Identity and divergence of protein domain architectures after the yeast-whole genome duplication event

Gene duplication is a key mechanism in evolution for generating new functionality, and it is known to have produced a large proportion of genes. Duplication mechanisms include small-scale, or "local", events such as unequal crossing over and retroposition, together with global events, such as chromosomal or whole genome duplication (WGD). In particular, different studies confirmed that the yeast S. cerevisiae arose from a 100-150 million-year old whole-genome duplication. Detection and study of duplications are usually based on sequence alignment, synteny and phylogenetic techniques, but protein domains are also useful in assessing protein homology. We develop a simple and computationally efficient protein domain architecture comparison method based on the domain assignments available from public databases. We test the accuracy and the reliability of this method in detecting instances of gene duplication in the yeast S. cerevisiae. In particular, we analyze the evolution of WGD and non-WGD paralogs from the domain viewpoint, in comparison with a more standard functional analysis of the genes. A large number of domains is shared by genes that underwent local and global duplications, indicating the existence of a common set of "duplicable" domains. On the other hand, WGD and non-WGD paralogs tend to have different functions. We find evidence that this comes from functional migration within similar domain superfamilies, but also from the existence of small sets of WGD and non-WGD specific domain superfamilies with largely different functions. This observation gives a novel perspective on the finding that WGD paralogs tend to be functionally different from small-scale paralogs. WGD and non-WGD superfamilies carry distinct functions. Finally, the Gene Ontology similarity of paralogs tends to decrease with duplication age, while this tendency is weaker or not observable by the comparison of the domain architectures of paralogs. This suggests that the set of domains composing a protein tends to be maintained, while its function, cellular process or localization diversifies. Overall, the gathered evidence gives a different viewpoint on the biological specificity of the WGD and at the same time points out the validity of domain architecture comparison as a tool for detecting homology

Dynamic Modeling of miRNA-mediated Feed-Forward Loops.

Given the important role of microRNAs (miRNAs) in genome-wide regulation of gene expression, increasing interest is devoted to mixed transcriptional and post-transcriptional regulatory networks analyzing the combinatorial effect of transcription factors (TFs) and miRNAs on target genes. In particular, miRNAs are known to be involved in feed-forward loops (FFLs), where a TF regulates a miRNA and they both regulate a target gene. Different algorithms have been proposed to identify miRNA targets, based on pairing between the 5' region of the miRNA and the 3'UTR of the target gene, and correlation between miRNA host genes and target mRNA expression data. Here we propose a quantitative approach integrating an existing method for mixed FFL identification based on sequence analysis with differential equation modeling approach that permits us to select active FFLs based on their dynamics. Different models are assessed based on their ability to properly reproduce miRNA and mRNA expression data in terms of identification criteria, namely: goodness of fit, precision of the estimates, and comparison with submodels. In comparison with standard approaches based on correlation, our method improves in specificity. As a case study, we applied our method to adipogenic differentiation gene expression data providing potential novel players in this regulatory network

Bioengineered tumoral microtissues recapitulate desmoplastic reaction of pancreatic cancer

Many of the existing three-dimensional (3D) cancer models in vitro fail to represent the entire complex tumor microenvironment composed of cells and extra cellular matrix (ECM) and do not allow a reliable study of the tumoral features and progression. In this paper we reported a strategy to produce 3D in vitro microtissues of pancreatic ductal adenocarcinoma (PDAC) for studying the desmoplastic reaction activated by the stroma-cancer crosstalk. Human PDAC microtissues were obtained by co-culturing pancreatic cancer cells (PT45) and normal or cancer-associated fibroblasts within biodegradable microcarriers in a spinner flask bioreactor. Morphological and histological analyses highlighted that the presence of fibroblasts resulted in the deposition of a stromal matrix rich in collagen leading to the formation of tumor microtissues composed of a heterotypic cell population embedded in their own ECM. We analyzed the modulation of expression of ECM genes and proteins and found that when fibroblasts were co-cultured with PT45, they acquired a myofibroblast phenotype and expressed the desmoplastic reaction markers. This PDAC microtissue, closely recapitulating key PDAC microenvironment characteristics, provides a valuable tool to elucidate the complex stroma\u2013cancer interrelationship and could be used in a future perspective as a testing platform for anticancer drugs in Tissue-on-chip technology

Dynamic Modeling of miRNA-mediated Feed-Forward Loops

Given the important role of microRNAs (miRNAs) in genome-wide regulation of gene expression, increasing interest is devoted to mixed transcriptional and post-transcriptional regulatory networks analyzing the combinatorial effect of transcription factors (TFs) and miRNAs on target genes. In particular, miRNAs are known to be involved in feed-forward loops (FFLs), where a TF regulates a miRNA and they both regulate a target gene. Different algorithms have been proposed to identify miRNA targets, based on pairing between the 5' region of the miRNA and the 3'UTR of the target gene, and correlation between miRNA host genes and target mRNA expression data. Here we propose a quantitative approach integrating an existing method for mixed FFL identification based on sequence analysis with differential equation modeling approach that permits us to select active FFLs based on their dynamics. Different models are assessed based on their ability to properly reproduce miRNA and mRNA expression data in terms of identification criteria, namely: goodness of fit, precision of the estimates, and comparison with submodels. In comparison with standard approaches based on correlation, our method improves in specificity. As a case study, we applied our method to adipogenic differentiation gene expression data providing potential novel players in this regulatory network

Molecular and morphologic characterization of superficial- and deep-subcutaneous adipose tissue subdivisions in human obesity.

OBJECTIVE: Human abdominal subcutaneous white adipose tissue (SAT) is composed of two different subcompartments: a "superficial" SAT (SSAT), located between the skin and a fibrous-fascia plane; and a deeper SAT, located under this fibrous fascia plane, indicated as "deep" SAT (DSAT). DESIGN AND METHODS: In order to investigate whether SSAT and DSAT have different molecular and morphological features, paired SSAT/DSAT biopsies were collected from 10 female obese patients and used for microarray and morphologic analysis. The stroma-vascular fraction cells were also isolated from both depots and cultured in vitro to assess the lipid accumulation rate. RESULTS: SSAT and DSAT displayed different patterns of gene expression, mainly for metabolic and inflammatory genes, respectively. Detailed gene expression analysis indicated that several metabolic genes, including adiponectin, are preferentially expressed in SSAT, whereas inflammatory genes are over-expressed in DSAT. Despite a similar lipid accumulation rate in vitro, in vivo SSAT showed a significant adipocyte hypertrophy together with a significantly lower inflammatory infiltration and vascular vessel lumen mean size, when compared to DSAT. CONCLUSIONS: These data show that, SSAT and DSAT are functionally and morphologically different and emphasize the importance of considering independent these two adipose depots when investigating SAT biology and obesity complications. Copyright 2013 The Obesity Society

A molecularly annotated platform of patient-derived xenografts (\u2018xenopatients\u2019) identifies HER2 as an effective therapeutic target in cetuximab-resistant colorectal cancer

Only a fraction of patients with metastatic colorectal cancer (mCRC) have clinical benefit from therapy with anti-EGFR antibodies, which calls for the identification of novel biomarkers for better personalized medicine. Direct transfer xenografts of tumor surgical specimens conserve the interindividual diversity and the genetic heterogeneity typical of the tumors of origin, potentially combining the flexibility of preclinical analysis with the informative value of population-based studies. We generated cohorts of 85 distinct, genetically characterized mCRC \u2018xenopatients\u2019 to discover novel determinants of therapeutic response and new druggable driver oncoproteins. Serially passaged tumors retained the morphological and genomic features of their original counterparts. A validation trial confirmed the robustness of this approach: xenopatients responded to the anti-EGFR antibody cetuximab with rates and extents analogous to those observed in the clinic and could be prospectively stratified as responders or non-responders based on several predictive biomarkers. Genotype-response correlations revealed HER2 amplification/overexpression in 2.7% of unselected tumors and in 36% of cetuximab-resistant, KRAS/NRAS/BRAF/PIK3CA wild-type cases. Importantly, HER2 amplification was also strongly enriched in clinically nonresponsive KRAS wild-type patients. A proof-of-concept, multi-arm study in HER2-amplified xenopatients revealed that the dual EGFR/ERBB2 inhibitor lapatinib led to disease stabilization, whereas pertuzumab, which blocks HER2 heterodimerization, was ineffective. Combinations of lapatinib and pertuzumab or lapatinib and cetuximab induced overt, long-lasting tumor regression. Our suite of patient-derived mCRC xenografts reliably mimicked disease response in humans and prospectively recapitulated biomarker-based case stratification. The impressive efficacy of combined EGFR/HER2 inhibition in HER2-amplified cases suggests promising therapeutic opportunities in cetuximab-resistant mCRC patients, whose medical treatment in the chemorefractory setting remains an unmet clinical need