Forensic Human Identification for Cutaneous Microbiome, a Brief Review

Forensic Science compounds many study areas in context of solving crimes, one of which is the forensic microbiology. Combined with genomic approaches, microbiology has shown strong performance in studies regarding the relationship between microorganisms present on human skin and environment. The Human Microbiome Project (HMP) has contributed significantly to characterization of microbial complexity and their connection to human being. The purpose of this work consists of a historical overview of scientific articles, demonstrating the growth and possibility of using skin microbiome in forensic identification. Studies about use of cutaneous microbiome in human identification, as well its forensic approaches, were looked into for writing of this review. Comparisons among cutaneous microbial communities and manipulated objects have been tested using 16S rRNA, as well as a thorough sequencing of the bacterial genome. From use of ecological measures of distance to genetic markers with nucleotide variants and predictive algorithms, research has shown promising results for advances in field of forensic identification. The development of metagenomic microbial panel markers, named hidSkinPlax for targeted sequencing has been designed and tested with great results. Research results show satisfactory potential in human identification by cutaneous microbiome and the possibility for contributive use in elucidating crimes

EcDBS1R4, an antimicrobial peptide effective against Escherichia coli with in vitro fusogenic ability

©2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open accessarticle distributed under the terms and conditions of the Creative Commons Attribution(CC BY) license (http://creativecommons.org/licenses/by/4.0/)Discovering antibiotic molecules able to hold the growing spread of antimicrobial resistance is one of the most urgent endeavors that public health must tackle. The case of Gram-negative bacterial pathogens is of special concern, as they are intrinsically resistant to many antibiotics, due to an outer membrane that constitutes an effective permeability barrier. Antimicrobial peptides (AMPs) have been pointed out as potential alternatives to conventional antibiotics, as their main mechanism of action is membrane disruption, arguably less prone to elicit resistance in pathogens. Here, we investigate the in vitro activity and selectivity of EcDBS1R4, a bioinspired AMP. To this purpose, we have used bacterial cells and model membrane systems mimicking both the inner and the outer membranes of Escherichia coli, and a variety of optical spectroscopic methodologies. EcDBS1R4 is effective against the Gram-negative E. coli, ineffective against the Gram-positive Staphylococcus aureus and noncytotoxic for human cells. EcDBS1R4 does not form stable pores in E. coli, as the peptide does not dissipate its membrane potential, suggesting an unusual mechanism of action. Interestingly, EcDBS1R4 promotes a hemi-fusion of vesicles mimicking the inner membrane of E. coli. This fusogenic ability of EcDBS1R4 requires the presence of phospholipids with a negative curvature and a negative charge. This finding suggests that EcDBS1R4 promotes a large lipid spatial reorganization able to reshape membrane curvature, with interesting biological implications herein discussed.This research was funded by Fundação para a Ciência e a Tecnologia—Ministério da Ciência, Tecnologia e Ensino Superior (FCT-MCTES, Portugal), Marie Skłodowska-Curie Research and Innovation Staff Exchange (MSCA-RISE, European Union) project INPACT (call H2020-MSCA-RISE-2014, grant agreement 644167), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brazil), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Brazil), Fundação de Amparo a Pesquisa do Distrito Federal (FAPDF, Brazil) and Fundação de Apoio ao Desenvolvimento do Ensino, Ciência e Tecnologia do Estado de Mato Grosso do Sul (FUNDECT, Brazil). M.M. and M.R.F. also acknowledge FCT-MCTES fellowships SPRH/BD/128290/2017 and SPRH/BD/100517/2014, respectively.info:eu-repo/semantics/publishedVersio

Structural studies of a lipid-binding peptide from tunicate hemocytes with anti-biofilm activity

This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/Clavanins is a class of peptides (23aa) histidine-rich, free of post-translational modifications. Clavanins have been studied largely for their ability to disrupt bacterial membranes. In the present study, the interaction of clavanin A with membranes was assessed by dynamic light scattering, zeta potential and permeabilization assays. We observed through those assays that clavanin A lysis bacterial cells at concentrations corresponding to its MIC. Further, the structure and function of clavanin A was investigated. To better understand how clavanin interacted with bacteria, its NMR structure was elucidated. The solution state NMR structure of clavanin A in the presence of TFE-d3 indicated an α-helical conformation. Secondary structures, based on circular dichroism measurements in anionic sodium dodecyl sulfate (SDS) and TFE (2,2,2-trifluorethanol), in silico lipid-peptide docking and molecular simulations with lipids DPPC and DOPC revealed that clavanin A can adopt a variety of folds, possibly influencing its different functions. Microcalorimetry assays revealed that clavanin A was capable of discriminating between different lipids. Finally, clavanin A was found to eradicate bacterial biofilms representing a previously unrecognized function.We would like to thank CNPq, CAPES (Ciências sem Fronteiras), FAPDF and FUNDECT. D.G. acknowledges Fundação para a Ciência e a Tecnologia - Ministério da Educação e Ciência (FCT-MEC, Portugal) for fellowship SFRH/BPD/73500/2010 and A.S.V. for funding within the FCT Investigator Programme (IF/00803/2012).info:eu-repo/semantics/publishedVersio

Evaluation of the in vitro antitumor activity of nanostructured cyclotides in polymers of Eudragit (R) L 100-55 and RS 30 D

Background: Cancer is a major cause of mortality and morbidity and given the limitations of many current cancer drugs, there is great need to discover and develop novel treatments. An alternative to the conventional drug discovery path is to exploit new classes of natural compounds such as cyclotides. This peptide family is characterized by linked C- and N-termini and a structural fold called the cyclic cystine knot (CCK). The CCK fold is responsible for the exceptional enzymatic, chemical and thermal stability of cyclotides.Methods: In the present study, an alternative to traditional cancer treatments, involving new nano-materials and nanocarriers allowing efficient cyclotide delivery, is proposed. Using the polymers Eudragit (R) L 100-55 and RS 30 D, the cyclotides kalata B2 and parigidin-br1 ( PBR1) were nanocapsulated, and nanoparticles 91 nm and 188 nm in diameter, respectively, were produced.Results: An encapsulation rate of up to 95% was observed. In vitro bioassays showed that the nanostructured cyclotides were partially able to control the development of the colorectal adenocarcinoma cell line CACO2 and the breast cancer cell line MCF-7.Conclusion: Data reported herein indicate that nanoformulated cyclotides exhibit antitumor activity and sustained drug release. Thus, the system using Eudragit (R) nanocapsules seems to be efficient for cyclotide encapsulation and probably could be used to target specific tumors in future studies

Cyclotides

In recent years, a number of peptides containing a cyclic structural fold have been described. Among them, the cyclotides family was widely reported in different plant tissues, being composed of small cyclic peptides containing 6 conserved cysteine residues connected by disulfide bonds and forming a cysteine-binding cyclic structure known as a cyclic cysteine knot. This structural scaffold is responsible for an enhanced structural stability against chemical, thermal, and proteolytic degradation. Because of the observed stability and multifunctionality, including insecticidal, antimicrobial, and anti-HIV (human immunodeficiency virus) action, much effort has gone into trying to elucidate the structural-function relations of cyclotide compounds. This review focuses on the novelties involving gene structure, precursor formation and processing, and protein folding of the cyclotide family, shedding some light on molecular mechanisms of cyclotide production. Because cyclotides are clear targets for drug development and also biotechnology applications, their chemical synthesis, heterologous systems production, and protein grafting are also addressed

Effects of cyclotides against cutaneous infections caused by Staphylococcus aureus

The main bacterium associated with skin infection is Staphylococcus aureus, occurring especially in infections acquired via surgical wounds, commonly leading to lethal hospital-acquired infections, emphasizing the importance of identifying new antimicrobial compounds. Among them, cyclotides have gained interest due to their high stability and multifunctional properties. Here, cycloviolacin 2 (CyO2) and kalata B2 (KB2) were evaluated to determinate their anti-staphylococcal activities using a subcutaneous infection model. Anti-staphylococcal activities of 50 mM for KB2 and 25 mM for CyO2 were detected with no cytotoxic activities against RAW 264.7 monocytes. In the in vivo assays, both cyclotides reduced bacterial load and CyO2 demonstrated an increase in the phagocytosis index, suggesting that the CyO2 in vivo anti-staphylococcal activity may be associated with phagocytic activity, additionally to direct anti-pathogenicactivity. (C) 2014 Elsevier Inc. All rights reserved

Characterization of a bioactive acyclotide from Palicourea rigida

The extraction and purification of parigidin-br3, a cyclotide analogue belonging to the "bracelet" subfamily, from Palicourea rigida leaves is discussed. Unlike conventional cyclotides, parigidin-br3 has free N- and C-termini, as identified by MALDI-TOP/TOP analysis and confirmed by gene structure elucidation, and is one of a small number of acyclotides discovered during recent years. Parigidin-br3 showed cytotoxic activity against MCP-7 (breast cancer) and CACO2 (colorectal adenocarcinoma) cells, with IC50 values of similar to 2.5 mu M and less than 10% hemolytic activity. Overall, parigidin-br3 is a promising new molecule with cytotoxic properties against tumor cell lines and, unlike many synthetic acyclic analogues, demonstrates that cytotoxic activity is not limited to conventional (i.e., cyclic) cyclotides

Membrane-active macromolecules kill antibiotic-tolerant bacteria and potentiate antibiotics towards Gram-negative bacteria

<div><p>Chronic bacterial biofilms place a massive burden on healthcare due to the presence of antibiotic-tolerant dormant bacteria. Some of the conventional antibiotics such as erythromycin, vancomycin, linezolid, rifampicin etc. are inherently ineffective against Gram-negative bacteria, particularly in their biofilms. Here, we report membrane-active macromolecules that kill slow dividing stationary-phase and antibiotic tolerant cells of Gram-negative bacteria. More importantly, these molecules potentiate antibiotics (erythromycin and rifampicin) to biofilms of Gram-negative bacteria. These molecules eliminate planktonic bacteria that are liberated after dispersion of biofilms (dispersed cells). The membrane-active mechanism of these molecules forms the key for potentiating the established antibiotics. Further, we demonstrate that the combination of macromolecules and antibiotics significantly reduces bacterial burden in mouse burn and surgical wound infection models caused by <i>Acinetobacter baumannii</i> and Carbapenemase producing <i>Klebsiella pneumoniae</i> (KPC) clinical isolate respectively. Colistin, a well-known antibiotic targeting the lipopolysaccharide (LPS) of Gram-negative bacteria fails to kill antibiotic tolerant cells and dispersed cells (from biofilms) and bacteria develop resistance to it. On the contrary, these macromolecules prevent or delay the development of bacterial resistance to known antibiotics. Our findings emphasize the potential of targeting the bacterial membrane in antibiotic potentiation for disruption of biofilms and suggest a promising strategy towards developing therapies for topical treatment of Gram-negative infections.</p></div

Disruption of <i>E</i>. <i>coli</i> biofilms.

<p>(A) Confocal laser scanning microscopy (CLSM) images of biofilms either treated with QCybuAP (50 μg mL^-1), erythromycin (Ery, 50 μg mL^-1) and erythromycin + QCybuAP (50 μg mL^-1 + 50 μg mL^-1) or left untreated for 24 h. Biofilms were stained with SYTO 9 dye and each image is a 3D reconstruction of z-stack images; (B) Reduction in bacterial cell counts in biofilms with and without treatment of compounds; (C) Absorbance of the crystal violet (CV) staining of the biofilms.</p