Earticle

In the past several decades, there have been significant advances in understanding of protein sequences, structures and functions which in turn have aided engineering of proteins with novel structures and/or functions. In addition, recent work from several laboratories has shown that an expanded repertoire of amino acid analogues can be incorporated into proteins. The recruitment of new amino acid constituents provides the protein engineering field with new tools and raises the prospects for creating novel proteins. In this talk, I will present a high-throughput screening method for engineering aminoacyl-tRNA synthetases to activate noncanonical amino acids and show examples how the engineered enzymes are utilized to study biology, especially to interrogate cellular protein synthesis.

High quality single crystal inorganic materials for high performance electronics and optoelectronics are intrinsically stiff and brittle, while biological systems are soft and curvilinear. To achieve flexible and stretchable bio-integrated electronic systems with comparable performance to state-of-the-art, wafer-based devices, high quality single crystal inorganic materials should be incorporated with substrates of mechanical properties of biosystem. Deterministic assembly method using transfer printing inorganic nanomaterials onto flexible and stretchable substrates solves this fundamental mechanical mismatch between electronic devices and bio-system [1].By designing unconventional shapes of nanomaterials and controlling pre-strains, stretchable systems with wavy and serpentine layout are developed. This system minimizes the induced strain in active area while successfully isolates strain to highly stretchable interconnection regions. The concepts of flexible and stretchable bio-integrated devices are illustrated through application examples including soft and conformable electrophysiological monitors integrated with hearts and brains[2-4], and tattoo-like epidermal electronics with mechanical properties matched to the epidermis[5]. The value of this approach is demonstrated in in-vivo and in-vitro experiments. The resulting bio-integrated electronic technologies realize important improvements in diagnostic and therapeutic surgical tools, including electrophysiological mapping systems, prosthetic devices and electrical stimulation therapies.

As part of an effort to develop an innovative biomolecular detection platform, we have investigated a new biomolecular detection strategy that employs a touchscreen, which is widely used in current mobile devices. Based on changes in the location of touch signals on a surface capacitive touchscreen or the intensities of touch signals on a projected capacitive touchscreen, concentrations of unknown DNA samples, serving as model targets, were accurately determined in a manner that is comparable to the detection performance of a conventional absorbancebased method. Furthermore, the detection capability of touchscreen method was verified by using a polypyrrole, conducting polymer. The results suggest that the new technique should be generally applicable to other biosensing devices. The capacitive touchscreen-based system should serve as a new platform for the development of various systems targeted at the detection of biomolecules having compatible dielectric properties. Since this technology should be readily integrated into touchscreen-equipped smart phones or smart pads, it could truly accelerate the development of real POCT or personalized medical devices.

In vitro techniques are efficient tools for evaluating the effects of newly developed chemicals, drugs and nanomaterials on human beings. We have recently developed a cell-based chip that enables the sensitive detection of cell viability using electrochemical methods. Peptide nanopatterned array was fabricated on the electrode surface as nanodots, nanorods or nanopillar structures to enhance cell adhesion, spreading and proliferation. The enhanced cell adhesion on the electrode surface accelerates the electron transfer rate at the cell-electrode interfaces, which increases the sensitivity of fabricated cell chip. The redox signals obtained from neural cells via voltammetric tools were proportional to the cell viability which was used for assessing the toxicity of environmental toxins, as well as for evaluating the efficiency of anticancer drugs. The nanofabrication technology also enabled the integration of a powerful analysis tool, Surface-enhanced Raman spectroscopy (SERS), which can investigate the changes of intracellular composition and the effects of anticancer drugs on single cancer cells precisely. Proposed cell chip based on nanofabrication technology can be applied as an invitro analysistoolinvariouskindsofneural cell researches.

An integrated allele-specific (AS) polymerase chain reaction (PCR) and resultant amplicon detection system has been developed for multiplex single nucleotide polymorphism (SNP) genotyping on a portable instrumentation, which was applied for on-site identification of HANWOO. As for the detection method, micro-capillary electrophoresis (CE) and microarray were employed, and the integrated PCR-μCE and PCR-microarray devices were constructed. Twelve sets of primers were designed for targeting eleven SNP loci and one primer set for a positive PCR control. The operation of microdevices were automated with a portable genetic analyzer, and the detection was performed on a miniaturized fluorescence detector for CE separation or a fluorescence scanner for microarray. Successful HANWOO verification was conducted with 99.37% fidelity in 90 min. Thus, our genetic analysis system provides rapid, accurate, and on-site multiplex SNP typing platform.

A new sunscreen agent, methoxycinnamidopropyl polysilsesquioxane (MCP-PSQ), w hich contains an ultraviolet (UV)-absorbing pmethoxycinnamoyl group, has been developed and evaluated. MCPPSQ has composed by polymeric particles, number average size of 600-700 nm, with an organic/inorganic hybrid composition. We investigated the physical characteristics and analyzed organic part of MCP-PSQ in qualitatively and quantitatively. MCP-PSQ is an UVB blocking agent by absorbing of UVB ray, but unlike of other UVB sunscreen agent, UVA/UVB ratio is high with a 0.3 to 0.4, and critical wavelength of 350-360 nm also seems to have characteristics that MCP-PSQ has inorganic properties in addition to regular organic UV-absorbing parts. MCP-PSQ has a raising or boosting effect on the sun protection factor (SPF) of other sunscreen agents.We evaluated photostabilities of MCP-PSQ that has the same functional group as a typical organic sunscreen agent, ethylhexyl methoxycinnamate (EHMC). Using the relation of UV absorbance and fluorescence, we studied photostabilizers to enhance the photostability of MCP-PSQ that has the similar photochemical properties to EHMC. We confirmed that octocrylene, ethylhexyl methoxycrylene, and bis-ethylhexyloxyphenol methoxyphenyl triazine were good photostabilizers for MCP-PSQ.

한-미 FTA가 발효되고 곧 나고야의정서 또한 발효되는 등 최근 대내외 환경의 변화로 인해 국내 화장품 산업의 미래에 대한 불학실성이 증가하고 있다. 이러한 환경변화에 대응해 중장기적인 발전 전략을 수립해 수출사업화 및 미래 성장동력으로 육성할 필요가 있다. 보건복지부는 그동안 수출진흥 및 R&D 지원 등 화장품 산업의 경쟁력 강화를 위해 다양한 정책들을 추진해 왔다. 한편으로는 현재까지 추진되었던 정책들의 성과를 점검하고 추가적인 과제 발굴을 통해 화장품 산업의 중장기 발전전략과 지원 정책들을 모색하고 있다.

Generally, tissue-cultured plant has a benefit to solve the drawbacks such as a rareness, long time and high cost for growing herb. In this study, We have studied possibilities of applying cosmetic material about the two kinds of tissue culture obtained by using plant tissue culture techniques. One of these is a Anoectochilus formosanus. Anoectochilus formosanus, commonly known as "Jewel Orchids," which has been used in traditional folk medicines for fever, pain, and diseases of the lung and liver in Taiwan. The other one is the Echinacea angustifolia,.commonly known as compositae of dicotyledonous plant. It has been used in Indian traditional folk medicines for the treatment of disease like colds or other infections in North America. We artificially cultured Anoectochilus formosanus plantlet and Echinacea angustifolia adventitious roots by using the bioreactor culture system for this study. Previously, several studies have been reported on the efficacy (pharma –cological activities of lipid-metabolism, hepatoprotective activity, anti-tumor activity, neogenesis, wound healing, inflammation, hypertension, diabetes, liver disease, anti-tumor and immuno-stimulating effects) about these plants. Although these plants have been well known as herbs to remedy various diseases as a traditional medicine, we investigated whether the plantlet and adventitious roots cultured from these plants have efficacy as a cosmetic ingredient for skin care.

Novel transdermal delivery systems were developed for improving skin penetration of natural substances. Polygonum aviculare L. (P. aviculare) extracts were selected to encapsulate in delivery system because P. aviculare extracts could be applicable to antiaging and whitening agents by in vitro biological activity assay. Liposome and polymeric micelle were developed for improving skin penetration of P.aviculare extracts. The entrapment efficiency of liposome was higher than polymeric micelle. Therefore we investigated further experiment using liposome system. Franz diffusion cell and CLSM studies showed that this system promoting skin penetration of P. aviculare extracts. In vivo studies using guinea pigs and hairless mice indicated liposome system was better than control in their effectiveness on the wrinkle and whitening improvement by promoting skin penetration of P. aviculare extracts.

TiO2 nanoparticles have long been utilized in various industrial application (e.g. semiconductiors ,photocatalyst, optical coating) due to its photonic band structure. [1,2] Especially TiO2 nanoparticle complex can be utilized in sun care products because of its UV-ray shielding (or absorbing) performance and low absorption in the visible region. In the present work, we synthesized mesoporous TiO2 sphere with a crystalline framework and a high surface area. In order to enhance the UV light blocking ability, various materials (e.g. Tinosorb) were loaded within the mesoporous of TiO2 materials. The physicochemical properties of TiO2 based composite materials thus obtained have been investigated by X-ray powder diffraction (XRD), scanning electron microscopy (SEM), and nitrogen adsorption-desorption isotherm, and UV-visible spectroscopy. UV-blocking materials loaded on mesoporous TiO2 materials exhibit higher UV-light blocking effects than the TiO2 maerials without other metal oxides, which is probably due to increase in reflective index and enhanced of UV-Vis absorption ability.

Skin color is determined by the amount of melanin present. Many enzymes have a relationship in m elanin synthesis. Especially, tyrosinase is well known as key enzyme for melanin synthesis. MITF (Microphthalmia-associated transcription factor), a bHLH (basic helix-loop-helix) transcription factor with a leucine zipper, is a key protein in regulation of the pigmentation process in melanocytes. Binding of the MITF to E-box induces transcription of several genes associated with pigmentation, including the tyrosinase gene. The purpose of this study is to evaluate the interaction of MITF with respective DNA binding site (E-box, CATGTG) by SPR (surface plasmon resonance). SPR, a label-free gold chip based DNA-protein interaction assay was used to evaluate the interactions. The MITF showed almost a twice higher resonance signal after binding with E-box, when compared to mutated E-box. We have also evaluated various parameters to stabilize the assay conditions and to reduce the false signals. We anticipate that evaluation of interaction provide a quantitative, comparative, highthroughput screening of MITF inhibitors, which will help to develop an effective methodology of screening of potential depigmenting agent.

To evaluate safety of novel products for the cosmetic and pharmaceutical uses, various animal models have been conventionally used. However, in near future, all animal tests are scheduled to be banned in European cosmetic industry and therefore alternative skin toxicity tests should be required to evaluate the safety of newly introduced cosmetic ingredients. Currently, artificial epidermal equivalents or artificial skins are suggested to be used for skin irritation testing as alternative methods. However, it is practically impossible that the three dimensional tissue engineered products are introduced to the early developmental stages of active ingredients as a method for testing skin irritation due to their higher costs and low-throughputs in performance. In this regard, we are developing novel and high-throughput alternative test methods for evaluating skin irritation through the omics-based systems toxicology approaches. In preliminary results, in order to evaluate the feasibility to develop mechanism-based toxicity testings related to skin barrier, we evaluated the toxicological effect of Asian dust storm particles on transcriptional changes of skin barrier development-associated proteins. In addition, we studied the cosmeceutical whitening effect of kojic acid with a melanocyte-keratinocyte co-culture system to evaluate whether co-culture system is effective as an alternative testing system. Genome-wide transcriptional profiles for cosmetic ingredients, such as retinol, kojic acid, and UV filters, etc., have been evaluated in cultured normal human keratinocytes to discover novel toxicity biomarkers. In addition, to differentiate between pharmacological and toxicological components in omics data, we also performed genome-wide transcriptional profile studies for a representative skin irritant, sodium lauryl sulfate (SLS), and a skin sensitizer, urushiol in human keratinocytes. We will present current advancements of the omics-based development of novel alternative test methods for evaluating skin irritation as one of projects in the National Coordination Center for Global Cosmetic R&D.

The objective of this research was to investigate that NAADP can stimulate the differentiation of human keratinocytes and to build a foundation of the business based on the dermatological functions of NAADP.NAADP can stimulate to increase Ca2+ for cell signaling which synthesizes insulin from β-cell of pancreas, and now this signaling is very similar to the one for the differentiation of human keratinocytes. So we hypothesized that NAADP can stimulate the differentiation of human keratinocytes through cell signaling of Ca2+. To prove this hypothesis, we tested to increase Ca2+ and to express six kinds of differentiation marker in human keratinocytes by NAADP.As a result, Keratin1/10, Involucrin, Loricrin, Filaggrin, and Transglutaminase showed to be increased in western blot assay dependent on the concentration of NAADP, and also Keratin1/10, Filaggrin, and Loricrin showed to be increased by NAADP in immunohistochemistry by using hairless mice. Then we confirmed the function of NAADP for the differentiation of human keratinocytes. To investigate the efficacy of NAADP, Migration assay of human keratinocytes, repair assay of damaged human skin which induced by tape-stripping, moisturizing assay were carried out, and NAADP showed higher migration capacity than positive control group(growth factors), and excellent wound healing effect and moisturizing effect. When considered these results, we held a conviction for the business of NAADP, and wanted to build a foundation for this good substance, NAADP. So we have optimized the condition of NAADP production by enzyme reaction, and have done up-scale test for mass production. We concluded that NAADP can stimulate the differentiation of keratinocytes and give human skin the dermatological effects. If practice further studies of NAADP for signal transduction and genetic assay, we expect that NAADP will be valuable more and more.

The inflammatory responses are complicated events that involve many immune cells from resident tissue and infiltrated cells from blood vessel. In skin and cornea, epidermal lining provides a first line of defense against microbial pathogens as well as physical and chemical insult. To evaluate cosmetic ingredients for inflammation and allergy, a hypersensitivity of immune reaction, we conventionally utilized either in vivo animal models or in vitro immune cells. In this study, we developed a new alternative method for inflammation and allergy testing using co-culture of 3D- reconstructed skin and cornea with monitoring immune cells which represents in vitro substitute for functional epidermal barrier and multiple immune responses in the tissue. Firstly, we generated 3D- reconstructed skin and cornea with cells from human skin and rabbit cornea using transwell. Specific markers for basal and differentiated layer in normal skin or cornea, either K5 and K10 or K5 and K3+12, were well expressed in 3D skin or cornea, respectively. We applied RAW264.7 and RBL-2H3 as monitoring immune cells for inflammatory and allergic responses in co-culture system. LPS-induced immune responses in RAW 264.7 cells were examined. Decreased expression levels of IL-6, TNF-alpha, IL-10, iNOS, and COX-2 were observed in co-culture system compared to those of the direct administration in RAW 264.7 cells. In addition, DNP-IgE-mediated allergic responses were attenuated in co-culture system which was monitored by b-hexosaminisases activity in RBL-2H3. These data suggests that epidermal barriers of 3D-reconstruced skin and cornea provide a protective role under inflammatory environment in a similar mechanism as with intact in vivo condition. Especially, RAW 264.7 cells played dual roles mediating inflammatory responses and acted as anti- inflammatory dermal macrophages in the skin. These results suggest that this in vitro co-culture model system can be used as a novel alternative method for testing immune responses in the skin and cornea.

A biodegradable and biocompatible material plays an important role in increasing demand for eco-friendly materials. Microfibrilated cellulose (MFC), an eco-friendly material for composite hydrogels, was obtained by acid hydrolysis of bacterial cellulose produced by Gluconacetobacteria xylinus strain in Kombucha. The effect of growth of ice crystals in composite hydrogels was studied by freeze drying. The composite hydrogel was prepared by introducing MFC fibers within cross-linked poly (N-isopropyl acrylamide) using freeze drying then subsequent polymerization. PNIPAM was cross-linked in a frozen state by photopolymerization with methylenebisacrylamide (MBA). The hydrogel film was purified by soaking in distilled water. The morphology of fibers and its influence were confirmed by Field Emission Scanning Electron Microscopy, equilibrium swelling ratio and rheological properties. This study reveals that freeze drying can induce fiber alignment and also influence the equilibrium swelling ratio of hydrogels.

Keratinocytes can be divided into stem cells, transit amplifying cells, and postmitotic differentiating cells. Epidermal stem cells can contribute to the maintenance of the epidermis via their self-renewing ability. Recently, it was suggested that cell-ECM interactions influence these findings. Thus, strategies for manipulating cell-ECM interactions hold promise in controlling skin disease or preventing aging effects. Epidermal cells are surrounded by neighboring cells and tissues. Thus, it is possible that epidermal cells are under the control of surrounding environment. Previously, we reported the effects of dermal sheath cells (DSC) on the maintenance of stemness of interfollicular epidermal stem cells and recovering effects on basement membrane. In that study, we found that IGFBP-2 is a major factor from DSCs which affects epidermal regenerative capacity of skin. Thus, we proposed stem cell activation as a new concept for the development of cosmetic or therapeutic agents. There are diverse cells in the skin and fibroblasts are also an essential component of skin. Thus, it can be hypothesized that fibroblasts also may affect the stemness of epithelial cells. The epidermal regenerative effect of both neonatal and child fibroblasts were compared in a skin equivalent model. These findings clearly showed that stemness of interfollicular stem cells can be regulated by surrounding factors of skin and stem cell activator can be useful in controlling skin aging and disease. Importantly, skin equivalent needs to be consistent to be used as a model for in vitro model. In this study, the use of Cervi Cornus Colla will be presented which greatly improve the consistency of skin equivalent to be used in experiment.

In order to identify plant natural products as anti-MRSA constituents, bioassay- guided solvent fractionation of methanolic extract of Eisenia bicyclis led to the isolation of active phlorotannins along with six known compounds eckol (EK), fucofuroeckol-A (FF), 7-phloroeckol (7-P), dioxinodehydroeckol (DD), phlorofucofuroeckol-A (PFF) and dieckol (DE), respectively. All the isolated compounds were tested for their anti-MRSA activities and the results were compared to each other. Among them, PFF showed the highest anti-MRSA activity compared to those of other phlorotannins tested. The MIC of PFF was determined to be in a range of 32 μg/mL against MRSA strains. Also, PFF was assessed its synergistic effects in combination with β- lactams, ampicillin, penicillin and oxacillin, against MRSA, suggesting PFF will disturb a drug-resistant mechanism in MRSA cells. In MRSA strain, it has been well known that penicillin binding protein 2a (PBP2a) plays a key role to acquire a drug resistance against β-lactam antibiotics. Therefore, it was investigated the effect of PFF on the expression of genes and protein related the drug resistance. PFF exhibited a significant direct inhibition on mRNA expression of mecI, mecR1 and mecA gene and synthesis of PBP2a protein in dose-dependent manner. These results provide a possible anti-MRSA mechanism of PFF. Thus, PFF interferes with the mRNA expression of PBP2a gene or regulator genes not the synthesis of protein. The results obtained in this study suggest that the compounds derived from E. bicyclis can be a good source of natural antibacterial agents against MRSA.

Quantification of mRNA provides information crucial for various biological studies. Real-time PCR is known to be the most accurate method for quantifying mRNA, and thus represents the state-of-the-art for gene expression analysis. However, the use of real-time PCR for mRNA quantification is limited to a single target per analytical run because of reductions in quantification power and limitations of fluorescence dyes associated with multiplex applications. Multiplex ligation-dependent probe amplification (MLPA) is an alternative multiplex analysis method. However, MLPA has not been widely used for expression analysis because it uses DNA-size-dependent electrophoretic separation, which complicates probe design process and compromises accuracy of the analysis. In this study, we developed a new version of MLPA, which uses a conformation-sensitive electrophoretic separation. We have demonstrated that our method could be used to monitor expression of genes in Escherichia coli, Caenorhabditis elegans and Arabidopsis thaliana.

In this study, sequential strategy based design was applied to optimize the microalgae, Pavlova lutherimass culture conditions and fermentation conditions of the cultured algae by proteolytic yeast Candidiarugopelliculosa to obtain small peptide chains. This optim ization of culture and fermentation conditionsby response surface methodology (RSM) finally leads to effective purification of a bioactive peptideMPGPLSPL (793.01 Da) with hydroxyl radical scavenging activity. Collectively, these results indicated thatmicroalgae P. lutheri can enhance the hydroxyl radical inhibiting effect through protein hydrolysis processunder RSM optimal condition.

Because human tumors have an acidic extracellular environment, nanoparticles (NP) undergoing physicochemical changes to release the enclosed drug at acidic pH conditions are promising vehicles for antitumor drug delivery. Among various drug carriers, high density lipoprotein (HDL)-like NPs have been shown to be beneficial for cancer chemotherapy, but have not been designed to be pH-responsive. In this study, we developed a pH-responsive HDL-like NP which selectively releases paclitaxel (PTX), a model antitumor drug, at acidic pH. While the well-known HDL-like NP that contained phospholipids phosphatidylcholine as well as apolipoprotein A-I (apoA-I) and PTX (PTX-PL-NP) was structurally robust at a wide range of pHs (3.8~10.0), the PTX-NP that only contained PTX and apoA-I as constituents uncoated its apoA-I shell and released the PTX into medium selectively at low pH. The pH-sensitivity was correlated to structural disorder of apoA-I shell. The PTX-NP was stable at physiological and basic pHs, and over a wide range of temperatures, which is a required feature for efficient cancer chemotherapy. The homogeneous assembly enabled high PTX loading per NP, which was 62.2 % (w/w). The molar ratio of apoA-I and PTX was 1:55, suggesting that a NP contains ~110 PTX in a spherical structure with a 9.2 nm diameter. Among several reconstitution methods applied, simple dilution following sonication enhanced reconstitution yield of a soluble PTX-NP, which was 0.66. This is the first pH-responsive NP that does not contain any synthetic polymer.

Retroviral integration provides us with a powerful tool to realize prolonged gene expressions that are often critical to gene therapy. However, the perturbation of gene regulations in host cells by viral genome integration can lead to detrimental effects, yielding cancer. The oncogenic potential of retroviruses is linked to the preference of retroviruses to integrate into genomic regions that are enriched in gene regulatory elements. To better navigate the double-edged sword of retroviral integration we need to understand how retroviruses select their favored genomic loci during infections. In this study we showed that in addition to host proteins that tether retroviral pre-integration complexes to specific genomic regions, the epigenetic architecture of host genome might strongly affect retroviral integration patterns. Specifically, retroviruses showed their characteristic integration preference in differentiated somatic cells. In contrast, retroviral infections of hES cells, which are known to display decondensed chromatin, produced random-like integration patterns lacking of strong preference for regulatory-element-rich genomic regions. Better identification of the cellular and viral factors that determine retroviral integration patterns will facilitate the design of retroviral vectors for safer use in gene therapy.

ecent development of microbial platforms for the production of bisabolene, a precursor of bisabolane, biosynthetic alternative to D2 diesel fuel, has been stimulated due to rising petroleum costs and environmental concerns (1). Microbial engineering based on Synthetic biology boosts the efforts to produce ‘Drop-In’ biofuels from renewable feedstocks. Synthetic biology aims to develop the foundational understanding and technologies to build intelligent biological systems to accomplish the breakthrough especially of metabolic engineering. In this presentation, I will present the IPTG-free bisabolene production system using engineered E. coli. Quorum-sensing (QS) mediated E. coli enables to induce neighboring cells as well as itself to initiate the carbon flux to the designated metabolic pathway without using expensive Isopropyl-b-D-1- thiogalactopyranoside (IPTG), a widely-used inducer, and showed the similar capability of genetic controls compared to the IPTG-induced system. Several genetic modules were constructed to link QS-system and biofuel pathway. Direct QS-system depending on cell density led the same level of bisabolene productions in E. coli. This QS-system for biofuels production could be a cost-effective and promising strategy to develop microbial platforms by reducing the usage of the exogenous inducer. (1) Peralta-Yahya et al., 2011 Nat. Commun.

It is often desirable to sequester cells in specific locations on the surface and to integrate sensing elements next to the cells. In the present study, surfaces were fabricated so as to position cytokine sensing domains inside non-fouling poly(ethylene glycol) (PEG) hydrogel microwells. Our aim was to increase sensitivity of micropatterned cytokine immunoassays through covalent attachment of biorecognition molecules. To achieve this, glass substrates were functionalized with a binary mixture of acrylate- and thiol-terminated methoxysilanes. During subsequent hydrogel photopatterning step acrylate moieties served to anchor hydrogel microwells to glass substrates. Importantly, glass attachment sites within the microwells contained thiol groups that could be activated with a hetero-bifunctional cross-linker for covalent immobilization of proteins. After incubation with fluorescently-labeled avidin, microwells fabricated on a mixed acryl/thiol silane layer emitted ~6 times more fluorescence compared to microwells fabricated on an acryl silane alone. This result highlighted the advantages of covalent attachment of avidin inside the microwells. To create cytokine immunoassays, micropatterned surfaces were incubated with biotinylated IFN-g or TNF-a antibodies (Abs). Micropatterned immunoassays prepared in this manner were sensitive down to 1 ng/ml or 60 pM IFN-g. To further prove utility of this bionterface design, macrophages were seeded into 30 mm diameter microwells fabricated on either bi-functional (acryl/thiol) or monofunctional silane layers. Both types of microwells were coated with avidin and biotin-anti-TNF-a prior to cell seeding. Short mitogenic activation followed by immunostaining for TNF-a revealed that microwells created on bi-functional silane layer had 3 times higher signal due to macrophage-secreted TNF-a compared to microwells fabricated on mono-functional silane. The rational design of cytokine-sensing surfaces described here will be leveraged in the future for rapid detection of multiple cytokines secreted by individual immune cells.

Skin homeostasis is interrupted during UV-A irradiation. How the UV-A altered skin components influences photoaging of skin should be investigated using human in vitro models that are important for understand skin aging. In the present study, chromene compound was isolated from Sargassum horneri and its potency inhibition of photoaging was investigated in UV-A irradiated dermal fibroblasts. Effects of compound on the prevention of photoaging were evaluated by measuring ROS production, membrane protein oxidation, lipid peroxidation and aging related gene expression in UV-A irradiated human skin dermal fibroblasts. The results indicated that treatment with compound suppressed the collagen degrading MMPs, MMP-1, MMP-2 and MMP-9 expression without any cytotoxicity and phototoxicity. It was further found that these inhibitions were due to increase in the expression of TIMP-1 and TIMP-2 genes. Furthermore, we confirmed that the UV-A induced transcriptions of AP-1 signaling pathway was regulated by compound treatment in UV-A irradiated dermal fibroblasts. Therefore, this study suggests that compound isolated from S. horneri can be developed as topical application of natural anti-aging cosmeceuticals as it reduced the skin collagen decomposition caused by UV-A irradiation.

Precise prediction of prokaryotic translation efficiency can provide valuable information for optimizing bacterial host for the production of biochemical compounds or recombinant proteins. However, dynamic changes in mRNA folding throughout translation make it difficult to assess translation efficiency. Here, we systematically determined the universal folding regions that affect the efficiency of translation arising via two different initiation mechanisms in Escherichia coli. By assessing the exquisite regions for mRNA folding, we could construct a predictive design method, UTR Designer, and demonstrate that proper codon optimization around the 5’-proximal coding sequence is necessary to achieve a broad range of expression levels. Finally, we applied our method to control the threshold value of input signals switching on a genetic circuit. This should increase our understanding of the processes underlying gene expression and provide an efficient design principle for optimizing various biological systems, thereby facilitating future efforts in metabolic engineering and synthetic biology.

Osteoclasts are responsible for bone resorption on bone erosive surfaces in the marrow, which are key contributors to the bone pathogenesis of osteoporosis and rheumatoid arthritis. The induction of primary signals for osteoclast development and maturation depends on receptor activator of NF-κB ligand and macrophage-colony stimulating factor. Membrane bound signal adaptors such as FcRγ and DAP12 are also associated with osteoclastogenesis, supporting the cell surface expression of the activating-type immunoreceptors of the Ig superfamily or C-type lectin family. Anti-CLEC5A monoclonal antibodies to stimulate osteoclast formation were generated by rat immunization with mouse CLEC5A extracellular domain from E. coli and 5 antibodies showed their binding specificity with CLEC5A in western blot. Murine osteoclast differentiation model was established with RAW 264.7 cells to evaluate the antibody effect on osteoclastogenesis. Anti-CLEC5A monoclonal antibodies enhanced in vitro osteoclastogenesis and cell fusion as agonists and bound with U937 cells expressing human CLEC5A to suggest a potential cross-reactivity towards human CLEC5A.

A typical butanol producing clostridium such as Clostridium acetobutylicum ATCC 824 rarely yields higher than 13 g/L of butanol in the batch culture due to solvent toxicity, feedback inhibition, etc. In this study, the pfkA (6- phosphofructokinase) and pykA (pyruvate kinase) genes of C. acetobutylicum ATCC 824 were over-expressed in the same strain in order to enhance the butanol production possibly by lowering the inhibitory effect of the solvent. These genes were PCR-amplified from the genomic DNA of C. acetobutylicum ATCC 824 and inserted into the pSOS94 plasmid under the control of phosphate butyryltransferase promoter. The recombinant plasmids (pSO94/pfkA, pSO94/pykA, and pSO94/ pfkA+pykA) were then electroporated back into C. acetobutylicum ATCC 824. In a 5 liter fed-batch bioreactor, butanol and ethanol concentrations of ATCC 824/ pSO94/pfkA+pykA reached 19.03 g/L and 2.93 g/L, respectively, which were 32.7% and 1.08% higher than those from the wild type C. acetobutylicum ATCC 824. The total solvent concentration also increased by 25.7%. Interestingly, the acetone concentration of ATCC824/pSO94/pfkA+pykA was lower by 12.1% than that of the wild type. The intracellular NADH and ATP concentrations in the solventogenesis of the strain ATCC 824/pSO94/pfkA+pykA were also higher than the wild type C. acetobutylicum ATCC 824, which were thought to improve butanol production and tolerance of the recombinant strain to solvent toxicity.

Droplet-based microfluidic systems have emerged as a powerful platform for performing high-throughput biological experimentation. Fluorescence polarization(FP) measurements have been used to probe many biological reactions such as receptor-ligand binding, DNA hybridization, DNA-protein binding, peptide-protein binding, immunoassays and enzymatic degradation. FP detection is particularly advantageous since it is truly homogeneous assay format, allows for real-time (kinetic) measurements and is insensitive to concentration variations. For these reasons, we report herein the use of FP detection in a droplet-based microfluidic system.

Large and complex biomolecules are becoming increasingly important in new biotherapeutics. They include very large complex proteins, conjugates, immunoglobulins, pDNA, viruses, viral nanoparticles and virus-like-particles. These large biomolecules ranging from a few hundred to millions of kDa and up to hundreds of nanometers in size, pose great challenges not only in biomanufacturing but also in-process bioprocess monitoring and analysis of such biomolecules. Monolithic bioseparation media, in contrast to conventional particle-based media which is largely dependent on pore diffusion for mass transfer, consist of a single piece of highly porous polymer material with highly interconnected network of large channels (diameter > 1500nm), within which the biomolecules are instantly transported to the binding sites by convective flow, resulting in very efficient and fast mass transfer between the mobile and stationary phase. As a consequence, the separation of these large proteins, viruses, VLPs and pDNA can be achieved within seconds to a few minutes with very high resolution and binding capacity independent of flow rate. Even very short column just a few mm in height and 100ul in volume, provides extremely good separations in less than 10 minutes making it very suitable for analysis, in-process monitoring as well as process development of large and complex biomolecules

Recent biomimetic engineering has been driving toward a multifunctional cellular mimic system, which contain an agglomeration for all living metabolites: a mimicry of a nuclear pore transporter for selective mass transfer at the interface of nucleus and cytosol1,2; a virus-like assembly for effective drug delivery3; an artificial dendritic cell for tunable immune system.4 Inspired by these achievements, we sought to design a genenetworking hydrogel (G-net-gel), resembling an organized chromosome in the cell nucleus. It was spherodically manufactured using both simple lithography and DNA engineering methods. Stable transgene crossnetworks have been optimized in various parameters to accomplish enhanced in vitro protein expression efficiency. Furthermore, once with lipid capping, it completely reproduced the natural nucleus system, demonstrating the improved level of messenger RNAs relative to solution phase vectors. It is speculated that the proto-nucleus biomimetics would be a substitute to the conventional gene therapy, by which it overcome several barriers such as impaired transgene incorporations, and be a ground-breaking stratagem for gene-associated molecular therapeutics.