An efficient transformation method for genome editing of elite bread wheat cultivars.

An efficient genetic transformation protocol is necessary to edit genes for trait improvement directly in elite bread wheat cultivars. We used a protein fusion between a wheat growth-regulating factor 4 (GRF4) and its interacting factor (GIF1) to develop a reproducible genetic transformation and regeneration protocol, which we then used to successfully transform elite bread wheat cultivars Baj, Kachu, Morocco, Reedling, RL6077, and Sujata in addition to the experimental cultivar Fielder. Immature embryos were transformed with the vector using particle bombardment method. Transformation frequency increased nearly 60-fold with the GRF4-GIF1-containing vectors as compared to the control vector and ranged from ~5% in the cultivar Kachu to 13% in the cultivar RL6077. We then edited two genes that confer resistance against leaf rust and powdery mildew directly in the aforementioned elite cultivars. A wheat promoter, TaU3 or TaU6, to drive the expression of guide RNA was effective in gene editing whereas the OsU3 promoter failed to generate any edits. Editing efficiency was nearly perfect with the wheat promoters. Our protocol has made it possible to edit genes directly in elite wheat cultivars and would be useful for gene editing in other wheat varieties, which have been recalcitrant to transformation thus far.

Animal models of atherosclerosis.

Cardiovascular disease is currently the predominant cause of mortality worldwide and its incidence is expected to increase significantly during the next decades owing to the unhealthy effects of modern lifestyle habits (e.g., obesity and lack of physical exercise). Cardiovascular death is frequently associated with acute myocardial infarction or stroke, which are generally the ultimate consequence of an underlying atherosclerotic process. Small and big animal models are valuable tools to understand the molecular mechanisms underlying atherosclerotic plaque formation and progression, as well as the occurrence of associated ischemic events. Moreover, animal models of atherosclerosis are pivotal for testing mechanistic hypothesis and for translational research, including the assessment of dietary and/or pharmacological interventions and the development of imaging technologies and interventional devices. In this chapter, we will describe the most widely used animal models that have permitted major advances in atherosclerosis research and significant improvements in the treatment and diagnosis of atherosclerotic disease.

Neuroprotective effects of the nonpsychoactive cannabinoid cannabidiol in hypoxic-ischemic newborn piglets.

To test the neuroprotective effects of the nonpsychoactive cannabinoid cannabidiol (CBD), piglets received i.v. CBD or vehicle after hypoxia-ischemia (HI: temporary occlusion of both carotid arteries plus hypoxia). Nonhypoxic-ischemic sham-operated piglets remained as controls. Brain damage was studied by near-infrared spectroscopy (NIRS) and amplitude-integrated electroencephalography (aEEG) and by histologic assessment (Nissl and FluoroJadeB staining). In HI+vehicle, HI led to severe cerebral hemodynamic and metabolic impairment, as reflected in NIRS by an increase in total Hb index (THI) and a decrease in the fractional tissue oxygenation extraction (FTOE); in HI+CBD the increase of THI was blunted and FTOE remained similar to SHAM. HI profoundly decreased EEG amplitude, which was not recovered in HI+vehicle, indicating cerebral hypofunction; seizures were observed in all HI+vehicle. In HI+CBD, however, EEG amplitude recovered to 46.4 7.8% baseline and seizures appeared only in 4/8 piglets (both p < 0.05). The number of viable neurons decreased and that of degenerating neurons increased in HI+vehicle; CBD reduced both effects by more than 50%. CBD administration was free from side effects; moreover, CBD administration was associated with cardiac, hemodynamic, and ventilatory beneficial effects. In conclusion, administration of CBD after HI reduced short-term brain damage and was associated with extracerebral benefits.

Vitamin D-dependent recruitment of corepressors to vitamin D/retinoid X receptor heterodimers.

Transcriptional regulation by nuclear receptors is mediated by recruitment of coactivators and corepressors. In the classical model, unliganded nonsteroidal receptors bind corepressors, such as the silencing mediator of thyroid and retinoid receptors (SMRT) or nuclear corepressor (NCoR), that are released upon ligand binding. We show here that, unlike other receptors, the heterodimer of the vitamin D receptor (VDR) with the retinoid X receptor (RXR) recruits NCoR and SMRT strictly in a VDR agonist-dependent manner. Binding of an agonist to VDR allows its partner receptor, RXR, to bind the corepressors. The RXR ligand has the opposite effect and induces corepressor release from the heterodimer. 1,25-Dihydroxy-vitamin D(3) (VD3) causes recruitment of SMRT and NCoR to a VDR target promoter. Down-regulation of corepressors by means of small interfering RNA enhances transcriptional responses to VD3. These data reveal a new paradigm of SMRT and NCoR binding to nuclear receptors and demonstrate that these corepressors can function as physiological negative regulators of VD3-mediated transcription.

A high-throughput study in melanoma identifies epithelial-mesenchymal transition as a major determinant of metastasis.

Metastatic disease is the primary cause of death in cutaneous malignant melanoma (CMM) patients. To understand the mechanisms of CMM metastasis and identify potential predictive markers, we analyzed gene-expression profiles of 34 vertical growth phase melanoma cases using cDNA microarrays. All patients had a minimum follow-up of 36 months. Twenty-one cases developed nodal metastatic disease and 13 did not. Comparison of gene expression profiling of metastatic and nonmetastatic melanoma cases identified 243 genes with a >2-fold differential expression ratio and a false discovery rate of <0.2 (206 up-regulated and 37 down-regulated). This set of genes included molecules involved in cell cycle and apoptosis regulation, epithelial-mesenchymal transition (EMT), signal transduction, nucleic acid binding and transcription, protein synthesis and degradation, metabolism, and a specific group of melanoma- and neural-related proteins. Validation of these expression data in an independent series of melanomas using tissue microarrays confirmed that the expression of a set of proteins included in the EMT group (N-cadherin, osteopontin, and SPARC/osteonectin) were significantly associated with metastasis development. Our results suggest that EMT-related genes contribute to the promotion of the metastatic phenotype in primary CMM by supporting specific adhesive, invasive, and migratory properties. These data give a better understanding of the biology of this aggressive tumor and may provide new prognostic and patient stratification markers in addition to potential therapeutic targets.

The retinoid X receptor ligand restores defective signalling by the vitamin D receptor.

It is assumed that the retinoid X receptor (RXR) acts as a silent partner to the vitamin D receptor (VDR) with its only function to increase affinity of VDR/RXR to its DNA recognition site. In this study, we show that the RXR ligand 9-cis-retinoic acid (9-cis-RA) induces recruitment of coactivators by the DNA-bound heterodimer and potentiates vitamin D-dependent transcriptional responses. The presence of 9-cis-RA increases induction of cyp24 transcripts and differentiation of colon cancer cells by vitamin D, confers significant agonistic activity to a VDR ligand with very low agonistic activity and can even restore transcriptional activity of an AF-2 mutant VDR that causes hereditary rickets. This study shows that, in VDR/RXR heterodimers, allosteric communication triggered by the RXR ligand has a previously unrecognized role in vitamin D signalling, with important physiological and therapeutic implications.

A permissive retinoid X receptor/thyroid hormone receptor heterodimer allows stimulation of prolactin gene transcription by thyroid hormone and 9-cis-retinoic acid.

Heterodimers of the retinoid X receptor (RXR) with the thyroid hormone receptor (TR) are considered to be nonpermissive. It is believed that within these complexes RXR acts as a "silent partner." We demonstrate here that a permissive heterodimer mediates stimulation of prolactin expression by the thyroid hormone T3 and by 9-cis retinoic acid (9-cis-RA). A response element located in the prolactin distal enhancer mediates transactivation by both ligands in pituitary cells, and RXR recruits coactivators when bound to this element as a heterodimer with TR. Furthermore, transcription by the RXR agonist can be obtained in CV-1 cells only after overexpression of coactivators, and overexpression of corepressors inhibits the response in pituitary cells. Thus, cell type-specific differences in coregulator recruitment can determine the cellular response to both ligands. Coactivator recruitment by 9-cis-RA requires the ligand-dependent transactivation domains (AF-2) of both heterodimeric partners. Interestingly, the presence of the RXR ligand can overcome the deleterious effect of the AF-2 mutation E401Q on association with coactivators and transactivation. These results demonstrate an unexpected role for RXR in TR signaling and show that in particular cellular environments this receptor can act as a "nonsilent" partner of TR, allowing stimulation by RXR agonists.