Flavonoids from Engineered Tomatoes Inhibit Gut Barrier Pro-inflammatory Cytokines and Chemokines, via SAPK/JNK and p38 MAPK Pathways.

Flavonoids are a diverse group of plant secondary metabolites, known to reduce inflammatory bowel disease symptoms. How they achieve this is largely unknown. Our study focuses on the gut epithelium as it receives high topological doses of dietary constituents, maintains gut homeostasis, and orchestrates gut immunity. Dysregulation leads to chronic gut inflammation, via dendritic cell (DC)-driven immune responses. Tomatoes engineered for enriched sets of flavonoids (anthocyanins or flavonols) provided a unique and complex naturally consumed food matrix to study the effect of diet on chronic inflammation. Primary murine colonic epithelial cell-based inflammation assays consist of chemokine induction, apoptosis and proliferation, and effects on kinase pathways. Primary murine leukocytes and DCs were used to assay effects on transmigration. A murine intestinal cell line was used to assay wound healing. Engineered tomato extracts (enriched in anthocyanins or flavonols) showed strong and specific inhibitory effects on a set of key epithelial pro-inflammatory cytokines and chemokines. Chemotaxis assays showed a resulting reduction in the migration of primary leukocytes and DCs. Activation of epithelial cell SAPK/JNK and p38 MAPK signaling pathways were specifically inhibited. The epithelial wound healing-associated STAT3 pathway was unaffected. Cellular migration, proliferation, and apoptosis assays confirmed that wound healing processes were not affected by flavonoids. We show flavonoids target epithelial pro-inflammatory kinase pathways, inhibiting chemotactic signals resulting in reduced leukocyte and DC chemotaxis. Thus, both anthocyanins and flavonols modulate epithelial cells to become hyporesponsive to bacterial stimulation. Our results identify a viable mechanism to explain the in vivo anti-inflammatory effects of flavonoids.

The positive transcriptional elongation factor (P-TEFb) is required for neural crest specification.

Regulation of gene expression at the level of transcriptional elongation has been shown to be important in stem cells and tumour cells, but its role in the whole animal is only now being fully explored. Neural crest cells (NCCs) are a multipotent population of cells that migrate during early development from the dorsal neural tube throughout the embryo where they differentiate into a variety of cell types including pigment cells, cranio-facial skeleton and sensory neurons. Specification of NCCs is both spatially and temporally regulated during embryonic development. Here we show that components of the transcriptional elongation regulatory machinery, CDK9 and CYCLINT1 of the P-TEFb complex, are required to regulate neural crest specification. In particular, we show that expression of the proto-oncogene c-Myc and c-Myc responsive genes are affected. Our data suggest that P-TEFb is crucial to drive expression of c-Myc, which acts as a 'gate-keeper' for the correct temporal and spatial development of the neural crest.

A Database of microRNA Expression Patterns in Xenopus laevis.

MicroRNAs (miRNAs) are short, non-coding RNAs around 22 nucleotides long. They inhibit gene expression either by translational repression or by causing the degradation of the mRNAs they bind to. Many are highly conserved amongst diverse organisms and have restricted spatio-temporal expression patterns during embryonic development where they are thought to be involved in generating accuracy of developmental timing and in supporting cell fate decisions and tissue identity. We determined the expression patterns of 180 miRNAs in Xenopus laevis embryos using LNA oligonucleotides. In addition we carried out small RNA-seq on different stages of early Xenopus development, identified 44 miRNAs belonging to 29 new families and characterized the expression of 5 of these. Our analyses identified miRNA expression in many organs of the developing embryo. In particular a large number were expressed in neural tissue and in the somites. Surprisingly none of the miRNAs we have looked at show expression in the heart. Our results have been made freely available as a resource in both XenMARK and Xenbase.

Chemical genetics and drug discovery in Xenopus.

Chemical genetics uses small molecules to modulate protein function and has the potential to perturb any biochemical event in a complex cellular context. The application of chemical genetics to dissect biological processes has become an attractive alternative to mutagenesis screens due to its technical simplicity, inexpensive reagents, and low-startup costs. Xenopus embryos are particularly amenable to whole organism chemical genetic screens. Here we describe the basic protocols we have developed to screen small compound libraries on Xenopus laevis embryos. We score embryos either by observing phenotypic changes in the whole tadpole or by changes in gene expression pattern using automated wholemount in situ hybridization.

DHODH modulates transcriptional elongation in the neural crest and melanoma.

Melanoma is a tumour of transformed melanocytes, which are originally derived from the embryonic neural crest. It is unknown to what extent the programs that regulate neural crest development interact with mutations in the BRAF oncogene, which is the most commonly mutated gene in human melanoma. We have used zebrafish embryos to identify the initiating transcriptional events that occur on activation of human BRAF(V600E) (which encodes an amino acid substitution mutant of BRAF) in the neural crest lineage. Zebrafish embryos that are transgenic for mitfa:BRAF(V600E) and lack p53 (also known as tp53) have a gene signature that is enriched for markers of multipotent neural crest cells, and neural crest progenitors from these embryos fail to terminally differentiate. To determine whether these early transcriptional events are important for melanoma pathogenesis, we performed a chemical genetic screen to identify small-molecule suppressors of the neural crest lineage, which were then tested for their effects on melanoma. One class of compound, inhibitors of dihydroorotate dehydrogenase (DHODH), for example leflunomide, led to an almost complete abrogation of neural crest development in zebrafish and to a reduction in the self-renewal of mammalian neural crest stem cells. Leflunomide exerts these effects by inhibiting the transcriptional elongation of genes that are required for neural crest development and melanoma growth. When used alone or in combination with a specific inhibitor of the BRAF(V600E) oncogene, DHODH inhibition led to a marked decrease in melanoma growth both in vitro and in mouse xenograft studies. Taken together, these studies highlight developmental pathways in neural crest cells that have a direct bearing on melanoma formation.

Chemical genomics identifies compounds affecting Xenopus laevis pigment cell development.

A forward chemical genomic screen was carried out using Xenopus laevis embryos to identify compounds disrupting pigmented cell development, including the retinal pigment epithelial (RPE) layer of the eye and the melanophores (melanocytes). Phenotypes showing changes in cell migration, morphology and pigmentation were observed. The screen also identified compounds affecting other aspects of Xenopus development including general patterning and morphogenesis, eye development and edema formation. Evidence is presented for the molecular targets of three of the compounds identified. Xenopus melanophore and human melanoma cell lines were also utilised in follow-up cell morphology assays. Chemical genomic screens of this type have an important role to play in the identification of novel compounds providing new molecular tools, and biological information, along with identification of new protein targets and leads for potential therapeutic agents.

A chemical genomic approach identifies matrix metalloproteinases as playing an essential and specific role in Xenopus melanophore migration.

To dissect the function of matrix metalloproteinases (MMPs) involved in cellular migration in vivo, we undertook both a forward chemical genomic screen and a functional approach to discover modulators of melanophore (pigment cell) migration in Xenopus laevis. We identified the 8-quinolinol derivative NSC 84093 as affecting melanophore migration in the developing embryo and have shown it to act as a MMP inhibitor. Potential targets of NSC 84093 investigated include MMP-14 and MMP-2. MMP-14 is expressed in migrating neural crest cells from which melanophores are derived. MMP-2 is expressed at the relevant time of development and in a pattern that suggests it contributes to melanophore migration. Morpholino-mediated knockdown of both MMPs demonstrates they play a key role in melanophore migration and partially phenocopy the effect of NSC 84093.

Three matrix metalloproteinases are required in vivo for macrophage migration during embryonic development.

Macrophages are essential in development, repair and pathology of a variety of tissues via their roles in tissue remodelling, wound healing and inflammation. These biological functions are also associated with a number of human diseases, for example tumour associated macrophages have well defined functions in cancer progression. Xenopus embryonic macrophages arise from a haematopoietic stem cell population by direct differentiation and act as the main mechanism of host defence, before lymphoid cells and a circulatory system have developed. This function is conserved in mouse and human development. Macrophages express a number of matrix metalloproteinases (MMPs), which are central to their function. MMPs are a large family of zinc-dependent endoproteases with multiple roles in extracellular matrix remodelling and the modulation of signalling pathways. We have previously shown MMP-7 to be expressed by Xenopus embryonic macrophages. Here we investigate the role of MMP-7 and two other MMPs (MMP-18 and MMP-9) that are also expressed in the migrating macrophages. Using morpholino (MO) mediated knockdown of each of the MMPs we demonstrate that they are necessary for normal macrophage migration in vivo. The loss-of-function effect can be rescued using the specific MMPs, altered to be resistant to morpholinos but not by overexpression of the other MMPs. Double and triple morpholino knockdowns further suggest that these MMPs act combinatorily to promote embryonic macrophage migration. Thus, our results imply that these three MMPs have distinct functions, which together are crucial to mediate macrophage migration in the developing embryo. This demonstrates conclusively that MMPs are required for normal macrophage cell migration in the whole organism.

Xenopus as a model organism in developmental chemical genetic screens.

Chemical genetics is a potentially powerful tool for studying developmental processes in vertebrate systems. We present data showing Xenopus laevis as a model organism in which systematic chemical genetic screens can be carried out. Previous forward chemical genetic screens, including those with developing zebrafish embryos, have demonstrated the nature and value of biological information gained with this approach. We show how amenable Xenopus is to chemical genetics by investigating a series of compounds either with known biochemical effects, or previously identified to give developmental phenotypes, on a range of biological functions, including the development of pigmentation, the heart and the central nervous system in zebrafish. We have found that the compounds give comparable phenotypes when applied to developing Xenopus embryos. We have also studied the penetrance and expressivity of these chemical genetic phenotypes in relation to genetic variation and the developmental window during which the compound is present. Finally, we assess the feasibility and the potential throughput of a screen in this vertebrate species.

High-throughput protein localization in Arabidopsis using Agrobacterium-mediated transient expression of GFP-ORF fusions.

We describe a streamlined and systematic method for cloning green fluorescent protein (GFP)-open reading frame (ORF) fusions and assessing their subcellular localization in Arabidopsis thaliana cells. The sequencing of the Arabidopsis genome has made it feasible to undertake genome-based approaches to determine the function of each protein and define its subcellular localization. This is an essential step towards full functional analysis. The approach described here allows the economical handling of hundreds of expressed plant proteins in a timely fashion. We have integrated recombinational cloning of full-length trimmed ORF clones (available from the SSP consortium) with high-efficiency transient transformation of Arabidopsis cell cultures by a hypervirulent strain of Agrobacterium. To demonstrate its utility, we have used a selection of trimmed ORFs, representing a variety of key cellular processes and have defined the localization patterns of 155 fusion proteins. These patterns have been classified into five main categories, including cytoplasmic, nuclear, nucleolar, organellar and endomembrane compartments. Several genes annotated in GenBank as unknown have been ascribed a protein localization pattern. We also demonstrate the application of flow cytometry to estimate the transformation efficiency and cell cycle phase of the GFP-positive cells. This approach can be extended to functional studies, including the precise cellular localization and the prediction of the role of unknown proteins, the confirmation of bioinformatic predictions and proteomic experiments, such as the determination of protein interactions in vivo, and therefore has numerous applications in the post-genomic analysis of protein function.

iASPP oncoprotein is a key inhibitor of p53 conserved from worm to human.

We have previously shown that ASPP1 and ASPP2 are specific activators of p53; one mechanism by which wild-type p53 is tolerated in human breast carcinomas is through loss of ASPP activity. We have further shown that 53BP2, which corresponds to a C-terminal fragment of ASPP2, acts as a dominant negative inhibitor of p53 (ref. 1). Hence, an inhibitory form of ASPP resembling 53BP2 could allow cells to bypass the tumor-suppressor functions of p53 and the ASPP proteins. Here, we characterize such a protein, iASPP (inhibitory member of the ASPP family), encoded by PPP1R13L in humans and ape-1 in Caenorhabditis elegans. iASPP is an evolutionarily conserved inhibitor of p53; inhibition of iASPP by RNA-mediated interference or antisense RNA in C. elegans or human cells, respectively, induces p53-dependent apoptosis. Moreover, iASPP is an oncoprotein that cooperates with Ras, E1A and E7, but not mutant p53, to transform cells in vitro. Increased expression of iASPP also confers resistance to ultraviolet radiation and to cisplatin-induced apoptosis. iASPP expression is upregulated in human breast carcinomas expressing wild-type p53 and normal levels of ASPP. Inhibition of iASPP could provide an important new strategy for treating tumors expressing wild-type p53.