.D. (Gates Cambridge Scholarship) and conference funding.This is the final published version. It first appeared at http://pubs.acs.org/doi/abs/10.1021/ja5042397