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Mechanically durable transparent electrodes are essential for achieving long-term stability in flexible optoelectronic devices. Furthermore, they are crucial for applications in the fields of energy, display, healthcare, and soft robotics. Conducting meshes represent a promising alternative to traditional, brittle, metal oxide conductors due to their high electrical conductivity, optical transparency, and enhanced mechanical flexibility. In this paper, we present a simple method for fabricating an ultra-transparent conducting metal oxide mesh electrode using self-cracking-assisted templates. Using this method, we produced an electrode with ultra-transparency (97.39%), high conductance (R s  = 21.24 Ω sq -1 ), elevated work function (5.16 eV), and good mechanical stability. We also evaluated the effectiveness of the fabricated electrodes by integrating them into organic photovoltaics, organic light-emitting diodes, and flexible transparent memristor devices for neuromorphic computing, resulting in exceptional device performance. In addition, the unique porous structure of the vanadium-doped indium zinc oxide mesh electrodes provided excellent flexibility, rendering them a promising option for application in flexible optoelectronics., (© 2024. The Author(s).)

Key Message: The novel gene CaAN3 encodes an R2R3 MYB transcription factor that regulates fruit-specific anthocyanin accumulation. The key regulatory gene CaAN2 encodes an R2R3 MYB transcription factor that regulates anthocyanin biosynthesis in various tissues in pepper (Capsicum annuum). However, CaAN2 is not expressed in certain pepper accessions showing fruit-specific anthocyanin accumulation. In this study, we identified the novel locus CaAN3 as a regulator of fruit-specific anthocyanin biosynthesis, using an F 2 population derived from a hybrid cultivar with purple immature fruits and segregating for CaAN3. We extracted total RNA, assembled two RNA pools according to fruit color, and carried out bulked segregant RNA sequencing. We aligned the raw reads to the pepper reference genome Dempsey and identified 6,672 significant single nucleotide polymorphisms (SNPs) by calculating the Δ(SNP-index) between the two pools. We then conducted molecular mapping to delimit the target region of CaAN3 to the interval 184.6-186.4 Mbp on chromosome 10. We focused on Dem.v1.00043895, encoding an R2R3 MYB transcription factor, as the strongest candidate gene. Sequence analysis revealed four insertion/deletion polymorphisms in the promoter region of the green CaAN3 allele. We employed virus-induced gene silencing and transient overexpression assays to characterize the function of the candidate gene. When Dem.v1.00043895 was silenced in pepper, anthocyanin accumulation decreased in the pericarp, while the transient overexpression of Dem.v1.00043895 in Nicotiana benthamiana leaves resulted in the accumulation of anthocyanins around the infiltration sites. These results showed that Dem.v1.00043895 is CaAN3, an activator of anthocyanin biosynthesis in pepper fruits., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

A novel device structure for thermally activated delayed fluorescence (TADF) top emission organic light-emitting diodes (TEOLEDs) that improves the viewing angle characteristics and reduces the efficiency roll-off is presented. Furthermore, we describe the design and fabrication of a cavity-suppressing electrode (CSE), Ag (12 nm)/WO 3 (65 nm)/Ag (12 nm) that can be used as a transparent cathode. While the TADF-TEOLED fabricated using the CSE exhibits higher external quantum efficiency (EQE) and improved angular dependency than the device fabricated using the microcavity-based Ag electrode, it suffers from low color purity and severe efficiency roll-off. These drawbacks can be reduced by using an optimized multi-quantum well emissive layer (MQW EML). The CSE-based TADF-TEOLED with an MQW EML fabricated herein exhibits a high EQE (18.05%), high color purity (full width at half maximum ~ 59 nm), reduced efficiency roll-off (~ 46% at 1000 cd m -2 ), and low angular dependence. These improvements can be attributed to the synergistic effect of the CSE and MQW EML. An optimized transparent CSE improves charge injection and light outcoupling with low angular dependence, and the MQW EML effectively confines charges and excitons, thereby improving the color purity and EQE significantly. The proposed approach facilitates the optimization of multiple output characteristics of TEOLEDs for future display applications., (© 2022. The Author(s).)

Ultrathin film-based transparent conductive oxides (TCOs) with a broad work function (WF) tunability are highly demanded for efficient energy conversion devices. However, reducing the film thickness below 50 nm is limited due to rapidly increasing resistance; furthermore, introducing dopants into TCOs such as indium tin oxide (ITO) to reduce the resistance decreases the transparency due to a trade-off between the two quantities. Herein, we demonstrate dopant-tunable ultrathin (≤ 50 nm) TCOs fabricated via electric field-driven metal implantation (m-TCOs; m = Ni, Ag, and Cu) without compromising their innate electrical and optical properties. The m-TCOs exhibit a broad WF variation (0.97 eV), high transmittance in the UV to visible range (89-93% at 365 nm), and low sheet resistance (30-60 Ω cm -2 ). Experimental and theoretical analyses show that interstitial metal atoms mainly affect the change in the WF without substantial losses in optical transparency. The m-ITOs are employed as anode or cathode electrodes for organic light-emitting diodes (LEDs), inorganic UV LEDs, and organic photovoltaics for their universal use, leading to outstanding performances, even without hole injection layer for OLED through the WF-tailored Ni-ITO. These results verify the proposed m-TCOs enable effective carrier transport and light extraction beyond the limits of traditional TCOs., (© 2021. The Author(s).)

Background: Preservation of the spleen in distal pancreatectomy has recently attracted considerable attention. Our current study aimed in the first instance to define the safety of lap-WT in relation to the capacity of this technique to achieve preservation of the spleen and secondly to investigate the effectiveness of a planned lap-WT procedure or early conversion to lap-WT in selected patients with a large tumor attached to the splenic vessels., Methods: Among 1056 patients who underwent a laparoscopic distal pancreatectomy between January 2005 and December 2014 at our hospital, 122 (24.6 %) underwent lap-WT which were analyzed. The 122 patients were categorized into two groups chronologically (early group: 2005-2012, late group: 2013-2014)., Results: The median follow-up was 35 months, and the median operation time was 181 min. The median postoperative hospital stay was 7 days, and the median estimated blood loss was 316 ml. Postoperative complications occurred in 9 patients (7.3 %), including 4 patients (3.2 %) with major pancreatic fistula (ISGPF grade B, C). A reoperation to address postoperative bleeding was needed in one patient. During a median follow-up of 35 months, there were no clinical significant splenic infarctions or gastric varices in any case. All patients were observed conservatively. In patients in the late group who underwent the lap-WT, the mean operating time (171 vs. 205 min, p = 0.001) and mean estimated blood loss (232.1 vs. 370.0 ml, p = 0.017) were significantly less than the early group cases who received lap-WT., Conclusions: A lap-WT is a safe treatment strategy in select cases when used as a way of preserving the spleen. When splenic vessel preservation is technically challenging, for example when the tumor is enlarged or is attached to the splenic vessels, planned lap-WT or early conversion to lap-WT may be a feasible option.

Porcine epidemic diarrhea virus (PEDV) belongs to the Coronaviridae family and causes acute enteritis in pigs. A fragment of the large spike glycoprotein, termed the S1D epitope (aa 636-789), alone and fused with cholera toxin B subunit, were independently cloned into plant expression vectors, yielding plasmids pMYV717 and pMYV719, respectively. Plant expression vectors were transformed into Agrobacterium tumefaciens and subsequently infiltrated into Nicotiana benthamiana leaves. The highest expression level of S1D was found at 2 days post infiltration (dpi), reached 0.04 % of total soluble protein, and rapidly decreased thereafter. The expression and assembly of CTB-S1D fusion protein were confirmed by Western blot and G M1 -ELISA. The highest expression level of CTB-S1D fusion protein was 0.07 % of TSP at 4 dpi, with a rapid decrease thereafter. In the presence of p19 protein from tomato bushy stunt virus, the S1D and CTB-S1D protein levels peaked at 6 dpi and were fourfold to sevenfold higher than in the absence of p19, respectively. After oral administration of transiently expressed CTB-S1D fusion protein, or with bacterial cholera toxin or rice callus expressing mutant cholera toxin 61F, mice exhibited significantly greater serum IgG and sIgA levels against bacterial CTB and S1D antigen, peaking at week 6. Transiently expressed CTB-S1D fusion protein will be administered orally to pigs to assess the immune response against PEDV., (© Springer Science+Business Media Dordrecht 2016.)

Although many studies reported improvement of back pain after osteoporosis treatment, there is insufficient evidence to determine whether osteoporosis is painful. We investigated whether bone mineral density correlated with musculoskeletal pain in postmenopausal Korean women aged ≥50 years. Data for postmenopausal women aged ≥50 years were obtained from the fifth Korea National Health and Nutrition Examination Survey database. Demographics, Kellgren-Lawrence grade, and numeric rating scale for pain in the hip and knee joints, presence of back pain, and activity level were analyzed. Only subjects with dual-energy X-ray absorptiometry scans and hip and knee radiographs were included. Those with malignancy, pain medication use, or a history of fragility fractures were excluded. After univariate analysis, multiple linear regression analysis was performed to examine the significant factors correlated with the degree of hip and knee pain. Binary logistic regression analysis was performed to identify factors significantly associated with the presence of back pain. In total, 387 women were included in the data analysis. Age (p = 0.005) was the only significant factor correlated with the intensity of hip pain, while Kellgren-Lawrence grade (p < 0.001) was the only significant factor correlated with knee pain intensity in multiple regression analysis. Binary logistic regression analysis showed that age (p = 0.002) was the only significant factor associated with the presence of back pain. Musculoskeletal pain was not affected by or associated with the bone mineral density (BMD) of the affected body part in postmenopausal Korean women aged ≥50 years after adjusting for the degree of osteoarthritis.

Dengue is a disease caused by dengue virus and represents the most important arthropod-borne viral disease in humans. Dengue virus enters host cells via binding of envelope glycoprotein (E) to a receptor. In this study, plant expression vectors containing native and synthetic glycoprotein E genes (sE) modified based on plant-optimized codon usage and fused with an ER retention signal were constructed under control of the rice amylase 3D promoter expression system. Plant expression vectors were introduced into rice callus (Oryza sativa L. cv. Dongin) via particle bombardment-mediated transformation. The integration and expression of target genes were confirmed in the transgenic callus by genomic DNA PCR and Northern blot analyses, respectively. The plant-codon optimized sE gene with an ER retention signal showed high protein production levels based on Western blot analysis of approximately 18.5 ug/g dried calli weight by immunoblot-based densitometric analysis. These results suggest that the plant-codon optimized sE gene with an ER retention signal was highly produced in the transgenic rice callus.

We propose a transparent conductive oxide electrode scheme of gallium oxide nanoparticle mixed with a single-walled carbon nanotube (Ga2O3 NP/SWNT) layer for deep ultraviolet light-emitting diodes using spin and dipping methods. We investigated the electrical, optical and morphological properties of the Ga2O3 NP/SWNT layers by increasing the thickness of SWNTs via multiple dipping processes. Compared with the undoped Ga2O3 films (current level 9.9 × 10-9 A @ 1 V, transmittance 68% @ 280 nm), the current level flowing in the Ga2O3 NP/SWNT increased by approximately 4 × 105 times and the transmittance improved by 9% after 15 times dip-coating (current level 4 × 10-4 A at 1 V; transmittance 77.0% at 280 nm). These improvements result from both native high transparency of Ga2O3 NPs and high conductivity and effective current spreading of SWNTs.

The spread of dengue (DEN) virus is becoming a major concern due to the possibility of primary infection with one of the four dengue serotypes (DEN 1-4) and secondary infection with other heterotypes, which can further aggravate clinical manifestations. A gene encoding consensus envelope protein domain III (cEDIII) of dengue virus with neutralizing activity against four dengue virus serotypes was fused to M cell-targeting peptide ligand (Co1) to increase its mucosal immunogenicity and was introduced into rice calli under the control of the inducible rice amylase 3D promoter expression system. The integration and expression of scEDIII-Co1 fusion gene in transgenic rice calli were confirmed by genomic DNA PCR amplification, Northern and Western blot analyses, respectively. The deliveries of cEDIII-Co1 fusion proteins into mucosal immune inductive site (including M cells) were confirmed by in vitro and in vivo antigen uptake assays. These results showed that plant-produced M cell-targeting peptide ligand, Co1, fusion antigen proteins have the potential to be targeted to the mucosal immune system for improvement of immune responses.

To increase immune responses of plant-based vaccines in intestine mucosal immune systems, a synthetic neutralizing epitope (sCOE) gene of porcine epidemic diarrhea virus (PEDV) was fused with M cell-targeting ligand (Co1) and introduced into a plant expression vector under the control of rice amylase 3D promoter. The sCOE-Co1 fusion gene was introduced into rice calli via the particle bombardment-mediated transformation method. The stable integration and transcriptional expression of the sCOE-Co1 fusion gene was confirmed by genomic DNA PCR amplification and Northern blot analysis, respectively. The expression of the COE-Co1 fusion protein was confirmed by immunoblot analysis. The highest expression level of the COE-Co1 fusion protein reached 0.083 % of the total soluble protein according to quantitative densitometry of Western blot analysis. Mice immunized with transgenic rice calli protein extracts induced significant serum IgG and fecal IgA antibody levels against purified bacterial COE. The systemic and mucosal immune responses were confirmed by measuring COE-specific IgG and IgA antibody-secreting cells in the lymphocytes extracted from the spleen and COE-specific IgA antibody-secreting cells in the Peyer's patches from immunized mice. These results indicated that oral immunization of plant-produced COE-Co1 fusion protein could elicit efficient systemic and mucosal immune responses against the COE antigen. Key message Neutralizing epitope from porcine epidemic diarrhea virus-M cell targeting ligand fusion protein was produced in transgenic rice calli and elicited systemic and mucosal immune responses by oral administration in mice.

Transgenic plants have been used as a safe and economic expression system for the production of edible vaccines. A synthetic cholera toxin B subunit gene (CTB) was fused with a synthetic neutralizing epitope gene of the porcine epidemic diarrhea virus (sCTB-sCOE), and the sCTB-sCOE fusion gene was introduced into a plant expression vector under the control of the ubiquitin promoter. This plant expression vector was transformed into lettuce (Lactuca sativa L.) using the Agrobacterium-mediated transformation method. Stable integration and transcriptional expression of the sCTB-sCOE fusion gene was confirmed using genomic DNA PCR analysis and northern blot analysis, respectively. The results of western blot analysis with anti-cholera toxin and anti-COE antibody showed the synthesis and assembly of CTB-COE fusion protein into oligomeric structures with pentameric sizing. The biological activity of CTB-COE fusion protein to its receptor, G(M1)-ganglioside, in transgenic plants was confirmed via G(M1)-ELISA with anti-cholera toxin and anti-COE antibody. Based on G(M1)-ELISA, the expression level of CTB-COE fusion proteins reached 0.0065% of the total soluble protein in transgenic lettuce leaf tissues. Transgenic lettuce successfully expressing CTB-COE fusion protein will be tested to induce efficient immune responses against porcine epidemic diarrhea virus infection by administration with raw material.

The aim of this study was to develop wireless Picture Archiving and Communication System (PACS) device and to analyze its effect on image transfer from portable imaging modalities to the main PACS server. Using a laptop computer equipped with wireless local area network (LAN), the authors developed a wireless PACS device with DICOM modality worklist and DICOM storage server modules. This laptop computer could be easily fixed to portable imaging modalities such as ultrasound machines. From May to August 2007, 112 portable examinations were evaluated. Of these, 62 were done with wireless LAN-based PACS device, and 50 were done without wireless PACS device. To evaluate the impact of the wireless LAN-based PACS device on productivity and workflow, we analyzed the mean time delay and standard deviations (SD) both in cases where wireless LAN-based PACS device was used and in cases where it was not used. Statistical analysis was performed using a t test. The mean time interval from image acquisition to storage in the main PACS when the wireless LAN-based PACS device was used was 342.4 s (5 min and 42.4 s, SD = 509.2 s). When the wireless PACS was not used, the mean time interval was 2,305.5 s (38 min and 25.5 s, SD = 1,371.8 s). The mean time interval was statistically different between the two groups (t test, p < 0.001). The wireless LAN-based PACS device could help in reducing the storage intervals of images obtained by portable machines and in promoting effective and rapid treatment of patients who have undergone portable imaging examinations.

Enterotoxigenic Escherichia coli is one of the leading causes of diarrhea in developing countries, and the disease may be fatal in the absence of treatment. Enterotoxigenic E. coli heat-labile toxin B subunit (LTB) can be used as an adjuvant, as a carrier of fused antigens, or as an antigen itself. The synthetic LTB (sLTB) gene, optimized for plant codon usage, has been introduced into rice cells by particle bombardment-mediated transformation. The integration and expression of the sLTB gene were observed via genomic DNA PCR and western blot analysis, respectively. The binding activity of LTB protein expressed in transgenic rice callus to G(M1)-ganglioside, a receptor for biologically active LTB, was confirmed by G(M1)-ELISA. Oral inoculation of mice with lyophilized transgenic rice calli containing LTB generated significant IgG antibody titers against bacterial LTB, and the sera of immunized mice inhibited the binding of bacterial LTB to G(M1)-ganglioside. Mice orally immunized with non-transgenic rice calli failed to generate detectable anti-LTB IgG antibody titers. Mice immunized with plant-produced LTB generated higher IgG1 antibody titers than IgG2a, indicating a Th2-type immune response. Mice orally immunized with lyophilized transgenic rice calli containing LTB elicited higher fecal IgA antibody titers than mice immunized with non-transgenic rice calli. These experimental results demonstrate that LTB proteins produced in transgenic rice callus and given to mice by oral administration induce humoral and secreted antibody immune responses. We suggest that transgenic rice callus may be suitable as a plant-based edible vaccine to provide effective protection against enterotoxigenic E. coli heat-labile toxin.

Background: Efficient RNA transfer to dendritic cell and T cells by electroporation have been successfully applied for immunotherapy. Herein, RNA electroporation was used to transfer antigen-specific receptor (scFv) gene to cytokine-induced killer cells (CIK)., Methods: CIK was generated from peripheral blood mononuclear cells with anti-CD3 antibody, interleukin-2, and interferon (IFN)-gamma for 14 days and showed typical characteristics of CIK expressing both CD3+ and CD56+ markers and NKG2D+. CIK could lyse K562 cells, but not SKOV3 and MCF7/Her-2/neu., Results: After RNA encoding anti-Her-2/neu chimeric immune receptor (CIR) with signaling portion of CD28 and CD3zeta was electroporated to CIK, more than 95% of CIK expressed anti-Her-2/neu CIR (CIR-CIK). CIR-CIK was able to produce cytokines including IFN-gamma, granulocyte-macrophage colony-stimulating factor, and tumor necrosis factor-alpha, and show cytotoxicity specific to tumor cell lines expressing Her-2/neu, SKOV3, and MCF7/Her-2/neu. Adoptive transfer of CIR-CIK in SKOV3 xenograft nude mice model led to significant inhibition of tumor growth compared with transfer of mock-transduced CIK and showed higher inhibition than that of Herceptin, humanized monoclonal antibody specific for Her-2/neu. These results suggest that RNA transfer is the convenient and efficient strategy to introduce antigen-specificity into CIK and provide potential therapeutic value of CIR-CIK in the treatment of tumors.

The gram-negative anaerobic oral bacterium Porphyromonas gingivalis initiates periodontal disease through fimbrial attachment to saliva-coated oral surfaces. To study the effects of immunomodulation on enhancement of subunit vaccination, the expression in E. coli and immunogenicity of P. gingivalis fimbrial protein (FimA) linked to the C-terminus of the cholera toxin B subunit (CTB) were investigated. Complementary DNAs encoding the P. gingivalis 381 fimbrillin protein sequence FimA1 (amino acid residues 1-200) and FimA2 (amino acid residues 201-337) were cloned into an E. coli expression vector downstream of a cDNA fragment encoding the immunostimulatory CTB. CTB-FimA1 and CTB-FimA2 fusion proteins synthesized in E. coli BL21 (DE3) cells were purified under denaturing conditions by Ni2+-NTA affinity column chromatography. Renaturation of the CTB-FimA1 and CTB-FimA2 fusion proteins, permitted identification of CTB-FimA pentamers and restored CTB binding activity to GM1-ganglioside to provide a biologically active CTB-FimA fusion protein. Mice orally inoculated with purified CTB-FimA1 or CTB-FimA2 fusion proteins generated measurable FimA1 and FimA2 IgG antibody titers, while no serum fimbrial IgG antibodies were detected when mice were inoculated with FimA1 or FimA2 proteins alone. Immunoblot analysis confirmed that sera from mice immunized with CTB linked to FimA1 or FimA2 contained antibodies specific for P. gingivalis fimbrial proteins. In addition, mice immunized with FimA2 or CTB-FimA2 generated measurable intestinal IgA titers indicating the presence of fimbrial antibody class switching. Further, mice orally immunized with CTB-FimA1 generated higher IgA antibody titers than mice inoculated with FimA1 alone. The experimental data show that the immunostimulatory molecule CTB enhances B cell-mediated immunity against linked P. gingivalis FimA fusion proteins, in comparison to immunization with FimA protein alone. Thus, linkage of CTB to P. gingivalis fimbrial antigens can increase subunit vaccine immunogenicity to provide enhanced protection against periodontal disease.

We expressed the cholera toxin B subunit (CTB) fused to an endoplasmic reticulum retention signal (SEKDEL) in carrot roots using an Agrobacterium-mediated transformation method. Fourteen independent transgenic lines were regenerated via somatic embryogenesis after 6 months of culture. The sCTB gene was detected in the genomic DNA of transgenic carrot by PCR amplification. Expressions and assembly of sCTB protein into oligomeric structures of pentameric size were observed in transgenic plant extracts by Western blot analysis. The sCTB produced by transgenic carrot roots demonstrated strong affinity for GM1-ganglioside, suggesting that the sCTB conserved the antigenic sites for binding and proper folding of the pentameric sCTB structure. The expression level of sCTB comprised approximately 0.48% of total soluble protein (TSP) in root of transgenic carrot.

The synthetic cholera toxin B subunit (CTB) gene, modified according to the optimized codon usage of plant genes, was introduced into a plant expression vector and expressed under the control of the Bx17 HMW (high molecular weight) wheat endosperm-specific promoter containing an intron of the rice act1. The recombinant vector was transformed into rice plants using a biolistic-mediated transformation method. Stable integration of the synthetic CTB gene into the chromosomal DNA was confirmed by PCR amplification analysis. A high level of CTB (2.1% of total soluble protein) was expressed in the endosperm tissue of the transgenic rice plants. The synthetic CTB produced only in the rice endosperm demonstrated strong affinity for G(M1)-ganglioside, thereby suggesting that the CTB subunits formed an active pentamer. The successful expression of CTB genes in transgenic plants makes it a powerful tool for the development of a plant-derived edible vaccine.

Human growth hormone (hGH), a pituitary-derived polypeptide, evidences a wide range of biological functions, including protein synthesis, cell proliferation, and metabolism. A synthetic hGH gene (shGH) has been synthesized on the basis of plant-optimized codon usage via an overlap PCR strategy and located in a plant expression vector under the control of the rice amylase 3D (Ramy3D) promoter, which is induced by sugar starvation. The plant expression vector was introduced into rice calli (Oryza sativa L. cv. Donjin) via particle bombardment transformation methods. The integration of the shGH gene into the chromosome of the transgenic rice callus was verified via genomic DNA PCR amplification and shGH expression in transgenic rice suspension cells was confirmed via Northern blot analysis. The shGH protein was detected in the transgenic rice cell suspension culture medium following induction with sugar starvation, using Western blot analysis. The quantity of shGH that accumulated in the transgenic rice cell suspension medium was 57 mg/l. The shGH accumulated in the transgenic rice cell suspension culture medium evidenced a biological activity similar to that of Escherichia coli-derived recombinant hGH. These results indicate that the shGH was generated and accumulated in the transgenic rice cell suspension culture medium, and manifested biological activity.

In this study, a cell suspension culture system for zedoary (Curcuma zedoaria Roscoe) was developed, using 50 g/l of fresh weight inoculum in a batch culture. The highest cell biomass obtained from a 5-l bioreactor equipped with three impellers after 14 days of culture was utilized to extract secondary metabolites (essential oil and curcumin) and determine the activities of antioxidant enzymes (peroxidase, superoxide dismutase, and catalase). For essential oil and curcumin, zedoary extracts were recovered via a variety of methods: steam distillation, volatile solvents, and Soxhlet. After 14 days of culture using volatile solvents, the optimal yield of essential oil (1.78%) was obtained when using petroleum ether at 40 degrees C in 6 h of extraction, and the best curcumin yield (9.69%) was obtained at 60 degrees C in 6 h via extraction with 90% ethanol. The activities of antioxidant enzymes from zedoary cells were also assessed. The specific activities of peroxidase, superoxide-dismutase, and catalase reached maximum values of 0.63 U/mg of protein, 16.60 U/mg of protein, and 19.59 U/mg of protein after 14 days of culture, respectively.

Epitopes often require co-delivery with adjuvant and targeting proteins to enable recognition by the immune system, and this approach may also increase the efficacy of the antigen. In this study, we assess and describe the ability of transgenic rice plants to express a fusion protein consisting of the B-subunit of the Escherichia coli heat-labile enterotoxin (LTB) and a synthetic core-neutralizing epitope (COE) of porcine epidemic diarrhea virus (PEDV), inducing an enteric disease that is seen most predominantly in piglets. Both components of the fusion proteins were detected with Western blot analysis. The fusion protein was determined to assemble into pentamers, as was evidenced by its ability to bind to GM1 gangliosides, and evidenced an average level of expression in a transgenic rice endosperm. This indicates that the expression system of the plant is capable of generating a sizable amount of antigen, possibly allowing for the successful development of an edible vaccine.

To assess the immunomodulatory activity of the HIV Tat transduction peptide for enhancement of suppression of Type 1 autoimmune diabetes, the 11 amino acid HIV-1 Tat transduction peptide was genetically linked to the major islet autoantigens proinsulin (INS) and glutamic acid decarboxylase (GAD). The Tat-autoantigen fusion proteins were synthesized in Escherichia coli and characterized by acrylamide gel separation and immunoblot analysis. Histological examination of pancreatic islets isolated from juvenile NOD mice inoculated orally with the Tat-autoantigen conjugates revealed a significant reduction in islet inflammation (insulitis) in comparison with islets from unimmunized mice. Increased serum IgG1 antibody isotype titers detected in Tat-autoantigen inoculated mice suggest that the transduction peptide-autoantigen fusion proteins stimulate Th2 lymphocyte mediated bystander suppression. The reduction of islet insulitis observed in Tat-autoantigen inoculated mice suggests that the adjuvant effect of the Tat transduction peptide resides in Tat enhanced delivery of linked autoantigens through enterocytes to lymphocytes in the gut-associated lymphoid tissues.

A DNA encoding the 27-kDa domain I of anthrax lethal factor protein (LF), was linked to the carboxyl terminus of the cholera toxin B-subunit (CTB-LF). The CTB-LF fusion gene was transferred into Solanum tuberosum cells by Agrobacterium tumefaciens-mediated in vivo transformation methods and antibiotic-resistant plants were regenerated. The CTB-LF fusion gene was detected in transformed potato leaf genomic DNA by polymerase chain reaction (PCR)-mediated DNA amplification. Immunoblot analysis with anti-CTB and anti-LF primary antibodies verified the synthesis and assembly of biologically active CTB-LF fusion protein oligomers in transformed plant tuber tissues. Furthermore, the binding of CTB-LF fusion protein pentamers to intestinal epithelial cell membrane receptors measured by GM1-ganglioside enzyme-linked immunosorbent assay (GM1-ELISA) indicated that the CTB-LF fusion protein made up approx 0.002% of the total soluble tuber protein. Synthesis of CTB-LF monomers and their assembly into biologically active CTB-LF fusion protein pentamers in potato tuber tissues demonstrates the feasibility of using edible plants for production and delivery of adjuvanted LF protein for CTB-mediated immunostimulation of mucosal immune responses against anthrax toxin.

A deoxyribonucleic acid (DNA) fragment encoding the cholera toxin B subunit (CTB) was linked 5' to the simian immunodeficiency virus (SIVmac) Gag p27 capsid gene (CTB-Gag). The fusion gene was transferred into Solanum tuberosum cells by Agrobacterium tumefaciens-mediated transformation methods and transformed plants regenerated. The CTB-Gag gene fusion was detected in transformed potato leaf genomic DNA by polymerase chain reaction-mediated DNA amplification. The results of immunoblot analysis with anti-CTB and anti-Gag antibodies verified the synthesis of biologically active CTB-Gag fusion protein in transformed leaf and tuber tissues. Synthesis and assembly of the CTB-Gag fusion protein into oligomeric structures of pentamer size was confirmed by GM1-ganglioside-enzyme-linked immunosorbent assay (GM1-ELISA) of transformed potato tuber tissue extracts. The binding of CTB-Gag fusion protein oligomers to intestinal epithelial cell membrane receptors quantified by GM1-ELISA showed that CTB-Gag fusion protein made up approx 0.016-0.022% of the total soluble tuber protein. The synthesis of CTB-Gag monomers and their assembly into biologically active CTB-Gag fusion protein oligomers in potato tuber tissues provides the opportunity for employment of the carrier and adjuvant properties of CTB for the development of edible plant-based subunit mucosal vaccines for enhanced mucosal immunity against SIV in macaques.

A DNA fragment encoding a 12-amino acid (aa) HIV-1 Tat transduction peptide fused to a 90-aa murine rotavirus NSP4 enterotoxin protein (Tat-NSP4(90)) was transferred to Solanum tuberosum by Agrobacterium tumefaciens-mediated transformation. The fusion gene was detected in the genomic DNA of transformed plant leaf tissues by PCR DNA amplification. The Tat-NSP4(90 )fusion protein was identified in transformed tuber extracts by immunoblot analysis using anti-NSP4(90) and anti-Tat as the primary antibodies. Enzyme-linked immunosorbent assay results showed that the Tat-NSP4(90) fusion protein made up to 0.0015% of the total soluble tuber protein. The synthesis of Tat-NSP4(90) fusion protein in transformed potato tuber tissues demonstrates the feasibility of plant cell delivery of the HIV-1 Tat transduction domain as a carrier for non-specific targeting of fused antigens to the mucosal immune system.

A CTB-NSP4(175) fusion gene encoding the entire 175-aa murine rotavirus NSP4 enterotoxin protein was transferred into Solanum tuberosum cells by Agrobacterium tumefaciens-mediated transformation. The CTB-NSP4(175) enterotoxin fusion gene was detected in the genomic DNA of transformed leaves by PCR DNA amplification. Synthesis and assembly of the full-length CTB-NSP4(175) fusion protein into oligomeric structures of pentamer size was detected in transformed tuber extracts by immunoblot analysis. The binding of CTB-NSP4(175 )fusion protein pentamers to intestinal epithelial cell membrane receptors was quantified by G(M1)-ganglioside enzyme-linked immunosorbent assay (G(M1)-ELISA). The ELISA results showed that CTB-NSP4(175) fusion protein was 0.006-0.026% of the total soluble tuber protein. The synthesis of CTB-NSP4(175) monomers and their assembly into biologically active oligomers in transformed potato tubers demonstrates the feasibility of using edible plants for the synthesis of enterocyte-targeted full-length rotavirus enterotoxin antigens that retain all of their pathogenic epitopes for initiation of a maximum mucosal immune response.

The Sn-->S0 ultraviolet fluorescence spectra (270-380 nm) of 4-aminophthalimide (4AP) and its N-methyl derivative 4-amino-N-methylphthalimide (4ANMP) are reported, following the absorption of two laser pulses. In polar but non-hydrogen-bonding solvents, both molecules exhibit a principal emission maximum near 290 nm, whereas solutions in hydrogen-bonding solvents display two prominent emission bands, near 300 and 350 nm. The relative intensities of these bands depend on solvent type and, in a pump-probe experiment, on the wavelengths and temporal spacing of two ultrashort pulses. Experiments covering the range approximately 0.1-100 ps showed evidence for at least two distinct ultrashort relaxation processes, the rates of which depend on solvent. Fluorescence upconversion experiments at < 0.2 ps resolution have shown that the longer-duration process correlates with the fluorescence Stokes shift, and provide evidence that the solvent-dependent shift of the S1-->S0 fluorescence spectrum is reflected in the fluorescence quantum efficiency of a nearby electronic state.