Topography-Mediated Enhancement of Nonviral Gene Delivery in Stem Cells

Gene delivery holds great promise for bioengineering, biomedical applications, biosensors, diagnoses, and gene therapy. In particular, the influence of topography on gene delivery is considered to be an attractive approach due to low toxicity and localized delivery properties. Even though many gene vectors and transfection systems have been developed to enhance transfection potential and combining it with other forms of stimulations could even further enhance it. Topography is an interesting surface property that has been shown to stimulate differentiation, migration, cell morphology, and cell mechanics. Therefore, it is envisioned that topography might also be able to stimulate transfection. In this study, we tested the hypothesis "topography is able to regulate transfection efficiency", for which we used nano- and microwave-like topographical substrates with wavelengths ranging from 500 nm to 25 µm and assessed the transfectability of human bone marrow-derived mesenchymal stem cells (hBM-MSCs) and myoblasts. For transfection, Lipofectamine 2000 and a gene encoding plasmid for red-fluorescent protein (m-Cherry) were used and topography-induced cell morphology and transfection efficiency was analyzed. As a result, topography directs cell spreading, elongation, and proliferation as well as the transfection efficiency, which were investigated but were found not to be correlated and dependent on the cell type. A 55% percent improvement of transfection efficiency was identified for hBM-MSCs grown on 2 µm wrinkles (24.3%) as compared to hBM-MSCs cultured on flat controls (15.7%). For myoblast cells, the highest gene-expression efficiency (46.1%) was observed on the 10 µm topography, which enhanced the transfection efficiency by 64% as compared to the flat control (28.1%). From a qualitative assessment, it was observed that the uptake capacity of cationic complexes of TAMRA-labeled oligodeoxynucleotides (ODNs) was not topography-dependent but that the intracellular release was faster, as indicated by the positively stained nuclei on 2 μm for hBM-MSCs and 10 μm for myoblasts. The presented results indicate that topography enhances the gene-delivery capacity and that the responses are dependent on cell type. This study demonstrates the important role of topography on cell stimulation for gene delivery as well as understanding the uptake capacity of lipoplexes and may be useful for developing advanced nonviral gene delivery strategies

Impact of target site distribution for Type I restriction enzymes on the evolution of methicillin-resistant Staphylococcus aureus (MRSA) populations.

A limited number of Methicillin-resistant Staphylococcus aureus (MRSA) clones are responsible for MRSA infections worldwide, and those of different lineages carry unique Type I restriction-modification (RM) variants. We have identified the specific DNA sequence targets for the dominant MRSA lineages CC1, CC5, CC8 and ST239. We experimentally demonstrate that this RM system is sufficient to block horizontal gene transfer between clinically important MRSA, confirming the bioinformatic evidence that each lineage is evolving independently. Target sites are distributed randomly in S. aureus genomes, except in a set of large conjugative plasmids encoding resistance genes that show evidence of spreading between two successful MRSA lineages. This analysis of the identification and distribution of target sites explains evolutionary patterns in a pathogenic bacterium. We show that a lack of specific target sites enables plasmids to evade the Type I RM system thereby contributing to the evolution of increasingly resistant community and hospital MRSA

Topography-Mediated Fibroblast Cell Migration Is Influenced by Direction, Wavelength, and Amplitude

Biophysical stimuli including topography play a crucial role in the regulation of cell morphology, adhesion, migration, and cytoskeleton organization and have been known to be important in biomaterials design for tissue engineering. However, little is known about the individual effects of topographic direction, structure repetition, and feature size of the substrate on which wound healing occurs. We report on the design of a topographical gradient with wavelike features that gradually change in wavelength and amplitude, which provides an efficient platform for an in vitro wound healing assay to investigate fibroblast migration. The wound coverage rate was measured on selected areas with wavelength sizes of 2, 5, and 8 mu m in perpendicular and parallel orientations. Furthermore, a method was developed to produce independently controlled wavelength and amplitude and study which parameter has greater influence. Cell movement was guided by topographical properties, with a lower wrinkle wavelength (2 mu m) eliciting the fastest migration speed, and the migration speed increased with decreasing amplitude. However, when the amplitudes were matched, cells migrated faster on a larger wavelength. This study also highlights the sensitivity of fibroblasts to the topographic orientation, with cells moving faster in the parallel direction of the topography. The overall behavior indicated that the wavelength and amplitude both play an important role in directing cell migration. The collective cell migration was found not to be influenced by altered cell proliferation. These findings provide key insights into topography-triggered cell migration and indicate the necessity for better understanding of material-directed wound healing for designing bio-inductive biomaterials

Nanogels with Selective Intracellular Reactivity for Intracellular Tracking and Delivery

A multimodal approach for hydrogel-based nanoparticles was developed to selectively allow molecular conjugated species to either be released inside the cell or remain connected to the polymer network. Using the intrinsic difference in reactivity between esters and amides, nanogels with an amide-conjugated dye could be tracked intracellularly localizing next to the nucleus, while ester-conjugation allowed for liberation of the molecular species from the hydrogel network inside the cell, enabling delivery throughout the cytoplasm. The release was a result of particle exposure to the intracellular environment. The conjugation approach and polymer network building rely on the same chemistry and provide a diverse range of possibilities to be used in nanomedicine and theranostic approaches

Biomimetic Multiscale Hierarchical Topography Enhances Osteogenic Differentiation of Human Mesenchymal Stem Cells

The interface between materials and cells plays a critical role in many biomedical applications. Inspired by the hierarchical architecture of collagen, most abundant structure in the extracellular matrix (ECM), a multiscale hierarchical topography is designed to mimic the collagen nano/micro hierarchical topography. It is hypothesized that the ECM topography affects osteogenesis of human mesenchymal stem cells but until now, it cannot be studied without the biochemical and mechanical influences of the ECM. The multiscale hierarchical topography is achieved by innovatively using sequentially aligned topography preparation via a silicone stretch-oxidation-release method and imprinting lithography. The anisotropically hierarchical topography influences stem cell morphology, orientation, and osteogenic differentiation. Intriguingly, the design resembling that of assembled collagen, exhibits the highest degree of osteogenesis. The hierarchical topotaxis effects are further exemplified by the enhanced vinculin expression, cell contractility, and more pronounced nuclear translocation of Yes-associated protein with the collagen-mimicking topography, indicative for enhanced osteogenesis. The developed multiscale hierarchical system provides insights into the importance of specific biological ECM-like topography by decoupling the biochemical influence. Various diseases, cancer, osteoarthritis, and fibrosis display impaired ECM structures, and therefore this system may have a great potential for tissue engineering approaches and developing in vitro disease models

Topography induced stiffness alteration of stem cells influences osteogenic differentiation

Topography-driven alterations in cell morphology tremendously influence cell biological processes, particularly stem cell differentiation. Aligned topography is known to alter the cell shape, which we anticipated to also induce altered physical properties of the cell. Here, we show that topography has a significant influence on single cell stiffness of human bone marrow derived-Mesenchymal Stem Cells (hBM-MSCs) and the osteogenic differentiation of these. Aligned topographies were used to control the cell elongation, depicted as the cell aspect ratio (C-AR). Intriguingly, an equal C-AR elicited from different topographies, resulted in highly altered differentiation behavior and the underlying single cell mechanics was found to be critical. The cell behavior was found to be focal adhesion-mediated and induced stiffness alterations rather than just influencing the cell elongation. The effect was further corroborated by investigations of the transcriptional regulators YAP. Our study provides insight into how mechanical properties of the cell, which are stimulated by topography, modulate the osteogenesis of hBM-MSCs, which is beneficial for improving the understanding of interactions between stem cells and topography for developing applications of tissue engineering and regenerative medicine

A Universal Nanogel-Based Coating Approach for Medical Implant Materials

Coatings are essential for biomedical applications antifouling and antimicrobialproperties, supporting cell adhesion and tissue integration and particularlyinteresting in this field are nanogel (nGel)-based coatings. Since biomaterialsdiffer in physiochemical properties, specific nGel-coating strategies need to bedeveloped for every distinct material, leading to complex coating strategies.Hence, the solution lies in adopting a universal strategy to apply the same nGelcoating with the same function on a wide range of implant surfaces. To this end, auniversal nGel-based coating approach provides the same coating using a singlemethod on implant materials including stiff polymer materials, metals, ceramics,glass, and elastomers. The coating formation is achieved by electrostatic interactionsbetween oxygen plasma–activated surfaces and positively charged nGelsusing a spray-deposition method. Fluorescent labels are introduced into thenGels as a model for post-modification capabilities to increase the functionality ofthe coating. The coating is highly stable under in vitro physiological conditionswith the retention of its function on different clinically relevant materials.Meanwhile, the in vivo study indicates that the nGel coating on a polyvinylidenefluoride hernia mesh is stable and biocompatible, therefore, making the coatingand the coating strategy, a highly impactful approach for future clinicaldevelopments

Full humanization of the glycolytic pathway in Saccharomyces cerevisiae

Although transplantation of single genes in yeast plays a key role in elucidating gene functionality in metazoans, technical challenges hamper humanization of full pathways and processes. Empowered by advances in synthetic biology, this study demonstrates the feasibility and implementation of full humanization of glycolysis in yeast. Single gene and full pathway transplantation revealed the remarkable conservation of glycolytic and moonlighting functions and, combined with evolutionary strategies, brought to light context-dependent responses. Human hexokinase 1 and 2, but not 4, required mutations in their catalytic or allosteric sites for functionality in yeast, whereas hexokinase 3 was unable to complement its yeast ortholog. Comparison with human tissues cultures showed preservation of turnover numbers of human glycolytic enzymes in yeast and human cell cultures. This demonstration of transplantation of an entire essential pathway paves the way for establishment of species-, tissue-, and disease-specific metazoan models

Exploring contested authenticity among speakers of a contested language: the case of ‘Francoprovençal'

This paper explores the notion of speaker authenticity in the context of obsolescent ‘Francoprovençal’: a highly fragmented grouping of Romance varieties spoken in parts of France, Italy, and Switzerland by less than 1% of the total regional population. While Francoprovençal has long been losing ground to the dominant language(s) with which it is in contact, new speakers have begun to emerge within the context of revitalisation movements and activities geared more favourable language planning policies and increased literacy. The emergence of these new speakers has polarised native-speaker communities, and has blurred the lines associated with the traditional view of sociolinguistic authenticity. Through an analysis of qualitative data collected in 2012, this article argues in particular that it may not be sufficient to simply examine contested authenticities from a native–non-native perspective, but rather it is important to consider how new speakers might themselves form a complex spectrum of speaker types with new sets of tensions as has been argued elsewhere

Characterisation of proteins in excretory/secretory products collected from salmon lice, Lepeophtheirus salmonis

Background  The salmon louse, Lepeophtheirus salmonis, is an ectoparasitic copepod which feeds on the mucus, skin and blood of salmonid fish species. The parasite can persist on the surface of the fish without any effective control being exerted by the host immune system. Other ectoparasitic invertebrates produce compounds in their saliva, excretions and/or secretions which modulate the host immune responses allowing them to remain on or in the host during development. Similarly, compounds are produced in secretions of L. salmonis which are thought to be responsible for immunomodulation of the host responses as well as other aspects of crucial host-parasite interactions.  Methods  In this study we have identified and characterised the proteins in the excretory/secretory (E/S) products of L. salmonis using LC-ESI-MS/MS.  Results  In total 187 individual proteins were identified in the E/S collected from adult lice and pre-adult sea lice. Fifty-three proteins, including 13 serine-type endopeptidases, 1 peroxidase and 5 vitellogenin-like proteins were common to both adult and pre-adult E/S products. One hundred and seven proteins were identified in the adult E/S but not in the pre-adult E/S and these included serine and cysteine-type endopeptidases, vitellogenins, sphingomyelinase and calreticulin. A total of 27 proteins were identified in pre-adult E/S products but not in adult E/S.  Conclusions  The assigned functions of these E/S products and the potential roles they play in host-parasite interaction is discussed