Multiplexed labeling system for high-throughput cell sorting.

Flow cytometry and fluorescence activated cell sorting techniques were designed to realize configurable classification and separation of target cells. A number of cell phenotypes with different functionalities have recently been revealed. Before simultaneous selective capture of cells, it is desirable to label different samples with the corresponding dyes in a multiplexing manner to allow for a single analysis. However, few methods to obtain multiple fluorescent colors for various cell types have been developed. Even when restricted laser sources are employed, a small number of color codes can be expressed simultaneously. In this study, we demonstrate the ability to manifest DNA nanostructure-based multifluorescent colors formed by a complex of dyes. Highly precise self-assembly of fluorescent dye-conjugated oligonucleotides gives anisotropic DNA nanostructures, Y- and tree-shaped DNA (Y-DNA and T-DNA, respectively), which may be used as platforms for fluorescent codes. As a proof of concept, we have demonstrated seven different fluorescent codes with only two different fluorescent dyes using T-DNA. This method provides maximum efficiency for current flow cytometry. We are confident that this system will provide highly efficient multiplexed fluorescent detection for bioanalysis compared with one-to-one fluorescent correspondence for specific marker detection.

mRNA-Producing Pseudo-nucleus System.

A pseudo-eukaryotic nucleus (PEN) system consisting of a gene-containing DNA hydrogel encapsulated in a liposome is fabricated. Owing to the structural characteristics of gene-containing DNA hydrogel, mRNA transcription efficiency is promoted 2.57-fold. Through the use of PEN as a platform for mRNA delivery to the cytosol, prolonged protein translation is achieved.

Stretchable, wireless sensors and functional substrates for epidermal characterization of sweat.

This paper introduces materials and architectures for ultrathin, stretchable wireless sensors that mount on functional elastomeric substrates for epidermal analysis of biofluids. Measurement of the volume and chemical properties of sweat via dielectric detection and colorimetry demonstrates some capabilities. Here, inductively coupled sensors consisting of LC resonators with capacitive electrodes show systematic responses to sweat collected in microporous substrates. Interrogation occurs through external coils placed in physical proximity to the devices. The substrates allow spontaneous sweat collection through capillary forces, without the need for complex microfluidic handling systems. Furthermore, colorimetric measurement modes are possible in the same system by introducing indicator compounds into the depths of the substrates, for sensing specific components (OH(-) , H(+) , Cu(+) , and Fe(2+) ) in the sweat. The complete devices offer Young's moduli that are similar to skin, thus allowing highly effective and reliable skin integration without external fixtures. Experimental results demonstrate volumetric measurement of sweat with an accuracy of 0.06 µL/mm(2) with good stability and low drift. Colorimetric responses to pH and concentrations of various ions provide capabilities relevant to analysis of sweat. Similar materials and device designs can be used in monitoring other body fluids.

Neural stem cell delivery using brain-derived tissue-specific bioink for recovering from traumatic brain injury.

Traumatic brain injury is one of the leading causes of accidental death and disability. The loss of parts in a severely injured brain induces edema, neuronal apoptosis, and neuroinflammation. Recently, stem cell transplantation demonstrated regenerative efficacy in an injured brain. However, the efficacy of current stem cell therapy needs improvement to resolve issues such as low survival of implanted stem cells and low efficacy of differentiation into respective cells. We developed brain-derived decellularized extracellular matrix (BdECM) bioink that is printable and has native brain-like stiffness. This study aimed to fabricate injured cavity-fit scaffold with BdECM bioink and assessed the utility of BdECM bioink for stem cell delivery to a traumatically injured brain. Our BdECM bioink had shear thinning property for three-dimensional (3D)-cell-printing and physical properties and fiber structures comparable to those of the native brain, which is important for tissue integration after implantation. The human neural stem cells (NSCs) (F3 cells) laden with BdECM bioink were found to be fully differentiated to neurons; the levels of markers for mature differentiated neurons were higher than those observed with collagen bioinkin vitro. Moreover, the BdECM bioink demonstrated potential in defect-fit carrier fabrication with 3D cell-printing, based on the rheological properties and shape fidelity of the material. As F3 cell-laden BdECM bioink was transplanted into the motor cortex of a rat brain, high efficacy of differentiation into mature neurons was observed in the transplanted NSCs; notably increased level of MAP2, a marker of neuronal differentiation, was observed. Furthermore, the transplanted-cell bioink suppressed reactive astrogliosis and microglial activation that may impede regeneration of the injured brain. The brain-specific material reported here is favorable for NSC differentiation and suppression of neuroinflammation and is expected to successfully support regeneration of a traumatically injured brain.

Derivation of primitive neural stem cells from human-induced pluripotent stem cells.

Human-induced pluripotent stem cells (hiPSCs) have facilitated studies on organ development and differentiation into specific lineages in in vitro systems. Although numerous studies have focused on cellular differentiation into neural lineage using hPSCs, most studies have initially evaluated embryoid body (EB) formation, eventually yielding terminally differentiated neurons with limited proliferation potential. This study aimed to establish human primitive neural stem cells (pNSCs) from exogene-free hiPSCs without EB formation. To derive pNSCs, we optimized N2B27 neural differentiation medium through supplementation of two inhibitors, CHIR99021 (GSK-3 inhibitor) and PD0325901 (MEK inhibitor), and growth factors including basic fibroblast growth factor (bFGF) and human leukemia inhibitory factor (hLIF). Consequently, pNSCs were efficiently derived and cultured over a long term. pNSCs displayed differentiation potential into neurons, astrocytes, and oligodendrocytes. These early NSC types potentially promote the clinical application of hiPSCs to cure human neurological disorders.

Wuweizisu C from Schisandra chinensis decreases membrane potential in C6 glioma cells.

AIM: To study the effects of dibenzocyclooctadiene lignans isolated from Schisandra chinensis, such as wuweizisu C, gomisin N, gomisin A, and schisandrin, on the membrane potential in C6 glioma cells. METHODS: The membrane potential was estimated by measuring the fluorescence change in DiBAC-loaded glioma cells. RESULTS: Wuweizisu C decreased the membrane potential in a concentration-dependent manner. Gomisin N and gomisin A, however, showed differential modulation and no change was induced by schisandrin or dimethyl- 4,4'-dimethoxy-5,6,5',6'-dimethylene dioxybiphenyl-2,2'-dicarboxylate, a synthetic drug derived from dibenzocyclooctadiene lignans. We found no involvement of G(i/o ) proteins, phospholipase C, and extracellular Na(+) on the wuweizisu C-induced decrease of the membrane potential. Wuweizisu C by itself did not change the intracellular Ca(2+)[Ca(2+)](i) concentration, but decreased the ATP-induced Ca(2+) increase in C6 glioma cells. The 4 lignans at all concentrations used in this study did not induce any effect on cell viability. Furthermore, we found a similar decrease of the membrane potential by wuweizisu C in PC12 neuronal cells. CONCLUSION: Our results suggest that the decrease in the membrane potential and the modulation of [Ca(2+)](i) concentration by wuweizisu C could be important action mechanisms of wuweizisu C.

Optimization of ascorbic acid-2-phosphate production from ascorbic acid using resting cell of Brevundimonas diminuta.

With the aim to produce ascorbic acid-2-phosphate (AsA-2-P) from L-ascorbic acid (AsA, Vitamin C), nine bacteria conferring the ability to transform AsA to AsA-2-P were isolated from soil samples alongside known strains from culture collections. Most isolates were classified to the genus Brevundimonas by 16S phylogenetic analysis. Among them, Brevundimonas diminuta KACC 10306 was selected as the experimental strain because of its the highest productivity of AsA-2-P. The optimum set of conditions for the AsA-2-P production from AsA using resting cells as the source of the enzyme was also investigated. The optimum cultivation time was 16 h and the cell concentration was 120 g/l (wet weight). The optimum concentrations of AsA and pyrophosphate were 550 mM and 450 mM, respectively. The most effective buffer was 50 mM sodium formate. The optimum pH was 4.5 and temperature was 40 degrees C. Under the above conditions, 27.5 g/l of AsA-2-P was produced from AsA after 36 h of incubation, which corresponded to a 19.7% conversion efficiency based on the initial concentration of AsA.

CpG and Non-CpG Methylation in Epigenetic Gene Regulation and Brain Function.

DNA methylation is a major epigenetic mark with important roles in genetic regulation. Methylated cytosines are found primarily at CpG dinucleotides, but are also found at non-CpG sites (CpA, CpT, and CpC). The general functions of CpG and non-CpG methylation include gene silencing or activation depending on the methylated regions. CpG and non-CpG methylation are found throughout the whole genome, including repetitive sequences, enhancers, promoters, and gene bodies. Interestingly, however, non-CpG methylation is restricted to specific cell types, such as pluripotent stem cells, oocytes, neurons, and glial cells. Thus, accumulation of methylation at non-CpG sites and CpG sites in neurons seems to be involved in development and disease etiology. Here, we provide an overview of CpG and non-CpG methylation and their roles in neurological diseases.

Hyaluronic acid-supported combination of water insoluble immunostimulatory compounds for anti-cancer immunotherapy.

A novel powder-form combination adjuvant system containing two immunostimulatory compounds was firstly developed and evaluated as a therapeutic intervention for cancer immunotherapy. With the help of hyaluronic acid (HA), water insoluble monophosphoryl lipid A (MPL), QS21 and imiquimod (R837), could be easily dispersed in aqueous solution and lyophilized as powder-form, which have an advantage in room-temperature storage stability compared with those conventional liquid formulation that requires cold storage. Two kinds of HA-based combination vaccine adjuvants (HA/MPL/QS21, HMQ and HA/MPL/R837, HMR) contributed to the increase of both humoral and cellular immunity, which is very important for efficient cancer immunotherapy. Through the challenge experiments in EG7-OVA (mouse lymphoma-expressing OVA) tumor-bearing mice model, we found out that the immunostimulatory effects of HMQ and HMR were successful in the inhibition of tumor proliferation. Taken together, both HA-based powder-form combination adjuvant systems are expected to be used as potent prophylactic and therapeutic cancer vaccine.

Cell Surface Nano-modulation for Non-invasive in†vivo Near-IR Stem Cell Monitoring.

A stem cell tracking system is in high demand for the determination of cell destinations and for the validation of cell therapeutic efficacy in regenerative transplantation. To date, near-infrared (NIR) imaging technology has received considerable attention in cell behavior monitoring, owing to its patient compatibility, easy accessibility and cost effectiveness. Conventionally, in vivo cell tracking has been visualized by direct in-cell staining with NIR, where it may be achieved by complicated genetic engineering. Such genetic amendment techniques have suffered from serious challenges, which can destroy a cell's metabolism and can accidentally incur unexpected carcinoma. Herein we demonstrate a novel cell nano-modulation method for noninvasive stem cell monitoring. It is simply achieved by conjugating stem cells with lipid-supported, NIR-tagged, polymeric nanoparticles. These engineered cells, which are designated as NIR-labeled light-emitting stem cells (LESCs), maintain their biochemical functionality (i.e., differentiation, quantum efficacy, etc.) even after conjugation. LESCs were used for in situ stem cell monitoring at inoculation sites. It is speculated that the LESC technique could provide a new preparative methodology for in vivo cell tracking in advanced diagnostic medicine, where cell behavior is a critical issue.

Gomisin A from Schisandra chinensis induces endothelium-dependent and direct relaxation in rat thoracic aorta.

Schisandra chinensis (SC), a member of the Magnoliaceae family, has been used to improve the vascular health for postmenopausal women in Korea. In order to provide some scientific rationales for such effectiveness, this study investigated the vascular effects of gomisin A (GA) from SC. In the endothelium (ED)-intact rings of rat thoracic aorta, GA (1 x 10 (-6) to 3 x 10 (-4) M) caused a concentration-dependent relaxation which was markedly attenuated not only by removal of ED but also by pretreatment with N(G)-nitro- L-arginine (10 (-4) M) or 1 H-[1,2,4]oxadiazol[4,3- a]quinoxalin-1-one (3 x 10 (-5) M). Direct measurement of nitrite, a metabolite of nitric oxide (NO), confirmed that NO production in isolated aorta was increased by GA. In the ED-denuded specimens, the relaxation by GA was not abolished but reduced significantly. The relaxation by GA in ED-denuded aortic rings were clearly inhibited by calyculin A (3 x 10 (-8) M), an inhibitor of MLC phosphatase. Furthermore, the phenylephrine-enhanced phosphorylation ratio of MLC was significantly attenuated by GA. Based on these results, it is suggested that GA induced vascular relaxation by partially activating ED-dependent NO pathway, and partially dephosphorylation of MLC.