Earticle

In this study, we report the structure and shape of PS-PSMA nanofibers with entrapped QDs, that were prepared by electrospinning a homogenous mixture of polymers and QDs. Uniformly distributed QDs within the polymer matrix of nanofibers induced high degree of compactness and shape rigidity in the nanofibers and also showed efficient enzyme immobilization. The esterase-nanofiber composites were prepared by fabricating crosslinked enzyme aggregates on the surfaces of nanofibers. After immobilization, the Vmax value of the QD embedded esterasenanofibers system was higher compared to pristine nanofibers. Furthermore, the esterase enzyme coating on the QDs nanofibers retained good stability with no loss of enzyme activity even after recycling for more than ten times. The present study demonstrates the successful application of QDs PS-PSMA nanofibers for enzyme immobilization for the first time; it is anticipated that this technology would be employed in various enzyme applications due to the facile shape control of the nanofibers.

Rapid and efficient protein digestion is critical in proteomic analysis. However, the protein digestion step is tedious and time consuming due to poor stability of a digestive enzyme, trypsin. In the present work, we stabilized the trypsin activity in the form of enzyme coatings on polymer nanofibers [1]. The trypsin column was prepared by filling up the column with as-spun polystyrene and poly(styrene-co-maleic anhydride) (PS-PSMA) nanofibers, dispersing them upon ethanol treatment, and fabricating the trypsin coatings on ethanol-dispersed nanofibers in a column. The resulting trypsin column shows an efficient digestion performance and im proved performance stability under recycled uses. The combination of highly stable trypsin coatings and ethanol-dispersion of polymer nanofibers has opened up a new potential to develop a trypsin column for on-line and automated protein digestion [2].

Recently, numerous studies have attempted to search for new natural cosmeceuticals from marine resources. In this study, we focused on the protective effect of chitooligosaccharides (COS) with different molecular weight on cellular damage by ultraviolet mediated oxidative stress in human dermal fibroblast cells. Our results showed that the protective effect of COS on ultraviolet mediated dermal fibroblasts was dependent on their molecular weights. Additionally, expression of matrix metalloproteinases (MMPs) by ultraviolet ray might be result in aging process. COS suppressed ultraviolet mediated oxidative stress and DNA damage by generation of reactive oxygen species (ROS) in human dermal fibroblasts. Moreover, COS inhibited expression of MMPs including collagenases and gelatinases in the levels of mRNA and protein, respectively. Therefore, our results indicated that COS can be developed as anti-aging cosmeceuticals and their effects might be mediated via inhibition of MMPs expression and oxidative stress.

Biodegradable polymer/ceramic composite scaffolds for bone regeneration applications are advantageous over either biodegradable polymer or ceramic alone. In this study, a simple and fast method for fabricating a polymer/bioceramic composite scaffold by coating polydopamine (PD) to induce hydroxyapatite (HA) adsorption on polyglycolic acid (PGA) surfaces was developed. PGA meshes were coated with HA by immersing the scaffolds in a solution containing PD and HA for various periods of time. HA coating was examined by selective staining of ceramic particles, scanning electron microscopy, attenuated total reflectance Fourier transformed-infrared spectroscopy, X-ray photoelectron spectroscopy, and energy-dispersive spectroscopy. Substantial HA adsorption on PGA was achieved within 24 hours of incubation. To evaluate bone formation efficacy of scaffolds in vivo, PGA, PD-coated PGA, and HA/PD-coated PGA scaffolds were implanted to critical size defects in mouse skulls for 8 weeks. Soft X-ray radiography, microcomputed tomography and histological analyses showed that PD-induced HA-polymer scaffolds exhibited enhanced bone formation efficacy as compared to the polymer scaffolds.

Autologous chondrocyte transplantation is an effective treatment for articular cartilage damage but involves surgical procedures which may cause further cartilage degeneration. Human adipose-derived stem cells (hADSCs) are an alternative cell source for cartilage regeneration due to their multipotency, easy accessibility and expansion. In this study, we developed an efficient chondrogenic differentiation method and cartilage formation of hADSCs, elucidated mechanisms of enhanced chondrogenesis. Chondrogenesis was promoted in spheroids in spinner flasks. As a control, hADSCs were cultured in tissue culture dishes. Signal cascades for chondrogenesis were examined. To evaluate cartilage formation, hADSCs cultured as spheroids or in monolayer were mixed with fibrin gel and implanted subcutaneously into athymic mice for four weeks. Enhanced chondrogenic differentiation in spheroids versus monolayer culture was observed. The enhanced chondrogenesis is likely attributed to hypoxia-related cascades and enhanced cell-cell interactions. Spheroid culture in three-dimensional bioreactors is advantageous over monolayer culture for chondrogenic differentiation and subsequent cartilage formation.

Recently, numerous studies have attempted to search for new natural cosmeceuticals from marine resources. In this study, we focused on the protective effect of chitooligosaccharides (COS) with different molecular weight on cellular damage by ultraviolet mediated oxidative stress in human dermal fibroblast cells. Our results showed that the protective effect of COS on ultraviolet mediated dermal fibroblasts was dependent on their molecular weights. Additionally, expression of matrix metalloproteinases (MMPs) by ultraviolet ray might be result in aging process. COS suppressed ultraviolet mediated oxidative stress and DNA damage by generation of reactive oxygen species (ROS) in human dermal fibroblasts. Moreover, COS inhibited expression of MMPs including collagenases and gelatinases in the levels of mRNA and protein, respectively. Therefore, our results indicated that COS can be developed as anti-aging cosmeceuticals and their effects might be mediated via inhibition of MMPs expression and oxidative stress.

Owing to their biodegradability, biocompatibility and low toxicity, various biomaterials using chitosan and their derivatives have been developed, especially in biomedical applications [1, 2]. Among these, hollow spheres have attracted increasing interest as drug delivery systems since they enable high loading of hydrophilic, hydrophobic or even solid-type drugs [3]. The formation of hollow microspheres generally involves the use of organic solvent and high temperature or high pressure, which is harmful to the biological environment. In the present study, chitosan hollow hydrogels were simply prepared by ionotropic gelation of chitosan with sodium tripolyphosphate. The fabrication process and major parameters were discussed and optimized. The morphological characteristics and formation mechanisms were characterized by scanning electron microscopy and paraffin section analysis. The results revealed that the microspheres exhibited a very smooth and hollow structure. Also, the formation of the hollow inside microspheres could be controlled by varying fabrication conditions including types of chitosan, concentrations and pH of sodium tripolyphosphate. This method of production of hollow spheres can be considered a simple, fast but effective preparation procedure of hollow hydrogel-based devices in biomedical applications.

One stone, two birds: The Goal of the protein engineers and chemical biologist is to expand the building blocks of protein translation of eukaryotes and prokaryotes with amino acid surrogates (Unnatural amino acid). Currently, a number of unnatural amino acid was incorporated into protein with the help the methodology such as Expanding (sense codon reassignment or residue-specific incorporation) and engineering the genetic code (non-sense suppression or site-specific incorporation). These approaches can be applied to incorporate any desired functional group into protein which provides the protein with novel function properties such as bio-tagging, probing, enhanced functional and spectral properties which is inherited from the incorporated functional group. Despite the potential significance, there remain pitfalls such that these methods can incorporate only one functionality into the protein. Simultaneously, incorporating two different functionalities will provide the protein with a two different functions. One of the major significance is that it will open a new door in the peptidomimetics to synthesis novel antimicrobial peptides with different combination UAAs. To attain the challenging goal, here we have developed a simple, efficient and robust method for the incorporation of multiple unnatural amino acids (MUAA) in a single protein by coupling the above mentioned approaches. In this study, to prove the approach, we used GFP as model protein in which the methionine will be replaced globally with its analog L-homopropargylglycine through sense codon reassignment approach. Simultaneously, tyrosine analog 3,4-dihydroxy-L-phenylalanine (L-Dopa) will be incorporated in response to the non sense codon (Amber codon) inserted at Y66 (or) K15 with the help of an evolved Methanococcus jannaschii tRNA/synthetase pairs (mutant TyrRS). Now, the multi-functional protein has the potential ability for bio-orthogonal conjugation derived from the incorporated DOPA and L-Hpg can be chemoselectively modified with specific alkyne bearing reagent by means of a copper mediated azide-alkyne cycloaddition. This is the first study to demonstrate in vivo incorporation of MUAA into a recombinant protein through combination of two different methodologies. This combination will offer an extraordinary opportunity for protein engineers to create protein with novel functionality which expand the proteome of the cell. This approach will offer protein engineers to provide toolkits with unprecedented benefits and opportunities in the coming age of synthetic biotechnology.

Corydalis heterocarpa, a kind of salt-tolerant plant, has been employed as folk medicine to cure travail and spasm, and some studies have reported the antioxidant and anti-inflammatory activities of components isolated from Corydalis heterocarpa. However, whether compound isolated from this plant attenuates the UVB-induced cell damage in skin still remains largely unknown. Solar ultraviolet (UV) radiation, particularly the UVB (290-320 nm) radiation causes various harmful biological effects including sunburn, melanoma and photoaging. This stud focused on the investigation of anti-photodamage effects of compound isolated from corydalis heterocarpa on UVB induced human keratinocyte (HaCaT) cells. The presence of compound in UVB-exposed keratinocytes suppressed the reactive oxygen species (ROS) generation and DNA damage. In addition, the compound down regulated the increased MMP-2, MMP-9 and up regulated the reduced TIMP-1, TIMP-2 protein and gene expressions due to UVB exposure. Furthermore, the under lying signaling mechanisms of UVB protective effects of compound was elucidated through ASK1-MAPK-responsive signaling pathways.

MITF (microphthalmia-associated transcription factor), a bHLH (basic helix-loop-helix) transcription factor with a leucine zipper, is a key regulatory protein for the pigmentation process in melanocytes. Binding of MITF to E-box caused transcription of several pigmenting genes, including the tyrosinase gene. The purpose of this study is to analyze the interaction of MITF and its DNA binding site (E-box, CATGTG) by surface plasmon resonance. Label free DNA-protein interaction assay, SPR (surface plasmon resonance) was used to analyze their interactions on the gold surface. It is expected that SPR can be a useful method to study of MITF and E-box interactions. We are looking forward to applying this study as a screening tool for depigmenting agents in the cosmetic industry.

This study was carried out to evaluate the skin whitening efficacy of Vietnamese Robusta oil. Vietnamese Robusta green beans were roasted at six different levels and extracted oil by supercritical carbon dioxide. After measuring the radical scavenging activity and tyrosinase inhibition activity of coffee oils, the good roasting condition was chosen for in vitro test on B16F1 mouse melanoma cells. First, MTT assay was conducted to identify the toxicity of the coffee oil. After that, melanin content was measured to evaluate the inhibition of Vietnamese Robusta oil on melanin production. Finally, proteome analysis using 2-D was conducted to observe the changes of proteins that are involved in melanogenesis.

For the past decades, in vivo incorporation of Unnatural into recombinant protein expanded the application of protein with novel functions. Genetic code engineering (Codon reassignment) is one such methodology to design proteins with novel functional and biophysical properties. It is a recent developing method which engineer proteins with additional chemical moiety through incorporation of unnatural amino acids and helps in expanding the proteome of cell. To date, several groups successfully incorporated more than 100 unnatural amino acids into recombinant proteins through in vivo and in vitro approaches. Although, novel functional proteins were developed through this approach, we are the first to demonstrate the development of copper sensing fluorescent protein through unnatural amino acid mutagenesis. In this study we show the incorporation of unnatural amino acid, L-DOPA into Green fluorescent protein provides the protein with highly sensitive copper binding property. The Copper binding property of the engineered protein was characterized by the combination of fluorescent spectroscopy, absorption spectroscopy and circular dichroism. Overall this study demonstrates that the development of selective, sensitive and robust fluorescent based metal biosensor using unnatural amino acid mutagenesis.

Colorimetric detection of nucleic acids has attracted considerable research attention due to many advantages as compared to the conventional gel based assays, such as rapid visual detection, low cost and easy implementation to point-of-care testing (POCT) facilities. Along this line, we developed a new and simple label-free colorimetric detection method of target DNA utilizing the peroxidase mimicking activity of magnetic nanoparticles (MNPs), which relies on the target DNA-induced shielding action against the peroxidase activity. PCRamplified sample solution is mixed with unmodified MNPs followed by addition of colorimetric reaction solution containing colorimetric substrates and hydrogen peroxidase (H2O2). In the presence of target DNA, the am plified DNA products diminished the accessibility of positively charged substrate, OPD toward MNPs in a supernatant and the direct contact of the substrate with MNPs by adsorbing onto the surface of MNPs through electrostatic interactions. This simple but very efficient colorimetric strategy was successfully demonstrated by correctly diagnosing Chlamydia infection using a real human urine sample. Since the results can be clearly seen by the naked eye without any sophisticated instrumentation, this method has great potential as a POCT sensor for the verification of the amplified nucleic acids.

In this study, methanogenic reactor was operated along with the shift of hydraulic retention time (HRT) at 35 oC. In the methanogenic stage, COD was reduced by 65.8 (± 1.1)% compared to a 47.4 (± 2.9)% reduction in VS reduction efficiency, indicating the gradual increase in methane production and the gradual decrease in methane yield during a HRT between 25 and 10 days. This was probably due to the fact that methanogens consume a substantial amount of the VFAs produced as a result of the degradation of major organic materials by bacteria. Through denaruting gel gradient electrophoresis (DGGE), Methanoculleus bourgnesis, belonging to Mehtanomicrobiales (MMB) was predominant during a HRT between 25 and 10 days, while Methanobrevibacter sp. AbM4, belonging to Methanobacteriales (MBT) was predominant during a HRT between 10 and 5 days. Overall 16S rRNA gene concentration also gradually decreased with decline of HRT. Redundancy analysis was also carried out n order to understand correlation between methanogenic community and environmental variables. Acknowledgements This research was supported by the Korea Ministry of Knowledge and Economy (MKE) as a Manpower Development Program for Energy & Resources, the Ministry of Environment (MOE) as a Human Resource Development Project for Waste to Energy, and by the BK-21 program.

The construction of hepatic cDNA library of control Japanese medaka and fish under specific estrogenic and phenol compounds mediated stress responses was described. Accomplished characterization of the library provides an opportunity to identify novel genes that are potentially important for environmental toxicology. Information of 1509 high-quality ESTs including 260 new ESTs was added onto GenBank dbEST. The ESTs were clustered and assembled into 159 contigs and 372 singletons. Using homology comparison against the non-redundant (nr) protein database, 128 contigs and 163 singletons (54.8%) were functionally characterized, and 13 UniESTs (2.5%) were similar to unknown proteins. Annotation resulting 282 UniESTs with 2,102 GOs involved and functionally classified in one or more GOs. Among them, 93 sequences were retrieved enzyme codes (ECs) associated with 116 ECs in the Kyoto encyclopedia of genes and genomes (KEGG). The 535 cDNA probes originated from this library were used for constructing DNA microarray chips and gene expression analysis was conducted with 17-beta estradiol (E2), nonylphenol (NP), and 2-chlorophenol (2CP). The tentative biomarkers showed unique gene expression patterns depending on chemical concentration(s) and exposure duration in real time RT-PCR analysis. The microarray chips were also validated by assessing the toxicity of AgNO3 and Ag_NPs at equivalent metallic silver amount. The expression profiling analysis showed that the toxic impacts of both silver types were temporal, dose-dependent, and between these two silver forms exist a distinguishable toxic fingerprints. We have suggested a tentative biomarker screening strategy for aquatic environmental toxicology using Japanese medaka under specific chemical mediated stress responses.

This study aimed to investigate the changes in methanogen profiles with respect to the performance of a conventional anaerobic sludge digester at different hydraulic retention times (HRTs). Mesophilic anaerobic digesters, fed with secondary sludge, were operated at HRTs ranging from 25 to 7 days. The chemical oxygen demand (COD) removal efficiency was 12.4-33.7% and the methane yield was 0.21-0.39 L CH4/g CODremoved. Although the maximal COD removal efficiency was observed at 25 days HRT, the maximal COD removal rate, methane production rate and methane yield were observed at 7 days HRT. The methanogen community structure was analyzed using group-specific real-time PCR assay. Hydrogenotrophic Methanomicrobiales was the most abundant (>90%) methanogen group at all trials. These results suggest that anaerobic sludge digestion can be operated at HRTs shorter than conventional ones (20–0 days) without altering the core methanogen community structure significantly.

As today’ technology progresses and increment in industrial activities, a high volume of wastewater containing heavy metal is released to the natural environment. Heavy metal pose a significant threat to the environment and public health because of their toxicity, accumulation in the food chain and persistence in nature. According to the World Health Organization (WHO), the metals of most immediate concern are cadmium, chromium, cobalt, copper, lead, nickel, stainless steel, nickel electroplating, battery and accumulator manufacturing, pigments, ceramic and porcelain enameling industries wastewaters contain an undesirable amounts of nickel(II) ions. Nickel at trace amount maybe beneficial as an activator of some enzyme systems but if the nickel(II) ion intake over the permissible levels results in different types of diseases such as pulmonary fibrosis, lung cancer, renal edema, skin dermatitis and gastrointestinal disorder such as nausea, vomiting diarrhea [1-3]. Therefore, the elimination of these metals from water and wastewaters is important to protect public health. Traditional technologies for removal of heavy metals from wastewaters include chemical precipitation, ion-exchange, biosorption, reverse osmosis, evaporation and electrolysis [4-6].The present study explores the ability of T. versicolor (rainbow) biomass for the removal of Ni(II) under different experimental conditions. The effects of relevant parameters such as pH of solution, contact time, biomass dosage, initial metal ion concentration and temperature. The maximum Ni(II) adsorption was obtained at pH 4.0. Further, the biosorbent was characterized by Fourier Transform Infrared Spectroscopy (FTIR), BET Surface area analysis and Scanning Electron Microscopy (SEM). The biosorption data of Ni(II) ions at 303, 313 and 323 K are fitted to Langmuir and Freundlich isotherm models. Langmuir model fitted the equilibrium data better than the Freundlich isotherm. The calculated thermodynamic parameters, ΔGo, ΔHo and ΔSo showed that the biosorption of Ni(II) ions onto T. versicolor (rainbow) biomass was feasible, spontaneous and endothermic at 303-323 K. Experimental data were analysed in terms of pseudo-first-order, pseudo-second-order and intraparticle diffusion models. The results showed that the biosorption process of Ni(II) ions followed well pseudo-second-order kinetics. Based on the results, it can be concluded that the T. versicolor (rainbow) is an effective and alternative biomass for the removal of Ni(II) ions from aqueous solution.

Fermentative production of liquid biofuel as alternatives to gasoline such as ethanol and butanol has gained interest due to both economic and environmental reasons. Butanol that has better fuel properties than ethanol is now considered as one of the most promising next generation biofuel. At the same time, marine biomass such as macroalgae, can provide a high-yield source of biofuels without considering the food crisis or consumption of water and fertilizer. (1,2) Although the carbohydrate composition of macroalgae is more complicated, there is no lignin found in most of macroalgae, which can facilitate production of butanol trough a consolidated bioprocessing (CBP) which features hydrolase production, polymeric carbonhydrate hydrolysis and fermentation in one step. (3) As solventogenic clostridia are capable to produce a variety of extracellular enzymes to degrade polysaccharides, macroalgae fermentation seems to be more suitable for butanol production rather than ethanol production by yeast strains. In this study, red brown and grown macroalgae, Laminaria japonica, Pachymeniopsis elliptica, Chondrus ocellatus, Enteromorpha compressa and Gelidium amansii were used for butanol fermentation with different hydrolysis treatment. Acid or enzyme treatment was applied to the fermentation of L. japonica, P. elliptica, C. ocellatus and E. compressa, while acid pretreatment modified from NREL hydrolysis method for lignocellulosic materials (4) was applied prior to the fermentation of Gelidium amansii.Clostridium beijerinckii NCIMB 8052, a well-known solventogenic clostridium, was used in this research. L. japonica and P. elliptica produced the most reducing sugar of 6.27 ± 0.46 g/L and 6.22 ± 0.12 g/L, respectively. 4 ml and 20 ml fermentation tests were performed in the hydrolysate of each kind of macroalgae. In the case of L. japonica, it was noticeable that water soluble material without any pretreatment was found to be a fine culture medium for butanol production, giving a butanol product of 0.27 g/L in 20 ml culture. The supernatant produced the highest butanol production of 0.34 g/L in 20 ml culture was obtained from P. elliptica hydrolysates with combination treatment of acid and enzyme. Fermentation of the Gelidium amansii hydrolysate was performed in a 200 ml culture after 5 ml and 50 ml cultures were tested. In several runs of fermentation which were conducted under different concentrations of the hydrolysate, a maximum amount of 2.71 g/L of solvents (2.18 g/L of butanol) production, and the high level of acids (acetate and butyrate) production, 8.67 g/L, were obtained at 36 h after inoculation. It was also found that Clostridium beijerinckii can grow and metabolize brown algae Laminaria japonica, whose carbonhydrate composition are alginate, cellulose, laminarin and mannitol without any pretreatment or supplyment. 50 ml and 500 ml of fermentation were performed in a concentration of 100 g/L (dry wt/v) brown algae media resulting in 1.2 g/L of butanol at the end of the fermentation (36h).On the other hand, many different carbohydrates available in macroalgae were test for butanol production to examine carbohydrate preference or utilization by C. beijerinckii in order to provide more information on utilization of macroalgae as substrate. The aim of this study is to examine potential of butanol production using macroalgae and to provide a basis for biofuel production from marine biomass.

In the present study we found that the fruiting body of the medicinal and edible mushroom Sparassis crispa produces lignin peroxidase. Lignin peroxidase was measured with two assays: quantitation of the veratraldehyde produced on oxidation of veratryl alcohol. The assay was also used to monitor oxidation of the dye azure B involving absorbance measurements in the visible range. The S. crispa lignin peroxidase was purified using ammonium sulphate precipitation, anion exchange and gel filtration chromatography. The molecular weight of the purified enzyme was approximately 45 kDa, as determined by SDS-PAGE. A zymogram analysis using a pyrogallol substrate revealed that this enzyme is peroxidase. The substrate specificity, optimum pH and temperature of this enzyme were determined. The effect of nutrient nitrogen was also assessed because nitrogen sources are frequently affecting the enzyme production. We summarize the potential application of this fungal lignin peroxidase in chemical industry, coal chemistry and environmental protection, etc. References:1.Horie K, Rakwal R, Hirano M, Shibato J, Nam HW, Kim YS, Kouzuma Y, Agarwal GK, Masuo Y and Yonekura M. Proteomics of Two Cultivated Mushrooms Sparassis crispa and Hericium erinaceum Provides Insight into their Numerous Functional Protein Components and Diversity. Journal of Proteome Research. 2008;7:1819-35.

Light is the most important parameters in the mass cultivation of microalgae in photobioreactors (PBRs). Microalgae have the ability to utilize sunlight energy for the production of fine chemicals and for biofuels. Microalgal photosynthetic efficiency is higher than that of higher plants. Although algae can only use all the photons of visible light, other colored photons could have different effects and efficiency on cell growth and metabolism. Chlorella vulgaris UTEX 295 was cultivated in 0.4L bubble column PBRs with supplementation of three different monochromatic LEDs (470, 525, and 660 nm) in addition to white light from fluorescent lamps.The results indicated that fresh cell weight and total lipid concentration were positively increased by over 11% and 10%, respectively, under the condition of long wavelength supplement. In fatty acid methyl ester (FAME) composition, short and long wavelength supplement have the positive effects on linolenic acid and oleic acid methyl ester concentration, respectively. By analogy with the results, we hypothesized that the specific wavelength of light could affect the cell niche. In order to confirm this hypothesis, additional studies with Dunaliella tertiolecta were conducted with colored plastic filter film in red and blue in outdoor cultivation. The results clearly showed that final biomass concentrations under the red and the blue were 41% and 37% higher, respectively, than the control. Moreover, the major FAMEs were increased by 9-11% under the blue colored film and total FAME productivity was also increased by 49% and 34% in the red and the blue, respectively. Therefore, we propose that certain wavelength of light could induce high quality biodiesel.

Red algae have a low lignin content, higher growing rate than land plant biomass, and higher carbohydrates content than brown and green algae. Ceylon moss is one of the red algae. After Ceylon moss was hydrolyzed by acid, the hydrolyzate of Ceylon moss (hCM) contained galactose as main carbohydrate, glucose and 5-hydroxymethylfurfural. Three type strains, C. acetobutyricum ATCC824, C. beijerlinkii NCIMB8052 and C. aurantobutyricum NCIMB10659 were cultivated in modified reinforced clostridial medium containing a half concentration of hCM (hccCM) at 37°C and then productions of acetate, butyrate, acetone, ethanol and butanol were determined. Their growth and fermentation characteristics were influenced by substrate concentration. It seemed that cell growth and metabolic characteristics were affected by inhibitor(s) existed in hCM. In order to find alternative biochemical producing microbes instead of type strains, various environmental samples obtained from anoxic conditions were used as microbial sources. After successful isolation on the plate containing hccCM, colony PCR and 16S rDNA analysis were performed to identify isolates. Thirteen bacteria that showed 99~100% similarity with Clostridium strains were isolated. Characterization of two isolates was performed in the different substrates, hccCM and galactose/glucose (as same concentration of carbohydrates contents in hccCM). Isolated strains showed more feasibility as industrial strains compared to type strains in terms of galactose utility and biochemical productivity on hccCM.

Citrus peel waste is produced after the extraction of juice, from citrus fruits. The citrus peel waste contains about 27% solid while a 73% of liquid content. The juicy content was used as a substrate for fermentation to produce antimicrobial substance. The organism used for fermentation was Bacillus sp. LS 1-2, isolated from Korean transitional soybean paste foods. It was confirmed as Bacillus sp based on 16S rRNA sequence similarities. LS 1-2 was cultured in citrus-processing-waste containing five Brix as a culture medium and produced an antimicrobial agent against both gram-positives and negatives at adequate culture conditions (pH, Temperature, Culture period and, etc.). An antimicrobial agent produced by LS 1-2 was purified by the consecutive steps of the alcohol precipitation, C-18 cartridges, Recycle preparative HPLC and preparative HPLC. The active compound was not sensitive to lipase, proteinase K, amylase, catalase but was affected by pronase E. In order to evaluate its commercial value, the minimum inhibitory concentration of purified antim icrobial agent from Bacillus sp. LS 1-2 was compared with commercialized antimicrobial agents (Ampicillin) against Staphylococcus aureus and Escherichia coli O-157.

Toxicity of two common pesticides (e.g., glyphosate and methidathion) and their impacts on the gene expression in non-target organisms were studied in this work using a water flea, Daphnia magna. Particularly, we investigated both the acute (24h) and chronic (21d) effects of these chemicals on physiological responses such as viability, growth, and reproduction as well as molecular responses such as gene expression in D. magna. Through RT-PCR, the expression of five genes selected as representative biomarkers including Arnt, Vtg, CYP4, CYP314 and Dhb was analyzed in D. magna exposed to glyphosate and methidathion. Our results showed that the expression of these genes in D. magna were significantly down-regulated in response to the LC5, LC10, LC20, and LC50 concentrations of pesticides for short-time (24h) exposure. Among five examined genes, Cyp4 and Cyp314 genes, expressed differently in exposure of glyphosate and methidathion, respectively. This revealed that these two genes could be considered as potential biomarkers for the acute toxicity of glyphosate and methidathion. Interestingly, expression of these five genes in D. magna tended to increase when culturing neonates in the medium fed with some low concentrations (e.g., 1/10, 1/50, and 1/100 LC50) of both pesticides for a long-time (21d) experiment. Therefore, this finding suggested that the acute toxicity of glyphosate and methidathion may cause some damages on the DNA function in D. magna which lead to the decrease of expression level of the five selected genes while the chronic toxicity of these chemicals may stimulate some acclimation mechanism to help organisms to overcome the adverse impacts from the toxic environments.

Ttrypsin digestion is a critical step in the bottom-up proteomic analysis, but takes a long time due to the poor stability of trypsin itself. We prepared trypsin-coated magnetic nanoparticles (EC-TR/NPs) via a simple multilayer random crosslinking of the trypsin molecules onto magnetic nanoparticles. The resulting EC-TR/NPs were highly stable and could be easily captured using a magnet after the digestion was complete. EC-TR/NPs showed a negligible loss of trypsin activity after multiple uses and continuous shaking, while the conventional immobilization of covalently-attached trypsin on NPs resulted in a rapid inactivation under the same conditions due to the denaturation and autolysis of trypsin. A single model protein, a five protein mixture, and a whole mouse brain proteome were digested at atmospheric pressure and 37 ºC for 12 h or in combination with pressure cycling technology (PCT) at room temperature for 1 min. In all cases, EC-TR/NPs performed equally to or better than free trypsin in terms of both identified peptide/protein number and the digestion reproducibility. In addition, the concomitant use of EC-TR/NPs and PCT resulted in very rapid (~1 min) and efficient digestions with more reproducible digestion results.

Mussels inhabit seashore by attachment themselves using their foot proteins (fps) which has underwater adherent properties. L-3,4-dihydroxyphenyl alanine (Dopa) is regarded as a key factor for strong and submerged adhesion. Previously, we successfully produced hybrid fp proteins, fp-151 which is composed of six fp-1 decapeptide repeats at both termini of fp-5 and fp-131 which is composed of six fp-1 decapeptide repeats at both termini of fp-3 variant A, and confirmed that both fp-151 and fp-131 formed complex coacervation with hyaluronic acid (HA) as a negative partner. Complex coacervation process enhanced density and bulk adhesive force of fps in dried condition. Moreover, complex coacervated fps/HA material is watery liquid with no dispersion into water, thus it is expected having an adhesive property in even wet condition such as inside of body. In the present work, glutaraldehyde (GA) was added on fps and its amount was optimized to enhance the strength in dried condition. Then, optimized glutaraldehyde-added fps and dopa-adapted fps were compared in both dried and wet conditions.

Listeriosis is a form of serious infection that is caused by consumption of foods that have become contaminated with a type of bacteria known as, Listeria monocytogenes. It is one of the most virulent foodborne pathogens with 20 to 30 percent of clinical infections resulting in death. Traditionally, diagnosis of listeriosis and detection of L. monocytogenes is based on classical culturing and serological identification methods which are laborious and time-consuming, requiring up to ten days for completion. Therefore, the rapid, cost-effective, and automated detection of L. monocytogenes throughout the food chain continues to be a major concern. Here we describe directly cell detection aptasensor for L. monocytogenes based on DNA aptamers. For selected of cell binding aptamers, we performed Cell-SELEX (systematic evolution of ligands by exponential enrichment). Using Surface Plasmon Resonance (SPR), aptamers obtained after 8 to 10 rounds of selection demonstrated the high affinity and specific binding with L. monocytogenes cells, where as the selected aptamers not bind to other living cells. In conclusion, aptasensor based on specific cell-binding aptamers can be effectively applied for the rapid and sensitive detection of L. monocytogenes from the contaminated environments such as corrupt food products, polluted drinking water, and stable vegetables.

Recombinant spider and silkworm have been studied as silk materials for several decades. Even though bees and ants have been focused as new silk-bearing organisms, the research for silk proteins from other organisms is rare. Especially, there is no precedent about silk-like proteins from marine organisms. Comparing with the silk genes from spider and silkworm, we found that marine mussel has silk-like gene that consists of many repeats with abundance of glycine and alanine. We designed mussel-derived silk gene which contains alternating repeated (hydrophobic) and non-repeated regions. Because we observed prominent retardation of cell growth by expression of mussel silk-like protein, fusion strategy with the baculoviral polyhedrin was used to induce insoluble expression of target silk protein. Finally, we could express the mussel silk-like proteins as insoluble form with high cell growth.

In this study, we have developed a canine nose-mimicking bioelectronic sensor to detect low concentration of hexanal, a product of lipid oxidation. For mimicking a canine nose, nanovesicles containing canine olfactory receptor, cfOR5269, were produced from host HEK293 cells expressing cfOR5269 on their surface, and then the produced nanovesicles were immobilized on swCNT-FET platform. Since swCNT-FET amplified the signal from nanovesicles, we were able to develop a highly sensitive bioelectronic nose, which has the selectivity of the canine olfactory receptor. We obtained selective signals from hexanal treatment, and the detection lim it of our sensor was 1 fM. Our selective and sensitive bioelectronic nose can be used as an alternative sensing system for canine nose.

The efficient and safe gene delivery system using smart biomaterial has been a useful tool to enhance gene delivery for stem cell. We have demonstrated developed substrate-mediated gene delivery using Adeno-associated viral (AAV) vectors which have the capacity to mediate efficient and safe gene delivery and Elastin-like polypeptide (ELP) which has been recently employed as a macromolecular therapeutic delivery vehicle. A novel AAV variant, AAV r3.45, which was previously engineered by directed evolution to enhance transduction toward stem cell, was non-specifically bound onto ELPs on a tissue culture polystyrene surface (TCPS). The cellular transduction was modulated by mechanical properties as the presence of different ELP quantities on the TCPS. Importantly, ELP-mediated AAV delivery system enhanced delivery efficiency with substantially reduced viral quantities compared to bolus delivery in fibroblasts and human neural stem cells. Therefore this system with capacity to enhance gene delivery efficiency has strong potential for use in tissue engineering applications and neurodegenerative disorder treatments.

The aetiological agent of epizootic ulcerative syndrome (EUS) is Aphanomyces invadans in both estuarine and freshwater fish (1). The actual economic losses due to EUS in aquaculture industry were to be in excess of US$9 billion per year. Currently, no effective proplylatic measures are available for EUS disease, although adding lime and salt to the ponds has been used (2).In the present study, Cirrhina mrigala intramuscularly injected with Aphanomyces invadans (isolate B99C) at 2.3 x 107 CFU ml-1 was investigate the hematological and biochemical parameters. In infected untreated group, the white blood cell count (WBC: 106mm-3) is significantly increased (P <0.05) from the control, while in no change in groups fed with probiotics, herbal, and azadirachtin supplementation diets on 30 days. Similar trend was noted in the hemoglobin (Hb: g/dL) and hematocrit (Hct: %) levels. Interestingly, infected fish fed with probiotics, herbal and azadirachtin supplementation diets groups did not differ (P> 0.05) from the control. Asimilar trend prevailed in the percentage of lymphocytes (LYM), monocytes (MON), eosinophils (EOS) and neutrophils (NEU). The total protein (TP: g dl-1), glucose (GLU: mg dl-1), calcium (CAL: mmol l-1) and cholesterol (CHO mmol l-1) levels were affected (P <0.05) in the infected group whereas these values after probiotics, herbal and azadirachtin supplementation diet treated groups were restored near control group. The present study was suggested that the infected fish after administration with probiotics, herbal, and azadirachtin supplementation diets for 30 days protect the hematological and biochemical parameters in C. mrigala against A. invadans.