Interference of p53:Twist1 interaction through competing nanobodies.

Twist1 promote the bypass of p53 response by interacting with p53 and facilitating its MDM2-mediated degradation. We reasoned that reagents able to interfere with the p53:Twist1 complex might alleviate Twist1 inhibitory effect over p53, thus representing potential therapeutic tools in p53 wild type tumors. From a pre-immune library of llama nanobodies (VHH), we isolated binders targeting the p53 C-terminal region (p53-CTD) involved in the interaction with Twist1 by using recombinant Twist1 as an epitope-specific competitor during elution. Positive hits were validated by proving their capacity to immunoprecipitate p53 and to inhibit Twist1:p53 binding in vitro. Molecular modeling confirmed a preferential docking of positive hits with p53-CTD. D11 VHH activity was validated in human cell models, succeeded in immunoprecipitating endogenous p53 and, similarly to Twist1 knock-down, interfered with p53 turnover, p53 phosphorylation at Serine 392 and affected cell viability. Despite the limited functional effect determined by D11 expression in target cells, our results provide the proof of principle that nanobodies ectopically expressed within a cell, have the capacity to target the assembly of the pro-tumorigenic Twist1:p53 complex. These results disclose novel tools for dissecting p53 biology and lay down the grounds for the development of innovative targeted therapeutic approaches.

Expression pattern dysregulation of stress- and neuronal activity-related genes in response to prenatal stress paradigm in zebrafish larvae.

Maternal stress during pregnancy adversely affects developmental fetal programming. Glucocorticoid excess is one of those conditions that underlie the prenatal stress and can lead to many pathological disorders later in life. Beyond the obvious use of mammalian model organisms to uncover the different mechanisms at the basis of prenatal stress effects, zebrafish represents a complementary fruitful model for this research field. Here we demonstrated that the application of an experimental paradigm, which simulates prenatal stress by exposing embryos to cortisol excess, produced an alteration of gene expression pattern. In particular, the transcript level of hsd11b2, a gene involved in the cortisol catabolism, was affected in prenatally stressed larvae, even after many hours from the removal of cortisol excess. Interestingly, the expression pattern of c-fos, a marker gene of neural activity, was affected in prenatally stressed larvae even in response to a swirling and osmotic stress challenge. Our data corroborate the idea of zebrafish as a useful model organism to study prenatal stress effects on vertebrate development.

Right posterior cortical functions in a tumour patient series.

It is standardly believed that the localisation of cognitive function by means of impairments arising from cortical tumour is not possible as the functional defects that result are mild and unspecific. These assumptions were not supported in an investigation of four processes generally sensitive to right posterior cortical lesions, when patients with parieto-occipital lesions were compared with prefrontal ones. In three of the tests loading on the individual processes - Reaching Accuracy, Star Cancellation, Fragmented Letters and Cube Analysis - parieto-occipital impairments were found in the basic groups analysis and this was so in the right-hemisphere group. More critically, in these tests Lesion Behaviour Mapping showed the critical lesion site for the tests to have relatively little overlap with those of the other tests, indicating that the cognitive effects were not widespread and diffuse. In addition, in three of the tests the critical lesion sites fitted localisations arrived from other procedures. Patients with high-grade tumours performed considerably worse than those with low-grade tumours in only two of the tests (Star Cancellation, Cube Analysis) particularly in the right parieto-occipital group. In three (Reaching Accuracy, Star Cancellation, Cube Analysis) there was a deterioration with the operation specifically in the low-grade tumour patients. It is suggested that a tumour patient series may provide converging evidence for the localisation of a function initially obtained by some other procedure.