Dhvar5 antimicrobial peptide (AMP) chemoselective covalent immobilization results on higher antiadherence effect than simple physical adsorption

Bacterial colonization and subsequent biofilm formation is still one of the major problems associated with medical devices. Antimicrobial peptides (AMP) immobilization onto biomaterials surface is a promising strategy to avoid bacterial colonization. However, a correct peptide orientation and exposure from the surface is essential to maintain AMP antimicrobial activity. This work aims to evaluate the effect of the immobilization on antibacterial activity of Dhvar5 (LLLFLLKKRKKRKY), an AMP with a head-to-tail amphipathicity. Dhvar5 was linked to thin chitosan coatings in i) a controlled orientation and exposure, testing covalent immobilization of its N- or C-terminus and using spacers with different lengths and flexibilities or in ii) a random orientation by physical adsorption. Chitosan coating was chosen due to its antimicrobial properties and readiness to be functionalized. Surface characterization demonstrated the chemoselective immobilization of the peptide with different spacers in a similar concentration (∼2 ng/cm2). Efficacy assays demonstrated that covalent immobilization of Dhvar5 exposing its cationic end, improves the chitosan coating antimicrobial effect by decreasing Methicillin-resistant Staphylococcus aureus (MRSA) colonization. This effect was enhanced when longer spacers were used independently of their flexibility. In opposite, immobilized Dhvar5 exposing its hydrophobic end has no effect on bacterial adhesion to chitosan, and when adsorbed in a random orientation even induces bacterial adhesion to chitosan coating.This work was financed by FEDER funds through the Programa Operacional Factores de Competitividade (COMPETE) and by Portuguese funds through FCT (Fundacao para a Ciencia e a Tecnologia) in the framework of the projects: PTDC/CTM/101484/2008; PEst-C/SAU/LA0002/2013; Pest-C/QUI/UI0081/2013. Fabiola Costa acknowledges FCT for the PhD grant SFRH/BD/72471/2010

Prevention of urinary catheter-associated infections by coating antimicrobial peptides from crowberry endophytes

Urinary catheters are extensively used in hospitals, being responsible for about 75% of hospital-acquired infections. In this work, a de novo designed antimicrobial peptide (AMP) Chain201D was studied in the context of urinary catheter-associated infections. Chain201D showed excellent antimicrobial activity against relevant ATCC strains and clinical isolates of bacteria and yeast and demonstrated high stability in a wide range of temperatures, pH and salt concentrations. Moreover, the bactericidal activity of Chain201D immobilized on a model surface was studied against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus), some of the most prevalent strains found in urinary catheter-associated infections. Chain201D was successfully tethered to ((1-mercapto-11-undecyl)-(tetra(ethylene glycol) (EG4)) terminated self-assembled monolayers (SAMs), (EG4-SAMs), activated by 1,1'-Carbonyldiimidazole (CDI) at different concentrations. Chain201D surfaces can bind and kill by contact a high percentage of adherent bacteria. These achievements are obtained without any peptide modification (for chemoselective conjugation) and without the use of a spacer. Moreover, increased amounts of immobilized AMP lead to higher numbers of adhered/dead bacteria, revealing a concentration-dependent behaviour and demonstrating that Chain201D has excellent potential for developing antimicrobial urinary catheters.AntINFECT: Bioengineered Advanced Therapies for Problematic Infected Wounds POCI-01-0145-FEDER-031781) and Institute for Research and Innovation in Health Sciences (UID/BIM/04293/2019). C. Monteiro would like to acknowledge national funding through FCT – Fundação para a Ciência e a Tecnologia, I.P., provided by the contract-program and according to numbers 4, 5 and 6 of art. 23 of Law no. 57/2016 of 29th August, as amended by Law no. 57/2017 of 19th July. Professor Paula Gomes from Faculdade de Ciências da Universidade do Porto (FC-UP), Porto Peptide Synthesis Facility (POP-UP) for CEM 7 peptide. The funding received from Tekes, Finland (3331/31/2014) is thankfully acknowledged

Norbornene-chitosan spray-dried microspheres for peptide conjugation using thiol-ene “photoclick” chemistry

This work was financed by Portuguese funds through FCT/MCTES (Fundação para a Ciência e a Tecnologia/Ministério da Ciência, Tecnologia e Inovação) in the framework of the projects 2022.06048.PTDC (i3S), UIDB/50006/2020 (LAQV-REQUIMTE), LA/P/0045/2020 (ALiCE) and UIDB/00511/2020 (LEPABE). P.A. (SFRH/BD/145471/2019) and D.F. (SFRH/ BD/146890/2019) doctoral grants, were financially supported by national (FCT/Norte 2020 Framework) and European Union (ESF – European Social Fund) funds. B.E. acknowledges FCT for the contract based on the “Lei do Emprego Científico” (DL 57/2016). Maria Cristina L. Martins also acknowledges FCT (LA/P/0070/2020), project Bio2Skin Advanced (2021-24):NORTE-01-0247-FEDER-047225; and MOBILIsE Project, which has received funding from the European Union’s Horizon 2020 research and innovation program under grant agreement no. 951723.The action of bioactive peptides, such as antimicrobial peptides (AMP), in the human body is often compromised by limited residence time and stability in the target site. Bioconjugation of peptides to biomaterial surfaces is one of the strategies that may overcome these limitations. Herein, norbornene-chitosan (NorChit) microspheres were engineered to react with thiolated peptides by thiolene “photoclick” chemistry. NorChit microspheres were produced by spray drying and crosslinked with dithiothreitol (DTT) to prevent their solubilization. Microspheres with a diameter of 5 ± 2 µm showed round and smooth morphology with pockets over the surface that could be related with hydrophobic interactions between internal norbornene groups. Thiol-ene bioconjugation carried out using a fluorescent model peptide, showed a yield of 45%, whereas using the peptide but without UV exposure indicated a maximum of peptide adsorption of 30%. Altogether, NorChit microspheres show the potential for carrying bioactive peptides, which may open avenues for AMP activity onto harsh environments in the bod

Surface Grafted MSI-78A Antimicrobial Peptide has High Potential for Gastric Infection Management

As we approach the end of the antibiotic era, newer therapeutic options, such as antimicrobial peptides (AMPs), are in urgent demand. AMP surface grafting onto biomaterials has been described as a good strategy to overcome problems associated with their in vivo stability. Helicobacter pylori is among the bacteria that pose greatest threat to human health, being MSI-78A one of the few bactericidal AMPs against this bacterium. Here, we report that MSI-78A grafted onto model surfaces (Self-Assembled Monolayers –SAMs), in a concentration of 30.3 ± 1.2 ng/cm2 determined by quartz crystal microbalance with dissipation (QCM-D), was able to kill, by contact, 98% of planktonic H. pylori in only 2 h. This fact was not verified against the control bacteria (Staphylococcus epidermidis), although the minimal inhibitory concentration (MIC) of MSI-78A in solution is much lower for S. epidermidis (2 µg/mL) than for H. pylori (64 µg/mL). Our results also demonstrated that, in opposite to other bacteria, H. pylori cells were attracted to ethylene glycol terminated (antiadhesive) surfaces, which can explain the high bactericidal potential of grafted MSI-78A. This proof of concept study establishes the foundations for development of MSI-78A grafted nanoparticles for gastric infection management within a targeted nanomedicine concept.This article is a result of the project NORTE-01-0145-FEDER-00012, (NORTE 2020) and FCT/MCTES-through the UID/BIM/04293/2019, PTDC/CTM-BIO/4043/2014, UID/QUI/50006/2013 (LAQV-REQUIMTE) projects. Claudia Monteiro acknowledges FCT for the SFRH/BPD/79439/2011 grant

Influence of Immobilization Strategies on the Antibacterial Properties of Antimicrobial Peptide-Chitosan Coatings

It is key to fight bacterial adhesion to prevent biofilm establishment on biomaterials. Surface immobilization of antimicrobial peptides (AMP) is a promising strategy to avoid bacterial colonization. This work aimed to investigate whether the direct surface immobilization of Dhvar5, an AMP with head-to-tail amphipathicity, would improve the antimicrobial activity of chitosan ultrathin coatings. The peptide was grafted by copper-catalyzed azide-alkyne cycloaddition (CuAAC) chemistry by either its C- or N- terminus to assess the influence of peptide orientation on surface properties and antimicrobial activity. These features were compared with those of coatings fabricated using previously described Dhvar5-chitosan conjugates (immobilized in bulk). The peptide was chemoselectively immobilized onto the coating by both termini. Moreover, the covalent immobilization of Dhvar5 by either terminus enhanced the antimicrobial effect of the chitosan coating by decreasing colonization by both Gram-positive (Staphylococcus aureus, Staphylococcus epidermidis) and Gram-negative (Escherichia coli, Pseudomonas aeruginosa) bacteria. Relevantly, the antimicrobial performance of the surface on Gram-positive bacteria depended on how Dhvar5-chitosan coatings were produced. An antiadhesive effect was observed when the peptide was grafted onto prefabricated chitosan coatings (film), and a bactericidal effect was exhibited when coatings were prepared from Dhvar5-chitosan conjugates (bulk). This antiadhesive effect was not due to changes in surface wettability or protein adsorption but rather depended on variations in peptide concentration, exposure, and surface roughness. Results reported in this study show that the antibacterial potency and effect of immobilized AMP vary greatly with the immobilization procedure. Overall, independently of the fabrication protocol and mechanism of action, Dhvar5-chitosan coatings are a promising strategy for the development of antimicrobial medical devices, either as an antiadhesive or contact-killing surface.This work was financed by the FCT- Fundação para a Ciência e a Tecnologia through projects POCI-01-0145-FEDER-031781 (AntINFECT), UIB/50006/2020 (LAQV-REQUIMTE) and FEDER—Fundo Europeu de Desenvolvimento Regional through NORTE 2020—Programa Operacional Regional do Norte-Bio2Skin Advanced (NORTE-01-0247-FEDER-047225). M Barbosa (SFRH/BD/108966/2015) and Pedro Alves (SFRH/BD/145471/2019) Ph.D. grants were financially supported by national (FCT/Norte 2020 Framework) and European Union funds (ESF—European Social Fund). Paula Parreira (CEECIND/01210/2018) and Maria Cristina L. Martins (LA/P/0070/2020) also thank FCT for funding. Maria Cristina L. Martins also acknowledges the MOBILIsE Project, which has received funding from the European Union’s Horizon 2020 research and innovation program under grant agreement no. 951723

Climate services in Brazil: Past, present, and future perspectives

From the devastating effects of the 1877–1879 Great Drought in the Northeast region to the creation of the Center for Weather Forecast and Climate Studies (CPTEC) at the National Institute for Space Research (INPE) in the early 1990 s, Brazil went from a total absence of meteorological expertise to becoming a member of a select group of nations with the infrastructure and technical expertise to build and run a global general circulation model. This article reviews the most critical moments in the development of climate services in Brazil, addressing the evolution of its infrastructure for observation, monitoring, modeling, and prediction, the still incipient efforts in systematically understanding users’ perspectives and needs, and the work required to incorporate the usable science and co-production paradigms into the main centers of production of climate information. Advances and challenges are analyzed, and actions for strengthening the climate services framework are proposed