Transcriptome Profiling of Etridiazole-Exposed Zebrafish (Danio rerio) Embryos Reveals Pathways Associated with Cardiac and Ocular Toxicities.

Etridiazole (EDZ) is a thiadiazole-containing fungicide commonly used to control Pythium and Phytophthora spp. Although previous studies have shown that EDZ is teratogenic, the exact molecular mechanisms underlying its toxicity remain unknown. In this study, a zebrafish (Danio rerio; ZF) model was used to explore the molecular pathways associated with EDZ toxicity. The whole transcriptome of ZF embryos exposed to 96 h of EDZ was analyzed, along with developmental abnormalities. EDZ-induced malformations were primarily related to the eyes, heart, and growth of the ZF. Compared to untreated ZF, etridiazole-treated ZF had 2882 differentially expressed genes (DEGs), consisting of 1651 downregulated genes and 1231 upregulated genes. Gene ontology enrichment analysis showed that DEGs were involved in biological processes, such as sensory perception, visual perception, sensory organ development, and visual system development, and showed transmembrane transporter and peptidase regulator activities. Metabolism, phototransduction, aminoacyl-tRNA biosynthesis, MAPK signaling pathway, calcium signaling pathway, and vascular smooth muscle contraction were among the most enriched KEGG pathways. The qPCR analyses of the eight random genes were in good agreement with the transcriptome data. These results suggest several putative mechanisms underlying EDZ-induced developmental deformities in ZF.

Analysis of deoxyribonuclease activity by conjugation-free fluorescence polarisation in sub-nanolitre droplets.

We report the analysis of deoxyribonuclease (DNase) activity by conjugation-free fluorescence polarisation in a droplet-based microfluidic chip. DNase is a DNA cleaving enzyme and its activity is important in the maintenance of normal cellular functions. Alterations in DNase activity have been implicated as the cause of various cancers and autoimmune diseases. To date, various methods for the analysis of DNase activity have been reported. However, they are not cost effective due to the requirement of large sample volumes and the need for the conjugation of fluorescent dyes. In this study, we have used ethidium bromide (EtBr), a DNA intercalating reagent, as a fluorescent reporter without any prior conjugation or modification of DNA. Degradation of DNA by DNase 1 was monitored at a steady state by making changes in the fluorescence polarisation of EtBr in droplets with a volume of 330 picolitre at a 40 hertz frequency under visible light. Using this technique, we successfully determined the half-maximal inhibitory concentration (IC50) of ethylenediaminetetraacetic acid (EDTA) for the inhibition of DNase 1 activity to be 1.56 ± 0.91 mM.

Anthocyanins in Floral Colors: Biosynthesis and Regulation in Chrysanthemum Flowers.

Chrysanthemum (Chrysanthemum morifolium) is an economically important ornamental crop across the globe. As floral color is the major factor determining customer selection, manipulation of floral color has been a major objective for breeders. Anthocyanins are one of the main pigments contributing to a broad variety of colors in the ray florets of chrysanthemum. Manipulating petal pigments has resulted in the development of a vast range of floral colors. Although the candidate genes involved in anthocyanin biosynthesis have been well studied, the genetic and transcriptional control of floral color remains unclear. Despite advances in multi-omics technology, these methods remain in their infancy in chrysanthemum, owing to its large complex genome and hexaploidy. Hence, there is a need to further elucidate and better understand the genetic and molecular regulatory mechanisms in chrysanthemum, which can provide a basis for future advances in breeding for novel and diverse floral colors in this commercially beneficial crop. Therefore, this review describes the significance of anthocyanins in chrysanthemum flowers, and the mechanism of anthocyanin biosynthesis under genetic and environmental factors, providing insight into the development of novel colored ray florets. Genetic and molecular regulatory mechanisms that control anthocyanin biosynthesis and the various breeding efforts to modify floral color in chrysanthemum are detailed.

Muscarinic acetylcholine receptors regulate the dephosphorylation of eukaryotic translation elongation factor 2 in SNU-407 colon cancer cells.

Previously, we showed that muscarinic acetylcholine receptors (mAChRs) promote global protein biosynthesis in SNU-407 colon cancer cells. However, the molecular mechanisms underlying this event are poorly understood. Here, we asked whether mAChRs modulate the activity of eukaryotic translation elongation factor 2 (eEF2), which controls ribosomal translocation during the peptide elongation step. When SNU-407 cells were treated with the cholinergic agonist carbachol, eEF2 phosphorylation at T56 was decreased in a dose- and time-dependent manner. The muscarinic antagonist atropine almost completely blocked this effect of carbachol, demonstrating that mAChRs specifically regulate eEF2 dephosphorylation. We also investigated the signaling pathways that connect mAChR stimulation to eEF2 dephosphorylation using chemical inhibitors. Treating cells with U0126, a potent MEK1/2 inhibitor, decreased carbachol-stimulated eEF2 dephosphorylation. In contrast, the mTORC1 inhibitor rapamycin did not have a significant effect on eEF2 dephosphorylation. We also found that the protein kinase C (PKC) inhibitor GF109203X substantially reduced eEF2 dephosphorylation. Together, our experimental data indicate that the MEK1/2-ERK1/2 pathway and the PKC pathway, but not the mTORC1-S6K1 pathway, are involved in mAChR-mediated eEF2 dephosphorylation.

Analysis of ribonuclease activity in sub-nanoliter droplets by label-free fluorescence measurements.

We report the results of a label-free analysis of ribonuclease activity using droplet-based microfluidics. The ribonucleolytic activity of ribonucleases (RNases) plays a critical role in cellular functions such as development, survival, growth and differentiation. Altered ribonucleolytic activity and/or the expression level of the RNase A family are known to be associated with pancreatic, bladder, ovarian and thyroid cancers among others. For this reason, the RNase A family is a meaningful protein biomarker that can be used in the diagnosis of cancer and as a target for new drug screening. There are some successful traditional methods for analysing the RNase activity, such as radioactive label-based assay, methylene blue-based assay, gel zymography, as well as other more recently developed methods such as electrochemical assay and fluorescence resonance energy transfer (FRET). However, these methods require analytical samples with a volume ranging from microliters to milliliters, and are not suitable for high-throughput analysis. Therefore, we integrated ethidium bromide (EtBr), which intercalates the chemical itself to nucleic acid, to droplet-based microfluidics for a cost-effective, high-throughput analysis. Put simply, this method is dependent on the amount of intercalated EtBr molecules on RNA. Our assay also uses visible light that is harmless to humans, unlike previous methods that used harmful UV rays, to excite the EtBr molecules. Specifically, we monitored the ribonucleolytic activity of less than 10 nM RNase A in droplets of about 330 picoliters. Also, half the maximal inhibitory concentration (IC50) of the RNase inhibitor was successfully measured in the same volume of droplets at a frequency of 40 hertz.

The ERK1/2 and mTORC1 Signaling Pathways Are Involved in the Muscarinic Acetylcholine Receptor-Mediated Proliferation of SNU-407 Colon Cancer Cells.

Muscarinic acetylcholine receptors (mAChRs) regulate diverse cellular functions, including cell growth and proliferation, via multiple signaling pathways. Previously, we showed that mAChRs stimulate the MEK1/2-ERK1/2-RSK pathway in SNU-407 colon cancer cells and subsequently promote cell proliferation. In this study, we provide evidence that the PI3K-Akt-mTORC1-S6K1 pathway is activated by mAChRs in SNU-407 cells and that this pathway is associated with protein biosynthesis and cell proliferation. When the cells were treated with the cholinergic agonist carbachol, Akt was activated in a dose- and time-dependent fashion. This carbachol effect was almost completely blocked by the PI3K inhibitor LY294002, implying that PI3K is responsible for the Akt activation. S6K1, a major downstream target of mTORC1, was also activated by carbachol in a temporal profile similar to that of the Akt activation. This carbachol-stimulated S6K1 activation was abrogated by LY294002 or the mTORC1 inhibitor rapamycin, supporting the notion that mAChRs mediate S6K1 activation via the PI3K-Akt-mTORC1 pathway. We observed that global protein biosynthesis, monitored by puromycin incorporation, was strongly increased by carbachol in an atropine-sensitive manner. Inhibition experiments indicated that the ERK1/2 and mTORC1 signaling pathways may be involved in carbachol-stimulated global protein biosynthesis. We also found that treating SNU-407 cells with LY294002 or rapamycin significantly suppressed carbachol-stimulated cell proliferation. In the presence of the MEK1/2 inhibitor U0126, cell proliferation was further reduced by rapamycin treatment. Our data thus suggest that both the MEK1/2-ERK1/2 and mTORC1 pathways play important roles in mAChR-mediated cell proliferation in SNU-407 colon cancer cells. J. Cell. Biochem. 117: 2854-2863, 2016. © 2016 Wiley Periodicals, Inc.

Transcriptome Analysis of Thiram-Treated Zebrafish (Danio rerio) Embryos Reveals Disruption of Reproduction Signaling Pathways.

Thiram, a dithiocarbamate fungicide, is used for the treatment of various fungal infections affecting crops and ornamentals. However, thiram-associated toxicity has been reported in animals, including fish, and the underlying molecular mechanisms are unclear. Herein, we employed zebrafish (ZF) to gain further insights into thiram toxicity-associated molecular mechanisms. We studied developmental abnormalities and performed whole-transcriptome analysis of ZF embryos exposed to thiram for 96 h. Embryos exposed to 4.0 µg/L thiram exhibited several phenotypic abnormalities, including bradycardia, spinal curvature, hatching arrest, and growth retardation. Whole-transcriptome analysis revealed 1754 differentially expressed genes (DEGs), with 512 upregulated and 1242 downregulated DEGs. The majority of biological processes affected by thiram were metabolic. Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis yielded terms related to reproduction, such as steroid biosynthesis and steroid hormone biosynthesis. Quantitative real-time polymerase chain reaction validation results were in line with sequencing data for ten DEGs. The study results improve our current understanding of the effects of thiram exposure in ZF.

Cytotoxic Effects of Nanoliposomal Cisplatin and Diallyl Disulfide on Breast Cancer and Lung Cancer Cell Lines.

Dual drug delivery has become the choice of interest nowadays due to its increased therapeutic efficacy in targeting the tumor site precisely. As quoted in recent literature, it has been known to treat several cancers with an acute course of action. Even so, its use is restricted due to the drug's low pharmacological activity, which leads to poor bioavailability and increases first-pass metabolism. To overcome these issues, a drug delivery system using nanomaterials which would not only encapsulate the drugs of interest but also carry them to the target site of action is needed. Given all these attributes, we have formulated dual drug-loaded nanoliposomes with cisplatin (cis-diamminedichloroplatinum(II) (CDDP)), an effective anti-cancer drug, and diallyl disulfide (DADS), an organosulfur compound derived from garlic. The CDDP and DADS-loaded nanoliposomes (Lipo-CDDP/DADS) exhibited better physical characteristics such as size, zeta potential, polydispersity index, spherical shape, optimal stability, and satisfactory encapsulation percentage. The in vitro anti-cancer activity against MDA-MB-231 and A549 cell lines revealed that Lipo-CDDP/DADS showed significant efficacy against the cancer cell lines, depicted through cell nucleus staining. We conclude that Lipo-CDDP/DADS hold exceptional pharmacological properties with better anti-cancer activity and would serve as a promising formulation to treat various cancers.

Analysis of Phosphatase Activity in a Droplet-Based Microfluidic Chip.

We report analysis of phosphatase activity and inhibition on droplet-based microfluidic chips. Phosphatases are such attractive potential drug targets because abnormal phosphatase activity has been implicated in a variety of diseases including cancer, neurological disorders, diabetes, osteoporosis, and obesity. So far, several methods for assessing phosphatase activity have been reported. However, they require a large sample volume and additional chemical modifications such as fluorescent dye conjugation and nanomaterial conjugation, and are not cost-effective. In this study, we used an artificial phosphatase substrate 3-O-methylfluorescein phosphate as a fluorescent reporter and dual specificity phosphatase 22. Using these materials, the phosphatase assay was performed from approximately 340.4 picoliter (pL) droplets generated at a frequency of ~40 hertz (Hz) in a droplet-based microfluidic chip. To evaluate the suitability of droplet-based platform for screening phosphatase inhibitors, a dose-response inhibition study was performed with ethyl-3,4-dephostatin and the half-maximal inhibitory concentration (IC50) was calculated as 5.79 ± 1.09 µM. The droplet-based results were compared to microplate-based experiments, which showed agreement. The droplet-based phosphatase assay proposed here is simple, reproducible, and generates enormous data sets within the limited sample and reagent volumes.

Developmental Toxic Effects of Thiram on Developing Zebrafish (Danio rerio) Embryos.

Thiram, an oxidized dimer of dithiocarbamate, has fungicidal and ectoparasiticidal roles. This study aimed to determine the effects of thiram on the development of zebrafish (ZF) embryos. The developmental toxicity test was performed in accordance with the OECD 236 test guidelines, and ZF embryos were subjected to several thiram concentrations and a DMSO (0.01%) control. Subsequently, embryo mortalities and developmental anomalies were evaluated at different hours post fertilization (hpf). Thiram was highly toxic to ZF, with calculated median lethal concentrations (LC50) of thiram at 48 and 96 h as 13.10 ± 2.17 and 8.87 ± 2.09 µg/L, respectively. Thiram-treated embryos/larvae exhibited a variety of deformities, such as abnormal somites, reduced eye pigment, abnormal tail shape, yolk sac edema, hatching defects, and curved spines, with a median effective concentration (EC50) of 3.88 ± 1.23, 5.04 ± 1.82, 6.23 ± 0.92, 5.24 ± 2.22, 1.39 ± 0.25, and 2.60 ± 0.82 µg/L, respectively. Teratogenic index (TI) values ranged from 1.42 to 6.66 for the scored deformities. At 48 hpf, the average heartbeat of the control group was 177.20 ± 5.63 per minute, while the highest thiram-treated group (40 µg/L) was 99.50 ± 18.12 per minute. In addition, cardiac-related issues, such as pericardial edema and abnormal blood flow, were observed in thiram-treated ZF embryos. Overall, these findings suggest that thiram is teratogenic to ZF.

Transcriptome-Based Identification of Genes Responding to the Organophosphate Pesticide Phosmet in Danio rerio.

Organophosphate pesticides (OPPs) are one of the most widely used insecticides. OPPs exert their neurotoxic effects by inhibiting acetylcholine esterase (AChE). Most of the gross developmental abnormalities observed in OPP-treated fish, on the other hand, may not be explained solely by AChE inhibition. To understand the overall molecular mechanisms involved in OPP toxicity, we used the zebrafish (ZF) model. We exposed ZF embryos to an OPP, phosmet, for 96 h, and then analyzed developmental abnormalities and performed whole transcriptome analysis. Phenotypic abnormalities, such as bradycardia, spine curvature, and growth retardation, were observed in phosmet-treated ZF (PTZF). Whole transcriptome analysis revealed 2190 differentially expressed genes (DEGs), with 822 and 1368 significantly up-and downregulated genes, respectively. System process and sensory and visual perception were among the top biological pathways affected by phosmet toxicity. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed significant enrichment of metabolic pathways, calcium signaling pathway, regulation of actin cytoskeleton, cardiac muscle contraction, drug metabolism-other enzymes, and phototransduction. Quantitative real-time PCR results of six DEGs agreed with the sequencing data expression profile trend. Our findings provide insights into the consequences of phosmet exposure in ZF, as well as an estimate of the potential risk of OPPs to off-target species.

Morphometric analysis of alpha-1, 3-galactosyltransferase knockout pig kidney and heart.

We report a morphometric evaluation of α1,3-galactosyltransferase knockout (GTKO) pig heart and kidney (n = 9) at the end of one, three and seven months. Organs parameters gradually increased with the age (p < 0.05) and body weight (p < 0.05) of the pigs. Organs morphometries were significantly correlated to the age and body weight of the animal. We were able to conclude the average size of GTKO pig heart and kidney based on age and body weight, which could be helpful in estimating the size of these organs non-invasively for transplantation.

Validation of mouse phosphoprotein enriched in astrocyte 15 (mPEA15) expressing transgenic pig as a potential model in diabetes translational research.

The present study aimed to investigate the characteristics of mPEA15 expressing transgenic pig (TG pig) as a potential model for diabetes. Expression analysis confirmed the ubiquitous expression of mPEA15 in TG pigs at F4. Oral glucose tolerance test results showed that restoration of normal glucose levels was significantly delayed in the TG pigs when compared with that in the wild-type pigs (WT pigs). Primary skeletal muscle cells isolated from TG pigs demonstrated reduced glucose uptake and reduced GLUT4 translocation to the plasma membrane in response to insulin treatment. Combined, these results suggest that mPEA15 expressing pigs has a glucose intolerance and insulin resistance which are known to mediate the pathophysiology of type 2 diabetes mellitus. Thus, mPEA15 transgenic pigs would serve as a promising model for diabetes translational research.

Codon optimized membrane cofactor protein expression in α 1, 3 galactosyltransferase knockout pig cells improve protection against cytotoxicity of monkey serum.

In this study, we attempted to upgrade GT -MCP/-MCP pig genetically to express MCP at a higher level and additionally thrombomodulin (TBM), which have respective roles as a complement regulatory protein and a coagulation inhibitor. We constructed a dicistronic cassette consisting of codon-optimized MCP (mMCP) and TBM (m-pI2), designed for ubiquitous expression of MCP and endothelium specific expression of TBM. The cassette was confirmed to allow extremely increased MCP expression compared with non-modified MCP, and an endothelial-specific TBM expression. We thus transfected m-pI2 into ear-skin fibroblasts isolated from a GT -MCP/-MCP pig. By twice selection using magnetically activated cell sorting (MACS), and single-cell culture, we were able to obtain clones over 90% expressing MCP. The cells of a clone were provided as a donor for nuclear transfer resulting in the generation of a GT -MCP/-MCP /mMCP/TBM pig, which was confirmed to be carrying cells expressing MCP and functioning as an inhibitor against the cytotoxic effect of normal monkey serum, comparable with donor cells. Collectively, these results demonstrated an effective approach for upgrading transgenic pig, and we assumed that upgraded pig would increase graft survival.

VIG-1 is required for maintenance of genome stability in Caenorhabditis elegans.

To explore the function of VIG-1 in Caenorhabditis elegans, we analyzed the phenotypes of two vig-1 deletion mutants: vig-1(tm3383) and vig-1(ok2536). Both vig-1 mutants exhibited phenotypes associated with genome instability, such as a high incidence of males (Him) and increased embryonic lethality. These phenotypes became more evident in succeeding generations, implying that the germline of vig-1 accumulates DNA damage over generations. To examine whether vig-1 causes a defect in the DNA damage response, we treated worms with UV or camptothecin, a specific topoisomerase I inhibitor. We observed that the embryonic survival of the vig-1 mutants was reduced compared with that of the wild-type worms. Our results thus suggest that VIG-1 is required for maintaining genome stability in response to endogenous and exogenous genotoxic stresses.