Persistent expression of microRNA-125a targets is required to induce murine hematopoietic stem cell repopulating activity.

MicroRNAs (miRs) are small noncoding RNAs that regulate gene expression posttranscriptionally by binding to the 3' untranslated regions of their target mRNAs. The evolutionarily conserved microRNA-125a (miR-125a) is highly expressed in both murine and human hematopoietic stem cells (HSCs), and previous studies have found that miR-125 strongly enhances self-renewal of HSCs and progenitors. In this study we explored whether temporary overexpression of miR-125a would be sufficient to permanently increase HSC self-renewal or, rather, whether persistent overexpression of miR-125a is required. We used three complementary in vivo approaches to reversibly enforce expression of miR-125a in murine HSCs. Additionally, we interrogated the underlying molecular mechanisms responsible for the functional changes that occur in HSCs on overexpression of miR-125a. Our data indicate that continuous expression of miR-125a is required to enhance HSC activity. Our molecular analysis confirms changes in pathways that explain the characteristics of miR-125a overexpressing HSCs. Moreover, it provides several novel putative miR-125a targets, but also highlights the complex molecular changes that collectively lead to enhanced HSC function.

Water dynamics in MCF-7 breast cancer cells: a neutron scattering descriptive study.

Water mobility in cancer cells could be a powerful parameter to predict the progression or remission of tumors. In the present descriptive work, new insight into this concept was achieved by combining neutron scattering and thermal analyses. The results provide the first step to untangle the role played by water dynamics in breast cancer cells (MCF-7) after treatment with a chemotherapy drug. By thermal analyses, the cells were probed as micrometric reservoirs of bulk-like and confined water populations. Under this perspective we showed that the drug clearly alters the properties of the confined water. We have independently validated this idea by accessing the cellular water dynamics using inelastic neutron scattering. Finally, analysis of the quasi-elastic neutron scattering data allows us to hypothesize that, in this particular cell line, diffusion increases in the intracellular water in response to the action of the drug on the nanosecond timescale.

MiR-125a enhances self-renewal, lifespan, and migration of murine hematopoietic stem and progenitor cell clones.

Expansion of hematopoietic stem cells (HSCs) is a 'holy grail' of regenerative medicine, as successful stem cell transplantations depend on the number and quality of infused HSCs. Although many attempts have been pursued to either chemically or genetically increase HSC numbers, neither clonal analysis of these expanded cells nor their ability to support mature blood lineages has been demonstrated. Here we show that miR-125a, at the single cell level, can expand murine long-term repopulating HSCs. In addition, miR-125a increases clone longevity, clone size and clonal contribution to hematopoiesis. Unexpectedly, we found that miR-125a expanded HSCs clones were highly homogenously distributed across multiple anatomical sites. Interestingly, these miR-125a overexpressing cells had enhanced mobility and were more frequently detected in the spleen. Our study reveals a novel, cell-intrinsically controlled mechanism by which HSC migration is regulated.

Predicting the impact of Lynch syndrome-causing missense mutations from structural calculations.

Accurate methods to assess the pathogenicity of mutations are needed to fully leverage the possibilities of genome sequencing in diagnosis. Current data-driven and bioinformatics approaches are, however, limited by the large number of new variations found in each newly sequenced genome, and often do not provide direct mechanistic insight. Here we demonstrate, for the first time, that saturation mutagenesis, biophysical modeling and co-variation analysis, performed in silico, can predict the abundance, metabolic stability, and function of proteins inside living cells. As a model system, we selected the human mismatch repair protein, MSH2, where missense variants are known to cause the hereditary cancer predisposition disease, known as Lynch syndrome. We show that the majority of disease-causing MSH2 mutations give rise to folding defects and proteasome-dependent degradation rather than inherent loss of function, and accordingly our in silico modeling data accurately identifies disease-causing mutations and outperforms the traditionally used genetic disease predictors. Thus, in conclusion, in silico biophysical modeling should be considered for making genotype-phenotype predictions and for diagnosis of Lynch syndrome, and perhaps other hereditary diseases.