Earticle

A new covalent immobilization method for small proteins and short peptides is presented by using parylene-H film which, is a polymer of p-xylene having formyl groups. The covalent coupling of proteins to the parylene-H film is produced by only one step of incubation of proteins or peptides without additional coupling reagents. In this work, the parylene-H film is coated on a 96-well microplate for immunoassays. The immobilization efficiency to the parylene-H film was compared with the conventional physical adsorption by using human chorionic gonadotrophin protein and a small peptide called circulated citrullinated peptide as model molecules. Additionally, the applicability of this immobilization method for short peptides is demonstrated by detecting autoantibodies in rheumatoid arthritis patient serum.

Incorporating non canonical amino acids (NCAAs) into proteins is a powerful in vivo methodology that has been increasingly used. Currently, a number of NCAAs was incorporated into protein with the help the methodology such as reassignment of sense (residue specific incorporation) and non sense codon (site specific incorporation). Both methods balance one another in many ways. However, these two methods have a similar drawback in that they allow only incorporation of a single NCAA into the recombinant protein. Our group has developed an easy method to introduce two NCAAs containing different chemical moieties by coupling residue-specific and site-specific incorporation methods in a single protein. So far, all the techniques have demonstrated the possibility of introducing MNCAAs into a single protein; however, no studies have effectively utilized a multifunctional single protein in an effective way to show potential applications. Here, we used the MNCAA incorporation technique for self oriented immobilization (site- specific), to improve protein functionality, and for protein labeling (residue specific). For the purpose, we selected the surface exposed N-terminal residue (Lys15) of the green fluorescent protein (GFP) as a model protein. We introduced an amber codon (Lys15TAG) for the site- specific incorporation of L-DOPA using an evolved Methanococcus jannaschii tRNA/synthetase pairs and simultaneously (2S, 4S)-4-fluoroproline (4S-FPro) was selected for residue specific incorporation. In the next experiment, we prepared the MNCAAs protein with the methionine (Met) surrogate L-homopropargylglycine (L-HPG) (residue-specific incorporation) along with L -DOPA (site-specific incorporation) in GFP (GFPdphpg). The site-specific incorporation of L-DOPA into the protein will allow the protein to be immobilized in a controlled manner through Michael addition, the (2S, 4S)-4-fluoroproline (4S-F-Pro) was selected to improve protein stability. L-HPG contains an alkyne moiety and it will undergo a cycloaddition reaction with azide bearing molecules. We demonstrated that this method allows for site-specific attachment of the protein in a molecule for a subsequent labeling reaction. Incorporating MNCAA reduced the number of steps required for multiple labeling. We trust that the MNCAA protein engineering tool kit will occupy an important place in protein modification, as it offers numerous benefits and opportunities to develop synthetic biology research.

N,N′-diBoc-dityrosine (DBDY) was conjugated with two molecules of glycine and isoniazid (INH), and tested as a substrate for the protease assay. In DBDY-(Gly-INH)2 and DBDY-(Lys-INH)2, the fluorescence of DBDY (λex=284~320 nm; λem=400~420 nm) disappeared due to the quenching effect of INH. However, the fluorescence recovered upon the protease-catalyzed hydrolysis leading to the release of INH. When cysteine proteases (papain, chymopapain, and bromelain), serine proteases (chymotrypsin, trypsin, subtilisin, and proteinase K), metalloproteases (thermolysin and carboxypeptidase A, dispase, and collagenase), and aspartic proteases (pepsin and aspergillopepsin) were chosen and tested, DBDY-(Gly-INH)2 and DBDY-(Lys-INH)2 showed the selectivity to the cysteine proteases. Cathepsins B, L, and S are classified as the cysteine proteases and have gained lots of interest because they are found at high levels in various cancers. When DBDY-(Gly-INH)2 and DBDY- (Lys-INH)2 were tested as a substrate for these cathepsins, cathepsin B and cathepsin L catalyzed the hydrolysis reaction except cathepsin S.

In this study, we designed and applied molecular biosensors for heavy metals, zinc and copper, for use in bioremediation strategies. Bacteria utilize two component systems to sense changes in the environment by multiple signal components including heavy metals and control gene expression in response to changes in signal molecules. zraP and cusC promoters were selected from a genetic circuit of the ZraSR and CusSR two-component system and were fused to a dual-labeling reporter protein as an interactive biological component for zinc and copper to generate a signal from the constructed biosensor. The biosensor efficiently senses zinc and copper with a calculated detection limit of 16 μM and 26 μM respectively and was shown to be a sensitive and effective heavy metal monitoring bacterial system. To extend the application of the bacterial biosensor, we assembled a bioadsorption system that can trigger bacteria to sense and adsorb 13±0.3 mg/L of zinc and 11.4±0.42 mg/L of copper per gram of dry cell weight with induction at a concentration of 100 mg/L of the respective metal ion. This work was supported by a grant from the Next-Generation BioGreen 21 Program (SSAC, grant number: PJ008057), Rural Development Administration, Republic of Korea

Contractile force of myotubes is one of key parameters for studying diseases related to muscles. However, there is lack of an efficient and affordable device for generating single myotubes and quantifying the contractile force of each single myotube in vitro. In this study, we describe a novel cell culture device equipped with elastic posts for measuring contractile force generated by a single myotube in situ. The microchannel cell culture system is composed of an array of microchannels with a dimension of 50 μm (width) x 500 μm (length) x 100 μm (height) having PDMS (polydimethyl siloxane) pillars with a dimension of 30 μm (diameter) x 100 μm (height) located at both ends of each channel. When C2C12 myoblast cells were cultured in the microchannels for several days, they were fused to form multi-nucleated single myotubes and then started to be attached to pillars in the channel. Since the contractile force of each myotube can be calculated from the bending degree of micropillars pulled by a single myotube, it is expected that our device can be useful tool for studying the mechanical characteristics of myotubes in vitro.

Human adipose tissue-derived stem cells (ATSCs) can be differentiated into multiple mesodermal lineage cells through normal differentiation processes. They can be also trans-differentiated into some endodermal or ectodermal lineage cells through re-differentiation and de-differentiation processes. Here, we developed microfluidic culture system to induce neurogenic trans-differentiation potency of hATSCs. In this system, aggregates of neurospheres derived from hATSCs were trapped by arrays of micropillars in microchannels and cultured in neurogenic medium. After 3 day of the chip culture, HIF1α expression was significantly increased with upregulation of Oct4 and Nanog expressions, which indicated de-differentiation of hATSCs into more immature stem cells. After 7 days, cells actively spreaded out from the cell aggregates and started neural differentiation expressing TuJ-1. Then, they were fully differentiated into GABAergic neurons after 14 days. These results suggest that the microfluidic system can offer stem cells in-vivo like niches for cell proliferation as well as differentiation.

Ionic liquids(ILs) have many advantages like low-volatility, Nonflammability and Electrical conductivity, so the ILs are widely used in various fields including extraction, sensors, refining materials and chemical catalysis. Although ILs are called to green solvent due to their high recovery rate, micro-spill to the environment is inevitable. In this work, we study Shewanella oneidensis MR-1 to elucidate the toxic mechanism of ILs to resolve problem. We assume that the major cause of cell death is the perturbation of cell membranes, resulting in the physical disruption of its structure and the interferences of signaling pathway. To further study the membrane interaction of ILs, we use a model sell membrane composed of amphiphilic biopolymer, polydiacetylen e(PDA). In particular well ordered PCDA monomers in a liposome are crosslinked by 1,4-addition by UV, showing red color with the naked eyes. To ensure the membrane perturbation of ILs we employ other analytical tools including differential scanning calorimetry(DSC) and particle size analyzer. We expect that our work will provide more comprehensive understandings of the effects of ILs to biological organisms, particularly in cells at a molecular level.

Type I collagen is the structural and functional scaffold in the extracellular matrix in skin and other tissues. The catabolic and anabolic process for collagen is in a highly regulated and balancing order, inclination in either the process leads skin disease or aging. MMP-1 is mainly responsible for degradation of the collagen-1, and the expression of MMP-1 is influenced by several factors (extrinsic of intrinsic). Thus regulation in MMP-1 action either by enzyme inhibitor or down regulation could solution for delayed aging. Our focus in the present study is to screen the non-active site binding inhibitor which specifically bind to MMP-1. To evaluate the binding affinity and inhibitor properties, we used Surface Plasmon Resonance based biosensor. The collagen peptide was immobilized on gold nanochip while MMP-1 was used in flow, and the response observed in response to BSA (control), and MMP-1-Inhibitor (Putative) was compared and the relative affinity was calculated. The results encourage us to screen a wide range of molecule having a similar skeleton to develop new class of anti-aging agent, through this SPR based biosensor as a label free high-throughput screening.

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.

Recombinant Escherichia coli expressing organophosphorus hydrolase were directly immobilized onto mussel adhesive protein-coated surface of the wells of polystylene microplates. To enhance whole cell biocatalytic efficiency, we engineered E. coli to secrete organophosphorus hydrolase into periplasmic space by aid of chaperone GroEL/ES. Immobilized cell was confirmed by fluorescent microscopy using DeRed protein-expressing E.coli. Mussel adhesive protein can be effectively used to immobilize cell on surface: the strong adhesion ability of the MAP facilitates efficient and direct immobilization of cell on surface with no cross-linker and/or surface modifications. The recombinant E. coli-immobilized microplate was associated with optical transducer (microplate reader). Linear correlation between relative signal intensity and antigen concentration was shown with a high coefficient of determination (R2) of over 0.9933 in the antigen concentration range of 60–500 μM.

We herein introduce the development of a novel multiplexed single-nucleotide polymorphisms (SNPs) genotyping method based on amplification of separated ligation-dependent probe (ASLP) and RecA assisted hybridization (RAH) technology. In the ASLP technique, allele-specific ligation reaction was first performed between annealed allele-specific oligonucleotide (ASO) probe and locus-specific oligonucleotide (LSO) probe containing the unique regions of homology to target genomic DNA. The ASO probe covering up to SNP site is modified with universal forward primer site at the 5’ end while the LSO probe is extended with universal separation (US) site and poly A tail at the 3’ end. The two probes would be ligated when they are hybridized to a correctly matching sequence at a specific SNP locus. The separation probe that consists of universal reverse primer site labeled with biotin at its 5’ end and complimentary sequence of US site at its 3’ end was then applied to the resulting ligated product. During the following extension reaction of the separation probe, the ligated probes were dissociated from target genomic DNA as a form of double-stranded DNA (dsDNA) with the strand extended from the separation probe. By using streptavidin-coated magnetic beads, the dsDNA containing ligated probes could be simply separated from the reaction mixture including genomic DNA and unligated probes. PCR amplification of the purified ligation products was then carried out using a primer pair of Cy3-labeled universal forward primer and universal reverse primer, followed by RAH of the PCR products on DNA microarrays. Using this strategy we successfully genotyped fifteen SNPs markers for the cultivar identification of pepper in a single tube reaction within 3 hours.

An integrated microdevice of a reverse transcription-polymerase chain reaction (RT-PCR) reactor and an immunochromatographic strip (ICS) was constructed for colorimetric detection of gene expression of influenza A virus subtype H1N1. An RT-PCR cocktail, which included Texas Red-labeled primers, dNTP including biotin-labeled dUTP, and RNA templates of influenza A H1N1 virus, was filled in the PCR chamber through the micropump, and the RT-PCR was performed to amplify the target H1 gene (102 bp). The resultant amplicons bearing biotin moieties and Texas Red haptens were subsequently eluted to the ICS, in which they were first conjugated with the Au NP labeled anti-hapten antibody in the conjugation pad, and then captured on the streptavidin coated test line through the biotin–streptavidin interaction. By observing a violet color in the test line we confirmed the H1N1 target virus. The entire process on the integrated microdevice consisting of a micropump, a 2 μL PCR chamber, and an ICS was carried out on the portable genetic analyzer within 2.5 h, enabling on-site colorimetric pathogen identification with detection sensitivity of 14.1 pg RNA templates.

Capillary electrophoresis-single strand conformation polymorphism (CE-SSCP) analysis is known as a good molecular marker detection technique due to its sensitivity and reproducibility with conformation dependent separation of single strand DNA. For high resolution CE-SSCP analysis, new polymer matrix suitable for CE-SSCP, poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) triblock copolymer (PEO-PPO-PEO) was developed for separation of same-sized DNA fragments. The previous research showed improved resolution in the case of 15wt% condition of F108 polymer. In this research, effect of the heterogeneity in Pluronic polymer matrix was investigated with various blending method, ratio and length of each polymer. The two blending methods were used, Bi-molecular micelle blending and micelle mixture blending. As a result, heterogeneity generally reduced the resolution, except F108-F127 bi-molecular micelle blending which showed less heterogeneity in both blending method and polymer length. The result also implies that the character of micelle in the Pluronic polymer matrix is a critical factor, thus further optimization might give a better resolution.

Development of a rapid, sensitive, and multiplex pathogen DNA sensor enables early diagnosis and subsequently proper treatment of infectious diseases, increasing the possibility of saving lives of infected patients. Here we report the development of an ultrasensitive and multiplex pathogen DNA detection method by combining patterned Au nanowire (NW)-on-film surface-enhanced Resonance Raman scattering (SERRS) sensor with exonuclease III-assisted target DNA recycling reaction. Combining the high sensitivity of NW-on-film sensor and the target recycling reaction significantly improved DNA detection performance, resulting in the detection limit of 100 fM corresponding to 3 amole. By positioning Au NWs at specific addresses, multiple pathogen DNAs could be identified in a single step. Clinical sample tests with multiple genomic DNAs of pathogens showed the potential of this sensor for practical diagnosis of infectious diseases. [This research was supported by WCU program through the Korea Science and Engineering Foundation funded by the Ministry of Education, Science and Technology]

We described the fabrication method of microfluidic channels with circular-cross sectional geometries by reflow phenomenon of thick positive photoresist. Rectangular shaped positive photoresist micropatterns on a Si wafer were fabricated by a photolithography and then melted by heating at 105 °C, which leads to shape change from rectangular to semicircular. For the perfect cylindrical micropatterns, we optimized the reflow conditions by using positive photoresist and the resultant circular micropatterns were used as a template for generating a PDMS circular microchannels. The diameter can be varied from several to several hundred micrometers, and the channel routes can be easily patterend such as straight, S-curve, x-shape, cross shapes, Y and T-shape for mimicking the for vascular network.To form the artificial vascular network, we cultured the HUVECs inside the PDMS micro circular channels to make the endothelial layer. Similar to the in-vivo situations, HUVEC cells were adhered, proliferated and aligned to the direction of the microchannel, suggesting that the simple reflow method can produce a PDMS circular microchannels to mimick the in-vivo like 3D artificial blood vessel.

Genetic diseases were caused by genetic mutations in genes that change amino acid or splicing sites. For the effective treatment of these diseases, mutation analysis must meet the demands for rapid, accurate, simple, cost-effective factor. In this study, the sensitive, specific, and quantitative detection of single mutation causing genetic disease was accomplished by combining specificity of S1 nuclease reaction with well-defined Au nanowire (NW)-on-film sensor. We detected single base mismatch of target DNA at a detection limit of 100 pM through the simple experimental process. Wilson disease (WD) and Avelllino corneal dystrophy (ACD) relevant mutations from clinical samples were successfully identified using Au NW-on-film sensor. We anticipate that our assay could be improved to high throughput mutation detection by using the tremendous multiplexing capacity. [This research was supported by WCU program through the Korea Science and Engineering Foundation funded by the Ministry of Education, Science and Technology]

Nucleic acid extraction/purification is an essential step for many biomolecular diagnostics. In particular, the on-site virus detection system requires to obtain the DNA/RNA from biological samples with ease and high rapidity [1]. However, the conventional nucleic acid extraction method such as phenol extraction [2] or ethanol precipitation is highly labor intensive and time consuming due to numerous manual steps, and it is not adequate for the on-site sample preparation. To address this issue, we have developed a rotary microfluidic system to extract and purify the RNA without necessity of mechanical external syringe pumps to allow flow control. We designed three reservoirs for sample, washing buffer, and elution buffer which were connected with different dimensional microfluidic channels. By controlling RPM, we could dispense a RNA sample solution, a washing buffer, and an elution buffer successively, so that the RNA was captured in the sol-gel solid phase, purified, and eluted in the downstream. Such a novel rotary sample preparation system eliminates any complicated hardware and human intervention, and performs the RNA preparation with high speed and portability.

A method is described for the characterization of sexual transmitted diseases (STD) infections by restriction fragment mass fingerprint analysis. Through analysis of over 35,000 clinical cases, 14 pathogens which are most abundantly found among Koreans were selected and primers containing restriction enzyme region and mass fragment sequences according to each type of pathogen were designed by considering their sequences. A multiplex PCR method was established for the diagnosis of multiple infections to STD. Amplified genomic nucleic acids encoding the pathogenicity-determining region of all pathogens were digested with a restriction enzyme, and the restriction fragments were analyzed by matrix-assisted laser desorption-ionizationtime- of-flight mass spectrometry (MALDI-TOF MS). This method employed to diagnose reliably both single and multiple infections in clinical human samples for 14 target pathogens. The procedure described is rapid, inexpensive and compatible with automation.

Fluorescence polymers have received a great deal of interest due to their unique physical properties such as conductivity, electroluminescence. Moreover, polyphenylenevinylene (PPV) and their derivatives are currently the most popular class of conjugated polymers used in electronic materials, such as organic light-emitting diodes (OLEDs). Hybrid nanomaterials containing two or more functionalities have been received attention as attractive candidates for biomedical application. Furthermore, superparamagnetic iron oxide (Fe3O4) nanoparticles have been extensively studied for use as magnetic resonance imaging (MRI) contrast agents. In this study, we prepared multifunctional nanoparticles incorporating fluorescent polymer and Fe3O4 nanoparticles, and coated by ionic polymer layers. Properties of prepared multifunctional nanoparticles were characterized by transmission electron microscopy (TEM) and particle analyzer, UV spectrometer and PL spectrometer. The bioapplication of nanoparticles was evaluated by specific binding capability, cell permeability and viability by optical and MR imaging. Consequently, we expect that our nanoparticles will have considerable potential as a powerful novel material within the emerging field of biomedical, such as drug delivery, therapeutics and magnetic resonance imaging (MRI) contrast agents.

Among proteases, matrix metalloproteinases (MMPs) have been of significant interest because they are considered as one of promising biomarkers in association with cancer metastasis, inflammation and other degenerative diseases. Many attempts based on the optical sensing have been made to analyze the activity of MMPs, but most of them require an expensive fluorescent readout and a labor-intensive process. To circumvent this issue, we demonstrate here gold nanoparticle (AuNP)-based colorimetric assay which allows MMP activity to be monitored in a simple and rapid way. This approach is based on the self-aggregation of AuNPs through the coordination effect between the carboxyl groups of AuNPs and hexahistidine at both ends of peptide substrates in the presence of divalent metal ions. The AuNPs were easily aggregated and changed in color (from brown-red to violet) after adding the peptide substrates and metal ions without MMPs, whereas the presence of MMPs in solution prevented the NP aggregation by cleaving the peptide, thus retaining the original color of the AuNPs. Based on this principle, we analyzed the MMP activity in terms of metal ion species, buffer strength, assaying time, and MMP concentration. Given the specificity of targeting MMPs by controlling the peptide substrate, this approach is anticipated to dissect the physiological roles of many MMPs from complex biological media in a high-throughput manner.

In April 2009, H1N1 influenza (swine flu) was identified for the first time and the virus has spread worldwide to become pandemic. The infection with swine influenza can only be confirmed by polymerase chain reaction (PCR) assays. However, other special PCR assays are needed to sub-typing and further characterization. Here, we developed a new method to discriminate various types of influenza A, including H1N1, using stuffer-free multiplex ligation-dependent probe amplification based on a capillary electrophoresis–single-strand conformation polymorphism. Unlike conventional methods, this approach precisely detects five relevant gene markers permitting confirmation of infection. As a result, all the five genes were respectively identified using synthetic genes and also verified with total RNA of the H1N1 influenza virus. The method had a limit of detection of approximately 32.5 pfu/ml, which is similar to that of the PCR-based technique. These results indicate that the RT-MLPA-CE-SSCP system have considerable potential in clinical diagnosis.

Single nucleotide polymorphisms (SNPs) are one-base DNA sequence variations that are responsible for various inherited diseases. Moreover, SNPs can cause difference of drug response because SNPs have an effect on phenotype of human. So, there is increasing demand for accurate, simple, and cost-effective SNP genotyping method, particularly for method that is commensurate with clinical use. Conventional genotyping methods such as ligase-based methods are limited to apply genotyping for SNPs because mismatched-base hybridization is always generated, so mismatched hybridization and complementary hybridization is not discriminated in SNP site. Here, we report the simple and accurate multiplex genotyping method based on capillary electrophoresis-single strand conformation polymorphism (CE-SSCP) by reaction condition optimization. We tried to overcome mismatch occurred and discriminate SNP sites by controlling concentration of ligase, probe, and template and ligation time. And then ligated products are separated by CE-SSCP.

Protein kinases play a key role in numerous cellular functions and have been the targets for investigation of specific signaling pathways as well as for development of therapeutic drugs. Rapid and simple analysis of kinases activity, therefore, is prerequisite for understanding various physiological and pharmacological functions of the kinases. Here we demonstrate a rapid and simple method to detect the phosphorylation of peptide substrates by using the fluorescence resonance energy transfer (FRET) between quantum dot (QD) as a donor and the dye-tethered peptide as an acceptor. Compared to a nonphosphorylated peptide, a synthetic phosphorylated one was strongly attached on the surface of the carboxyl QD in the presence of ZnCl2, due to a metal-coordination effect, thus leading to higher energy transfer efficiency. Metal ion-free condition and other divalent ions (CoCl2, ZnCl2, and FeCl2) did not give rise to the change in the energy transfer. Upon kinase reaction, the energy transfer efficiency between the QD and the peptide substrate was modulated. Based on these observations, we anticipate that our developed method will have a great potential for analyzing protein kinase activity with high sensitivity and thus will be useful for real-time monitoring.

As late-stage cancer diagnosis results high death rate, it is desirable to develop methods for early diagnosis. As aberrant DNA methylation of CpG islands in promoter has been described in human cancer, it is suggested as a strong candidate for diagnostic biomarker. Though several studies have identified aberrant DNA methylation of many genes in cancer, critical gene target remain largely unknown. To find critical genes, validation through population study is needed. Methylation sensitive (MS-) multiplex ligation-dependent probe amplification (MLPA) has great potential as a screening tool for critical genes target. But this method has limitations: designing a custom MLPA probe is difficult due to stuffer sequence, and only limited target site can be analyzed due to sequence specific restriction enzyme. In this study, we developed a novel DNA methylation analysis method based on MS-MLPA, in which stuffer-free MLPA probes and three restriction enzymes are used for precise multiplex quantitative analysis. We tested our new method to analysis 28 cancer candidate genes related with hepatocellular carcinoma, which is common cancer type.

Wheat(Triticum aestivum) is widely cultivated as a cash crop because it produces a good yield per unit area, grows well in a temperate climate even with a moderately short growing season, and yields a versatile, high-quality flour that is widely used in baking. Recently wheat sprout gained much attention because of its nutritional and physiological properties. The amount of antioxidant compound strongly increases during the germination phase, while scantly present in the wheat germ, and virtually absent in the young wheat plant. Thus wheat sprouts contain a remarkable level of various antioxidants. In this study, based on these facts, aqueous and ethanolic extracts from wheat sprout powder were analyzed to determine its reduction and antioxidant activities and antimicrobial activities as well. In addition the effect of wheat sprout extract on hair growth stimulation and/or growth inhibition using C57BL/6 mice. The extracts and the control compounds were topically treated once a day for 4 weeks. The hair growth was photographically and historically examined during the period. Hair growth of the test group (3% extract) was better than that of positive (3% minoxidil) and negative control groups (cosmetic formulation without the extract and no treated groups). The whole data showed remarkable improvement of hair growth effect of wheat sprout extract but needs more statistical analysis. These results suggested that wheat sprout extract could be used in making scalp-related cosmetics and shampoo products in the near future.

Camellia japonica has gained many attentions because of cosmetic and medicinal ingredients in its leaf and seed. Thus C. japonica has been selected as a representative plant for commercial growth in Jeonnam region nowadays. In this presentation C. japonica L. has been used as a main functional raw material to make a skin care and hair essence product. C. japonica L. leaf extracts were used to prepare skin care products and seed oil for hair essence products. Based on clinical trials, skin care product showed an acne diminishing activities in 4 weeks and hair essence product for a hair cuticle recovering activities. These activities are considered to stem from several ingredients in camellia leaf and seed. To elucidate the skin effect, firstly, antioxidant activity assays were performed. For hydroxyl radical scavenging activity hot water extracts of camellia leaf showed better activity than that of 40% ethanol extracts. Several kinds of flavonoids such as quercetin and kaempferol as well as polyphenols such as (-)-Epicatechin (EC) and (-)-Epicatechin gallate (ECG) have been found in hot water extracts of Camellia japonica leaf. The average amounts are as follows: quercetin (46.165 mg/L), kaempferol (156.273 mg/L) and (-)-Epicatechin (EC) (21.266 mg/L), (-)-Epicatechin gallate (ECG) (7.019 mg/L). Next the antimicrobial activities were tested using ampicillin as a standard antibiotic. Comparing a clear zone diameter, hot water extracts of camellia leaf showed 56% of 100 μg/mL ampicillin. The ethyl acetate fraction showed less activity than that of water fractions throughout this study. For hair recovering effect, the oil composition of seed oil has been considered to play an important role. As a control experiment, olive oil was used to prepare the formula and the effect was less than using camellia seed oil. By comparing the oil compositions of olive and camelia oils, the amount of unsaturated fatty acids was different which might be crucial in hair cuticle recovery. As a result, the skin and hair cosmetic formula showed good stability with no change of color and appearance for several months. In summary, C. japonica L. and seed could be valuable raw materials for cosmetic applications and further studies including the purification of an effective single compound should be done before commercializing this kind of botanical products.

Delayed onset muscle soreness (DOMS), also called muscle fever, is the pain and stiffness felt in muscles several hours to days after unaccustomed and/or strenuous exercise. After strong exercise, the muscle adapts rapidly to prevent muscle damage, and thereby DOMS, in repeated bout. While, myofascia release and massage are forms of soft tissue therapy used to treat somatic dysfunction and accompanying pain and restriction of motion. This is accomplished by relaxing contracted muscles, increasing circulation, increasing venous and lymphatic drainage, and stimulating the stretch reflex of muscles and overlying fascia. In this work, the effects of myofascia massage on the reduction of myalgia and muscle relaxation, and recovery of DOMS of lower body have been examined using algometer and myotomometer analyses. Ten young male patients were participated in the study to analyze the difference of muscle tension and blood constituents after the massage treatment was applied. Visual analog scale showed the significant difference between control and treated groups (p<0.05). The myalgia decreased a lot after the massage. These data were supported by several assays of blood biomarkers such as cortisol, creatine, C-reactive protein and lactate dehydrogenase. All the blood factors concerning DOMS decreased after the massage treatment resulting in the positive aspect of the myofascia massage. In summary the myofascia massage has been considered to show the positive psychological effect on reduction of myalgia and muscle relaxation, and recovery of DOMS of lower body as well.

Poly-γ-glutamic acid (γ-PGA) is a bio-polymer traditionally produced during the fermentation of soy based nutrients by Bacillus subtilis, a naturally occurring microorganism fundamental to fermented soy foods known as “natto” in Japan and “chungkookjang” in Korea. The polymeric γ-PGA, which gives those foods their unique sticky texture, is essentially a chain of gamma-linked D and L glutamic acid peptides. This makes γ-PGA more resistant to proteases found on the skin that can rapidly breakdown protein and other peptide ingredients added to many skin care products. BioLeaders’s γ -PGA, HumaMAX brings significantly improved functionalities and has novel modes of action that can contribute to the formulation of truly differentiated skin care products improving the health of the skin including moisture and structure of the skin. Personal care formulators are constantly seeking ingredients that allow for innovative and differentiating products. Gamma-polyglutamic acid is one such material. Recent work has added to knowledge about its value in providing moisturizing and soothing effects of skin in conjunction with, or in place of hyaluronic acid.. This work discusses various aspects of a newly commercialized highly functional form of γ-PGA. [This work was supported by a grant from the Next-Generation BioGreen 21 Program (No. PJ0081642011)]

The present studies have been carried out to evaluate the variation of three ethnic groups (Malaysian, Korean and Vietnamese) in Asian stratum corneum lipid profile. Stratum corneum lipid plays a predominant role in maintaining the water barrier of the skin. In order to understand the biological variation in the levels and composition of cholesterol, fatty acids and ceramides, stratum corneum lipids collected from solvent extraction from inner forearm of three ethnic groups were analysed. In addition we studied the correlation of stratum corneum lipid composition on the skin barrier function and epidermal thickness from the same area. Stratum corneum lipids are separated into three main lipid classes: cholesterol, fatty acid and ceramide. In this study, a salicylic acid column chromatography (SACC) method was successfully developed to separate the three lipid classes from stratum corneum lipid samples. The main lipid species were quantified using high performance thin-layer chromatography (HPTLC) and individual fatty acids using gas chromatography (GC). Our finding demonstrated significantly lower amounts of total stratum corneum lipid and level of ceramide composition in Malaysian subjects compared to Korean and Vietnamese. In parallel, Malaysians were placed in the third position for skin barrier function, expressed in TEWL. In contrast, Malaysians had the thickest epidermis as measured by confocal reflectance microscopy (CRM). This pilot study suggests that among the Asian subject itself manifest skin conditions differently. Therefore, the individual fatty acid profile was carried out to provide valuable information on skin biological variations among the three ethnic groups.

Human skin is composed of epidermis, dermis and cutaneous fat, which protects human body in a physical and physiological way. Especially, stratum corneum that is outmost layer of epidermis contains corneocytes and lipids, thus it carries out skin barrier function. The skin barrier is sustained by epidermal homeostasis that comprises proliferation and differentiation of epidermal keratinocytes, controlling skin moisturization and preventing skin diseases. This study aimed to discover the effect of Chrysanthemum zawadskii and Mentha arvensis on skin barrier function since Chrysanthemum zawadskii and Mentha arvensis were used for the medicainal plants with anti inflammatory effect.HaCaT keratinocytes were cultured with C.zawaskii and M. arvensis EtOH extracts followed by real-time RT-PCR. Resuts showed the increased mRNA expression of Involucrin and Transglutaminase-1 as epidermal differentiation marker in a dose dependent manner. The same results were obtained from an experiment using normal human keratinoctes(NHK). These in vitro data strongly indicate that C.zawaskii and M.arvensis extract may have an efficacy of enhancing skin barrier function. In vivo skin stress test by tape stripping dorsal skin of hairless mice were also performed and the results confirmed that C.zawaskii and M.arvensis extract stimulate recovery of the damaged skin barrier function.