A designed cyclic analogue of gomesin has potent activity against Staphylococcus aureus biofilms

© The Author(s) 2022. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy. This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (https:// creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact [email protected]: Infections caused by bacterial biofilms are very difficult to treat. The use of currently approved antibiotics even at high dosages often fails, making the treatment of these infections very challenging. Novel antimicrobial agents that use distinct mechanisms of action are urgently needed. Objectives: To explore the use of [G1K,K8R]cGm, a designed cyclic analogue of the antimicrobial peptide gomesin, as an alternative approach to treat biofilm infections. Methods: We studied the activity of [G1K,K8R]cGm against biofilms of Staphylococcus aureus, a pathogen associated with several biofilm-related infections. A combination of atomic force and real-time confocal laser scanning microscopies was used to study the mechanism of action of the peptide. Results: The peptide demonstrated potent activity against 24 h-preformed biofilms through a concentration-dependent ability to kill biofilm-embedded cells. Mechanistic studies showed that [G1K,K8R]cGm causes morphological changes on bacterial cells and permeabilizes their membranes across the biofilm with a half-time of 65 min. We also tested an analogue of [G1K,K8R]cGm without disulphide bonds, and a linear unfolded analogue, and found both to be inactive. Conclusions: The results suggest that the 3D structure of [G1K,K8R]cGm and its stabilization by disulphide bonds are essential for its antibacterial and antibiofilm activities. Moreover, our findings support the potential application of this stable cyclic antimicrobial peptide to fight bacterial biofilms.This project received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 828774. This work was supported by project grants funded by Fundação para a Ciência e a Tecnologia (FCT-MCTES, Portugal; UIDB/04565/2020 and PPBI-POCI-01-0145-FEDER-022122). S.A.D. acknowledges FCT for the fellowship PD/BD/114425/2016. S.T.H. is an Australian Research Council (ARC) Future Fellow (FT150100398) and is supported by the ARC Centre of Excellence for Innovations in Peptide & Protein Science (CE200100012).info:eu-repo/semantics/publishedVersio

The Health-Promoting Potential of Salix spp. Bark Polar Extracts: Key Insights on Phenolic Composition and In Vitro Bioactivity and Biocompatibility

Salix spp. have been exploited for energy generation, along with folk medicine use of bark extracts for antipyretic and analgesic benefits. Bark phenolic components, rather than salicin, have demonstrated interesting bioactivities, which may ensure the sustainable bioprospection of Salix bark. Therefore, this study highlights the detailed phenolic characterization, as well as the in vitro antioxidant, anti-hypertensive, Staphylococcus aureus growth inhibitory effects, and biocompatibility of Salix atrocinerea Brot., Salix fragilis L., and Salix viminalis L. bark polar extracts. Fifteen phenolic compounds were characterized by ultra-high-performance liquid chromatography-ultraviolet detection-mass spectrometry analysis, from which two flavan-3-ols, an acetophenone, five flavanones, and a flavonol were detected, for the first time, as their bark components. Salix bark extracts demonstrated strong free radical scavenging activity (5.58–23.62 µg mL−1 IC50 range), effective inhibition on angiotensin-I converting enzyme (58–84%), and S. aureus bactericidal action at 1250–2500 µg mL−1 (6–8 log CFU mL−1 reduction range). All tested Salix bark extracts did not show cytotoxic potential against Caco-2 cells, as well as S. atrocinerea Brot. and S. fragilis L. extracts at 625 and 1250 µg mL−1 against HaCaT and L929 cells. These valuable findings can pave innovative and safer food, nutraceutical, and/or cosmetic applications of Salix bark phenolic-containing fractions.info:eu-repo/semantics/publishedVersio

Mechanisms of bacterial membrane permeabilization by crotalicidin Ctn and its fragment Ctn 15–34, antimicrobial peptides from rattlesnake venom

© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.Crotalicidin (Ctn), a cathelicidin-related peptide from the venom of a South American rattlesnake, possesses potent antimicrobial, antitumor, and antifungal properties. Previously, we have shown that its C-terminal fragment, Ctn(15-34), retains the antimicrobial and antitumor activities but is less toxic to healthy cells and has improved serum stability. Here, we investigated the mechanisms of action of Ctn and Ctn(15-34) against Gram-negative bacteria. Both peptides were bactericidal, killing ∼90% of Escherichia coli and Pseudomonas aeruginosa cells within 90-120 and 5-30 min, respectively. Studies of ζ potential at the bacterial cell membrane suggested that both peptides accumulate at and neutralize negative charges on the bacterial surface. Flow cytometry experiments confirmed that both peptides permeabilize the bacterial cell membrane but suggested slightly different mechanisms of action. Ctn(15-34) permeabilized the membrane immediately upon addition to the cells, whereas Ctn had a lag phase before inducing membrane damage and exhibited more complex cell-killing activity, probably because of two different modes of membrane permeabilization. Using surface plasmon resonance and leakage assays with model vesicles, we confirmed that Ctn(15-34) binds to and disrupts lipid membranes and also observed that Ctn(15-34) has a preference for vesicles that mimic bacterial or tumor cell membranes. Atomic force microscopy visualized the effect of these peptides on bacterial cells, and confocal microscopy confirmed their localization on the bacterial surface. Our studies shed light onto the antimicrobial mechanisms of Ctn and Ctn(15-34), suggesting Ctn(15-34) as a promising lead for development as an antibacterial/antitumor agent.This work was supported by Spanish Ministry of Economy and Competitiveness (MINECO) Grants SAF2011-24899 and AGL2014-52395-C2, by Fundação para a Ciência e a Tecnologia (FCT, Portugal) Grants PTDC/QEQ-MED/4412/2014, and by EU Marie Skłodowska-Curie Research and Innovation Staff Exchange (RISE) program Grant 644167, 2015–2019.info:eu-repo/semantics/publishedVersio

SARS-CoV-2 introductions and early dynamics of the epidemic in Portugal

Genomic surveillance of SARS-CoV-2 in Portugal was rapidly implemented by the National Institute of Health in the early stages of the COVID-19 epidemic, in collaboration with more than 50 laboratories distributed nationwide. Methods By applying recent phylodynamic models that allow integration of individual-based travel history, we reconstructed and characterized the spatio-temporal dynamics of SARSCoV-2 introductions and early dissemination in Portugal. Results We detected at least 277 independent SARS-CoV-2 introductions, mostly from European countries (namely the United Kingdom, Spain, France, Italy, and Switzerland), which were consistent with the countries with the highest connectivity with Portugal. Although most introductions were estimated to have occurred during early March 2020, it is likely that SARS-CoV-2 was silently circulating in Portugal throughout February, before the first cases were confirmed. Conclusions Here we conclude that the earlier implementation of measures could have minimized the number of introductions and subsequent virus expansion in Portugal. This study lays the foundation for genomic epidemiology of SARS-CoV-2 in Portugal, and highlights the need for systematic and geographically-representative genomic surveillance.We gratefully acknowledge to Sara Hill and Nuno Faria (University of Oxford) and Joshua Quick and Nick Loman (University of Birmingham) for kindly providing us with the initial sets of Artic Network primers for NGS; Rafael Mamede (MRamirez team, IMM, Lisbon) for developing and sharing a bioinformatics script for sequence curation (https://github.com/rfm-targa/BioinfUtils); Philippe Lemey (KU Leuven) for providing guidance on the implementation of the phylodynamic models; Joshua L. Cherry (National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health) for providing guidance with the subsampling strategies; and all authors, originating and submitting laboratories who have contributed genome data on GISAID (https://www.gisaid.org/) on which part of this research is based. The opinions expressed in this article are those of the authors and do not reflect the view of the National Institutes of Health, the Department of Health and Human Services, or the United States government. This study is co-funded by Fundação para a Ciência e Tecnologia and Agência de Investigação Clínica e Inovação Biomédica (234_596874175) on behalf of the Research 4 COVID-19 call. Some infrastructural resources used in this study come from the GenomePT project (POCI-01-0145-FEDER-022184), supported by COMPETE 2020 - Operational Programme for Competitiveness and Internationalisation (POCI), Lisboa Portugal Regional Operational Programme (Lisboa2020), Algarve Portugal Regional Operational Programme (CRESC Algarve2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF), and by Fundação para a Ciência e a Tecnologia (FCT).info:eu-repo/semantics/publishedVersio

Deficient Endoplasmic Reticulum-Mitochondrial Phosphatidylserine Transfer Causes Liver Disease

Non-alcoholic fatty liver is the most common liver disease worldwide. Here, we show that the mitochondrial protein mitofusin 2 (Mfn2) protects against liver disease. Reduced Mfn2 expression was detected in liver biopsies from patients with non-alcoholic steatohepatitis (NASH). Moreover, reduced Mfn2 levels were detected in mouse models of steatosis or NASH, and its re-expression in a NASH mouse model ameliorated the disease. Liver-specific ablation of Mfn2 in mice provoked inflammation, triglyceride accumulation, fibrosis, and liver cancer. We demonstrate that Mfn2 binds phosphatidylserine (PS) and can specifically extract PS into membrane domains, favoring PS transfer to mitochondria and mitochondrial phosphatidylethanolamine (PE) synthesis. Consequently, hepatic Mfn2 deficiency reduces PS transfer and phospholipid synthesis, leading to endoplasmic reticulum (ER) stress and the development of a NASH-like phenotype and liver cancer. Ablation of Mfn2 in liver reveals that disruption of ER-mitochondrial PS transfer is a new mechanism involved in the development of liver disease.This study was supported by the MINECO (SAF2016-75246R), the Generalitat de Catalunya (2014SGR48, 2017SGR696, ICREA Acadèmia), INFLAMES (PIE-14/00045, ISCIII), CIBERDEM, ISCIII, INTERREG IV-B-SUDOE-FEDER (DIOMED, SOE1/P1/E178), and “la Caixa” Foundation. S.F.-V. acknowledges support from the Miguel Servet tenure-track program (CP10/00438 and CPII16/00008) from the Fondo de Investigación Sanitaria, co-financed by the ERD. We gratefully acknowledge institutional funding from the MINECO through the Centres of Excellence Severo Ochoa Award and from the CERCA Programme of the Generalitat de Catalunya

Importance of the cell membrane on the mechanism of action of cyclotides

Their distinctive structures, diverse range of bioactivities, and potential for pharmaceutical or agricultural applications make cyclotides an intriguing family of cyclic peptides. Together with the physiological role in plant host defense, cyclotides possess antimicrobial, anticancer, and anti-HIV activities. In all of the reported activities, cell membranes seem to be the primary target for cyclotide binding. This article examines recent literature on cyclotide-membrane studies and highlights the hypothesis that the activity of cyclotides is dependent on their affinity for lipid bilayers and enhanced by the presence of specific lipids, i.e., phospholipids containing phosphatidylethanolamine headgroups. There is growing evidence that the lipid composition of target cell membranes dictates the amount of cyclotides bound to the cell and the extent of their activity. After membrane targeting and insertion in the bilayer core, cyclotides induce disruption of membranes by a pore formation mechanism. This proposed mechanism of action is supported by biophysical studies with model membranes and by studies on natural biological membranes of known lipid compositions

Characterisation of microbial attack on archaeological bone

As part of an EU funded project to investigate the factors influencing bone preservation in the archaeological record, more than 250 bones from 41 archaeological sites in five countries spanning four climatic regions were studied for diagenetic alteration. Sites were selected to cover a range of environmental conditions and archaeological contexts. Microscopic and physical (mercury intrusion porosimetry) analyses of these bones revealed that the majority (68%) had suffered microbial attack. Furthermore, significant differences were found between animal and human bone in both the state of preservation and the type of microbial attack present. These differences in preservation might result from differences in early taphonomy of the bones. © 2003 Elsevier Science Ltd. All rights reserved