CRISPR/Cas9-Mediated Generation of Pathogen-Resistant Tomato against Tomato Yellow Leaf Curl Virus and Powdery Mildew

Tomato is one of the major vegetable crops consumed worldwide. Tomato yellow leaf curl virus (TYLCV) and fungal Oidium sp. are devastating pathogens causing yellow leaf curl disease and powdery mildew. Such viral and fungal pathogens reduce tomato crop yields and cause substantial economic losses every year. Several commercial tomato varieties include Ty-5 (SlPelo) and Mildew resistance locus o 1 (SlMlo1) locus that carries the susceptibility (S-gene) factors for TYLCV and powdery mildew, respectively. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) is a valuable genome editing tool to develop disease-resistant crop varieties. In this regard, targeting susceptibility factors encoded by the host plant genome instead of the viral genome is a promising approach to achieve pathogen resistance without the need for stable inheritance of CRISPR components. In this study, the CRISPR/Cas9 system was employed to target the SlPelo and SlMlo1 for trait introgression in elite tomato cultivar BN-86 to confer host-mediated immunity against pathogens. SlPelo-knockout lines were successfully generated, carrying the biallelic indel mutations. The pathogen resistance assays in SlPelo mutant lines confirmed the suppressed accumulation of TYLCV and restricted the spread to non-inoculated plant parts. Generated knockout lines for the SlMlo1 showed complete resistance to powdery mildew fungus. Overall, our results demonstrate the efficiency of the CRISPR/Cas9 system to introduce targeted mutagenesis for the rapid development of pathogen-resistant varieties in tomato.     

Identification of Cyclophilin A as a Potential Anticancer Target of Novel Nargenicin A1 Analog in AGS Gastric Cancer Cells

We recently discovered a novel nargenicin A1 analog, 23-demethyl 8,13-deoxynargenicin (compound 9), with potential anti-cancer and anti-angiogenic activities against human gastric adenocarcinoma (AGS) cells. To identify the key molecular targets of compound 9, that are responsible for its biological activities, the changes in proteome expression in AGS cells following compound 9 treatment were analyzed using two-dimensional gel electrophoresis (2-DE), followed by MALDI/TOF/MS. Analyses using chemical proteomics and western blotting revealed that compound 9 treatment significantly suppressed the expression of cyclophilin A (CypA), a member of the immunophilin family. Furthermore, compound 9 downregulated CD147-mediated mitogen-activated protein kinase (MAPK) signaling pathway, including c-Jun N-terminal kinase (JNK) and extracellular signal-regulated protein kinase 1/2 (ERK1/2) by inhibiting the expression of CD147, the cellular receptor of CypA. Notably, the responses of AGS cells to CypA knockdown were significantly correlated with the anticancer and antiangiogenic effects of compound 9. CypA siRNAs reduced the expression of CD147 and phosphorylation of JNK and ERK1/2. In addition, the suppressive effects of CypA siRNAs on proliferation, migration, invasion, and angiogenesis induction of AGS cells were associated with G2/M cell cycle arrest, caspase-mediated apoptosis, inhibition of MMP-9 and MMP-2 expression, inactivation of PI3K/AKT/mTOR pathway, and inhibition of hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) expression. The specific interaction between compound 9 and CypA was also confirmed using the drug affinity responsive target stability (DARTS) and cellular thermal shift assay (CETSA) approaches. Moreover, in silico docking analysis revealed that the structure of compound 9 was a good fit for the cyclosporin A binding cavity of CypA. Collectively, these findings provide a novel molecular basis for compound 9-mediated suppression of gastric cancer progression through the targeting of CypA.     

Optimization and evaluation of reduced graphene oxide hydrogel composite as a demulsifier for heavy crude oil-in-water emulsion

The rising production of produced water from oilfields had been proven to bring detrimental environmental effects. In this study, an efficient, recyclable, and environmental-friendly reduced graphene oxide immobilized κ-Carrageenan hydrogel composite(κCa GO) was fabricated as an alternative sorbent for crude oil-in-water demulsification. Polyethyleneimine(PEI) was employed to form a stable hydrogel composite. The conditions for the immobilization of graphene oxide(GO) on PEI-modified κ-Carrageenan(κC) beads were optimized appropriately. An immobilization yield of 77% was attained at 2% PEI, 2 h immobilization activation time, and p H6.5. Moreover, the synthesized κCa GO is capable of demulsification with an average demulsification efficiency of 70%. It was found that the demulsification efficiency increases with salinity and κCa GO dosage, and it deteriorates under alkaline condition. These phenomena can be attributed to the interfacial interactions between κCa GO and the emulsion. Furthermore, the κCa GO can be recycled to use for up to six cycles without significant leaching and degradation. As such, the synthesized κCa GO could be further developed as a potential sorbent substitute for the separation of crude oil from produced water.     

Design and Synthesis of a Novel PLK1 Inhibitor Scaffold Using a Hybridized 3D-QSAR Model

Polo-like kinase 1 (PLK1) plays an important role in cell cycle progression and proliferation in cancer cells. PLK1 also contributes to anticancer drug resistance and is a valuable target in anticancer therapeutics. To identify additional effective PLK1 inhibitors, we performed QSAR studies of two series of known PLK1 inhibitors and proposed a new structure based on a hybridized 3D-QSAR model. Given the hybridized 3D-QSAR models, we designed and synthesized 4-benzyloxy-1-(2-arylaminopyridin-4-yl)-1H-pyrazole-3-carboxamides, and we inspected its inhibitory activities to identify novel PLK1 inhibitors with decent potency and selectivity.     

Apoptotic Effects of Anthocyanins from Vitis coignetiae Pulliat Are Enhanced by Augmented Enhancer of the Rudimentary Homolog (ERH) in Human Gastric Carcinoma MKN28 Cells

Evidence suggests that augmented expression of a certain gene can influence the efficacy of targeted and conventional chemotherapies. Here, we tested whether the high expression of enhancer of the rudimentary homolog (ERH), which serves as a prognostic factor in some cancers, can influence the efficacy of anthocyanins isolated from fruits of Vitis coignetiae Pulliat, Meoru in Korea (AIMs) on human gastric cancer cells. The anticancer efficacy of AIMs was augmented in ERH-transfected MKN28 cells (E-MKN28 cells). Molecularly, ERH augmented AIM-induced caspase-dependent apoptosis by activating caspase-3 and -9. The ERH-augmented apoptotic effect was related to mitochondrial depolarization and inhibition of antiapoptotic proteins, XIAP, and Bcl-2. In addition, reactive oxygen species (ROS) generation was augmented in AIMs-treated E-MKN28 cells compared to AIMs-treated naïve MKN28 cells. In conclusion, ERH augmented AIM-induced caspase-dependent mitochondrial-related apoptosis in MKN28 cells. A decrease in expression of Bcl-2 and subsequent excessive ROS generation would be the mechanism for ERH-augmented mitochondrial-related apoptosis in AIMs-treated MKN28 cells. A decrease in expression of XIAP would be another mechanism for ERH-augmented caspase-dependent apoptosis in AIMs-treated MKN28 cells.     

Exploring the Biomaterial-Induced Secretome: Physical Bone Substitute Characteristics Influence the Cytokine Expression of Macrophages

In addition to their chemical composition various physical properties of synthetic bone substitute materials have been shown to influence their regenerative potential and to influence the expression of cytokines produced by monocytes, the key cell-type responsible for tissue reaction to biomaterials in vivo. In the present study both the regenerative potential and the inflammatory response to five bone substitute materials all based on β-tricalcium phosphate (β-TCP), but which differed in their physical characteristics (i.e., granule size, granule shape and porosity) were analyzed for their effects on monocyte cytokine expression. To determine the effects of the physical characteristics of the different materials, the proliferation of primary human osteoblasts growing on the materials was analyzed. To determine the immunogenic effects of the different materials on human peripheral blood monocytes, cells cultured on the materials were evaluated for the expression of 14 pro- and anti-inflammatory cytokines, i.e., IL-6, IL-10, IL-1β, VEGF, RANTES, IL-12p40, I-CAM, IL-4, V-CAM, TNF-α, GM-CSF, MIP-1α, Il-8 and MCP-1 using a Bio-Plex^® Multiplex System. The granular shape of bone substitutes showed a significant influence on the osteoblast proliferation. Moreover, smaller pore sizes, round granular shape and larger granule size increased the expression of GM-CSF, RANTES, IL-10 and IL-12 by monocytes, while polygonal shape and the larger pore sizes increased the expression of V-CAM. The physical characteristics of a bone biomaterial can influence the proliferation rate of osteoblasts and has an influence on the cytokine gene expression of monocytes in vitro. These results indicate that the physical structure of a biomaterial has a significant effect of how cells interact with the material. Thus, specific characteristics of a material may strongly affect the regenerative potential in vivo.     

Dynamics of Bacterial Community Structure in the Rhizosphere and Root Nodule of Soybean: Impacts of Growth Stages and Varieties

Bacterial communities in rhizosphere and root nodules have significant contributions to the growth and productivity of the soybean (Glycine max (L.) Merr.). In this report, we analyzed the physiological properties and dynamics of bacterial community structure in rhizosphere and root nodules at different growth stages using BioLog EcoPlate and high-throughput sequencing technology, respectively. The BioLog assay found that the metabolic capability of rhizosphere is in increasing trend in the growth of soybeans as compared to the bulk soil. As a result of the Illumina sequencing analysis, the microbial community structure of rhizosphere and root nodules was found to be influenced by the variety and growth stage of the soybean. At the phylum level, Actinobacteria were the most abundant in rhizosphere at all growth stages, followed by Alphaproteobacteria and Acidobacteria, and the phylum Bacteroidetes showed the greatest change. But, in the root nodules Alphaproteobacteria were dominant. The results of the OTU analysis exhibited the dominance of Bradyrhizobium during the entire stage of growth, but the ratio of non-rhizobial bacteria showed an increasing trend as the soybean growth progressed. These findings revealed that bacterial community in the rhizosphere and root nodules changed according to both the variety and growth stages of soybean in the field.