Development of Systems for the Production of Plant-Derived Biopharmaceuticals.

Over the last several decades, plants have been developed as a platform for the production of useful recombinant proteins due to a number of advantages, including rapid production and scalability, the ability to produce unique glycoforms, and the intrinsic safety of food crops. The expression methods used to produce target proteins are divided into stable and transient systems depending on applications that use whole plants or minimally processed forms. In the early stages of research, stable expression systems were mostly used; however, in recent years, transient expression systems have been preferred. The production of the plant itself, which produces recombinant proteins, is currently divided into two major approaches, open-field cultivation and closed-indoor systems. The latter encompasses such regimes as greenhouses, vertical farming units, cell bioreactors, and hydroponic systems. Various aspects of each system will be discussed in this review, which focuses mainly on practical examples and commercially feasible approaches.

A comparative study of the deviation of the menton on posteroanterior cephalograms and three-dimensional computed tomography.

PURPOSE: Facial asymmetry has been measured by the severity of deviation of the menton (Me) on posteroanterior (PA) cephalograms and three-dimensional (3D) computed tomography (CT). This study aimed to compare PA cephalograms and 3D CT regarding the severity of Me deviation and the direction of the Me. MATERIALS AND METHODS: PA cephalograms and 3D CT images of 35 patients who underwent orthognathic surgery (19 males and 16 females, with an average age of 22.1±3.3 years) were retrospectively reviewed in this study. By measuring the distance and direction of the Me from the midfacial reference line and the midsagittal plane in the cephalograms and 3D CT, respectively, the x-coordinates (x1 and x2) of the Me were obtained in each image. The difference between the x-coordinates was calculated and statistical analysis was performed to compare the severity of Me deviation and the direction of the Me in the two imaging modalities. RESULTS: A statistically significant difference in the severity of Me deviation was found between the two imaging modalities (Δx=2.45±2.03 mm, p<0.05) using the one-sample t-test. Statistically significant agreement was observed in the presence of deviation (k=0.64, p<0.05) and in the severity of Me deviation (k=0.27, p<0.05). A difference in the direction of the Me was detected in three patients (8.6%). The severity of the Me deviation was found to vary according to the imaging modality in 16 patients (45.7%). CONCLUSION: The measurement of Me deviation may be different between PA cephalograms and 3D CT in some patients.

The location of midfacial landmarks according to the method of establishing the midsagittal reference plane in three-dimensional computed tomography analysis of facial asymmetry.

PURPOSE: The purpose of this study was to evaluate the influence of methods of establishing the midsagittal reference plane (MRP) on the locations of midfacial landmarks in the three-dimensional computed tomography (CT) analysis of facial asymmetry. MATERIALS AND METHODS: A total of 24 patients (12 male and 12 female; mean age, 22.5 years; age range, 18.2-29.7 years) with facial asymmetry were included in this study. The MRP was established using two different methods on each patient's CT image. The x-coordinates of four midfacial landmarks (the menton, nasion, upper incisor, and lower incisor) were obtained by measuring the distance and direction of the landmarks from the MRP, and the two methods were compared statistically. The direction of deviation and the severity of asymmetry found using each method were also compared. RESULTS: The x-coordinates of the four anatomic landmarks all showed a statistically significant difference between the two methods of establishing the MRP. For the nasion and lower incisor, six patients (25.0%) showed a change in the direction of deviation. The severity of asymmetry also changed in 16 patients (66.7%). CONCLUSION: The results of this study suggest that the locations of midfacial landmarks change significantly according to the method used to establish the MRP.

Plant regeneration of Korean wild ginseng (Panax ginseng Meyer) mutant lines induced by γ-irradiation ((60)Co) of adventitious roots.

An efficient in vitro protocol has been established for somatic embryogenesis and plantlet conversion of Korean wild ginseng (Panax ginseng Meyer). Wild-type and mutant adventitious roots derived from the ginseng produced calluses on Murashige and Skoog (MS) medium supplemented with 0.5 mg/L 2,4-dichlorophenoxyacetic acid and 0.3 mg/L kinetin; 53.3% of the explants formed callus. Embryogenic callus proliferation and somatic embryo induction occurred on MS medium containing 0.5 mg/L 2,4-dichlorophenoxyacetic acid. The induced somatic embryos further developed to maturity on MS medium with 5 mg/L gibberellic acid, and 85% of them germinated. The germinated embryos were developed to shoots and elongated on MS medium with 5 mg/L gibberellic acid. The shoots developed into plants with well-developed taproots on one-third strength Schenk and Hildebrandt basal medium supplemented with 0.25 mg/L 1-naphthaleneacetic acid. When the plants were transferred to soil, about 30% of the regenerated plants developed into normal plants.

Overexpression of phytochrome A and its hyperactive mutant improves shade tolerance and turf quality in creeping bentgrass and zoysiagrass.

Phytochrome A (phyA) in higher plants is known to function as a far-red/shade light-sensing photoreceptor in suppressing shade avoidance responses (SARs) to shade stress. In this paper, the Avena PHYA gene was introduced into creeping bentgrass (Agrostis stolonifera L.) and zoysiagrass (Zoysia japonica Steud.) to improve turf quality by suppressing the SARs. In addition to wild-type PHYA, a hyperactive mutant gene (S599A-PHYA), in which a phosphorylation site involved in light-signal attenuation was removed, was also transformed into the turfgrasses. Phenotypic traits of the transgenic plants were compared to assess the suppression of SARs under a simulated shade condition and outdoor field conditions after three growth seasons. Under the shade condition, the S599A-PhyA transgenic creeping bentgrass plants showed shade avoidance-suppressing phenotypes with a 45 % shorter leaf lengths, 24 % shorter internode lengths, and twofold increases in chlorophyll concentrations when compared with control plants. Transgenic zoysiagrass plants overexpressing S599A-PHYA also showed shade-tolerant phenotypes under the shade condition with reductions in leaf length (15 %), internode length (30 %), leaf length/width ratio (19 %) and leaf area (22 %), as well as increases in chlorophyll contents (19 %) and runner lengths (30 %) compared to control plants. The phenotypes of transgenic zoysiagrass were also investigated in dense field habitats, and the transgenic turfgrass exhibited shade-tolerant phenotypes similar to those observed under laboratory shade conditions. Therefore, the present study suggests that the hyperactive phyA is effective for the development of shade-tolerant plants, and that the shade tolerance nature is sustained under field conditions.

Ginsenoside Production and Morphological Characterization of Wild Ginseng (Panax ginseng Meyer) Mutant Lines Induced by γ-irradiation ((60)Co) of Adventitious Roots.

With the purpose of improving ginsenoside content in adventitious root cultures of Korean wild ginseng (Panax ginseng Meyer), the roots were treated with different dosages of γ-ray (5, 10, 25, 50, 75, 100, and 200 Gy). The growth of adventitious roots was inhibited at over 100 Gy. The irradiated adventitious roots showed significant variation in the morphological parameters and crude saponin content at 50 to100 Gy. Therefore, four mutant cell lines out of the propagation of 35 cell lines treated with 50 Gy and 100 Gy were selected on the basis of phenotypic morphology and crude saponin contents relative to the wild type control. The contents of 7 major ginsenosides (Rg1, Re, Rb1, Rb2, Rc, Rf, and Rd) were determined for cell lines 1 and 3 from 100 Gy and lines 2 and 4 from 50 Gy treatments. Cell line 2 showed more secondary roots, longer length and superior growth rate than the root controls in flasks and bioreactors. Cell line 1 showed larger average diameter and the growth rate in the bioreactor was comparable with that of the control but greater in the flask cultured roots. Cell lines 1 and 2, especially the former, showed much more ginsenoside contents than the control in flasks and bioreactors. Therefore, we chose cell line 1 for further study of ginsenoside contents. The crude saponin content of line 1 in flask and bioreactor cultures increased by 1.4 and 1.8-fold, respectively, compared to the control. Total contents of 7 ginsenoside types (Rg1, Re, Rb1, Rb2, Rc, Rf, and Rd) increased by 1.8 and 2.3-fold, respectively compared to the control. Crude saponin and ginsenoside contents in the bioreactor culture increased by about 1.4-fold compared to that the flask culture.

Expression of the protective antigen for PEDV in transgenic duckweed, Lemna minor.

Duckweeds are small, floating aquatic plants with a number of useful characteristics, including edibility, fast-growing, and a clonal proliferation. Duckweed is also fed to animals as a diet complement because of its high nutritional value. Porcine epidemic diarrhea virus (PEDV) is a major causative agent of fatal diarrhea in piglets and is a serious problem in the hog-raising industry. In this study, we assessed the feasibility of producing a protective antigen for the PEDV spike protein 1 using duckweed, Lemna minor. Stably transformed Lemna were obtained by co-cultivation with A. tumefaciens EHA105 harboring the PEDV spike protein gene. Transgene integration and expression of the PEDV spike protein 1 gene were confirmed by genomic PCR and RT-PCR and western blot analysis of transgenic Lemna, respectively. This is the first report of the expression of a vaccine antigen against an animal infectious disease in duckweed.

The RAV1 transcription factor positively regulates leaf senescence in Arabidopsis.

Leaf senescence is a developmentally programmed cell death process that constitutes the final step of leaf development and involves the extensive reprogramming of gene expression. Despite the importance of senescence in plants, the underlying regulatory mechanisms are not well understood. This study reports the isolation and functional analysis of RAV1, which encodes a RAV family transcription factor. Expression of RAV1 and its homologues is closely associated with leaf maturation and senescence. RAV1 mRNA increased at a later stage of leaf maturation and reached a maximal level early in senescence, but decreased again during late senescence. This profile indicates that RAV1 could play an important regulatory role in the early events of leaf senescence. Furthermore, constitutive and inducible overexpression of RAV1 caused premature leaf senescence. These data strongly suggest that RAV1 is sufficient to cause leaf senescence and it functions as a positive regulator in this process.

Spatiotemporal expression of duplicate AGAMOUS orthologues during floral development in Phalaenopsis.

The AGAMOUS (AG) family of MADS-box genes plays important roles in controlling the development of the reproductive organs of flowering plants. To understand the molecular mechanisms behind the floral development in the orchid, we isolated and characterized two AG-like genes from Phalaenopsis that we denoted PhalAG1 and PhalAG2. Phylogenetic analysis indicated that PhalAG1 and PhalAG2 fall into different phylogenetic positions in the AG gene family as they belong to the C- and D-lineages, respectively. Reverse transcription-polymerase chair reaction (RT-PCR) analyses showed that PhalAG1 and PhalAG2 transcripts were detected in flower buds but not in vegetative organs. Moreover, in situ hybridization experiments revealed that PhalAG1 and PhalAG2 hybridization signals were observed in the lip, column, and ovule during the floral development of Phalaenopsis, with little difference between the expression patterns of the two genes. These results suggest that both AG-like genes in Phalaenopsis act redundantly with each other in floral development.

Development and evaluation of the Web-based wound care course for undergraduate nursing students.

PURPOSE: This study developed a web-based wound care course for undergraduate nursing students and evaluated the course's content, system, and student-satisfaction. METHODS: This study was done in three stages, the development of the web-based wound care course, the implementation and evaluation of the course. The course was developed based on the ARCS model. 80 undergraduate nursing students to Y University in Korea used the web-based wound care program during four weeks. After that, they completed questionnaires, evaluating the contents, system, and their satisfaction. RESULTS: Eighteen learning objectives were used to create the web-based wound care course and the course was developed with 7 chapters and 20 subsections. The analysis of the questionnaires showed a mean score for content and system-related items of 3.04 each, out of a possible 4 points. Student satisfaction items had a mean score of 2.89. CONCLUSIONS: The web-based course allowed students access to the course anytime and anywhere, and according to their own learning abilities. However this advantage would only be possible when nurse educators develop qualitative web-based course to meet the demand of a complex health care system as well as the needs of the students and the effectively incorporate it into traditional lectures.

Characterization of TrcMADS1 gene of Trillium camtschatcense (Trilliaceae) reveals functional evolution of the SOC1/TM3-like gene family.

Plant MADS-box genes encode transcriptional regulators that are critical for a number of developmental processes, such as the establishment of floral organ identity, flowering time, and fruit development. It appears that the MADS-box gene family has undergone considerable gene duplication and divergence within various angiosperm lineages. SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1)/Tomato MADS-box gene 3 (TM3)-like genes are members of the MADS-box gene family and have undergone repeated duplication events. Here, we isolated and characterized the SOC1/TM3-like gene TrcMADS1 from Trillium camtschatcense (Trilliaceae) to infer the ancestral function of SOC1/TM3-like genes. The alignment of SOC1/TM3-like genes revealed the presence of a highly conserved region in the C-terminal of predicted protein sequences, designated the SOC1 motif. Phylogenetic analysis indicated that TrcMADS1 is at the basal position of the SOC1/TM3-like gene family. The TrcMADS1 mRNA was detected in both vegetative and reproductive organs by RT-PCR. Our results suggest that duplicated copies of SOC1/TM3-like gene evolved to become variously functionally specialized.

Molecular phylogeny and evolution of alcohol dehydrogenase (Adh) genes in legumes.

BACKGROUND: Nuclear genes determine the vast range of phenotypes that are responsible for the adaptive abilities of organisms in nature. Nevertheless, the evolutionary processes that generate the structures and functions of nuclear genes are only now be coming understood. The aim of our study is to isolate the alcohol dehydrogenase (Adh) genes in two distantly related legumes, and use these sequences to examine the molecular evolutionary history of this nuclear gene. RESULTS: We isolated the expressed Adh genes from two species of legumes, Sophora flavescens Ait. and Wisteria floribunda DC., by a RT-PCR based approach and found a new Adh locus in addition to homologues of the Adh genes found previously in legumes. To examine the evolution of these genes, we compared the species and gene trees and found gene duplication of the Adh loci in the legumes occurred as an ancient event. CONCLUSION: This is the first report revealing that some legume species have at least two Adh gene loci belonging to separate clades. Phylogenetic analyses suggest that these genes resulted from relatively ancient duplication events.