Physicochemical cues are not potent regulators of human dermal fibroblast trans-differentiation.

Due to their inherent plasticity, dermal fibroblasts hold great promise in regenerative medicine. Although biological signals have been well-established as potent regulators of dermal fibroblast function, it is still unclear whether physiochemical cues can induce dermal fibroblast trans-differentiation. Herein, we evaluated the combined effect of surface topography, substrate rigidity, collagen type I coating and macromolecular crowding in human dermal fibroblast cultures. Our data indicate that tissue culture plastic and collagen type I coating increased cell proliferation and metabolic activity. None of the assessed in vitro microenvironment modulators affected cell viability. Anisotropic surface topography induced bidirectional cell morphology, especially on more rigid (1,000 kPa and 130 kPa) substrates. Macromolecular crowding increased various collagen types, but not fibronectin, deposition. Macromolecular crowding induced globular extracellular matrix deposition, independently of the properties of the substrate. At day 14 (longest time point assessed), macromolecular crowding downregulated tenascin C (in 9 out of the 14 groups), aggrecan (in 13 out of the 14 groups), osteonectin (in 13 out of the 14 groups), and collagen type I (in all groups). Overall, our data suggest that physicochemical cues (such surface topography, substrate rigidity, collagen coating and macromolecular crowding) are not as potent as biological signals in inducing dermal fibroblast trans-differentiation.

Water-Soluble Saccharina latissima Polysaccharides and Relation of Their Structural Characteristics with In Vitro Immunostimulatory and Hypocholesterolemic Activities.

Brown macroalgae are an important source of polysaccharides, mainly fucose-containing sulphated polysaccharides (FCSPs), associated with several biological activities. However, the structural diversity and structure-function relationships for their bioactivities are still undisclosed. Thus, the aim of this work was to characterize the chemical structure of water-soluble Saccharina latissima polysaccharides and evaluate their immunostimulatory and hypocholesterolemic activities, helping to pinpoint a structure-activity relationship. Alginate, laminarans (F1, neutral glucose-rich polysaccharides), and two fractions (F2 and F3) of FCSPs (negatively charged) were studied. Whereas F2 is rich in uronic acids (45 mol%) and fucose (29 mol%), F3 is rich in fucose (59 mol%) and galactose (21 mol%). These two fractions of FCSPs showed immunostimulatory activity on B lymphocytes, which could be associated with the presence of sulphate groups. Only F2 exhibited a significant effect in reductions in in vitro cholesterol's bioaccessibility attributed to the sequestration of bile salts. Therefore, S. latissima FCSPs were shown to have potential as immunostimulatory and hypocholesterolemic functional ingredients, where their content in uronic acids and sulphation seem to be relevant for the bioactive and healthy properties.

Macromolecular crowding in animal component-free, xeno-free and foetal bovine serum media for human bone marrow mesenchymal stromal cell expansion and differentiation.

Background: Cell culture media containing undefined animal-derived components and prolonged in vitro culture periods in the absence of native extracellular matrix result in phenotypic drift of human bone marrow stromal cells (hBMSCs). Methods: Herein, we assessed whether animal component-free (ACF) or xeno-free (XF) media formulations maintain hBMSC phenotypic characteristics more effectively than foetal bovine serum (FBS)-based media. In addition, we assessed whether tissue-specific extracellular matrix, induced via macromolecular crowding (MMC) during expansion and/or differentiation, can more tightly control hBMSC fate. Results: Cells expanded in animal component-free media showed overall the highest phenotype maintenance, as judged by cluster of differentiation expression analysis. Contrary to FBS media, ACF and XF media increased cellularity over time in culture, as measured by total DNA concentration. While MMC with Ficoll™ increased collagen deposition of cells in FBS media, FBS media induced significantly lower collagen synthesis and/or deposition than the ACF and XF media. Cells expanded in FBS media showed higher adipogenic differentiation than ACF and XF media, which was augmented by MMC with Ficoll™ during expansion. Similarly, Ficoll™ crowding also increased chondrogenic differentiation. Of note, donor-to-donor variability was observed for collagen type I deposition and trilineage differentiation capacity of hBMSCs. Conclusion: Collectively, our data indicate that appropriate screening of donors, media and supplements, in this case MMC agent, should be conducted for the development of clinically relevant hBMSC medicines.

Alkali-Doped Nanopaper Membranes Applied as a Gate Dielectric in FETs and Logic Gates with an Enhanced Dynamic Response.

The market for flexible, hybrid, and printed electronic systems, which can appear in everything from sensors and wearables to displays and lighting, is still uncertain. What is clear is that these systems are appearing every day, enabling devices and systems that can, in the near future, be crumpled up and tucked in our pockets. Within this context, cellulose-based modified nanopapers were developed to serve both as a physical support and a gate dielectric layer in field-effect transistors (FETs) that are fully recyclable. It was found that the impregnation of those nanopapers with sodium (Na+) ions allows for low operating voltage FETs (<3 V), with mobility above 10 cm2 V-1 s-1, current modulation surpassing 105, and an improved dynamic response. Thus, it was possible to implement those transistors into simple circuits such as inverters, reaching a clear discrimination between logic states. Besides the overall improvement in electrical performance, these devices have shown to be an interesting alternative for reliable, sustainable, and flexible electronics, maintaining proper operation even under stress conditions.

The synergistic effect of physicochemical in vitro microenvironment modulators in human bone marrow stem cell cultures.

Modern bioengineering utilises biomimetic cell culture approaches to control cell fate during in vitro expansion. In this spirit, herein we assessed the influence of bidirectional surface topography, substrate rigidity, collagen type I coating and macromolecular crowding (MMC) in human bone marrow stem cell cultures. In the absence of MMC, surface topography was a strong modulator of cell morphology. MMC significantly increased extracellular matrix deposition, albeit in a globular manner, independently of the surface topography, substrate rigidity and collagen type I coating. Collagen type I coating significantly increased cell metabolic activity and none of the assessed parameters affected cell viability. At day 14, in the absence of MMC, none of the assessed genes was affected by surface topography, substrate rigidity and collagen type I coating, whilst in the presence of MMC, in general, collagen type I α1 chain, tenascin C, osteonectin, bone sialoprotein, aggrecan, cartilage oligomeric protein and runt-related transcription factor were downregulated. Interestingly, in the presence of the MMC, the 1000 kPa grooved substrate without collagen type I coating upregulated aggrecan, cartilage oligomeric protein, scleraxis homolog A, tenomodulin and thrombospondin 4, indicative of tenogenic differentiation. This study further supports the notion for multifactorial bioengineering to control cell fate in culture.

Adesão à preparação pré-operatória em cirurgia de ambulatório: Influência da consulta da enfermagem pré-operatória presencial / Adherence to preoperative preparation in outpatient surgery: Influence of face-to-face preoperative nursing consultation

Em 2019, a atividade cirúrgica programada cresceu, destacando-se a realizada em regime de ambulatório, que se traduziu em cerca de 400 mil intervenções cirúrgicas. Consequentemente, contribuiu para melhorar a gestão dos serviços de internamento hospitalar, e o regresso a casa dos utentes em segurança. O incumprimento das orientações pré-operatórias é uma problemática relevante, que compromete a segurança do utente e está associado a complicações no pós-operatório. Partindo da questão qual a influência da consulta de enfermagem presencial (CEP) pré-operatória no cumprimento das orientações para a preparação pré-operatória na pessoa em CA? Foi desenvolvido um estudo quantitativo, descritivo-correlacional. A colheita de dados foi realizada entre março e agosto de 2019, por questionário-entrevista a utentes de uma Unidade de Cirurgia Ambulatória do centro do país. Teve como objetivos comparar a informação percecionada pelos utentes sobre as recomendações pré-operatórias, consoante o contacto tenha sido por CEP ou consulta de enfermagem telefónica (CET); analisar a adesão às orientações pré-operatórias dos utentes com CEP e CET; comparar a satisfação dos utentes com a informação recebida na CEP e na CET; analisar a relação entre a adesão às orientações pré-operatórias e variáveis sociodemográficas dos utentes e, analisar a relação entre a adesão às orientações pré-operatórias e a satisfação com a informação recebida, a presença de pessoa significativa na CEP e a entrega de informação escrita. Os resultados desta investigação permitiram concluir que os utentes com CEP cumprem mais as orientações pré-operatórias do que os com CET, embora globalmente os níveis de adesão fossem baixos. Permitiu identificar, entre as recomendações pré-operatórias, quais as que tiveram menor adesão dos participantes. Não se encontrou relação entre a adesão às orientações pré-operatórias e as hipóteses de investigação formuladas, embora aqui seja de salientar que o tamanho das amostras parcelares pode ter influenciado os resultados. Conclui-se que a CEP tem impacto no cumprimento das orientações, embora seja necessário melhorar as práticas de enfermagem, nomeadamente, com programas de ensinos mais estruturados e com mais entrega da informação por escrito; e, melhorar a capacitação dos enfermeiros em técnicas de comunicação e avaliação da informação fornecida.

The antimetastatic breast cancer activity of the viral protein-derived peptide vCPP2319 as revealed by cellular biomechanics.

The incidence of metastatic breast cancer (MBC) is increasing and the therapeutic arsenal available to fight it is insufficient. Brain metastases, in particular, represent a major challenge for chemotherapy as the impermeable nature of the blood-brain barrier (BBB) prevents most drugs from targeting cells in the brain. For their ability to transpose biological membranes and transport a broad spectrum of bioactive cargoes, cell-penetrating peptides (CPPs) have been hailed as ideal candidates to deliver drugs across biological barriers. A more ambitious approach is to have the CPP as a drug itself, capable of both killing cancer cells and interacting with the blood/brain interface, therefore blocking the onset of brain metastases. vCPP2319, a viral protein-derived CPP, has both properties as it: (a) is selective toward human breast cancer cells (MDA-MB-231) and increases cell stiffness compared to breast epithelial cells (MCF 10A) hindering the progression of metastases; and (b) adsorbs at the surface of human brain endothelial cells potentially counteracting metastatic cells from reaching the brain. Overall, the results reveal the selective anticancer activity of the peptide vCPP2319, which is also able to reside at the blood-brain interface, therefore counteracting brain penetration by metastatic cancer cells.

The nurse's professional identity on the Primary Health Care users perception / La identidad profesional de la enfermera en la percepción de usuarios de la Atención Primaria / A identidade profissional da enfermeira na percepção de usuários da Atenção Básica

ABSTRACT Objectives: to understanding the perception of Primary Health Care users about the professional identity of nurse. Methods: this is an exploratory, descriptive, cross-sectional and quantitative study, using the STROBE instrument. The sample included 94 users grouped according to the Family Health Strategy coverage. Results: the idealized identity of nurses dates back to the past of the profession. The subjects, in 81.9%, associate the female gender to the profession. 63.8% of them believed that the nurse is a subordinate, and from these, 90% believed they were subordinate to physicians. The analysis of the perception of the activities under responsibility of the nurse showed the predominance of assistance activities that are not exclusive to them. Conclusions: the findings indicate partial awareness about the identity and the role of nurses in Primary Health Care. The main deficits are related to the competences of this professional. The understanding of the role of the nurse was higher in the population under the coverage of the Family Health Strategy.

RESUMEN Objetivos: comprender la percepción de usuarios de la Atención Primaria sobre la identidad profesional de la enfermera. Métodos: estudio exploratorio, descriptivo, transversal y abordaje cuantitativo norteado por la STROBE. Muestreo consistió en 94 usuarios agrupados de acuerdo con la cobertura de Estrategia de Salud Familiar. Resultados: identidad de la enfermera idealizada por los participantes remonta al pasado de la profesión. En 81,9%, vincularon el género femenino a profesión. Del total, 63,8% acreditaban haber subordinación de la enfermera, de los cuales 90% entendían subordinación a médicos. Análisis de la percepción sobre actividades de responsabilidad de la enfermera reveló el predominio de actividades no privativas y de cuño asistencial. Conclusiones: hallados indican consciencia parcial sobre la identidad y papel de la enfermera en Atención Primaria. Principales déficits relacionaron a atribuciones de esa profesional. Apropiación del papel de la enfermera fue mayor en la población con cobertura por la Estrategia de Salud Familiar.

RESUMO Objetivos: compreender a percepção de usuários da Atenção Básica sobre a identidade profissional da enfermeira. Métodos: trata-se de um estudo exploratório, descritivo, transversal e de abordagem quantitativa norteado pela ferramenta STROBE. A amostra consistiu em 94 usuários agrupados de acordo com a cobertura de Estratégia Saúde da Família. Resultados: a identidade da enfermeira idealizada pelos participantes remonta ao passado da profissão. Os sujeitos, em 81,9%, vincularam o gênero feminino à profissão. Do total, 63,8% acreditavam haver subordinação da enfermeira, dos quais 90% entendiam subordinação aos médicos. A análise da percepção sobre atividades de responsabilidade da enfermeira revelou o predomínio de atividades assistenciais e não privativas. Conclusões: os achados indicam consciência parcial sobre a identidade e papel da enfermeira na Atenção Básica. Os principais déficits relacionaram às atribuições dessa profissional. A apropriação do papel da enfermeira foi maior na população com cobertura pela Estratégia Saúde da Família.

The nurse's professional identity on the Primary Health Care users perception.

OBJECTIVES: to understanding the perception of Primary Health Care users about the professional identity of nurse. METHODS: this is an exploratory, descriptive, cross-sectional and quantitative study, using the STROBE instrument. The sample included 94 users grouped according to the Family Health Strategy coverage. RESULTS: the idealized identity of nurses dates back to the past of the profession. The subjects, in 81.9%, associate the female gender to the profession. 63.8% of them believed that the nurse is a subordinate, and from these, 90% believed they were subordinate to physicians. The analysis of the perception of the activities under responsibility of the nurse showed the predominance of assistance activities that are not exclusive to them. Conclusions: the findings indicate partial awareness about the identity and the role of nurses in Primary Health Care. The main deficits are related to the competences of this professional. The understanding of the role of the nurse was higher in the population under the coverage of the Family Health Strategy.

Protonectin peptides target lipids, act at the interface and selectively kill metastatic breast cancer cells while preserving morphological integrity.

Despite the need for innovative compounds as antimicrobial and anticancer agents, natural sources of peptides remain underexplored. Protonectin (PTN), a cationic dodecapeptide of pharmacological interest, presents large hydrophobicity that is associated with the tendency to aggregate and supposedly influences bioactivity. A disaggregating role was assigned to PTN' N-terminal fragment (PTN1-6), which enhances the bioactivity of PTN in a 1:1 mixture (PTN/PTN1-6). Spectroscopic techniques and model membranes (phospholipid bilayers and SDS micelles) revealed that environment-dependent aggregation is reduced for PTN/PTN1-6, but cytotoxicity of PTNs on MDA-MB-231 breast cancer showed the same CC50 values around 16 µM and on MCF-10A epithelial breast cells 6 to 5-fold higher values, revealing a selective interaction. Since PTN1-6 lacks activity on breast cells, its presence should differently affect PTN activity, suggesting that aggregation could modulate activity depending on the membrane characteristics. Indeed, increased partitioning and lytic activity of PTN/PTN1-6 were found in model membranes independently of charge density, but affected by the curvature tendency. PTN and PTN/PTN1-6 do not alter morphology and roughness of cancer cells, indicating a superficial interaction with membranes and consistent with results obtained in NMR experiments. Our results indicate that aggregation of PTNs depends on the membrane characteristics and modulates the activity of the peptides.

Ionic Conductive Cellulose Mats by Solution Blow Spinning as Substrate and a Dielectric Interstrate Layer for Flexible Electronics.

Renewable cellulose substrates with submicron- and nanoscale structures have revived interest in paper electronics. However, the processes behind their production are still complex and time- and energy-consuming. Besides, the weak electrolytic properties of cellulose with submicron- and nanoscale structures have hindered its application in transistors and integrated circuits with low-voltage operation. Here, we report a simple, low-cost approach to produce flexible ionic conductive cellulose mats using solution blow spinning, which are used both as dielectric interstrate and substrate in low-voltage devices. The electrochemical properties of the cellulose mats are tuned through infiltration with alkali hydroxides (LiOH, NaOH, or KOH), enabling their application as dielectric and substrate in flexible, low-voltage, oxide-based field-effect transistors and pencil-drawn resistor-loaded inverters. The transistors exhibit good transistor performances under operation voltage below 2.5 V, and their electrical performance is strictly related to the type of alkali ionic specie incorporated. Devices fabricated on K+-infiltrated cellulose mats present the best characteristics, indicating pure capacitive charging of the semiconductor. The pencil-drawn load resistor inverter presents good dynamic performance. These findings may pave the way for a new generation of low-power, wearable electronics, enabling concepts such as the "Internet of Things".

Scalable Production of Human Mesenchymal Stromal Cell-Derived Extracellular Vesicles Under Serum-/Xeno-Free Conditions in a Microcarrier-Based Bioreactor Culture System.

Mesenchymal stromal cells (MSC) hold great promise for tissue engineering and cell-based therapies due to their multilineage differentiation potential and intrinsic immunomodulatory and trophic activities. Over the past years, increasing evidence has proposed extracellular vesicles (EVs) as mediators of many of the MSC-associated therapeutic features. EVs have emerged as mediators of intercellular communication, being associated with multiple physiological processes, but also in the pathogenesis of several diseases. EVs are derived from cell membranes, allowing high biocompatibility to target cells, while their small size makes them ideal candidates to cross biological barriers. Despite the promising potential of EVs for therapeutic applications, robust manufacturing processes that would increase the consistency and scalability of EV production are still lacking. In this work, EVs were produced by MSC isolated from different human tissue sources [bone marrow (BM), adipose tissue (AT), and umbilical cord matrix (UCM)]. A serum-/xeno-free microcarrier-based culture system was implemented in a Vertical-WheelTM bioreactor (VWBR), employing a human platelet lysate culture supplement (UltraGROTM-PURE), toward the scalable production of MSC-derived EVs (MSC-EVs). The morphology and structure of the manufactured EVs were assessed by atomic force microscopy, while EV protein markers were successfully identified in EVs by Western blot, and EV surface charge was maintained relatively constant (between -15.5 ± 1.6 mV and -19.4 ± 1.4 mV), as determined by zeta potential measurements. When compared to traditional culture systems under static conditions (T-flasks), the VWBR system allowed the production of EVs at higher concentration (i.e., EV concentration in the conditioned medium) (5.7-fold increase overall) and productivity (i.e., amount of EVs generated per cell) (3-fold increase overall). BM, AT and UCM MSC cultured in the VWBR system yielded an average of 2.8 ± 0.1 × 1011, 3.1 ± 1.3 × 1011, and 4.1 ± 1.7 × 1011 EV particles (n = 3), respectively, in a 60 mL final volume. This bioreactor system also allowed to obtain a more robust MSC-EV production, regarding their purity, compared to static culture. Overall, we demonstrate that this scalable culture system can robustly manufacture EVs from MSC derived from different tissue sources, toward the development of novel therapeutic products.

Rifabutin-Loaded Nanostructured Lipid Carriers as a Tool in Oral Anti-Mycobacterial Treatment of Crohn's Disease.

Oral anti-mycobacterial treatment of Crohn's disease (CD) is limited by the low aqueous solubility of drugs, along with the altered gut conditions of patients, making uncommon their clinical use. Hence, the aim of the present work is focused on the in vitro evaluation of rifabutin (RFB)-loaded Nanostructured lipid carriers (NLC), in order to solve limitations associated to this therapeutic approach. RFB-loaded NLC were prepared by hot homogenization and characterized in terms of size, polydispersity, surface charge, morphology, thermal stability, and drug payload and release. Permeability across Caco-2 cell monolayers and cytotoxicity and uptake in human macrophages was also determined. NLC obtained were nano-sized, monodisperse, negatively charged, and spheroidal-shaped, showing a suitable drug payload and thermal stability. Furthermore, the permeability profile, macrophage uptake and selective intracellular release of RFB-loaded NLC, guarantee an effective drug dose administration to cells. Outcomes suggest that rifabutin-loaded NLC constitute a promising strategy to improve oral anti-mycobacterial therapy in Crohn's disease.

Synthesis, Structure, and Activity of the Antifungal Plant Defensin PvD1.

Available treatments for invasive fungal infections have limitations, including toxicity and the emergence of resistant strains. Therefore, there is an urgent need for alternative solutions. Because of their unique mode of action and high selectivity, plant defensins (PDs) are worthy therapeutic candidates. Chemical synthesis remains a preferred method for the production of many peptide-based therapeutics. Given the relatively long sequence of PDs, as well as their complicated posttranslational modifications, the synthetic route can be considered challenging. Here, we describe a total synthesis of PvD1, the defensin from the common bean Phaseolus vulgaris. Analytical, structural, and functional characterization revealed that both natural and synthetic peptides fold into a canonical CSαß motif stabilized by conserved disulfide bonds. Moreover, synthetic PvD1 retained the biological activity against four different Candida species and showed no toxicity in vivo. Adding the high resistance of synthetic PvD1 to proteolytic degradation, we claim that conditions are now met to consider PDs druggable biologicals.

Antibodies for the Treatment of Brain Metastases, a Dream or a Reality?

The incidence of brain metastases (BM) in cancer patients is increasing. After diagnosis, overall survival (OS) is poor, elicited by the lack of an effective treatment. Monoclonal antibody (mAb)-based therapy has achieved remarkable success in treating both hematologic and non-central-nervous system (CNS) tumors due to their inherent targeting specificity. However, the use of mAbs in the treatment of CNS tumors is restricted by the blood-brain barrier (BBB) that hinders the delivery of either small-molecules drugs (sMDs) or therapeutic proteins (TPs). To overcome this limitation, active research is focused on the development of strategies to deliver TPs and increase their concentration in the brain. Yet, their molecular weight and hydrophilic nature turn this task into a challenge. The use of BBB peptide shuttles is an elegant strategy. They explore either receptor-mediated transcytosis (RMT) or adsorptive-mediated transcytosis (AMT) to cross the BBB. The latter is preferable since it avoids enzymatic degradation, receptor saturation, and competition with natural receptor substrates, which reduces adverse events. Therefore, the combination of mAbs properties (e.g., selectivity and long half-life) with BBB peptide shuttles (e.g., BBB translocation and delivery into the brain) turns the therapeutic conjugate in a valid approach to safely overcome the BBB and efficiently eliminate metastatic brain cells.

Translational Research Symposium-collaborative efforts as driving forces of healthcare innovation.

The 5th Translational Research Symposium was organised at the annual meeting of the European Society for Biomaterials 2018, Maastricht, the Netherlands, with emphasis on the future of emerging and smart technologies for healthcare in Europe. Invited speakers from academia and industry highlighted the vision and expectations of healthcare in Europe beyond 2020 and the perspectives of innovation stakeholders, such as small and medium enterprises, large companies and Universities. The aim of the present article is to summarise and explain the main statements made during the symposium, with particular attention on the need to identify unmet clinical needs and their efficient translation into healthcare solutions through active collaborations between all the participants involved in the value chain.

Influence of Post-UV/Ozone Treatment of Ultrasonic-Sprayed Zirconium Oxide Dielectric Films for a Low-Temperature Oxide Thin Film Transistor.

Solution-processed metal oxides require a great deal of thermal budget in order to achieve the desired film properties. Here, we show that the deposition temperature of sprayed zirconium oxide (ZrOx) thin film can be lowered by exposing the film surface to an ultraviolet (UV) ozone treatment at room temperature. Atomic force microscopy reveals a smooth and uniform film with the root mean square roughness reduced from ~ 0.63 nm (UVO-O) to ~ 0.28 nm (UVO-120) in the UV-ozone treated ZrOx films. X-ray photoelectron spectroscopy analysis indicates the formation of a Zr-O network on the surface film, and oxygen vacancy is reduced in the ZrOx lattice by increasing the UV-ozone treatment time. The leakage current density in Al/ZrOx/p-Si structure was reduced by three orders of magnitude by increasing the UV-ozone exposure time, while the capacitance was in the range 290-266 nF/cm2, corresponding to a relative permittivity (k) in the range 5.8-6.6 at 1 kHz. An indium gallium zinc oxide (IGZO)-based thin film transistor, employing a UV-treated ZrOx gate dielectric deposited at 200 °C, exhibits negligible hysteresis, an Ion/Ioff ratio of 104, a saturation mobility of 8.4 cm2 V-1S-1, a subthreshold slope of 0.21 V.dec-1, and a Von of 0.02 V. These results demonstrate the potentiality of low-temperature sprayed amorphous ZrOx to be applied as a dielectric in flexible and low-power-consumption oxide electronics.

Plant defensin PvD1 modulates the membrane composition of breast tumour-derived exosomes.

One of the most important causes of failure in tumour treatment is the development of resistance to therapy. Cancer cells can develop the ability to lose sensitivity to anti-neoplastic drugs during reciprocal crosstalk between cells and their interaction with the tumour microenvironment (TME). Cell-to-cell communication regulates a cascade of interdependent events essential for disease development and progression and can be mediated by several signalling pathways. Exosome-mediated communication is one of the pathways regulating these events. Tumour-derived exosomes (TDE) are believed to have the ability to modulate TMEs and participate in multidrug resistance mechanisms. In this work, we studied the effect of the natural defensin from common bean, PvD1, on the formation of exosomes by breast cancer MCF-7 cells, mainly the modulatory effect it has on the level of CD63 and CD9 tetraspanins. Moreover, we followed the interaction of PvD1 with biological and model membranes of selected composition, by biophysical and imaging techniques. Overall, the results show that PvD1 induces a dual effect on MCF-7 derived exosomes: the peptide attenuates the recruitment of CD63 and CD9 to exosomes intracellularly and binds to the mature exosomes in the extracellular environment. This work uncovers the exosome-mediated anticancer action of PvD1, a potential nutraceutical agent.

Cell Membrane Composition Drives Selectivity and Toxicity of Designed Cyclic Helix-Loop-Helix Peptides with Cell Penetrating and Tumor Suppressor Properties.

The tumor suppressor protein p53 is inactive in a large number of cancers, including some forms of sarcoma, breast cancer, and leukemia, due to overexpression of its intrinsic inhibitors MDM2 and MDMX. Reactivation of p53 tumor suppressor activity, via disruption of interactions between MDM2/X and p53 in the cytosol, is a promising strategy to treat cancer. Peptides able to bind MDM2 and/or MDMX were shown to prevent MDM2/X:p53 interactions, but most possess low cell penetrability, low stability, and/or high toxicity to healthy cells. Recently, the designed peptide cHLH-p53-R was reported to possess high affinity for MDM2, resistance toward proteases, cell-penetrating properties, and toxicity toward cancer cells. This peptide uses a stable cyclic helix-loop-helix (cHLH) scaffold, which includes two helices connected with a Gly loop and cyclized to improve stability. In the current study, we were interested in examining the cell selectivity of cHLH-p53-R, its cellular internalization, and ability to reactivate the p53 pathway. We designed analogues of cHLH-p53-R and employed biochemical and biophysical methodologies using in vitro model membranes and cell-based assays to compare their structure, activity, and mode-of-action. Our studies show that cHLH is an excellent scaffold to stabilize and constrain p53-mimetic peptides with helical conformation, and reveal that anticancer properties of cHLH-p53-R are mediated by its ability to selectively target, cross, and disrupt cancer cell membranes, and not by activation of the p53 pathway. These findings highlight the importance of examining the mode-of-action of designed peptides to fully exploit their potential to develop targeted therapies.

Lidocaine turns the surface charge of biological membranes more positive and changes the permeability of blood-brain barrier culture models.

The surface charge of brain endothelial cells forming the blood-brain barrier (BBB) is highly negative due to phospholipids in the plasma membrane and the glycocalyx. This negative charge is an important element of the defense systems of the BBB. Lidocaine, a cationic and lipophilic molecule which has anaesthetic and antiarrhytmic properties, exerts its actions by interacting with lipid membranes. Lidocaine when administered intravenously acts on vascular endothelial cells, but its direct effect on brain endothelial cells has not yet been studied. Our aim was to measure the effect of lidocaine on the charge of biological membranes and the barrier function of brain endothelial cells. We used the simplified membrane model, the bacteriorhodopsin (bR) containing purple membrane of Halobacterium salinarum and culture models of the BBB. We found that lidocaine turns the negative surface charge of purple membrane more positive and restores the function of the proton pump bR. Lidocaine also changed the zeta potential of brain endothelial cells in the same way. Short-term lidocaine treatment at a 10 µM therapeutically relevant concentration did not cause major BBB barrier dysfunction, substantial change in cell morphology or P-glycoprotein efflux pump inhibition. Lidocaine treatment decreased the flux of a cationic lipophilic molecule across the cell layer, but had no effect on the penetration of hydrophilic neutral or negatively charged markers. Our observations help to understand the biophysical background of the effect of lidocaine on biological membranes and draws the attention to the interaction of cationic drug molecules at the level of the BBB.