This study explored the application of tobacco plant expression system for production of dual target monoclonal antibody (mAb) proteins and computational analysis to evaluate their biochemical characteristics. The murine anti-colorectal cancer large single-chain antibody (CL) and H-13F6 human anti-Ebola virus full-size monoclonal antibody (EF) or anti-Ebola large single-chain antibody (EL) co-expressed in F1 plants through crossbreeding transgenic plants expressing each specific antibody. The binding activities of these antibodies to their respective target antigens were then examined. PCR analysis confirmed the presence of the CL gene and the heavy (HC) and light chain (LC) genes of EF in F1 plants resulting from the crossbreeding. Additionally, both CL and EL genes were identified in F1 plants produced through the crossbreeding of transgenic plants expressing each CL and EL. RT-PCR and immunoblot analyses confirmed the mRNA and protein expression of all transgenes in F1 plants. CL, EF, and EL were successfully purified from F1 plants. Indirect ELISA analysis revealed that CL × EF proteins exhibited binding activity to Ebola virus-specific antigenic protein, whereas CL × EL lost this binding activity. Conversely, both CL × EF and CL × EL proteins exhibited enhanced binding activity to the colorectal cancer-specific antigenic protein GA733. In addition, computational analyses [Molecular Dynamics (MD) and Molecular Mechanics Poisson-Boltzmann Surface Area (MM/PBSA) approach] were performed for Ebola virus (EBOV) antigen-antibody complexes and possible three combinations of Fc consistent with experimental results, supporting our assumption and interpretation.

Non-thermal plasma has recently gained popularity in agriculture for their potential applications in precultivation, cultivation, and postharvest processes. Plasma-treated seeds exhibit enhanced plant growth, and their fruits can be stored for extended periods. However, limited research has been conducted to confirm the effects of plasma-activated water (PAW) treatment on plant cultivation from germination to harvest. In this study, we aimed to investigate the use of PAW, generated using a surface dielectric barrier discharge (SDBD) device, for tomato cultivation from germination to harvest. PAW irrigation significantly improved seedling development, increasing cotyledon area by up to 4-times and seedling biomass by up to 3.6-times compared to the untreated control. During the reproductive phase, PAW treatment doubled the number of flowers and increased chlorophyll content and leaf area. At harvest, PAW irrigation led to a 3-times increase in fruit number and up to a 3.9-times increase in plant biomass. Moreover, the characteristics of fruits produced by PAW-treated plants were normal. These results highlight the potential of PAW in future agricultural practices as an alternative ecofriendly nutrient source for plant irrigation under nutrient-limiting conditions, during all developmental stages.

IntroductionRecent advancements in agricultural technology have highlighted the potential of eco-friendly innovations, such as plasma-activated water (PAW), for enhancing seed germination, growth, and biomass production.MethodsIn this study, we investigated the effects of PAW irrigation on young sorghum seedlings through phenotypic and transcriptional analyses. We measured growth parameters, including seedling height, stem thickness, and biomass, across five sorghum varieties: BTx623, Sodamchal, Noeulchal, Baremae, and Hichal. Additionally, we performed detailed analyses of stem cross-sections to evaluate the structural changes induced by PAW. Whole transcriptome analysis was conducted to identify differentially expressed genes (DEGs) and to perform Gene Ontology (GO) analysis.ResultsPhenotypic analysis revealed significant growth enhancements in PAW-treated seedlings compared to the control group, with notable increases in seedling height, stem thickness, and biomass. Stem cross-section analysis confirmed that PAW treatment led to the enlargement of primordia tissue, leaf sheath (LS1 and LS2), and overall stem tissue area. Transcriptomic analysis revealed that 78% of the DEGs were upregulated in response to PAW, indicating that PAW acts as a positive regulator of gene expression. Gene Ontology (GO) analysis further showed that PAW treatment predominantly upregulated genes associated with transmembrane transport, response to light stimulus, oxidoreductase activity, and transcriptional regulation. Additionally, an enriched AP2/EREBP transcription binding motif was identified.ConclusionThese findings suggest that PAW not only enhances sorghum seedling growth through transcriptional regulation but also has the potential to optimize agricultural practices by increasing crop yield. The upregulation of genes involved in critical biological processes underscores the need for further exploration of PAW’s potential in improving the productivity of sorghum and possibly other crops.

Peanut sprouts have recently been reported to be rich in some nutrients, including vitamin C, resveratrol, antioxidants, and folate, and are consumed as functional food. However, the effect of indirect plasma treatment, such as plasma-activated water (PAW), on the germination rate and growth of peanut remains largely unknown. Here, we evaluated the effect of PAW produced using a surface dielectric barrier discharge (SDBD) device on germination and vegetative growth in peanut. SDBD-generated PAW had significantly higher nitrate content than tap water (TW). Compared with the TW group, PAW treatment increased the germination rate of peanut seeds by more than twofolds at 3 days after planting; however, there was no significant difference in final germination rate between the PAW and TW groups at 7 days after seeding. In addition, PAW treatment significantly improved biomass-related morphological traits in peanut sprout compared with TW treatment. At the cellular level, PAW treatment increased cell size in the hypocotyl in both the horizontal (115

Abstract Plasma agriculture has recently gained popularity owing to its potential applications in precultivation, cultivation, and postharvest processes. Plasma treatments are beneficial seed-priming agents for improving seed germination. The increase plant growth during the seedling stage and enhance fruit storage duration during postharvest treatment. However, little research has been conducted to confirm the long-term effects of plasma-activated water (PAW) treatment on plant cultivation during stages from germination to harvest. Here, we demonstrate the utilization of PAW, generated from a single dielectric barrier discharge (SDBD) device, to understand the functional role of PAW in the cultivation of tomato ‘Micro-Tom’, from germination to harvest. PAW treatment enhanced the growth of young seedlings and increased the number of flowers, plant biomass, and yield compared with the control. Moreover, no adverse effects on the characteristics of fruits produced by PAW-treated plants were observed. These results highlight the potential of PAW as an alternative ecofriendly nutrient source for plant irrigation under nutrient-limiting conditions, during all the stages from seeds to fruits, and offer promising implications for future agricultural practices.

Circulating tumor cells (CTCs) are an indicator of metastatic progression and relapse. Since non-CTC cells such as red blood cells outnumber CTCs in the blood, the separation and enrichment of CTCs is key to improving their detection sensitivity. The ATP luminescence assay can measure intracellular ATP to detect cells quickly but has not yet been used for CTC detection in the blood because extracellular ATP in the blood, derived from non-CTCs, interferes with the measurement. Herein, we report on the improvement of the ATP luminescence assay for the detection of CTCs by separating and concentrating CTCs in the blood using a 3D printed immunomagnetic concentrator (3DPIC). Because of its high-aspect-ratio structure and resistance to high flow rates, 3DPIC allows cancer cells in 10 mL to be concentrated 100 times within minutes. This enables the ATP luminescence assay to detect as low as 10 cells in blood, thereby being about 10 times more sensitive than when commercial kits are used for CTC concentration. This is the first time that the ATP luminescence assay was used for the detection of cancer cells in blood. These results demonstrate the feasibility of 3DPIC as a concentrator to improve the detection limit of the ATP luminescence assay for the detection of CTCs.

Transgenic Arabidopsis thaliana expressing an anti-rabies monoclonal antibody (mAb), SO57, was obtained using Agrobacterium-mediated floral dip transformation. The endoplasmic reticulum (ER) retention signal Lys-Asp-Glu-Leu (KDEL) was tagged to the C-terminus of the anti-rabies mAb heavy chain to localize the mAb to the ER and enhance its accumulation. When the inaccurately folded proteins accumulated in the ER exceed its storage capacity, it results in stress that can affect plant development and growth. We generated T1 transformants and obtained homozygous T3 seeds from transgenic Arabidopsis to investigate the effect of KDEL on plant growth. The germination rate did not significantly differ between plants expressing mAb SO57 without KDEL (SO plant) and mAb SO57 with KDEL (SOK plant). The primary roots of SOK agar media grown plants were slightly shorter than those of SO plants. Transcriptomic analysis showed that expression of all 11 ER stress-related genes were not significantly changed in SOK plants relative to SO plants. SOK plants showed approximately three-fold higher mAb expression levels than those of SO plants. Consequently, the purified mAb amount per unit of SOK plant biomass was approximately three times higher than that of SO plants. A neutralization assay revealed that both plants exhibited efficient rapid fluorescent focus inhibition test values against the rabies virus relative to commercially available human rabies immunoglobulins. KDEL did not upregulate ER stress-related genes; therefore, the enhanced production of the mAb did not affect plant growth. Thus, KDEL fusion is recommended for enhancing mAb production in plant systems.

Plant molecular biofarming has been increasingly studied in recent decades. Many kinds of recombinant immunotherapeutic proteins have been produced in transgenic tobacco plants. However, tobacco mosaic virus (TMV) can damage tobacco plants and cause pathogenic symptoms, which affects plant biomass production. Cigarette sap solution has been used to infect TMV during tobacco cultivation. In this study, we obtained TMV-infected transgenic plants expressing a prostatespecific antigen (PSA) fused to an IgG Fc with KDEL ER retention motif (PSA-IgG FcK). The typical TMV-associated symptoms appeared and increased on plant leaves 5 days after infection. mRNA expression levels and the presence of TMV were confirmed by RT-PCR and transmission electron microscopy analyses. The expression level and purity of the target protein were not significantly different between noninfected and infected transgenic plants. TMV was not found in the purified protein samples from infected plants. Our study showed that TMV pathogen-infected plant biomass can be harvested and processed to obtain therapeutic vaccine proteins.

The anti-colorectal cancer monoclonal antibody CO17-1A (mAb CO), which recognizes the tumor-associated antigen EpCAM, was expressed in transgenic Arabidopsis plants. PCR and western blot analyses showed the insertion and expression of heavy chain (HC)/HC fused to the KDEL ER retention modif (HCK) and light chain (LC) of mAb CO and mAb CO with HCK (mAb COK) in Arabidopsis transformants. Both plant-derived mAbP CO and mAbP COK were purified from a biomass of approximately 1,000 seedlings grown in a greenhouse. In sandwich ELISA, both mAbP CO showed a slightly higher binding affinity for the target, EpCAM, compared to mAbM CO. In cell ELISA, both mAbsP COs showed binding affinity to the human colorectal cancer cell line SW480. Furthermore, mAbM CO, mAbP CO, and mAbP COK exhibited dose and time-dependent regression effects on SW480 cells in vitro. In summation, both mAbP CO and mAbP COK, expressed in Arabidopsis, recognized the target antigen EpCAM and showed anti-proliferative activity against human colorectal cancer cells.

Won Seok Ju · Ilchan Song · Se-Ra Park · Sang Young Seo · Jin Hyoung Cho · Sung-Hun Min · Dae-Heon Kim· Ji-Su Kim· Sun-Uk Kim · Soon Ju Park · Kisung Ko · Young-Kug Choo. J Plant Biotechnol 2019;46:217-27. https://doi.org/10.5010/JPB.2019.46.3.217

The main goal of this research was to determine optimum pH conditions for coupling between protein A and epoxy-activated Sepharose beads for purification of monoclonal antibodies (mAbs) expressed in plants. To confirm the effect of pH conditions on purification efficacy, epoxy-activated agarose beads were coupled to protein A under the pH conditions of 8.5, 9.5, 10.5, and 11.5 (8.5R, 9.5R, 10.5R, and 11.5R, respectively). A total of 300 g of fresh leaf tissue of transgenic Arabidopsis expressing human anti-rabies mAb (mAbP) SO57 were harvested to isolate the total soluble protein (TSP). An equal amount of TSP solution was applied to five resin groups including commercial protein A resin (GR) as a positive control. The modified 8.5R, 9.5R, 10.5R, and 11.5R showed delayed elution timing compared to the GR control resin. Nano-drop analysis showed that the total amount of purified mAbPSO57 mAbs from 60 g of fresh leaf mass were not significantly different among 8.5R (400 μg), 9.5R (360 μg), 10.5R (380 μg), and GR (350 μg). The 11.5R (25 μg) had the least mAbPSO57. SDS–PAGE analysis showed that the purity of mAbPSO57 was not significantly different among the five groups. Rapid fluorescent focus inhibition tests revealed that virus-neutralizing efficacies of purified mAbPSO57 from all the five different resins including the positive control resin were similar. Taken together, both pH 8.5 and 10.5 coupling conditions with high recovery rate should be optimized for purification of mAbPSO57 from transgenic Arabidopsis plant, which will eventually reduce down-stream cost required for mAb production using the plant system.

Plants have emerged as one of the most attractive systems for producing human therapeutic proteins against viral diseases. These include diagnostic reagents, vaccines, and antibodies. This process is known as molecular biofarming. The objective of this study was to develop and evaluate tobacco and Arabidopsis as plant platforms for producing human anti-rabies monoclonal antibody (mAb). Both tobacco and Arabidopsis transgenic plants were generated by Agrobacterium-mediated transformation. Purification of mAb SO57K from each plant was performed with ammonium sulfate-mediated precipitation and protein A affinity columns. SDS–PAGE analysis showed that the purity of mAb SO57K obtained from each transgenic plant was similar, whereas Arabidopsis showed approximately twofold greater protein expression than tobacco. The N-glycosylation was not significantly different between proteins from the two plant species, with both showing oligo-mannose glycan structures. The mAbs SO57 derived from both the model plants had similar neutralizing efficacy against target virus strain CVS-11. Taken together, tobacco and Arabidopsis are both promising platforms for producing a human anti-rabies mAb.

The endoplasmic reticulum (ER) is the main site of protein synthesis, folding, and secretion to other organelles. The capacity of the ER to process proteins is limited, and excessive accumulation of unfolded and misfolded proteins can induce ER stress, which is associated with plant diseases. Here, a transgenic Arabidopsis system was established to express anti-cancer monoclonal antibodies (mAbs) that recognize the tumor-associated antigen GA733-2. Monoclonal antibody (mAb) CO17-1A recognize a tumor-associated epitope expressed on the colorectal cancer cell surface. The ER retention Lys-Asp-Glu-Leu (KDEL) motif sequence was added to the C-terminus of the heavy chain to retain anti-colorectal cancer mAbs in the ER, consequently boosting mAb production. Agrobacterium-mediated floral dip transformation was used to generate T1 transformants, and homozygous T4 seeds obtained from transgenic Arabidopsis plants expressing anti-colorectal cancer mAbs were used to confirm the physiological effects of KDEL tagging. Germination rates were not significantly different between both plants expressing mAb CO without KDEL mAb CO (CO plant) and mAb CO with KDEL mAb COK (COK plant). However, COK plants primary root lengths were shorter than those of CO plants and non-transgenic Arabidopsis plants in in vitro media. Most ER stress-related genes, with the exception of bZIP28 and IRE1a, were upregulated in COK plants compared to CO plants. Western blot and SDS-PAGE analyses showed that COK plants exhibited up to five times higher expression and mAb amounts than plants. Enhanced expression in mAb COK plants was confirmed by immunohistochemical analyses. mAb COK was distributed across most of the area of leaf tissues, whereas mAb CO was mainly distributed in extracellular areas. Surface plasmon resonance analyses revealed that mAb CO and mAb COK possessed equivalent or slightly better binding activities to antigen EpCAM compared to a commercially available parental antibody. N-glycosylation analysis showed that mAb CO had plant specific residues whereas mAb COK mainly showed an oligo-mannose N-glycan structure without the plant specific glycan residues. In this study, the reduction of plant growth and biomass induced by ER retention signal peptide might be only in in vitro conditions, and thus should be carefully considered for the initial screening for transgenic lines on culture media. Taken together, nevertheless the fusion of ER retention signal peptide is an effective approach for enhancing the yields of recombinant proteins in vivo.

An epithelial cell adhesion molecule (EpCAM) is a type I transmembrane protein expressed in the majority of normal epithelial tissues and overexpressed in the human epithelial cancers including breast cancer. Treatment with plant-derived anti-EpCAM mAb (mAbr COK) and Raw264. 7 cells inhibited cell growth in the human breast cancer cell line MCF -7. In MCF-7 treated with mAbr COK and Raw264.7 cells, expression of p53 and p21 increased, whereas the expression of G1 phase-related protein, cyclin D1, CDK4, cyclin E and CDK2 decreased. In addition of treatment with mAbP COK and Raw264. 7 cells decreased the expressiOn of anti-apoptotic protein Bcl-2, but the expression of pro-apoptotic proteins Bax, TNF-u, caspase-3, caspase-6 and caspase-9, increased. Moreover, cells treated with mAbr COK and Raw264. 7 cells expressed metastasis-related gangliosides GM1 and GD1a. These results suggest that mAbr COK has an effect on anti-cancer activity.

There is concern about the potential for transgene contamination in wild type (WT) plants via pollen-mediated gene flow from transgenic plants. In this study, we investigate pollen-mediated gene flow using tobacco transgenic lines carrying recombinant protein GA733-FcK, which is a putative candidate for producing colorectal cancer vaccine. Transgenic and WT plants were grown in greenhouse, with WT plants placed 0.3, 1, 5, 10, and 20m away from transgenic plants. Seeds were harvested from randomly selected WT plants and sown on germination media supplemented with or without kanamycin. At 30days after sowing, none of the WT seedlings produced true leaves and roots when grown on selective media. Polymerase chain reaction analysis revealed that GA733-FcK and nptII genes were expressed in shoots of transgenic plants but not of WT plants. Expression of GA733-FcK and nptII proteins was also abolished in WT leaves when compared to that of transgenic plants. Our findings suggested that there is low risk of pollen-mediated gene flow from transgenic plants expressing GA733-FcK when grown in greenhouse conditions.

We performed seed germination tests to investigate the effects of seed sowing orientation on germination viability on peanut (Arachis hypogaea L.) sprouts. Specifically, we assessed the influence of seed sowing orientation on germination rate, seedling weight, and seedling length, as well as the seedling vigor index. The seeds were sown in oak tree sawdust at 3.0 cm depth. Four seed orientations were tested: vertical with the hypocotyl end down, vertical with the hypocotyl end up, horizontal with the hypocotyl end down, and horizontal with the hypocotyl end up. The mean seed germination percentages of the four seed orientations were significantly different (p < 0.01) and ranged from 25 to 91.7%. The vertical orientation with hypocotylend- down and hypocotyl-end-up orientations showed the highest (91.7%) and lowest (25%) germination rates, respectively. The vertical orientation with the hypocotyl end down produced the heaviest (4.9 g) seedlings and the longest hypocotyls (4.65 cm). This orientation also produced the longest true leaf + epycotyl (2.15 cm) and had the highest seedling vigor index (197.1). The seedlings had a straight growth pattern, whereas seedlings from seeds sown with the hypocotyl up had an awkward plumular hook shape. Taken together, to produce peanut sprouts, we recommend placing the seeds vertically with the hypocotyl end down because this orientation leads to a high germination rate, high biomass production, and high overall seedling quality.

The expression and glycosylation patterns of anti-colorectal cancer therapeutic monoclonal antibody (mAb) CO17-1A recognizing the tumor-associated antigen GA733-2, expressed in human colorectal carcinoma cells, were observed in the leaf and stem tissues of primary (0 cycle), secondary (1 cycle), and tertiary (2 cycle) growths of seedlings obtained from the stem cut of T2 plants. The bottom portion of the stem of T2 seedlings was cut to induce the 1 cycle shoot growth, which was again cut to induce the 2 cycle shoot growth. In the 1 and 2 cycle growths, the periods for floral organ formation (35 days) was shorter than that (100 days) for the 0 cycle growth. The genes of heavy and light chains of mAb CO17-1A existed at the top, middle, and basal portions of the leaves and stem obtained from the 0, 1, and 2 cycle plants. The protein levels in the leaves and stem tissues from the 1 and 2 cycles were similar to those in the tissues from the 0 cycle. The glycosylation level and pattern in the leaf and stem did not alter dramatically over the different cycles. Surface plasmon resonance (SPR) confirmed that mAbs CO17-1A obtained from leaf and stem tissues of the 0, 1, and 2 cycles had similar binding affinity for the GA733-2 antigen. These data suggest that the shoot growth by bottom stem cutting is applicable to speed up the growth of plant biomass expressing anti-colorectal cancer mAb without variation of expression, glycosylation, and functionality.

Various horticultural crops have been considered as potential heterologous expression systems for plant-made recombinant pharmaceutical proteins. However, there is little information concerning the total soluble protein (TSP) levels of major horticultural crops. Ten major horticultural crops-Chinese cabbage, broccoli, garlic, onion, cabbage, bunching onion, cucumber, zucchini, radish, and carrot, along with tobacco and Arabidopsis , were selected to investigate the TSP levels in their individual tissues. In tobacco, SDS-PAGE assay showed that leaf tissues had stronger protein bands than stem tissues, and freshly harvested samples had slightly stronger band density than the −70°C frozen samples, suggesting that fresh leaf should be used to measure the total soluble proteins without any protein loss or degradation. Bicinchoninic acid (BCA) protein assay revealed that among various horticultural crops, garlic (41.4 mg·g −1 FW), broccoli (21.9 mg·g −1 FW), and Chinese cabbage (11.9 mg·g −1 FW) had the highest TSP levels suggesting that these horticultural crops could be good candidates for plant molecular biofarming to produce highly valuable recombinant proteins. The inner clove tissue in garlic, the flower tissue in broccoli, and the green leaf tissue in Chinese cabbage showed the strongest protein band density as compared to other tissues. The TSP of Arabidopsis tissues was quantified by SDS-PAGE, BCA, and Nano-drop methods. In general, the middle leaf tissue showed the highest TSP levels. To evaluate TSP levels of various horticultural crops, these three different methods were compared. The correlation and regression analyses between SDS-PAGE and BCA, and SDS-PAGE and Nano-drop suggested that there were significant correlations between SDS-PAGE and BCA protein assays as compared to SDS-PAGE and Nano-drop assays, indicating that BCA assay is reliable to quantify TSP levels. In conclusion, the TSP levels varied depending on the horticultural crops and their tissue types, and BCA assay could be applied to quantify the TSP.