Peptide-based self-assembled monolayers (SAMs): what peptides can do for SAMs and vice versa.

Self-assembled monolayers (SAMs) represent highly ordered molecular materials with versatile biochemical features and multidisciplinary applications. Research on SAMs has made much progress since the early begginings of Au substrates and alkanethiols, and numerous examples of peptide-displaying SAMs can be found in the literature. Peptides, presenting increasing structural complexity, stimuli-responsiveness, and biological relevance, represent versatile functional components in SAMs-based platforms. This review examines the major findings and progress made on the use of peptide building blocks displayed as part of SAMs with specific functions, such as selective cell adhesion, migration and differentiation, biomolecular binding, advanced biosensing, molecular electronics, antimicrobial, osteointegrative and antifouling surfaces, among others. Peptide selection and design, functionalisation strategies, as well as structural and functional characteristics from selected examples are discussed. Additionally, advanced fabrication methods for dynamic peptide spatiotemporal presentation are presented, as well as a number of characterisation techniques. All together, these features and approaches enable the preparation and use of increasingly complex peptide-based SAMs to mimic and study biological processes, and provide convergent platforms for high throughput screening discovery and validation of promising therapeutics and technologies.

Enhancement of hMSC In Vitro Proliferation by Surface Immobilization of a Heparin-Binding Peptide.

The use of human Mesenchymal Stem Cells (hMSC) as therapeutic agents for advanced clinical therapies relies on their in vitro expansion. Over the last years, several efforts have been made to optimize hMSC culture protocols, namely by mimicking the cell physiological microenvironment, which strongly relies on signals provided by the extracellular matrix (ECM). ECM glycosaminoglycans, such as heparan-sulfate, sequester adhesive proteins and soluble growth factors at the cell membrane, orchestrating signaling pathways that control cell proliferation. Surfaces exposing the synthetic polypeptide poly(L-lysine, L-leucine) (pKL) have previously been shown to bind heparin from human plasma in a selective and concentration-dependent manner. To evaluate its effect on hMSC expansion, pKL was immobilized onto self-assembled monolayers (SAMs). The pKL-SAMs were able to bind heparin, fibronectin and other serum proteins, as demonstrated by quartz crystal microbalance with dissipation (QCM-D) studies. hMSC adhesion and proliferation were significantly increased in pKL-SAMs compared to controls, most probably related to increased heparin and fibronectin binding to pKL surfaces. This proof-of-concept study highlights the potential of pKL surfaces to improve hMSC in vitro expansion possible through selective heparin/serum protein binding at the cell-material interface.

Broad-Spectrum Anti-Adhesive Coating Based on an Extracellular Polymer from a Marine Cyanobacterium.

Medical device-associated infections are a major health threat, representing about half of all hospital-acquired infections. Current strategies to prevent this problem based on device coatings with antimicrobial compounds (antibiotics or antiseptics) have proven to be insufficient, often toxic, and even promoting bacterial resistance. Herein, we report the development of an infection-preventive coating (CyanoCoating) produced with an extracellular polymer released by the marine cyanobacterium Cyanothece sp. CCY 0110. CyanoCoating was prepared by spin-coating and its bacterial anti-adhesive efficiency was evaluated against relevant etiological agents (Staphylococcus aureus, S. epidermidis, Pseudomonas aeruginosa and Escherichia coli) and platelets, both in the presence or absence of human plasma proteins. CyanoCoating cytotoxicity was assessed using the L929 fibroblasts cell line. CyanoCoating exhibited a smooth topography, low thickness and high hydrophilic properties with mild negative charge. The non-cytotoxic CyanoCoating prevented adhesion of all the bacteria tested (≤80%) and platelets (<87%), without inducing platelet activation (even in the presence of plasma proteins). The significant reduction in protein adsorption (<77%) confirmed its anti-adhesive properties. The development of this anti-adhesive coating is an important step towards the establishment of a new technological platform capable of preventing medical device-associated infections, without inducing thrombus formation in blood-contacting applications.

Haplotype analysis of the germacrene A synthase gene and association with cynaropicrin content and biological activities in Cynara cardunculus.

Cynara cardunculus: L. represents a natural source of terpenic compounds, with the predominant molecule being cynaropicrin. Cynaropicrin is gaining interest since it has been correlated to anti-hyperlipidaemia, antispasmodic and cytotoxicity activity against leukocyte cancer cells. The objective of this work was to screen a collection of C. cardunculus, from different origins, for new allelic variants in germacrene A synthase (GAS) gene involved in the cynaropicrin biosynthesis and correlate them with improved cynaropicrin content and biological activities. Using high-resolution melting, nine haplotypes were identified. The putative impact of the identified allelic variants in GAS protein was evaluated by bioinformatic tools and polymorphisms that putatively lead to protein conformational changes were described. Additionally, cynaropicrin and main pentacyclic triterpenes contents, and antithrombin, antimicrobial and antiproliferative activities were also determined in C. cardunculus leaf lipophilic-derived extracts. In this work we identified allelic variants with putative impact on GAS protein, which are significantly associated with cynaropicrin content and antiproliferative activity. The results obtained suggest that the identified polymorphisms should be explored as putative genetic markers correlated with biological properties in Cynara cardunculus.

Lipophilic Fraction of Cultivated Bifurcaria bifurcata R. Ross: Detailed Composition and In Vitro Prospection of Current Challenging Bioactive Properties.

Macroalgae have been seen as an alternative source of molecules with promising bioactivities to use in the prevention and treatment of current lifestyle diseases. In this vein, the lipophilic fraction of short-term (three weeks) cultivated Bifurcaria bifurcata was characterized in detail by gas chromatography-mass spectrometry (GC-MS). B. bifurcata dichloromethane extract was composed mainly by diterpenes (1892.78 ± 133.97 mg kg-1 dry weight (DW)), followed by fatty acids, both saturated (550.35 ± 15.67 mg kg-1 DW) and unsaturated (397.06 ± 18.44 mg kg-1 DW). Considerable amounts of sterols, namely fucosterol (317.68 ± 26.11 mg kg-1 DW) were also found. In vitro tests demonstrated that the B. bifurcata lipophilic extract show antioxidant, anti-inflammatory and antibacterial activities (against both Gram-positive and Gram-negative bacteria), using low extract concentrations (in the order of µg mL-1). Enhancement of antibiotic activity of drug families of major clinical importance was observed by the use of B. bifurcata extract. This enhancement of antibiotic activity depends on the microbial strain and on the antibiotic. This work represents the first detailed phytochemical study of the lipophilic extract of B. bifurcata and is, therefore, an important contribution for the valorization of B. bifurcata macroalgae, with promising applications in functional foods, nutraceutical, cosmetic and biomedical fields.

Helicobacter pylori infection: A brief overview on alternative natural treatments to conventional therapy.

Helicobacter pylori is a human gastric pathogen considered as the etiologic agent of several gastric disorders, that may range from chronic gastritis to more severe outcomes, including gastric cancer. The current therapeutic scheme relies on the combination of several pharmacological substances, namely antibiotics and proton pump inhibitors. However, the cure rates obtained have been declining over the years, mostly due to bacterial resistance to antibiotics. In this context, the use of non-antibiotic substances is of the utmost importance regarding H. pylori eradication. In this review, we present different classes of compounds obtained from natural sources that have shown to present anti-H. pylori potential; we briefly highlight their possible use in the context of developing new therapeutic approaches.

Engineering resveratrol-loaded chitosan nanoparticles for potential use against Helicobacter pylori infection.

Helicobacter pylori (H. pylori) is a microorganism directly linked to severe clinical conditions affecting the stomach. The virulence factors and its ability to form biofilms increase resistance to conventional antibiotics, growing the need for new substances and strategies for the treatment of H. pylori infection. The trans-resveratrol (RESV), a bioactive polyphenol from natural sources, has a potential activity against this gastric pathogen. Here, Chitosan nanoparticles (NP) containing RESV (RESV-NP) were developed for H. pylori management. The RESV-NP were prepared using the ionic gelation method and characterized by Dynamic Light Scattering (DLS), Nanoparticle Tracking Analysis (NTA) and, Cryogenic Transmission Electron Microscopy (Cryo - TEM). The encapsulation efficiency (EE) and in vitro release rate of RESV were quantified using high-performance liquid chromatography (HPLC). RESV-NP performance against H. pylori was evaluated by the quantification of the minimum inhibitory/bactericidal concentrations (MIC/MBC), time to kill, alterations in H. pylori morphology in its planktonic form, effects against H. pylori biofilm and in an in vitro infection model. RESV-NP cytotoxicity was evaluated against AGS and MKN-74 cell lines and by hemolysis assay. Acute toxicity was tested using Galleria mellonella model assays. RESV-NP showed a spherical shape, size of 145.3 ± 24.7 nm, polydispersity index (PDI) of 0.28 ± 0.008, and zeta potential (ZP) of + 16.9 ± 1.81 mV in DLS, while particle concentration was 3.12 x 1011 NP/mL (NTA). RESV-NP EE was 72 %, with full release within the first 5 min. In microbiological assays, RESV-NP presented a MIC/MBC of 3.9 µg/mL, a time to kill of 24 h for complete eradication of H. pylori. At a concentration of 2xMIC (7.8 µg/mL), RESV-NP completely eradicated the H. pylori biofilm, and in an in vitro infection model, RESV-NP (4xMIC - 15.6 µg/mL) showed a significant decrease in bacterial load (1 Log10CFU/mL) when compared to the H. pylori J99 control. In addition, they did not demonstrate a toxic character at MIC concentration for both cell lines. The use of the RESV-NP with mucoadhesion profile is an interesting strategy for oral administration of substances targeting gastric disorders, linked to H. pylori infections.

One-Pot Microfluidics to Engineer Chitosan Nanoparticles Conjugated with Antimicrobial Peptides Using "Photoclick" Chemistry: Validation Using the Gastric Bacterium Helicobacter pylori.

Surface bioconjugation of antimicrobial peptides (AMP) onto nanoparticles (AMP-NP) is a complex, multistep, and time-consuming task. Herein, a microfluidic system for the one-pot production of AMP-NP was developed. Norbornene-modified chitosan was used for NP production (NorChit-NP), and thiolated-AMP was grafted on their surface via thiol-norbornene "photoclick" chemistry over exposure of two parallel UV LEDs. The MSI-78A was the AMP selected due to its high activity against a high priority (level 2) antibiotic-resistant gastric pathogen: Helicobacter pylori (H. pylori). AMP-NP (113 ± 43 nm; zeta potential 14.3 ± 7 mV) were stable in gastric settings without a cross-linker (up to 5 days in pH 1.2) and bactericidal against two highly pathogenic H. pylori strains (1011 NP/mL with 96 µg/mL MSI-78A). Eradication was faster for H. pylori 26695 (30 min) than for H. pylori J99 (24 h), which was explained by the lower minimum bactericidal concentration of soluble MSI-78A for H. pylori 26695 (32 µg/mL) than for H. pylori J99 (128 µg/mL). AMP-NP was bactericidal by inducing H. pylori cell membrane alterations, intracellular reorganization, generation of extracellular vesicles, and leakage of cytoplasmic contents (transmission electron microscopy). Moreover, NP were not cytotoxic against two gastric cell lines (AGS and MKN74, ATCC) at bactericidal concentrations. Overall, the designed microfluidic setup is a greener, simpler, and faster approach than the conventional methods to obtain AMP-NP. This technology can be further explored for the bioconjugation of other thiolated-compounds.

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.

Helicobacter pylori biofilms are disrupted by nanostructured lipid carriers: A path to eradication?

Bacterial biofilms account for 80% of all chronic infections, with cells being up to 1000 times more resistant to antibiotics than their planktonic counterparts. The recently discovered ability of Helicobacter pylori to form biofilms once again highlights why this bacterium is one of the most successful human pathogens. The current treatments failure rate reaches 40% of cases, emphasizing that new therapeutic options are a pressing need. Nanostructured lipid carriers (NLC), with and without docosahexaenoic acid (DHA), were very effective against H. pylori planktonic cells but their effect on H. pylori biofilms was unknown. Here, DHA-loaded NLC (DHA-NLC) and NLC without any drug (blank NLC) were tested on an optimized H. pylori in vitro floating mature biofilm model. DHA-NLC and blank NLC reduced the total biofilm biomass and had a bactericidal effect against both biofilm and planktonic bacteria in all the concentrations tested (0.125-2 mg/mL). DHA-NLC achieved biofilm biomass reduction in a concentration ~ 8 times lower than blank NLC (0.125 vs 1 mg/mL, respectively). Both NLC were bactericidal at the lowest concentration tested (0.125 mg/mL) although with different efficiency, i.e. a decrease of ∼6 log10 for DHA-NLC and ∼5 log10 for blank NLC. In addition, the equivalent amount of free DHA (3.1 µM) only reduced bacterial viability in ∼2 log10, demonstrating the synergistic effect of DHA and NLC in the treatment of H. pylori biofilms. Nevertheless, although viable bacteria were not detected by colony forming unit (CFU) counting after treatment with both NLC, confocal microscopy imaging highlighted that some H. pylori cells remained alive. In addition, scanning electron microscopy (SEM) analysis confirmed an increase in bacteria with a coccoid morphology after treatment, suggesting a transition to a viable but non-culturable (VBNC) state. Altogether, it is herein established that NLC, even without any drug, are promising for the management of H. pylori bacteria organized in biofilms, opening new perspectives for the eradication of this gastric pathogen.

Validation of Selective Capture of Fimbriated Uropathogenic Escherichia coli by a Label-free Engineering Detection System Using Mannosylated Surfaces.

Label-free detection of pathogens is of major concern to the microbiologist community. Most procedures require several steps and amplification techniques. Carbohydrates are well-established receptors for host-pathogen interactions, which can be amplified using glycodendritic architectures on the basis of multivalent binding interactions. Given that uropathogenic Escherichia coli bacterial FimH is based on such mannopyranoside-binding interactions, we demonstrate herein that synthetic monomeric and trimeric thiolated α-d-mannosides can be effectively bound to gold substrate-functionalized self-assembled monolayers (SAMs) preactivated with maleimide functionalities. Mannosides grafted onto SAMs were followed using Quartz Crystal Microbalance with Dissipation (QCM-D). Binding recognition efficiency was first evaluated using the plant lectin from Canavalia ensiformis (ConA) also using QCM-D. We showed a direct correlation between the amount of mannoside bound and the lectin attachment. Even though there was less trimer bound (nM/cm2) to the surface, we observed a 7-fold higher amount of lectin anchoring, thus further demonstrating the value of the multivalent interactions. We next examined the relative fimbriated E. coli selective adhesion/capture to either the monomeric or the trimeric mannoside bound to the surface. Our results established the successful engineering of the surfaces to show E. coli adhesion via specific mannopyranoside binding but unexpectedly, the monomeric derivative was more efficient than the trimeric analog, which could be explained by steric hindrance. This approach strongly suggests that it could be broadly applicable to other Gram-negative bacteria sharing analogous carbohydrate-dependent binding interactions.

How Charge, Size and Protein Corona Modulate the Specific Activity of Nanostructured Lipid Carriers (NLC) against Helicobacter pylori.

The major risk factor associated with the development of gastric cancer is chronic infection with Helicobacter pylori. The available treatments, based on a cocktail of antibiotics, fail in up to 40% of patients and disrupt their gut microbiota. The potential of blank nanostructured lipid carriers (NLC) for H. pylori eradication was previously demonstrated by us. However, the effect of NLC charge, size and protein corona on H. pylori-specific bactericidal activity herein studied was unknown at that time. All developed NLC formulations proved bactericidal against H. pylori. Although cationic NLC had 10-fold higher bactericidal activity than anionic NLC, they lacked specificity, since Lactobacillus acidophilus was also affected. Anionic NLC achieved complete clearance in both H. pylori morphologies (rod- and coccoid-shape) by inducing alterations in bacteria membranes and the cytoplasm, as visualized by transmission electron microscopy (TEM). The presence of an NLC protein corona, composed of 93% albumin, was confirmed by mass spectrometry. This protein corona delayed the bactericidal activity of anionic NLC against H. pylori and hindered NLC activity against Escherichia coli. Overall, these results sustain the use of NLC as a promising antibiotic-free strategy targeting H. pylori.

Antimicrobial peptide-based materials: opportunities and challenges.

The multifunctional properties of antimicrobial peptides (AMPs) make them attractive candidates for the treatment of various diseases. AMPs are considered as alternatives to antibiotics due to the increasing number of multidrug-resistant (MDR) bacteria. However, bare AMPs have limited therapeutic potentials due to a low residence time in the blood circulatory system and susceptibility to proteases and an alkaline wound environment. These limitations are the major hurdles for AMPs to succeed as commercial drugs. In contrast, AMP-based materials, for instance, NPs, hydrogels, electrospun fibres, dressings and implants, could overcome these challenges and provide therapeutic efficacies to the conjugated AMPs superior to those of bare AMPs in different disease models. In this review, we discuss the preparation of different compositions of AMP-based materials and their therapeutic potential for the treatment of microbial infections in the brain, eyes, mouth, skin, lungs, and gastrointestinal and urinary tracts. Apart from antimicrobial potential, the applications of AMP-based materials in the regeneration of skin/bone, prevention of implant-associated infections, detection/imaging of bacteria, cancer therapy and gene delivery are discussed in this review. Lastly, we discuss different challenges that hinder the commercialization of AMP-based materials. Overall, this review provides a comprehensive account of the current progress and prospects of AMP-based materials for clinical applications.

Grafting MSI-78A onto chitosan microspheres enhances its antimicrobial activity.

MSI-78A (Pexiganan A) is one of the few antimicrobial peptides (AMPs) able to kill Helicobacter pylori, a pathogenic bacterium that colonizes the gastric mucosa of half of the world's population. Antibiotics fail in 20-40% of H. pylori-infected patients, reinforcing the need for alternative treatments. Herein, a bioengineered approach was developed. MSI-78A with a C-terminal cysteine was grafted onto chitosan microspheres (AMP-ChMic) by thiol-maleimide (Michael-addition) chemistry using a long heterobifunctional spacer (NHS-PEG113-MAL). Microspheres with ∼4 µm diameter (near H. pylori length) and stable at low pH were produced by spray drying using a chitosan solution with an incomplete genipin crosslinking. A 3 × 10-5 µg AMP/microsphere grafting was estimated/confirmed by UV/Vis and FTIR spectroscopies. AMP-ChMic were bactericidal against H. pylori J99 (highly pathogenic human strain) at lower concentrations than the free peptide (∼277 µg grafted MSI-78A-SH/mL vs 512 µg free MSI-78A-SH/mL), even after pre-incubation in simulated gastric conditions with pepsin. AMP-ChMic killed H. pylori by membrane destabilization and cytoplasm release in a ratio of ∼10 bacteria/microsphere. This can be attributed to H. pylori attraction to chitosan, facilitating the interaction of grafted AMP with bacterium membrane. Overall, it was demonstrated that the peptide-microsphere conjugation chemistry did not compromise the MSI-78A antimicrobial activity, instead it boosted its bactericidal performance against H. pylori. STATEMENT OF SIGNIFICANCE: Half of the world's population is infected with Helicobacter pylori, a gastric bacterium that is responsible for 90% of non-cardia gastric cancers. Therefore, H. pylori eradication is now advocated in all infected individuals. However, available antibiotic therapies fail in up to 40% patients. Antimicrobial peptides (AMPs) are appealing alternatives to antibiotics, but their high susceptibility in vivo limits their clinical translation. AMP immobilization onto biomaterials surface will overcome this problem. Herein, we demonstrate that immobilization of MSI-78A (one of the few AMPs with activity against H. pylori) onto chitosan microspheres (AMP-ChMic) enhances its anti-H. pylori activity even at acidic pH (gastric settings). These results highlight the strong potential of AMP-ChMic as an antibiotic alternative for H. pylori eradication.

The biophysics of bacterial infections: Adhesion events in the light of force spectroscopy.

Bacterial infections are the most eminent public health challenge of the 21st century. The primary step leading to infection is bacterial adhesion to the surface of host cells or medical devices, which is mediated by a multitude of molecular interactions. At the interface of life sciences and physics, last years advances in atomic force microscopy (AFM)-based force spectroscopy techniques have made possible to measure the forces driving bacteria-cell and bacteria-materials interactions on a single molecule/cell basis (single molecule/cell force spectroscopy). Among the bacteria-(bio)materials surface interactions, the life-threatening infections associated to medical devices involving Staphylococcus aureus and Escherichia coli are the most eminent. On the other hand, Pseudomonas aeruginosa binding to the pulmonary and urinary tract or the Helicobacter pylori binding to the gastric mucosa, are classical examples of bacteria-host cell interactions that end in serious infections. As we approach the end of the antibiotic era, acquisition of a deeper knowledge of the fundamental forces involved in bacteria - host cells/(bio)materials surface adhesion is crucial for the identification of new ligand-binding events and its assessment as novel targets for alternative anti-infective therapies. This article aims to highlight the potential of AFM-based force spectroscopy for new targeted therapies development against bacterial infections in which adhesion plays a pivotal role and does not aim to be an extensive overview on the AFM technical capabilities and theory of single molecule force spectroscopy.

Effect of surface chemistry on hMSC growth under xeno-free conditions.

Human mesenchymal stem/stromal cells (hMSC) are promising therapeutic agents for regenerative medicine. However, therapeutic doses necessary for clinical application require in vitro expansion, ideally under Xeno-Free (XF) conditions to avoid the use of foetal bovine serum (FBS). We previously reported that hMSCs could be expanded using a pharmaceutical-grade human plasma-derived supplement for cell culture (SCC, Plastem®) combined with bFGF and TGFß1, on fibronectin (Fn)-coated surfaces. hMSCs expansion may also be affected by the chemistry of the culture surface, which modulates protein adsorption at the cell-material interface and, consequently, cell behavior. This work aimed to evaluate the effect of surface chemistry on hMSCs behavior in SCC-based XF media. For that, self-assembled monolayers (SAMs) with hydrophobic (-CH3) and hydrophilic (neutral -OH, positively charged -NH3+ and negatively charged -COO-) groups were used as model surfaces. Under XF conditions, Fn coating showed to be necessary to improve hMSC adhesion (4 h) onto all surfaces, except for OH-SAMs, probably due to a low protein adsorption capacity characteristic of this surface. In terms of cell metabolic activity (5 days) on Fn-coated surfaces, an increase over time under XF conditions was observed in all SAMs except in CH3-SAMs, which can be attributed to strong and irreversible protein adsorption characteristic of hydrophobic surfaces. This trend was also observed under FBS conditions. Nevertheless, none of the surfaces improved hMSC metabolic activity, as compared with tissue-cultured surfaces. Overall, this work describes the role of surface chemistry in XF hMSC expansion.

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.

An affinity-based approach to engineer laminin-presenting cell instructive microenvironments.

Laminin immobilization into diverse biological and synthetic matrices has been explored to replicate the microenvironment of stem cell niches and gain insight into the role of extracellular matrix (ECM) on stem cell behavior. However, the site-specific immobilization of this heterotrimeric glycoprotein and, consequently, control over its orientation and bioactivity has been a challenge that has limited many of the explored strategies to date. In this work, we established an affinity-based approach that takes advantage of the native high affinity interaction between laminin and the human N-terminal agrin (hNtA) domain. This interaction is expected to promote the site-selective immobilization of laminin to a specific substrate, while preserving the exposure of its key bioactive epitopes. Recombinant hNtA (rhNtA) domain was produced with high purity (>90%) and successfully conjugated at its N-terminal with a thiol-terminated poly(ethylene glycol) (PEG) without affecting its affinity to laminin. Self-assembled monolayers (SAMs) of mono-PEGylated rhNtA on gold (mPEG rhNtA-SAMs) were then prepared to evaluate the effectiveness of this strategy. The site-specific immobilization of laminin onto mPEG rhNtA-SAMs was shown to better preserve protein bioactivity in comparison to laminin immobilized on SAMs of thiol-PEG-succinimidyl glutaramide (HS-PEG-SGA), used for the non-selective covalent immobilization of laminin, as evidenced by its enhanced ability to efficiently self-polymerize and mediate cell adhesion and spreading of human neural stem cells. These results highlight the potential of this novel strategy to be used as an alternative to the conventional immobilization approaches in a wide range of applications, including engineered coatings for neuroelectrodes and cell culture, as well as biofunctionalization of 3D matrices.

N-acetylcysteine-functionalized coating avoids bacterial adhesion and biofilm formation.

N-acetyl cysteine (NAC) is an FDA-approved drug clinically applied on a broad range of pathologies. Further research has been conducted with this drug to benefit from its antimicrobial activity potential. However, NAC has a very short half-life and therefore strategies that accomplish high local concentrations would be beneficial. In this study, covalent immobilization of NAC was performed, in order to obtain long-lasting high local concentration of the drug onto a chitosan(Ch)-derived implant-related coating. For the development of NAC-functionalized Ch films, water-based carbodiimide chemistry was applied to avoid the use of toxic organic solvents. Here we report the optimization steps performed to immobilize NAC onto the surface of pre-prepared Ch coatings, to ensure full exposure of NAC. Surface characterization using ellipsometry, water contact angle measurements and X-ray photoelectron spectroscopy (XPS), demonstrated the success of NAC immobilization at 4 mg/mL. Quartz crystal microbalance with dissipation (QCM-D) demonstrated that surface immobilized NAC decreases protein adsorption to Ch coatings. Biological studies confirmed that immobilized NAC4 avoids methicillin-resistant Staphylococcus aureus adhesion to Ch coating, impairing biofilm formation, without inducing cytotoxic effects. This is particularly interesting towards further developments as a prevention coating.

Enfermagem transcultural no cuidado de Famílias ciganas: olhar do enfermeiro de família / Transcultural nursing in the care of Gypsy families: the family nurse's perspective

O presente relatório enquadra o Curso de Mestrado de Saúde Familiar conducente à atribuição do título de Enfermeiro Especialista em Saúde Comunitária na área de Saúde Familiar. A cultura é um fenómeno universal, com impacto na sociedade moderna, que impõe reajustes nomeadamente ao nível dos cuidados de enfermagem às famílias, considerando-as unidades sistémicas e em desenvolvimento. As famílias de cultura cigana são consideradas uma minoria étnica, com padrões culturais distintos; nem sempre compreendidas e aceites pela sociedade considerada maioritária. Os cuidados de enfermagem culturais às famílias ciganas assumem expressões e significados, que podem ser significativos na relação terapêutica enfermeiro-utente, fundamentada no respeito pela diferença, valores e crenças. Os enfermeiros de família têm a perceção de que precisam melhorar os cuidados e identificam determinantes que influenciam e promovem os cuidados culturalmente competentes. A Revisão Integrativa (II Parte) constitui uma estratégia de pesquisa subjacente à questão de investigação: quais os determinantes aos cuidados culturalmente competentes a desenvolver pelo enfermeiro de família, junto de famílias de cultura diversa, em especial cigana? Cujo objetivo configura da seguinte forma: identificar os determinantes da prática de enfermagem transcultural pelos enfermeiros de família, junto de famílias de cultura diversa, em especial cigana. A Revisão integra quatro estudos, publicados entre 2016 e 2022. Os resultados revelam que existem determinantes que condicionam os cuidados de enfermagem culturalmente competentes, uns que os condicionam outros que os promovem, conduzindo à discussão com base no Modelo de Sunrise de Madeleine Leininger. O Modelo de Sunrise relaciona-se com os determinantes promotores dos cuidados culturais e permite descobrir, conhecer e explicar a interdependência dos fenómenos culturais com os cuidados de enfermagem, conferindo satisfação das famílias, numa base de preservação, negociação e/ou RE padronização.

This report frames the Master's Degree in Family Health leading to the award of the title of Specialist Nurse in Community Health in the area of ​​Family Health. Culture is a universal phenomenon, with an impact on modern society, which imposes readjustments particularly in terms of nursing care for families, considering them to be systemic and developing units. Gypsy families are considered an ethnic minority, with distinct cultural patterns; not always understood and accepted by society considered the majority. Cultural nursing care for gypsy families takes on expressions and meanings, which can be significant in the nurse-user therapeutic relationship, based on respect for difference, values ​​and beliefs. Family nurses realize that they need to improve care and identify determinants that influence and promote culturally competent care. The Integrative Review (II Part) constitutes a research strategy underlying the research question: what are the determinants of culturally competent care to be developed by family nurses, with families from different cultures, especially Roma? The objective of which is as follows: to identify the determinants of transcultural nursing practice by family nurses, with families from different cultures, especially gypsies. The Review includes four studies, published between 2016 and 2022. The results reveal that there are determinants that condition culturally competent nursing care, some that condition it and others that promote it, leading to the discussion based on Madeleine Leininger's Sunrise Model. The Sunrise Model relates to the determinants that promote cultural care and allows discovering, knowing and explaining the interdependence of cultural phenomena with nursing care, providing family satisfaction, on a basis of preservation, negotiation and/or RE standardization.