Reliability Assessment of Eurocode 7 Retaining Structures Design Methodology

Codes and Standard

Effect of antifungal agents on non-Candida albicans Candida species enzymes secretion

The infective ability of Candida species depends on specific virulence mechanisms that confer the ability to colonize host surfaces, to invade deeper host tissue or to evade host defences. During the pathogenic process many virulence attributes may be involved including, production of extracellular proteases and haemolytic activity. Nevertheless, in vitro studies have indicated that antifungal agents could be able to influence the enzymatic activity of Candida species. Therefore, the purpose of this work was to investigate the action of antifungals on proteinase and haemolytic activity of Candida species. This study was conducted with C. albicans (1), C. glabrata (4), C. parapsilosis (5) and C. tropicalis (6) recovered from different body sites (blood, oral, vaginal and urinary tract). Four reference strains of C. albicans ATCC 90028, C. glabrata ATCC 2001, C. parapsilosis ATCC 22019 and C. tropicalis ATCC 750 were also examined. The susceptibility to fluconazole and amphotericin B was determined by the microdilution test in order to allow the determination of the minimal inhibitory concentrations (MIC) and the maximum antifungal concentration (MAC). Then, the proteinase and hemolytic activity was determined for yeasts grown at MIC and MAC. It was observed that all Candida species assayed were sensible to both antifungal agents. Concerning the antifungal effect on enzymatic activity of Candida species, C. parapsilosis from candiduria presented a decreased proteinase and haemolysin activity for both MIC and MAC of both antifungal agents. Moreover, the other species presented differences in terms of production of proteinase and haemolysin at MIC and MAC. Candida albicans reference strain presented lower proteinase activity at MIC of fluconazole (46.7%) but presented higher activity for MAC (61.9%) in comparison to the control (60%). Furthermore, regarding haemolysin activity there were isolates that expressed high levels of enzymes in the presence of both antifungals such as: C. glabrata from urine and from vaginal tract; and C. tropicalis from urine. Conversely, some clinical isolates, presented low levels of enzymatic activity after contact with the antifungal agents, such as: C. albicans (oral isolate); C. glabrata (oral isolate and vaginal isolate); C. parapsilosis (from urine) and also all C. tropicalis except one urinary isolate. It was possible to conclude that the proteinase and haemolysin activities were strain and species dependent and no correlation was found among activity profile and the site of isolation. Moreover, fluconazole and amphotericin B were able to influence the tested Candida species enzymatic activity

Effect of itraconazole on Candida glabrata biofilm matrix

The emergence of non-Candida albicans Candida (NCAC) species as a common cause of fungal infection is often associated with the increasing number of immunocompromised patients, the widespread use of indwelling medical devices and the decreased susceptibility to azoles. The ability of Candida species to adapt to a variety of different habitats and to form biofilms is also of major contribution to this increased incidence. Thus, the aim of this work was to study the influence of the antifungal agent itraconazole on the matrix composition of Candida glabrata biofilms. Biofilms of Candida glabrata vaginalstrain 534784 were formed in 6-well plates for 24h. Then, fresh RPMI1640/ MOPS medium (control biofilms) and itraconazole (256μg/mL) were added to the previously formed 24h biofilms. After 48h of exposure to these components, biofilms were scraped from the 6-well plates and the extracellular matrix extracted by sonication. The protein and carbohydrate content of the biofilm matrix were determined using a BCA kit and the Dubois method, respectively. The analysis of matrix composition of biofilms exposed to itraconazole showed an increase in both protein and carbohydrate content comparatively to the control. The results indicate that the presence of itraconazole leads to an increase in the production of extracellular matrix components in Candida glabrata biofilms

The role of antifungals agents on Candida glabrata biofilms matrix composition

Candida glabrata was considered, for years, a relatively non-pathogenic saprophyte of the normal flora of healthy individuals and as no causative agent of serious infection in humans. However, its high mortality rate and its quick spread confirm the opposite. In fact, due to the widespread and increased use of immunosuppressive therapy together with broad-spectrum antifungal treatments, the frequency of mucosal and systemic infections caused by C. glabrata has increased significantly. Furthermore, biofilms are described as surface associated communities of microorganisms within an extracellular matrix, generally composed of carbohydrate and proteins. Biofilm formation is an important virulence factor for a number of Candida species, as it confers significant resistance to antifungal therapy by limiting the penetration of substances through the matrix and protecting cells from host immune responses. Moreover, little is known about the role of antifungals on C. glabrata biofilms. Thus, the aim of this work was to study the role of fluconazole, itraconazole and amphotericin B on 24 h pre-formed C. glabrata biofilms and specially on their matrix composition. A total of 3 C. glabrata strains isolated from oral, urinary and vaginal tract were used, as well as a reference strain from ATCC (C. glabrata 2001). Biofilms were formed on 12-well plates on RPMI 1640, during 24h at 37ºC and 120 rpm. Then, the antifungal agents (fluconazole, amphotericin B and itraconazole) were added to the previously formed biofilms. After 48 h of action of each antifungal agent, the biofilms were evaluated in terms of total biomass by crystal violet staining and number of viable cells by colony forming units (CFUs). The role of itraconazole on biofilms of the clinical vaginal isolate (C. glabrata 534784) was also examined in terms of matrix composition. For this, biofilms were formed in 6-well plates during 24h and, after 48h of exposure to itraconazole, were scraped from the wells and the extracellular matrix was extracted by sonication. Biofilm matrix contents in proteins and carbohydrates were determined using the BCA kit and the Dubois method, respectively. The results showed that, amphotericin B and fluconazole were able to cause a significant decreased on total biomass and CFUs of C. glabrata. However, itraconazole was not able to affect biofilms, except for the clinical vaginal isolate (C. glabrata 534784) at 256 µg/mL point concentration, which presented an increase in total biofilm biomass. Candida glabrata 534784 biofilms matrix exposed to itraconazole (256 µg/mL) presented an increase in proteins content but not in carbohydrate comparatively to the control. In summary, fluconazole and amphotericin B were able to significantly decrease the pre-formed biofilms of C. glabrata strains. Furthermore, the highest amount of total biofilm biomass of the vaginal isolate seems to be due to the increased protein content in its matrix

Candida tropicalis biofilms : formation and virulence factors

Significance and objectives: A substantial proportion of Candida tropicalis infections is associated with biofilm formation, especially on catheters. Thus, the aim of this study was to investigate C. tropicalis biofilm formation on silicone and its effect on epithelial cells and enzyme production (hemolysins and proteinases). Methods and results: This study was performed with C. tropicalis (clinical isolate and reference strain ATCC 750). Biofilms formed on silicone coupons immersed in artificial urine, were quantified by crystal violet (CV) staining and by enumeration of colony forming units (CFU) and the matrix content in proteins and polysaccharides was also determined. Biofilm cells and matrix were assessed in terms of hemolysins and proteinases production and their effect on TCC-SUP urinary epithelial cells was evaluated as well. Biofilms of C. tropicalis ATCC 750 presented a higher number of cells than the clinical isolate although less biofilm biomass and less polysaccharides. Moreover C. tropicalis biofilm was able to express total hemolytic activity and higher proteinase but these factors were not detectable within the matrix. Additionally, C. tropicalis biofilm adhered in higher extent to epithelial cells than their planktonic counterparts. Moreover, epithelial cells showed low metabolic activity when in contact with biofilms. Conclusions: Therefore, it is possible to conclude that enzyme production was detected in C. tropicalis biofilm cells, but not in its matrix and that biofilm cells can cause more damage to epithelial cells than their placktonic counterparts. This highlights the importance of biofilm formation, associated to the use of urinary catheters, on C. tropicalis virulence

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

Propolis potential activity against Candida tropicalis adhered cells and its biofilms

Objectives: Invasive fungal infections, such as candidiasis, represent a public health problem of major importance, and Candida tropicalis has been highlighted among the main agents of candidiasis. One of the major contributions to C. tropicalis virulence is its versatility in adapting to a variety of different habitats and the formation of surface attached microbial communities known as biofilms. Moreover, from the clinical perspective, the most important feature of Candida biofilms is its role in increasing tolerance to conventional antifungal therapy. This scenario encourages the search for alternative therapies. Natural matrixes, such as propolis, compromise a multitude of bioactive properties, in particular phenolic extracts have evidenced significant antimicrobial properties against a multiple of opportunist invaders, including Candida species. Thus, the main objective of the present work was to evaluate the potential antifungal effect of propolis against Candida tropicalis biofilms. Methods: This study was conducted with four strains of C. tropicalis and one reference strain, from the American Type Culture Collection (ATCC 40042). Biofilm formation were carried out on 96-well microplates containing a cellular suspension of 1x105 cells/mL and incubated for 24 h at 37°C. Pre-formed C. tropicalis biofilms were treated with propolis (ranging from 0.47 to 1.42 mg/ml), during 24 h at 37°C and its effect assessed through quantification of the number of colony forming unit (CFU). Results: It was evident that all C. tropicalis strains tested were able to form biofilm and that propolis was able to reduce around 40% and 50% of the pre-formed biofilm. Moreover, in general the propolis effect was similar among all the C. tropicalis clinical isolates strains Conclusions: These data are promising, since they open important perspectives regarding new antifungal agents, much more effective and safer than the currently available to treat and to prevent C. tropicalis infections

Re-evaluating the role of strongly charged sequences in amphipathic cell-penetrating peptides: A fluorescence study using Pep-1

Cell-penetrating peptides (CPPs) are able to translocate across biological membranes and deliver bioactive proteins. Cellular uptake and intracellular distribution of CPPs is commonly evaluated with fluorescent labels, which can alter peptide properties. The effect of carboxyfluorescein label in the Lys-rich domain of the amphipathic CPP pep-1, was evaluated and compared with non-labelled pep-1 in vitro and in vivo. A reduced membrane affinity and an endosomal-dependent translocation mechanism, at variance with non-labelled pep-1, were detected. Therefore, the charged domain is not a mere enabler of peptide adsorption but has a crucial role in the translocation pathway of non-labelled pep-1. (c) 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved

Gating modifier toxin interactions with ion channels and lipid bilayers: Is the trimolecular complex real?

Spider peptide toxins have attracted attention because of their ability to target voltage-gated ion channels, which are involved in several pathologies including chronic pain and some cardiovascular conditions. A class of these peptides acts by modulating the gating mechanism of voltage-gated ion channels and are thus called gating modifier toxins (GMTs). In addition to their interactions with voltage-gated ion channels, some GMTs have affinity for lipid bilayers. This review discusses the potential importance of the cell membrane on the mode of action of GMTs. We propose that peptide–membrane interactions can anchor GMTs at the cell surface, thereby increasing GMT concentration in the vicinity of the channel binding site. We also propose that modulating peptide–membrane interactions might be useful for increasing the therapeutic potential of spider toxins. Furthermore, we explore the advantages and limitations of the methodologies currently used to examine peptide–membrane interactions. Although GMT–lipid membrane binding does not appear to be a requirement for the activity of all GMTs, it is an important feature, and future studies with GMTs should consider the trimolecular peptide–lipid membrane–channel complex. This article is part of the Special Issue entitled ‘Venom-derived Peptides as Pharmacological Tools.