A strong constitutive $P_{JH}$ promoter from Bacillus sp. was applied to overexpress the endoxylanase gene in Bacillus subtilis. The expression plasmid, pJHKJ4, was designed to contain the $P_{JH}$ promoter and open reading frame of endoxylanase including its own promoter. The plasmid was introduced into B. subtilis DB431 and the resulting transformant was grown on LB glucose medium. The endoxylanase activity in the culture supernatant reached about 140 unit/ml. The enzyme in the supernatant was efficiently concentrated to 70% by two-step treatments of ammonium sulfate saturation and ultrafiltration. The stabilization of concentrated enzyme solution at different storage temperatures was examined with various stabilizers such as NaCl, $CaCl_2$, sucrose, sorbitol, polyethylene glycol, and Tween-80.
Universities have recently introduced problem-based learning (PBL) to various subjects to enhance problem-solving skills (including self-directed learning and small-group learning) required in industry. The PBL module was applied to the personal production process in a draping class. A study was based on a questionnaire after conducting two PBL modules with a group of students. Each PBL module included 'design analysis', 'presentation of flat sketch and draping plan', 'discussion of the plan', 'evaluation of the draping result and correcting the problem', and 'final evaluation of the completed project'. Results showed that satisfaction with the PBL method and its activities was higher than satisfaction with existing teaching methods. In particular, among the various components, the 'design analysis' and 'the presentation step of flat sketch and draping plan' stages were more helpful to students compared to small-group discussion. Moreover, the effects of PBL were observed through student reflection essays, in which students suggested that PBL was very effective in enhancing problem-solving through self-directed and small-group learning. Despite the overall satisfaction with PBL, students expressed some minor difficulties associated with awkwardness with a novel learning method, lack of diverse perspectives among each group, and poor communication skills. Therefore, the study shows that PBL is highly likely to be useful to students when they are solving pattern drafting problems and making samples through self-directed learning and small-group learning.
Objective: Recapitulation of the spermatogenesis process in vitro is a tool for studying the biology of germ cells, and may lead to promising therapeutic strategies in the future. In this study, we attempted to transdifferentiate Wharton's jelly-derived mesenchymal stem cells (WJ-MSCs) into male germ cells using all-trans retinoic acid and Sertoli cell-conditioned medium. Methods: Human WJ-MSCs were propagated by the explant culture method, and cells at the second passage were induced with differentiation medium containing all-trans retinoic acid for 2 weeks. Putative germ cells were cultured with Sertoli cell-conditioned medium at $36^{\circ}C$ for 3 more weeks. Results: The gene expression profile was consistent with the stage-specific development of germ cells. The expression of Oct4 and Plzf (early germ cell markers) was diminished, while Stra8 (a premeiotic marker), Scp3 (a meiotic marker), and Acr and Prm1 (postmeiotic markers) were upregulated during the induction period. In morphological studies, approximately 5% of the cells were secondary spermatocytes that had completed two stages of acrosome formation (the Golgi phase and the cap phase). A few spermatid-like cells that had undergone the initial stage of tail formation were also noted. Conclusion: Human WJ-MSCs can be transdifferentiated into more advanced stages of germ cells by a simple two-step induction protocol using retinoic acid and Sertoli cell-conditioned medium.
Vascular endothelial growth factors (VEGFs) are a family of proteins that mediate angiogenesis. $VEGF_{165}$ is a VEGF-A isoform and has been extensively studied owing to its potential use in therapeutic angiogenesis. This study established Chinese hamster ovary (CHO) cells overexpressing recombinant human $VEGF_{165}$$(rhVEGF_{165})$ protein. The production rate of the established CHO cells was over 80mg/l of $rhVEGF_{165}$ protein from a 7-day batch culture process using a 7.5-l bioreactor with a 5-l working volume and serum-free medium. The $rhVEGF_{165}$ protein was purified to homogeneity from the culture supernatant using a two-step chromatographic procedure that resulted in a 48% recovery rate. The purified $rhVEGF_{165}$ protein was a glycosylated homodimeric protein with a higher molecular weight (MW) than the protein expressed from insect cells, suggesting that the glycosylation of the $rhVEGF_{165}$ protein in CHO cells differed from that in insect cells. The purified $rhVEGF_{165}$ protein in this study was functionally active with a half-maximal effective concentration of 3.8ng/ml and specific activity of $2.5{\times}10^5U/mg$.
Rodrigues, Maria Elisa;Costa, Ana Rita;Fernandes, Pedro;Henriques, Mariana;Cunnah, Philip;Melton, David W.;Azeredo, Joana;Oliveira, Rosario
Journal of Microbiology and Biotechnology
/
v.23
no.9
/
pp.1308-1321
/
2013
The emergence of microcarrier technology has brought a renewed interest in anchorage-dependent cell culture for high-yield processes. Well-known in vaccine production, microcarrier culture also has potential for application in other fields. In this work, two types of microcarriers were evaluated for small-scale monoclonal antibody (mAb) production by CHO-K1 cells. Cultures (5 ml) of microporous Cytodex 3 and macroporous CultiSpher-S carriers were performed in vented conical tubes and subsequently scaled-up (20 ml) to shake-flasks, testing combinations of different culture conditions (cell concentration, microcarrier concentration, rocking methodology, rocking speed, and initial culture volume). Culture performance was evaluated by considering the mAb production and cell growth at the phases of initial adhesion and proliferation. The best culture performances were obtained with Cytodex 3, regarding cell proliferation (average $1.85{\pm}0.11{\times}10^6$ cells/ml against $0.60{\pm}0.08{\times}10^6$ cells/ml for CultiSpher-S), mAb production ($2.04{\pm}0.41{\mu}g/ml$ against $0.99{\pm}0.35{\mu}g/ml$ for CultiSpher-S), and culture longevity (30 days against 10-15 days for CultiSpher-S), probably due to the collagen-coated dextran matrix that potentiates adhesion and prevents detachment. The culture conditions of greater influence were rocking mechanism (Cytodex 3, pulse followed by continuous) and initial cell concentration (CultiSpher-S, $4{\times}10^5$ cells/ml). Microcarriers proved to be a viable and favorable alternative to standard adherent and suspended cultures for mAb production by CHO-K1 cells, with simple operation, easy scale-up, and significantly higher levels of mAb production. However, variations of microcarrier culture performance in different vessels reiterate the need for optimization at each step of the scale-up process.
Leaves of Aloe vera Linne and bloods of domestic animal were composted in a soild state fermentation reactor (SSFR) by using microbial additive including a bulking and moisture controlling agent. From solid-culture of microbial additive, 10 species of bacteria and 10 species of fungi were isolated and, their enzyme activities including amylase, carboxy methyl cellulase CMCase, lipase and protease were detected. Optimum fermentation conditions of Aloe leaves and domestic animal bloods in SSFR were obtained from the studies of response surface analysis employing microbial additive content, initial moisture content, and fermentation temperature as the independent variables. The optimum conditions for SSFR using Aloe leaves were obtained at 9.45$\pm$73%(w/w) of microbial additives, 62.73$\pm$4.54%(w/w) of initial moisture content and 55.32$\pm$3.14$^{\circ}C$ of fermentation temperature while those for SSFR using domestic animal bloods were obtained at 10.25$\pm$2.04%, 58.68$\pm$4.97% and 57.85$\pm$5.$65^{\circ}C$, respectively. Composting process in SSFR was initially proceeded through fermentation and solid materials were decomposed within 24 hours by maintaining higher moisture level, and maturing and drying steps are followed later. After the fermentation step, the concentrations of solid phase inorganic components were increased while that of organic components were decreased. Also, concentrations of total organic carbon(TOC), peptides, amino acids, polysaccharides, and low fatty acids in water extracts were increased. As fermentation in composting process depends on initial C/N ratios in water extracts of two samples were increased because of increased water-soluble TOC. From these results, it was revealed that solid state fermentation reactor using microbial additives can be used in composting process of organic wastes with broad C/N ratio.
By examining the constitutive characteristics of Yon-Ko-Jeon and analyzing the lived meanings and experiencing essence of students, this study details the students' awareness, responses, and strategies with regards to the phenomenon of women's alienation from Yon-Ko-Jeon. Students from the two colleges were divided into two groups- one which was aware of women's alienation from Yon-Ko-Jeon, and another group that was unaware of such a phenomenon. Students without awareness tended to perceive the alienation of women from Yon-Ko-Jeon as a natural phenomenon. On the other hand, the members who had awareness tended to see it as a phenomenon that was either justified, unequal, or in need of change. In the latter group, those who saw women's alienation as a justified phenomenon responded with tolerance and adopted a bystander strategy. Conversely, those who saw it as an unequal phenomenon responded with rejection and instead chose to adopt anti-/alternative Yon-Ko-Jeon strategies. The last group, which perceived women's alienation as a phenomenon in need of change, adopted correcting responses as well as intercollegiate solidarity and cooperation strategies. A generalization of the findings reveals that the students' experiencing essence of the phenomenon extends from critical thinking to awareness, response, and strategy in a step-by-step and sequential process. Through this study, it was found that the students' experiencing essence all follow a continuous thinking pattern, as illustrated in the diagram provided.
Since the sensor nodes are randomly arranged in the region of interest, it may happen that the sensor network area is separated or there is no sensor node in some area. In addition, after the sensor nodes are deployed in the sensor network, a coverage hole may occur due to the exhaustion of energy or physical destruction of the sensor nodes. The coverage hole can greatly affect the overall performance of the sensor network, such as reducing the data reliability of the sensor network, changing the network topology, disconnecting the data link, and worsening the transmission load. Therefore, sensor network coverage hole recovery has been studied. Existing coverage hole recovery studies present very complex geometric methods and procedures in the two-step process of finding a coverage hole and recovering a coverage hole. This study proposes a method for discovering and recovering a coverage hole in a sensor network, discovering that the sensor node is a boundary node by itself, and determining the location of a mobile node to be added. The proposed method is expected to have better efficiency in terms of complexity and message transmission compared to previous methods.
Choi, Dong Jin;Park, Sang Jun;Gu, Bon Kang;Kim, Young-Jin;Chung, Seok;Kim, Chun-Ho
Journal of Industrial and Engineering Chemistry
/
v.67
/
pp.388-395
/
2018
Significant efforts have been applied toward fabricating three-dimensional (3D) scaffolds using 3D-bioprinting tissue engineering techniques. Gelatin has been used in 3D-bioprinting to produce designed 3D scaffolds; however, gelatin has a poor printability and is not useful for fabricating desired 3D scaffolds using 3D-bioprinting. In this study, we fabricated pore size controlled 3D gelatin scaffolds with two step 3D-bioprinting approach: a low-temperature ($-10^{\circ}C$) freezing step and a crosslinking process. The scaffold was crosslinked with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). The pore sizes of the produced 3D gelatin scaffolds were approximately 30% smaller than the sizes of the designed pore sizes. The surface morphologies and pore sizes of the 3D gelatin scaffolds were confirmed and measured using scanning electron microscopy (SEM). Human dermal fibroblasts (HDFs) were cultured on a 3D gelatin scaffold to evaluate the effect of the 3D gelatin scaffold pore size on the cell proliferation. After 14 days of culture, HDFs proliferation throughout the 3D gelatin scaffolds prepared with more than $580{\mu}m$ pore size was approximately 14% higher than proliferation throughout the 3D gelatin scaffold prepared with a $435{\mu}m$ pore size. These results suggested that control over the 3D gelatin scaffold pore size is important for tissue engineering scaffolds.
Journal of the Korean Institute of Landscape Architecture
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v.33
no.6
s.113
/
pp.109-126
/
2006
As the national interest in sports has been increased over time, sports facilities and sports complex are considered more than just physical environments for training athletes or watching games. Sports facilities now become symbolic spatial devices to represent the national and cultural pride of any countries. The existing national training camp located in Taenung, Seoul is gotting outdated and degraded. The polluted air in the city risks athletes' health condition. The government planned to construct the second training camp at Jincheon, Chungbuk at the area of $2,171,910m^2$. The Korea Sports Council called for proposals to envision the future training camp, and they held a design competition from August to November, 2005. The first phase was to present the masterplan for the entire site including training buildings, outdoor training facilities, dormitories, a visitor center, a research center, education and administration buildings. Considering the size of the site, the planning process required a strong relationship between landscape and architecture. This study tries to provide general explanations on the winning proposal focused on the landscape-related issues. It also attempts to have reference points for contemporary planning and design issues to situate the project in the stream of continuing design effort to avoid the dichotomy between nature and culture. The landscape proposal for the new national taming camp suggests four main strategies; 1) The camp has two intersecting and interweaving parks which represent the natural and urban order. 2) The entire site is organized and networked by the flow of landscape called landscape ribbon in terms of topography, vegetation, and water flow. 3) The landscape is choreographed through the time and process. 4) The ecological process and the digital contents are juxtaposed in the landscape. This winning proposal is the first step to portray the national vision for the sustainable environment coexisting with sports industry. Landscape in this proposal is an active agent to network various parts of the site which enables landscape to be infrastructure. Landscape design in this proposal should be considered open-ended strategies rather than determined concrete forms and its engaging further development will be tested in following Design-Build phase.
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