Abstract
Objectives
To investigate the mechanism of action at the molecular level of pepR, a multifunctional peptide derived from the Dengue virus capsid protein, against Staphylococcus aureus ...biofilms.
Methods
Biofilm mass, metabolic activity and viability were quantified using conventional microbiology techniques, while fluorescence imaging methods, including a real-time calcein release assay, were employed to investigate the kinetics of pepR activity at different biofilm depths.
Results
Using flow cytometry-based assays, we showed that pepR is able to prevent staphylococcal biofilm formation due to a fast killing of planktonic bacteria, which in turn resulted from a peptide-induced increase in the permeability of the bacterial membranes. The activity of pepR against pre-formed biofilms was evaluated through the application of a quantitative live/dead confocal laser scanning microscopy (CLSM) assay. The results show that the bactericidal activity of pepR on pre-formed biofilms is dose and depth dependent. A CLSM-based assay of calcein release from biofilm-embedded bacteria was further developed to indirectly assess the diffusion and membrane permeabilization properties of pepR throughout the biofilm. A slower diffusion and delayed activity of the peptide at deeper layers of the biofilm were quantified.
Conclusions
Overall, our results show that the activity of pepR on pre-formed biofilms is controlled by its diffusion along the biofilm layers, an effect that can be counteracted by an additional administration of peptide. Our study sheds new light on the antibiofilm mechanism of action of antimicrobial peptides, particularly the importance of their diffusion properties through the biofilm matrix on their activity.
Display omitted
•Zn-based materials functionalization with Cu, Fe and Mn oxides was achieved by electrodeposition.•CuOx functionalization resulted in fibroblasts cytotoxicity and vascularization ...inhibition.•MnOx, and in less extent FeOx, revelled good fibroblasts viability and hemocompatibility.•Antagonistic behaviours of these materials can be used to target distinct medical needs.
Zinc coated with nanostructured ZnO flowers has received increasing attention as a versatile biomaterial for medical applications. Whatsoever, the potential of these materials to meet specific medical requirements must be explored. Despite in its infancy, surface functionalization is the key strategy to achieve this goal. The functionalization, successfully achieved with cooper (Cu), iron (Fe) or manganese (Mn) oxides (Ox), was highly dependent on the presence of the flowered structures, with the deep physicochemical characterization of these new surfaces revealing specific metal oxide distributions. The functionalization with these metal oxides resulted in distinct biological and in vitro behaviours. The biological response, assessed by fibroblast viability, hemocompatibility, and chick chorioallantoic membrane (CAM), further supported by the in vitro degradation studies, evaluated by immersion and electrochemical techniques, revealed that the deleterious role of CuOx functionalization brought potential for anti-cancer applications; with an antagonist behaviour, the functionalization with MnOx, and in a less extent with FeOx, can be used to favour wound healing in traumatic processes. Despite the possible correlation between biocompatibility and hydroxyapatite precipitation, no correlation could be drawn with the corrosion activity of these surfaces. Overall, the minor addition of relevant physiological as Cu, Fe or Mn oxides resulted in antagonist in vitro responses that can be used as expedite strategies to modulate the behaviour of Zn-based materials, contributing in this way for the design of anti-cancer or wound healing therapies.
is an emerging fungal pathogen. Its increased prevalence is associated with its ability to rapidly develop antifungal drug resistance, particularly to azoles. In order to unravel new molecular ...mechanisms behind azole resistance, a transcriptomics analysis of the evolution of a
clinical isolate (isolate 044) from azole susceptibility to posaconazole resistance (21st day), clotrimazole resistance (31st day), and fluconazole and voriconazole resistance (45th day), induced by longstanding incubation with fluconazole, was carried out. All the evolved strains were found to accumulate lower concentrations of azole drugs than the parental strain, while the ergosterol concentration remained mostly constant. However, only the population displaying resistance to all azoles was found to have a gain-of-function mutation in the
gene, leading to the upregulation of genes encoding multidrug resistance transporters. Intermediate strains, exhibiting posaconazole/clotrimazole resistance and increased fluconazole/voriconazole MIC levels, were found to display alternative ways to resist azole drugs. Particularly, posaconazole/clotrimazole resistance after 31 days was correlated with increased expression of adhesin genes. This finding led us to identify the Epa3 adhesin as a new determinant of azole resistance. Besides being required for biofilm formation, Epa3 expression was found to decrease the intracellular accumulation of azole antifungal drugs. Altogether, this work provides a glimpse of the transcriptomics evolution of a
population toward multiazole resistance, highlighting the multifactorial nature of the acquisition of azole resistance and pointing out a new player in azole resistance.
Introduction:
Antisense oligonucleotides (ASOs) have been successfully utilized to silence gene expression for the treatment of many genetic human diseases, and particularly the locked nucleic acid ...(LNA) chemical modification is extensively used with this propose. However, LNA-modified ASOs have never been exploited for controlling virulence genes of
Candida
.
EFG1is an important determinant of virulence that is involved in the switch from yeast to filamentous forms in
C. albicans
. Thus, our main goal was to explore LNA antisense gapmers for controlling EFG1gene expression and to block
C. albicans
filamentation.
Methods:
A set of five LNA-modified gapmers were designed with different chemical modifications (phosphorothioate backbone (PS) and/or palmitoyl-2’-amino-LNA) and ASO length. The in vitro performance of the different ASOs was evaluatedon their ability to control EFG1 gene expression, by qRT-PCR, and to reduce
C. albicans
’ filamentation, through filaments’ enumeration by microscopy. The in vivo therapeutic potential of ASOs was assessed using a
G. mellonella
model of infection, through a survival assay.
Results:
In vitro results showed that all ASOs were able to reduce the levels of EFG1gene expression, consequently reducing the levels of
C. albicans
filamentation around 50%. Interestingly, in vivo tests showed that the LNA-modified gapmer with PS backbone and palmitoyl-2’-amino-LNA was more effective at preventing
G. mellonella
infections.
Conclusions:
Undeniably, this work promotes the development of a novel approach for the treatment of Candida infections based on the delivery of ASOs coupled with LNA chemical modification.
PDF
