Lactoferrin translocates to the nucleus of bovine rectal epithelial cells in the presence of Escherichia coli O157:H7

International audienceEnterohemorrhagic Escherichia coli (EHEC) O157:H7 is a foodborne pathogen which causes illness in humans. Ruminants are the main reservoirs and EHEC predominantly colonizes the epithelium of the recto-anal junction of cattle. Immunosuppression by EHEC promotes re-infection of cattle. However, bovine lactoferrin (bLF) apparently can overrule the immunosuppression by inducing EHEC-specific IgA responses at the mucosal site. The IgA responses are significantly correlated with reduced EHEC shedding and the absence of colonization at the rectal mucosa following re-infection. Therefore, to examine the interaction between bLF and bovine rectal epithelial cells, we first developed a method to establish a primary cell culture of epithelial cells of the rectum of cattle. Furthermore, we used LC–MS/MS to demonstrate the presence of secreted lactoferrin in bovine milk and the absence of a “delta” isoform which is known to translocate to the nucleus of cells. Nevertheless, lactoferrin derived from bovine milk was internalized by rectal epithelial cells and translocated to the nuclei. Moreover, nuclear translocation of bLF was significantly enhanced when the epithelial cells were inoculated with EHEC, as demonstrated by confocal fluorescence microscopy and confirmed by Raman microscopy and 3D imaging

Hierarchy of hybrid materials. Part-II: The place of organics-on-inorganics in it, their composition and applications

Hybrid materials or hybrids incorporating organic and inorganic constituents are emerging as a very potent and promising class of materials due to the diverse but complementary nature of their properties. This complementarity leads to a perfect synergy of properties of the desired materials and products as well as to an extensive range of their application areas. Recently, we have overviewed and classified hybrid materials describing inorganics-in-organics in Part-I (Saveleva, et al., Front. Chem., 2019, 7, 179). Here, we extend that work in Part-II describing organics–on-inorganics, i.e., inorganic materials modified by organic moieties, their structure and functionalities. Inorganic constituents comprise of colloids/nanoparticles and flat surfaces/matrices comprise of metallic (noble metal, metal oxide, metal-organic framework, magnetic nanoparticles, alloy) and non-metallic (minerals, clays, carbons, and ceramics) materials; while organic additives can include molecules (polymers, fluorescence dyes, surfactants), biomolecules (proteins, carbohydtrates, antibodies and nucleic acids) and even higher-level organisms such as cells, bacteria, and microorganisms. Similarly to what was described in Part-I, we look at similar and dissimilar properties of organic-inorganic materials summarizing those bringing complementarity and composition. A broad range of applications of these hybrid materials is also presented whose development is spurred by engaging different scientific research communities

Application of whey protein isolate in bone regeneration:Effects on growth and osteogenic differentiation of bone-forming cells

Recently, milk-derived proteins have attracted attention for applications in the biomedical field such as tissue regeneration. Whey protein isolate (WPI), especially its main component β-lactoglobulin, can modulate immunity and acts as an antioxidant, antitumor, antiviral, and antibacterial agent. There are very few reports of the application of WPI in tissue engineering, especially in bone tissue engineering. In this study, we tested the influence of different concentrations of WPI on behavior of human osteoblast-like Saos-2 cells, human adipose tissue-derived stem cells (ASC), and human neonatal dermal fibroblasts (FIB). The positive effect on growth was apparent for Saos-2 cells and FIB but not for ASC. However, the expression of markers characteristic for early osteogenic cell differentiation [type-I collagen (COL1) and alkaline phosphatase (ALP)] as well as ALP activity, increased dose-dependently in ASC. Importantly, Saos-2 cells were able to deposit calcium in the presence of WPI, even in a proliferation medium without other supplements that support osteogenic cell differentiation. The results indicate that, depending on the cell type, WPI can act as an enhancer of cell proliferation and osteogenic differentiation. Therefore, enrichment of biomaterials for bone regeneration with WPI seems a promising approach, especially due to the low cost of WPI

Phytase-mediated enzymatic mineralization of chitosan-enriched hydrogels

Hydrogels mineralized with calcium phosphate (CaP) are increasingly popular bone regeneration biomaterials. Mineralization can be achieved by phosphatase enzyme incorporation and incubation in calcium glycerophosphate (CaGP). Gellan gum (GG) hydrogels containing the enzyme phytase and chitosan oligomer were mineralized in CaGP solution and characterized with human osteoblast-like MG63 cells and adipose tissue-derived stem cells (ADSC). Phytase induced CaP formation. Chitosan concentration determined mineralization extent and hydrogel mechanical reinforcement. Phytase-induced mineralization promoted MG63 adhesion and proliferation, especially in the presence of chitosan, and was non-toxic to MG63 cells (with and without chitosan). ADSC adhesion and proliferation were poor without mineralization. Chitosan did not affect ADSC osteogenic differentiation

Enhancement of biomimetic enzymatic mineralization of gellan gum polysaccharide hydrogels by plant-derived gallotannins

Mineralization of hydrogel biomaterials with calcium phosphate (CaP) is considered advantageous for bone regeneration. Mineralization can be both induced by the enzyme alkaline phosphatase (ALP) and promoted by calcium-binding biomolecules, such as plant-derived polyphenols. In this study, ALP-loaded gellan gum (GG) hydrogels were enriched with gallotannins, a subclass of polyphenols. Five preparations were compared, namely three tannic acids of differing molecular weight (MW), pentagalloyl glucose (PGG), and a gallotannin-rich extract from mango kernel (Mangifera indica L.). Certain gallotannin preparations promoted mineralization to a greater degree than others. The various gallotannin preparations bound differently to ALP and influenced the size of aggregates of ALP, which may be related to ability to promote mineralization. Human osteoblast-like Saos-2 cells grew in eluate from mineralized hydrogels. Gallotannin incorporation impeded cell growth on hydrogels and did not impart antibacterial activity. In conclusion, gallotannin incorporation aided mineralization but reduced cytocompatibility

Effects of fibrillin mutations on the behavior of heart muscle cells in Marfan syndrome

Abstract: Marfan syndrome (MFS) is a systemic disorder of connective tissue caused by pathogenic variants in the fibrillin-1 (FBN1) gene. Myocardial dysfunction has been demonstrated in MFS patients and mouse models, but little is known about the intrinsic effect on the cardiomyocytes (CMs). In this study, both induced pluripotent stem cells derived from a MFS-patient and the line with the corrected FBN1 mutation were differentiated to CMs. Several functional analyses are performed on this model to study MFS related cardiomyopathy. Atomic force microscopy revealed that MFS CMs are stiffer compared to corrected CMs. The contraction amplitude of MFS CMs is decreased compared to corrected CMs. Under normal culture conditions, MFS CMs show a lower beat-to-beat variability compared to corrected CMs using multi electrode array. Isoproterenol-induced stress or cyclic strain demonstrates lack of support from the matrix in MFS CMs. This study reports the first cardiac cell culture model for MFS, revealing abnormalities in the behavior of MFS CMs that are related to matrix defects. Based on these results, we postulate that impaired support from the extracellular environment plays a key role in the improper functioning of CMs in MFS

Pectin-bioactive glass self-gelling, injectable composites with high antibacterial activity

The present work focuses on the development of novel injectable, self-gelling composite hydrogels based on two types of low esterified amidated pectins from citrus peels and apple pomace. Sol-gelderived, calcium-rich bioactive glass (BG) fillers in a particle form are applied as delivery vehicles for the release of Ca2+ ions to induce internal gelation of pectins. Composites were prepared by a relatively simple mixing technique, using 20% w/v BG particles of two different sizes (2.5 and <45 µm). Smaller particles accelerated pectin gelation slightly faster than bigger ones, which appears to result from the higher rate of Ca2+ ion release. µCT showed inhomogeneous distribution of the BG particles within the hydrogels. All composite hydrogels exhibited strong antibacterial activity against methicilin-resistant Staphylococcus aureus. The mineralization process of pectin-BG composite hydrogels occurred upon incubation in simulated body fluid for 28 days. In vitro studies demonstrated cytocompatibility of composite hydrogels with MC3T3-E1 osteoblastic cells

Phenolic plant extract enrichment of enzymatically mineralized hydrogels

Hydrogel mineralization with calcium phosphate (CaP) and antibacterial activity are desirable for applications in bone regeneration. Mineralization with CaP can be induced using the enzyme alkaline phosphatase (ALP), responsible for CaP formation in bone tissue. Incorporation of polyphenols, plant-derived bactericidal molecules, was hypothesized to provide antibacterial activity and enhance ALP-induced mineralization. Three phenolic rich plant extracts from: (i) green tea, rich in epigallocatechin gallate (EGCG) (herafter referred to as EGCG-rich extract); (ii) pine bark and (iii) rosemary were added to gellan gum (GG) hydrogels and subsequently mineralized using ALP. The phenolic composition of the three extracts used were analyzed by ultra-high-performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MSn). EGCG-rich extract showed the highest content of phenolic compounds and promoted the highest CaP formation as corroborated by dry mass percentage meassurements and ICP-OES de-termination of mass of elemental Ca and P. All three extracts alone exhibited antibacterial activity in the following order EGCG-rich > PI > RO, respectively. However, extract-loaded and mineralized GG hydro-gels did not exhibit appreciable antibacterial activity by diffusion test. In conclusion, only the EGCG-rich extract promotes ALP-mediated mineralization

Efficient long-range conduction in cable bacteria through nickel protein wires

Filamentous cable bacteria display long-range electron transport, generating electrical currents over centimeter distances through a highly ordered network of fibers embedded in their cell envelope. The conductivity of these periplasmic wires is exceptionally high for a biological material, but their chemical structure and underlying electron transport mechanism remain unresolved. Here, we combine high-resolution microscopy, spectroscopy, and chemical imaging on individual cable bacterium filaments to demonstrate that the periplasmic wires consist of a conductive protein core surrounded by an insulating protein shell layer. The core proteins contain a sulfur-ligated nickel cofactor, and conductivity decreases when nickel is oxidized or selectively removed. The involvement of nickel as the active metal in biological conduction is remarkable, and suggests a hitherto unknown form of electron transport that enables efficient conduction in centimeter-long protein structures

Mineralization of gellan gum hydrogels with calcium and magnesium carbonates by alternate soaking for bone regeneration applications

Mineralization of hydrogels is desirable prior to applications in bone regeneration. CaCO3 is a widely used bone regeneration material and Mg, when used as a component of calcium phosphate biomaterials, has promoted bone‐forming cell adhesion and proliferation and bone regeneration. In this study, gellan gum (GG) hydrogels were mineralized with carbonates containing different amounts of calcium (Ca) and magnesium (Mg) by alternate soaking in, firstly, a calcium and/or magnesium ion solution and, secondly, a carbonate ion solution. This alternate soaking cycle was repeated five times. Five different calcium and/or magnesium ion solutions, containing different molar ratios of Ca to Mg ranging from Mg‐free to Ca‐free were compared. Carbonate mineral formed in all sample groups subjected to the Ca:Mg elemental ratio in the carbonate mineral formed was higher than in the respective mineralizing solution. Mineral formed in the absence of Mg was predominantly CaCO3 in the form of a mixture of calcite and vaterite. Increasing the Mg content in the mineral formed led to the formation of magnesian calcite, decreased the total amount of the mineral formed and its crystallinity. Hydrogel mineralization and increasing Mg content in mineral formed did not obviously improve proliferation of MC3T3‐E1 osteoblast‐like cells or differentiation after 7 days