Short Communication: Enterotoxin Genes and Antibiotic Susceptibility of Bacillus cereus Isolated from Garlic Chives and Agricultural Environment

This study aims to investigate the enterotoxin profiles and antibiotic susceptibility of Bacillus cereus isolated from garlic chives and environmental samples. A total of 103 B. cereus isolates were used to identify enterotoxin genes, including hblA, hblC, hblD, nheA, nheB, and nheC. The hemolysin BL enterotoxin complex (hblACD) was detected in 38 isolates (36.9%), and the non-hemolytic enterotoxin complex (nheABC) was detected in 8 (7.8%) isolates. Forty-five isolates (43.7%) had hblACD and nheABC genes. B. cereus was resistant to β-lactam antibiotics and susceptible to non-β-lactam antibiotics. However, some B. cereus strains showed intermediate resistance to β-lactam and non-β-lactam antibiotics. B. cereus isolated from garlic chives showed intermediate resistance to cefotaxime (7.7%), rifampin (15.4%), clindamycin (30.8%), erythromycin (7.7%), and tetracycline (7.7%). B. cereus isolates from the agricultural environment were moderately resistant to cefotaxime (18.9%), rifampin (15.6%), clindamycin (12.2%), erythromycin (4.4%), and tetracycline (5.6%). Moreover, B. cereus isolates from garlic chives and cultivation environments could change their antibiotic resistance profile from susceptible to intermediate-resistant to rifampin, clindamycin, erythromycin, and tetracycline and exhibit multidrug resistance. These results indicate that continuous monitoring of B. cereus contamination in the produce and agricultural environment might be needed to ensure the safety of consuming fresh vegetables

Cellular Layer-by-Layer Coculture Platform Using Biodegradable, Nanoarchitectured Membranes for Stem Cell Therapy

Stem cells are regulated <i>in vivo</i> through interactions with the surrounding microenvironments in a three-dimensional (3D) manner. A coculture of stem cells with desired cell types, which recapitulates the complex <i>in vivo</i> cell–cell communications, has been reported as an effective method to direct stem cell differentiation into specific lineage. However, conventional bilayer coculture systems employ membranes of microscale thickness and low porosity, which limit interaction between cocultured cells for efficient stem cell differentiation. Furthermore, conventional coculture systems require cell-impairing enzyme treatment to harvest the cells from the membranes. Here, we developed a cellular layer-by-layer (cLbL) coculture platform using biodegradable, nanothin, highly porous (BNTHP) membranes. Equipped with more porous and thinner membranes, the cLbL coculture platform better mimicked the <i>in vivo</i> 3D microenvironment and promoted cellular cross-talks between cocultured cells which occurred in nanoscale, resulting in more efficient stem cell differentiation compared to the conventional bilayer coculture systems. Furthermore, biodegradibility, biocompatibility, and highly flexibility of BNTHP membranes enabled conversion of the cell-attached membranes into implantable 3D cell constructs, thus avoiding harmful enzymatic harvesting of the cells. The cLbL platform may be an effective method to induce stem cell differentiation and facilitate cell implantation for stem cell therapy

Covalent conjugation of mechanically stiff graphene oxide flakes to three-dimensional collagen scaffolds for osteogenic differentiation of human mesenchymal stem cells

Mesenchymal stem cells (MSCs) preferentially differentiate to osteogenic lineage when cultured on mechanically stiff substrates. However, collagen sponges, clinically approved scaffolds for bone regeneration, provide soft microenvironment to MSCs. Here, we demonstrate that the covalent conjugation of mechanically stiff graphene oxide (GO) flakes to three-dimensional (3D) collagen scaffolds improves the mechanical properties of the scaffolds and promotes the osteogenic differentiation of human MSCs (hMSCs) cultured on the scaffolds. The covalent conjugation of GO flakes to collagen scaffolds increased the scaffold stiffness by 3-fold and did not cause cytotoxicity. hMSCs cultured on the GO-collagen scaffolds demonstrated significantly enhanced osteogenic differentiation compared to cells cultured on non-modified collagen scaffolds. The enhanced osteogenic differentiation observed on the stiffer scaffolds was likely mediated by MSC mechanosensing because molecules that are involved in cell adhesion to stiff substrates were either up-regulated or activated. The 3D GO-collagen scaffolds could offer a powerful platform for stem cell research and orthopedic regenerative medicine. (C) 2014 Elsevier Ltd. All rights reserved

Graphene Potentiates the Myocardial Repair Efficacy of Mesenchymal Stem Cells by Stimulating the Expression of Angiogenic Growth Factors and Gap Junction Protein

Stem cell therapy has emerged as a potential modality for myocardial infarction treatment. Mesenchymal stem cells (MSCs) exert reparative actions in the injured myocardium mainly through the secretion of paracrine factors. In addition, the overexpression of connexin 43 (Cx43), a gap junction protein, promotes cardiac repair and function restoration. It is known that MSCs in a spheroid form, which have enhanced cell-cell interaction, exhibit enhanced expression of paracrine factors and Cx43. However, cell-extracellular matrix (ECM) interactions, which also contribute to growth factor expression, are very limited in MSC spheroids. Reduced graphene oxide (RGO) shows high affinity toward ECM proteins, such as fibronectin (FN), and high electrical conductivity. In this study, by incorporating FN-adsorbed RGO flakes into MSC spheroids, it is possible to enhance the cell-ECM interactions and, subsequently, the paracrine factor expression in the MSCs in spheroids. Cx43 is also upregulated likely due to the enhanced paracrine factor expression and electrical conductivity of RGO. The injection of MSC-RGO hybrid spheroids into the infarcted hearts enhances cardiac repair compared with the injection of RGO flakes or MSC spheroids. This study demonstrates that RGO can effectively improve the therapeutic efficacy of MSCs for ischemic heart diseases

Graphene Oxide Flakes as a Cellular Adhesive: Prevention of Reactive Oxygen Species Mediated Death of Implanted Cells for Cardiac Repair

Mesenchymal stem cell (MSC) implantation has emerged as a potential therapy for myocardial infarction (MI). However, the poor survival of MSCs implanted to treat MI has significantly limited the therapeutic efficacy of this approach. This poor survival is primarily due to reactive oxygen species (ROS) generated in the ischemic myocardium after the restoration of blood flow. ROS primarily causes the death of implanted MSCs by inhibiting the adhesion of the MSCs to extracellular matrices at the lesion site anoikis). In this study, we proposed the use of graphene oxide (GO) flakes to protect the implanted MSCs from ROS-mediated death and thereby improve the therapeutic efficacy of the MSCs. GO can adsorb extracellular matrix (ECM) proteins. The survival of MSCs, which had adhered to ECM protein-adsorbed GO flakes and were subsequently exposed to ROS in vitro or implanted into the ischemia-damaged and reperfused myocardium, significantly exceeded that of unmodified MSCs. Furthermore, the MSC engraftment improved by the adhesion of MSCs to GO flakes prior to implantation enhanced the paracrine secretion from the MSCs following MSC implantation, which in turn promoted cardiac tissue repair and cardiac function restoration. This study demonstrates that GO can effectively improve the engraftment and therapeutic efficacy of MSCs used to repair the injury of ROS-abundant ischemia and reperfusion by protecting implanted cells from anoikis

In situ hybridization of carbon nanotubes with bacterial cellulose for three-dimensional hybrid bioscaffolds

Carbon nanotubes (CNTs) have shown great potential in biomedical fields. However, in vivo applications of CNTs for regenerative medicine have been hampered by difficulties associated with the fabrication of three-dimensional (3D) scaffolds of CNTs due to CNTs' nano-scale nature. In this study, we devised a new method for biosynthesis of CNT-based 3D scaffold by in situ hybridizing CNTs with bacterial cellulose (BC), which has a structure ideal for tissue-engineering scaffolds. This was achieved simply by culturing Gluconacetobacter xylinus, BC-synthesizing bacteria, in medium containing CNTs. However, pristine CNTs aggregated in medium, which hampers homogeneous hybridization of CNTs with BC scaffolds, and the binding energy between hydrophobic pristine CNTs and hydrophilic BC was too small for the hybridization to occur. To overcome these problems, an amphiphilic comb-like polymer (APCLP) was adsorbed on CNTs. Unlike CNT-coated BC scaffolds (CNT-BC-Imm) formed by immersing 3D BC scaffolds in CNT solution, the APCLP-adsorbed CNT-BC hybrid scaffold (CNT-BC-Syn) showed homogeneously distributed CNTs throughout the 3D microporous structure of BC. Importantly, in contrast to CNT-BC-Imm scaffolds, CNT-BC-Syn scaffolds showed excellent osteoconductivity and osteoinductivity that led to high bone regeneration efficacy. This strategy may open a new avenue for development of 3D biofunctional scaffolds for regenerative medicine. (C) 2015 Elsevier Ltd. All rights reserved

Graphene- Regulated Cardiomyogenic Differentiation Process of Mesenchymal Stem Cells by Enhancing the Expression of Extracellular Matrix Proteins and Cell Signaling Molecules

The potential of graphene as a mesenchymal stem cell (MSC) culture substrate to promote cardiomyogenic differentiation is demonstrated. Graphene exhibits no sign of cytotoxicity for stem cell culture. MSCs are committed toward cardiomyogenic lineage by simply culturing them on graphene. This may be attributed, at least partially, to the regulation of expression levels of extracellular matrix and signaling molecules

Gold Nanoparticle/Graphene Oxide Hybrid Sheets Attached on Mesenchymal Stem Cells for Effective Photothermal Cancer Therapy

Cell-mediated nanoparticle delivery has been proposed for an effective cancer therapy. However, there are limitations in loading nanoparticles within cells as the internalized nanoparticles cause cytotoxicity and leak out of the cells via exocytosis. Here, we introduce hybrid sheets composed of gold nanoparticles (AuNPs) and graphene oxide (GO), which stably adhere to the cell surface and exhibit a remarkable photothermal effect. To form AuNP/GO sheets in which GO is sandwiched between two AuNP monolayers, AuNPs are coated with α-synuclein protein and subsequently adsorbed onto GO sheets. Attaching AuNP/GO sheets to the tumor-tropic mesenchymal stem cell (MSC) surface enhances the loading efficiency of AuNPs in MSCs by avoiding the cytotoxicity and exocytosis issues. Furthermore, the tight packing of AuNPs on microscaled GO sheets enhances the photothermal effect via strong plasmon coupling between AuNPs. The injection of AuNP/GO sheet-attached MSCs into tumor-bearing mice significantly improves the photothermal therapeutic efficacy by delivering larger amounts of AuNPs to the tumor and generating higher heat at the tumor region compared to injection of AuNP-internalized MSCs. The system of attaching AuNP/GO hybrid sheets to the tumor-tropic cell surface may be an effective platform for cancer therapy

Nanothin Coculture Membranes with Tunable Pore Architecture and Thermoresponsive Functionality for Transfer-Printable Stem Cell-Derived Cardiac Sheets

Coculturing stem cells with the desired cell type is an effective method to promote the differentiation of stem cells. The features of the membrane used for coculturing are crucial to achieving the best outcome. Not only should the membrane act as a physical barrier that prevents the mixing of the cocultured cell populations, but it should also allow effective interactions between the cells. Unfortunately, conventional membranes used for coculture do not sufficiently meet these requirements. In addition, cell harvesting using proteolytic enzymes following coculture impairs cell viability and the extracellular matrix (ECM) produced by the cultured cells. To overcome these limitations, we developed nanothin and highly porous (NTHP) membranes, which are ∼20-fold thinner and ∼25-fold more porous than the conventional coculture membranes. The tunable pore size of NTHP membranes at the nanoscale level was found crucial for the formation of direct gap junctions-mediated contacts between the cocultured cells. Differentiation of the cocultured stem cells was dramatically enhanced with the pore size-customized NTHP membrane system compared to conventional coculture methods. This was likely due to effective physical contacts between the cocultured cells and the fast diffusion of bioactive molecules across the membrane. Also, the thermoresponsive functionality of the NTHP membranes enabled the efficient generation of homogeneous, ECM-preserved, highly viable, and transfer-printable sheets of cardiomyogenically differentiated cells. The coculture platform developed in this study would be effective for producing various types of therapeutic multilayered cell sheets that can be differentiated from stem cells