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.

Multifactorial bottom-up bioengineering approaches for the development of living tissue substitutes.

Bottom-up bioengineering utilizes the inherent capacity of cells to build highly sophisticated structures with high levels of biomimicry. Despite the significant advancements in the field, monodomain approaches require prolonged culture time to develop an implantable device, usually associated with cell phenotypic drift in culture. Herein, we assessed the simultaneous effect of macromolecular crowding (MMC) and mechanical loading in enhancing extracellular matrix (ECM) deposition while maintaining tenocyte (TC) phenotype and differentiating bone marrow stem cells (BMSCs) or transdifferentiating neonatal and adult dermal fibroblasts toward tenogenic lineage. At d 7, all cell types presented cytoskeleton alignment perpendicular to the applied load independently of the use of MMC. MMC enhanced ECM deposition in all cell types. Gene expression analysis indicated that MMC and mechanical loading maintained TC phenotype, whereas tenogenic differentiation of BMSCs or transdifferentiation of dermal fibroblasts was not achieved. Our data suggest that multifactorial bottom-up bioengineering approaches significantly accelerate the development of biomimetic tissue equivalents.-Gaspar, D., Ryan, C. N. M., Zeugolis, D. I. Multifactorial bottom-up bioengineering approaches for the development of living tissue substitutes.

Mechanisms of bacterial membrane permeabilization by crotalicidin (Ctn) and its fragment Ctn(15-34), antimicrobial peptides from rattlesnake venom.

Crotalicidin (Ctn), a cathelicidin-related peptide from the venom of a South American rattlesnake, possesses potent antimicrobial, antitumor, and antifungal properties. Previously, we have shown that its C-terminal fragment, Ctn(15-34), retains the antimicrobial and antitumor activities but is less toxic to healthy cells and has improved serum stability. Here, we investigated the mechanisms of action of Ctn and Ctn(15-34) against Gram-negative bacteria. Both peptides were bactericidal, killing ∼90% of Escherichia coli and Pseudomonas aeruginosa cells within 90-120 and 5-30 min, respectively. Studies of ζ potential at the bacterial cell membrane suggested that both peptides accumulate at and neutralize negative charges on the bacterial surface. Flow cytometry experiments confirmed that both peptides permeabilize the bacterial cell membrane but suggested slightly different mechanisms of action. Ctn(15-34) permeabilized the membrane immediately upon addition to the cells, whereas Ctn had a lag phase before inducing membrane damage and exhibited more complex cell-killing activity, probably because of two different modes of membrane permeabilization. Using surface plasmon resonance and leakage assays with model vesicles, we confirmed that Ctn(15-34) binds to and disrupts lipid membranes and also observed that Ctn(15-34) has a preference for vesicles that mimic bacterial or tumor cell membranes. Atomic force microscopy visualized the effect of these peptides on bacterial cells, and confocal microscopy confirmed their localization on the bacterial surface. Our studies shed light onto the antimicrobial mechanisms of Ctn and Ctn(15-34), suggesting Ctn(15-34) as a promising lead for development as an antibacterial/antitumor agent.

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.

Microencapsulated Solid Lipid Nanoparticles as a Hybrid Platform for Pulmonary Antibiotic Delivery.

Solid lipid nanoparticles (SLN) containing rifabutin (RFB), with pulmonary administration purposes, were developed through a technique that avoids the use of organic solvents or sonication. To facilitate their pulmonary delivery, the RFB-loaded SLN were included in microspheres of appropriate size using suitable excipients (mannitol and trehalose) through a spray-drying technique. Confocal analysis microscopy showed that microspheres are spherical and that SLN are efficiently microencapsulated and homogeneously distributed throughout the microsphere matrices. The aerodynamic diameters observed an optimal distribution for reaching the alveolar region. The dry powder's performance during aerosolization and the in vitro drug deposition were tested using a twin-impinger approach, which confirmed that the microspheres can reach the deep lung. Isothermal titration calorimetry revealed that SLN have higher affinity for mannitol than for trehalose. Upon microsphere dissolution in aqueous media, SLN were readily recovered, maintaining their physicochemical properties. When these dry powders reach the deep lung, microspheres are expected to readily dissolve, delivering the SLN which, in turn, will release RFB. The in vivo biodistribution of microencapsulated RFB-SLN demonstrated that the antibiotic achieved the tested organs 15 and 30 min post pulmonary administration. Their antimycobacterial activity was also evaluated in a murine model of infection with a Mycobacterium tuberculosis strain H37Rv resulting in an enhancement of activity against M. tuberculosis infection compared to nontreated animals. These results suggest that RFB-SLN microencapsulation is a promising approach for the treatment of tuberculosis.

Apoptotic human neutrophil peptide-1 anti-tumor activity revealed by cellular biomechanics.

Cancer remains a major cause of morbidity and mortality worldwide. Although progress has been made regarding chemotherapeutic agents, new therapies that combine increased selectivity and efficacy with low resistance are still needed. In the search for new anticancer agents, therapies based on biologically active peptides, in particular, antimicrobial peptides (AMPs), have attracted attention for their decreased resistance development and low cytotoxicity. Many AMPs have proved to be tumoricidal agents against human cancer cells, but their mode of action is still controversial. The existence of common properties shared by the membranes of bacteria and tumor cells points to similar lipid-targeting mechanisms in both cases. On the other hand, anticancer peptides (ACPs) also induce apoptosis and inhibit angiogenesis. Human neutrophil peptide-1 (HNP-1) is an endogenous AMP that has been implicated in different cellular phenomena such as tumor proliferation. The presence of HNP-1 in the serum/plasma of oncologic patients turns this peptide into a potential tumor biomarker. The present work reveals the different effects of HNP-1 on the biophysical and nanomechanical properties of solid and hematological tumor cells. Studies on cellular morphology, cellular stiffness, and membrane ultrastructure and charge using atomic force microscopy (AFM) and zeta potential measurements show a preferential binding of HNP-1 to solid tumor cells from human prostate adenocarcinoma when compared to human leukemia cells. AFM also reveals induction of apoptosis with cellular membrane defects at very low peptide concentrations. Understanding ACPs mode(s) of action will certainly open innovative pathways for drug development in cancer treatment.

Monitoring antibacterial permeabilization in real time using time-resolved flow cytometry.

Despite the intensive study of antibiotic-induced bacterial permeabilization, its kinetics and molecular mechanism remain largely elusive. A new methodology that extends the concept of the live-dead assay in flow cytometry to real time-resolved detection was used to overcome these limitations. The antimicrobial activity of pepR was monitored in time-resolved flow cytometry for three bacterial strains: Escherichia coli (ATCC 25922), E. coli K-12 (CGSC Strain 4401) and E. coli JW3596-1 (CGSC Strain 11805). The latter strain has truncated lipopolysaccharides (LPS) in the outer membrane. This new methodology provided information on the efficacy of the antibiotics and sheds light on their mode of action at membrane-level. Kinetic data regarding antibiotic binding and lytic action were retrieved. Membrane interaction and permeabilization events differ significantly among strains. The truncation of LPS moieties does not hamper AMP binding but compromises membrane disruption and bacterial killing. We demonstrated the usefulness of time-resolved flow cytometry to study antimicrobial-induced permeabilization by collecting kinetic data that contribute to characterize the action of antibiotics directly on bacteria.

Bioorthogonal Strategy for Bioprocessing of Specific-Site-Functionalized Enveloped Influenza-Virus-Like Particles.

Virus-like particles (VLPs) constitute a promising platform in vaccine development and targeted drug delivery. To date, most applications use simple nonenveloped VLPs as human papillomavirus or hepatitis B vaccines, even though the envelope is known to be critical to retain the native protein folding and biological function. Here, we present tagged enveloped VLPs (TagE-VLPs) as a valuable strategy for the downstream processing and monitoring of the in vivo production of specific-site-functionalized enveloped influenza VLPs. This two-step procedure allows bioorthogonal functionalization of azide-tagged nascent influenza type A hemagglutinin proteins in the envelope of VLPs through a strain-promoted [3 + 2] alkyne-azide cycloaddition reaction. Importantly, labeling does not influence VLP production and allows for construction of functionalized VLPs without deleterious effects on their biological function. Refined discrimination and separation between VLP and baculovirus, the major impurity of the process, is achieved when this technique is combined with flow cytometry analysis, as demonstrated by atomic force microscopy. TagE-VLPs is a versatile tool broadly applicable to the production, monitoring, and purification of functionalized enveloped VLPs for vaccine design trial runs, targeted drug delivery, and molecular imaging.

Scaffold and scaffold-free self-assembled systems in regenerative medicine.

Self-assembly in tissue engineering refers to the spontaneous chemical or biological association of components to form a distinct functional construct, reminiscent of native tissue. Such self-assembled systems have been widely used to develop platforms for the delivery of therapeutic and/or bioactive molecules and various cell populations. Tissue morphology and functional characteristics have been recapitulated in several self-assembled constructs, designed to incorporate stimuli responsiveness and controlled architecture through spatial confinement or field manipulation. In parallel, owing to substantial functional properties, scaffold-free cell-assembled devices have aided in the development of functional neotissues for various clinical targets. Herein, we discuss recent advancements and future aspirations in scaffold and scaffold-free self-assembled devices for regenerative medicine purposes. Biotechnol. Bioeng. 2016;113: 1155-1163. © 2015 Wiley Periodicals, Inc.

Shifting gear in antimicrobial and anticancer peptides biophysical studies: from vesicles to cells.

Despite the intensive study on the mechanism of action of membrane-active molecules such as antimicrobial and anticancer peptides, most of the biophysical work has been performed using artificial model systems, mainly lipid vesicles. The use of these systems allows full control of the experimental parameters, and to obtain molecular-level detail on the action of peptides, the correlation with biological action is intangible. Recently, several biophysical methodologies have been translated to studies using bacterial and cancer cells. Here, we review biophysical studies on the mechanism of action of antimicrobial and anticancer peptides performed directly on cells. The data in these studies allow to correlate vesicle-based and cell-based studies and fill the vesicle-cell interdisciplinary gap.

Decoding distinct membrane interactions of HIV-1 fusion inhibitors using a combined atomic force and fluorescence microscopy approach.

Enfuvirtide and T-1249 are two potent HIV-1 fusion inhibitor peptides. Recent studies indicate that lipids play an important role in the mode of action of those bioactive molecules. Using a combined tandem atomic force microscopy (AFM)-epifluorescence microscopy approach, we studied the interaction of both enfuvirtide and T-1249 with supported lipid bilayers. Fluid (ld)-gel (so) and ld-liquid ordered (lo) phase-separated membrane systems were tested. Results, especially for T-1249, show significant lipid membrane activity at a 15µM peptide concentration. T-1249, in opposition to enfuvirtide, induces an increase in membrane surface roughness, decrease in membrane fluidity, bilayer thinning at ld domains and disruption of the so domain borders. In terms of structural properties, both enfuvirtide and T-1249 possess distinct functional hydrophobic and amphipathic domains of HIV gp41. While enfuvirtide only yields the tryptophan-rich domain (TRD), T-1249 possesses both TRD and pocket-binding domain (PBD). TRD increases the hydrophobicity of the peptide while PBD enhances the amphipathic characteristics. As such, the enhanced membrane activity of T-1249 may be explained by a synergism between its amphipathic N-terminal segment and its hydrophophic C-terminal. Our findings provide valuable insights on the molecular-level mode of action of HIV-1 fusion inhibitors, unraveling the correlation between their structural properties and membrane interactions as a factor influencing their antiviral activity. Ultimately, this work validates the applicability of a combined AFM and fluorescence approach to evaluate the mechanic and structural properties of supported lipid bilayers upon interaction with membrane-active peptides.

Design and characterization of novel antimicrobial peptides, R-BP100 and RW-BP100, with activity against Gram-negative and Gram-positive bacteria.

BP100 is a short cationic antimicrobial peptide with a mechanism of action dependent on peptide-lipid interactions and microbial surface charge neutralization. Although active against Gram-negative bacteria, BP100 is inactive against Gram-positive bacteria. In this study we report two newly designed BP100 analogues, RW-BP100 and R-BP100 that have the Tyr residue replaced with a Trp and/or the Lys residues replaced with an Arg. The new analogues in addition to being active against Gram-negative bacteria, possess activity against all tested Gram-positive bacteria. Mechanistic studies using atomic force microscopy, surface plasmon resonance and fluorescence methodologies reveal that the antibacterial efficiency follows the affinity for bacterial membrane. The studies suggest that the activity of BP100 and its analogues against Gram-negative bacteria is mainly driven by electrostatic interactions with the lipopolysaccharide layer and is followed by binding to and disruption of the inner membrane, whereas activity against Gram-positive bacteria, in addition to electrostatic attraction to the exposed lipoteichoic acids, requires an ability to more deeply insert in the membrane environment, which is favoured with Arg residues and is facilitated in the presence of a Trp residue. Knowledge on the mechanism of action of these antimicrobial peptides provides information that assists in the design of antimicrobials with higher efficacy and broader spectra of action, but also on the design of peptides with higher specificity if required.

The antimicrobial activity of Sub3 is dependent on membrane binding and cell-penetrating ability.

Because of their high activity against microorganisms and low cytotoxicity, cationic antimicrobial peptides (AMPs) have been explored as the next generation of antibiotics. Although they have common structural features, the modes of action of AMPs are extensively debated, and a single mechanism does not explain the activity of all AMPs reported so far. Here we investigated the mechanism of action of Sub3, an AMP previously designed and optimised from high-throughput screening with bactenecin as the template. Sub3 has potent activity against Gram-negative and Gram-positive bacteria as well as against fungi, but its mechanism of action has remained elusive. By using AFM imaging, ζ potential, flow cytometry and fluorescence methodologies with model membranes and bacterial cells, we found that, although the mechanism of action involves membrane targeting, Sub3 internalises inside bacteria at lethal concentrations without permeabilising the membrane, thus suggesting that its antimicrobial activity might involve both the membrane and intracellular targets. In addition, we found that Sub3 can be internalised into human cells without being toxic. As some bacteria are able to survive intracellularly and consequently evade host defences and antibiotic treatment, our findings suggest that Sub3 could be useful as an intracellular antimicrobial agent for infections that are notoriously difficult to treat.

Role of a disperse carbon interlayer on the performances of tandem a-Si solar cells.

We report the effect of a disperse carbon interlayer between the n-a-Si:H layer and an aluminium zinc oxide (AZO) back contact on the performance of amorphous silicon solar cells. Carbon was incorporated to the AZO film as revealed by x-ray photoelectron spectroscopy and energy-dispersive x-ray analysis. Solar cells fabricated on glass substrates using AZO in the back contact performed better when a disperse carbon interlayer was present in their structure. They exhibited an initial efficiency of 11%, open-circuit voltage Voc = 1.6 V, short-circuit current JSC = 11 mA cm-2 and a filling factor of 63%, that is, a 10% increase in the JSC and 20% increase in the efficiency compared to a standard solar cell.

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.

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.

Anticancer peptide SVS-1: efficacy precedes membrane neutralization.

Anticancer peptides are polycationic amphiphiles capable of preferentially killing a wide spectrum of cancer cells relative to noncancerous cells. Their primary mode of action is an interaction with the cell membrane and subsequent activation of lytic effects; however, the exact mechanism responsible for this mode of action remains controversial. Using zeta potential analyses we demonstrate the interaction of a small anticancer peptide with membrane model systems and cancer cells. Electrostatic interactions have a pivotal role in the cell killing process, and in contrast to the antimicrobial peptides action cell death occurs without achieving full neutralization of the membrane charge.

Anticancer ß-hairpin peptides: membrane-induced folding triggers activity.

Several cationic antimicrobial peptides (AMPs) have recently been shown to display anticancer activity via a mechanism that usually entails the disruption of cancer cell membranes. In this work, we designed an 18-residue anticancer peptide, SVS-1, whose mechanism of action is designed to take advantage of the aberrant lipid composition presented on the outer leaflet of cancer cell membranes, which makes the surface of these cells electronegative relative to the surface of noncancerous cells. SVS-1 is designed to remain unfolded and inactive in aqueous solution but to preferentially fold at the surface of cancer cells, adopting an amphiphilic ß-hairpin structure capable of membrane disruption. Membrane-induced folding is driven by electrostatic interaction between the peptide and the negatively charged membrane surface of cancer cells. SVS-1 is active against a variety of cancer cell lines such as A549 (lung carcinoma), KB (epidermal carcinoma), MCF-7 (breast carcinoma), and MDA-MB-436 (breast carcinoma). However, the cytotoxicity toward noncancerous cells having typical membrane compositions, such as HUVEC and erythrocytes, is low. CD spectroscopy, appropriately designed peptide controls, cell-based studies, liposome leakage assays, and electron microscopy support the intended mechanism of action, which leads to preferential killing of cancerous cells.