An Effective Way of Producing Fully Assembled Antibody in Transgenic Tobacco Plants by Linking Heavy and Light Chains via a Self-Cleaving 2A Peptide

Therapeutic monoclonal antibodies (mAbs) have evolved into an important class of effective medicine in treatment of various diseases. Since the antibody molecule consists of two identical heavy chains (HC) and two light chains (LC), each chain encoded by two different genes, their expressions at similar levels are critical for efficient assembly of functional recombinant mAbs. Although the plant-based expression system has been tested to produce fully assembled recombinant mAbs, coordinately expressing HC and LC at similar levels in a transgenic plant remains a challenge. A sequence coding for a foot-and-mouth disease virus (FMDV) 2A peptide has been successfully used to link two or more genes, which enable the translated polyprotein to be “self-cleaved” into individual protein in various genetically modified organisms. In the present study, we exploited the usage of F2A in Ebola virus monoclonal antibody (EBOV mAb) production. We found that transgenic tobacco plants carrying a transcription unit containing HC and LC linked by 2A not only produced similar levels of HC and LC but also rendered a higher yield of fully assembled EBOV mAb compared to those expressing HC and LC in two independent transcription units. Purified EBOV mAb bound to an Ebola epitope peptide with apparent Kd-values of 90.13–149.2 nM, indicating its proper assembly and high affinity binding to Ebola epitope peptide. To our knowledge, this is the first report showing mAb production by overexpressing a single transcription unit consisting of HC, LC and 2A in stable transformed tobacco plants

Ginger Stimulates Hematopoiesis via Bmp Pathway in Zebrafish

) has been widely used in traditional medicine; however, to date there is no scientific research documenting the potential of ginger to stimulate hematopoiesis. expression in the caudal hematopoietic tissue area. We further confirmed that Bmp/Smad pathway mediates this hematopoiesis promoting effect of ginger by using the Bmp-activated Bmp type I receptor kinase inhibitors dorsomorphin, LND193189 and DMH1.Our study provides a strong foundation to further evaluate the molecular mechanism of ginger and its bioactive components during hematopoiesis and to investigate their effects in adults. Our results will provide the basis for future research into the effect of ginger during mammalian hematopoiesis to develop novel erythropoiesis promoting agents

Methylglyoxal-Induced Retinal Angiogenesis in Zebrafish Embryo: A Potential Animal Model of Neovascular Retinopathy

Methylglyoxal (MG) is an intermediate of glucose metabolism and the precursor of advanced glycation end products (AGEs) found in high levels in blood or tissue of diabetic patients. MG and AGEs are thought to play a major role in the pathogenesis of diabetic retinopathy. In order to determine if zebrafish is valuable to help us understand more about retinopathy, we evaluate if MG induces abnormal vascular change and angiogenesis in zebrafish in a short incubation period. We also used an inhibitor of VEGFR (PTK787) to explore the mechanistic role of VEGF in MG-induced pathogenesis. A transgenic Tg(flk1:GFP) zebrafish line was used, and the embryos were incubated with MG solution and in combination with glucose (to mimic hyperglycemia). Retinal vascular structure visible with fluorescence signal was imaged using fluorescence microscopy. The percentage of vascular area was calculated and found elevated in the MG treatment groups than that in the control group (p<0.01) which indicated increased angiogenesis induced by MG treatment. PTK787 blocked the proangiogenic effects of MG treatment. This study suggests that MG has a potential proangiogenic effect via VEGF signaling in the retina of zebrafish embryos. Therefore, this zebrafish model may be used to study neovascular retinopathy

Inhibitory Effects of Euphorbia ebracteolata Hayata Extract ECB on Melanoma-Induced Hyperplasia of Blood Vessels in Zebrafish Embryos

Melanoma is a serious malignant form of skin cancer. Euphorbiaceae compound B (ECB, 2,4-dihydroxy-6-methoxy-3-methylacetophenone) is an acetophenone compound that is isolated from Euphorbia ebracteolata Hayata (EEH), an herbaceous perennial, and has antitumor activity. Here, we transplanted human melanoma cells into zebrafish embryos to establish a zebrafish/melanoma model. We showed that this model can be used to evaluate the therapeutic effect of EEH and ECB and discussed its potential mechanism of action. The results showed that ECB was an active ingredient of EEH in inhibiting melanoma-induced hyperplasia of blood vessels in zebrafish embryos, similar to the angiogenic inhibitor vatalanib. ECB inhibited the number and length of subintestinal veins (p<0.05), as well as the distribution of melanoma in zebrafish embryos (p<0.05). More importantly, unlike vatalanib, ECB only inhibited melanoma-induced abnormal and excessive growth of blood vessels in xenografts. In addition, ECB inhibited the mRNA expression of vegfr2 and vegfr3 in zebrafish. Both vegfr2 and vegfr3 are essential genes that regulate blood vessel formation and upregulate the expression of p53 and casp3a genes in zebrafish. Together, the above-mentioned results indicate that ECB has a potential antimelanoma effect in vivo, which may be mediated by inhibiting vascular endothelial growth factor receptors

Ginger/10-G treatment during gastrulation promotes <i>bmp2b/7a</i> and Bmp target gene expression in zebrafish embryos.

<p>(A) Treatment of late gastrulae with ginger at 15 or 20 µg/ml induces the <i>mercedes</i> mutant-like phenotype (partial duplication of the tail fin) at 1 dpf in 8% or 10% of the treated embryos, respectively. Thus, the zebrafish embryos exposed to ginger extract mimic the phenotype of the <i>ogon</i> mutant, which has a mutation in <i>sizzled</i>, a <i>bmp</i> suppressor gene, at 1 dpf. (B) <i>bmp7a</i> expression was strongly increased and extended to the entire blastoderm at 60% epiboly, following short-term exposure to ginger (5 µg/ml) or 10-G (1 µg/ml) from sphere (4 hpf) to 60% epiboly (7 hpf) stages. (C) Up-regulation and extension of the expression domain were observed for <i>bmp2b</i> at 60% epiboly. (D–E) Accordingly, BMP target genes were up-regulated after ginger/10G treatment from the sphere stage (4 hpf) to 7 hpf, as illustrated by enhanced <i>eve1</i> extended towards the dorsal side (arrow heads), a ventral mesoderm marker (D), and <i>gata2,</i> a non-neural ectoderm marker (E), in zebrafish embryos at 60% epiboly. Pictures on left panels show gastrulae, dorsal side to the right (B–E) and statistics tables (right panels) are representative of three independent experiments. N = number of embryos per group. Scale bars = 250 µm.</p

Ginger/10-G treatment after gastrulation promotes <i>bmp2b/7a</i> in the developing caudal hematopoietic tissue.

<p>(A–B) Zebrafish embryos were treated with ginger (5 µg/ml) or 10-G (2 µg/ml) from 10 to 48 hpf, followed by whole-mount in situ hybridization of <i>bmp2b</i> (A) and <i>bmp7a</i> (B). Both <i>bmp2b</i> and <i>bmp7a</i> were up-regulated locally in the CHT (and underlying fin) upon ginger or 10-G exposure (whereas they are not expressed in the CHT of control embryos at 48 hpf). Scale bars = 700 µm.</p

Ginger extract and its purified phenolic compounds promote <i>Tg(gata1:dsRed)</i> fluorescence and <i>gata1</i> mRNA expression.

<p>(A) Bright field (top left) and <i>Tg(gata1:dsRed)</i> fluorescence of zebrafish embryos at about 22 hpf, before the onset of circulation (anterior to the left). Exposure to ginger extract or its compounds 8-gingerol (8-G), 10-gingerol (10-G), 8-shogaol (8-S) and 10-shogaol (10-S) promoted <i>Tg(gata1:dsRed)</i> fluorescent erythroid cell development in the ICM and PBI (arrows), as compared to control embryos. N = 35 embryos per group. In this panel, we show an embryo treated with a lower concentration of 6-S (2 µg/ml) as this compound was toxic at higher doses. Scale bar = 400 µm. (B) Whole-mount in situ hybridization of ginger or 10-G treated embryos (8 hpf to 21 hpf exposure) revealed increased expression of <i>gata1</i> transcript at 22 hpf. N = 50 embryos per group. Scale bar = 350 µm. (C) At 48 hpf, control embryos at the top; ginger or 10-G treated embryos at the bottom. Scale bar = 500 µm. Fluorescent erythrocytes circulating in the axial vasculature (arrows) and in the pericardial space (arrow heads).</p