Assessing the limits of restraint-based 3D modeling of genomes and genomic domains

Restraint-based modeling of genomes has been recently explored with the advent of Chromosome Conformation Capture (3C-based) experiments. We previously developed a reconstruction method to resolve the 3D architecture of both prokaryotic and eukaryotic genomes using 3C-based data. These models were congruent with fluorescent imaging validation. However, the limits of such methods have not systematically been assessed. Here we propose the first evaluation of a mean-field restraint-based reconstruction of genomes by considering diverse chromosome architectures and different levels of data noise and structural variability. The results show that: first, current scoring functions for 3D reconstruction correlate with the accuracy of the models; second, reconstructed models are robust to noise but sensitive to structural variability; third, the local structure organization of genomes, such as Topologically Associating Domains, results in more accurate models; fourth, to a certain extent, the models capture the intrinsic structural variability in the input matrices and fifth, the accuracy of the models can be a priori predicted by analyzing the properties of the interaction matrices. In summary, our work provides a systematic analysis of the limitations of a mean-field restrain-based method, which could be taken into consideration in further development of methods as well as their applications.     

Superinfection prevents recombination of the alphaherpesvirus bovine herpesvirus 1

Homologous recombination between strains of the same alphaherpesvirus species occurs frequently both in vitro and in vivo. This process has been described between strains of herpes simplex virus type 1, herpes simplex virus type 2, pseudorabies virus, feline herpesvirus 1, varicella-zoster virus, and bovine herpesvirus 1 (BoHV-1). In vivo, the rise of recombinant viruses can be modulated by different factors, such as the dose of the inoculated viruses, the distance between inoculation sites, the time interval between inoculation of the first and the second virus, and the genes in which the mutations are located. The effect of the time interval between infections with two distinguishable BoHV-1 on recombination was studied in three ways: (i) recombination at the level of progeny viruses, (ii) interference induced by the first virus infection on β-galactosidase gene expression of a superinfecting virus, and (iii) recombination at the level of concatemeric DNA. A time interval of 2 to 8 h between two successive infections allows the establishment of a barrier, which reduces or prevents any successful superinfection needed to generate recombinant viruses. The dramatic effect of the time interval on the rise of recombinant viruses is particularly important for the risk assessment of recombination between glycoprotein E-negative marker vaccine and field strains that could threaten BoHV-1 control and eradication programs.     

Cryopreserved mesenchymal stromal cells display impaired immunosuppressive properties as a result of heat-shock response and impaired interferon-γ licensing

Human mesenchymal stromal cells (MSC) can suppress T-cell activation in vitro in an indoleamine 2,3-dioxygenase (IDO)-dependent manner. However, their clinical effects on immune ailments have been inconsistent, with a recent phase III study showing no benefit in acute graft-versus-host disease (GvHD). We here tested the hypothesis that the banked, cryopreserved MSC often used in clinical trials display biologic properties distinct from that of MSC in the log phase of growth typically examined in pre-clinical studies. In freshly thawed cryopreserved MSC derived from normal human volunteers, we observed that MSC up-regulate heat-shock proteins, are refractory to interferon (IFN)-γ-induced up-regulation of IDO, and are compromised in suppressing CD3/CD28-driven T cell proliferation. Immune suppressor activity, IFN-γ responsiveness and induction of IDO were fully restored following 24 h of MSC tissue culture post-thaw. These results highlight a possible cause for the inefficacy of MSC-based immunotherapy reported in clinical trials using cryopreserved MSC thawed immediately prior to infusion.     

Very-Long-Chain Fatty Acids Are Involved in Polar Auxin Transport and Developmental Patterning in Arabidopsis

Very-long-chain fatty acids (VLCFAs) are essential for many aspects of plant development and necessary for the synthesis of seed storage triacylglycerols, epicuticular waxes, and sphingolipids. Identification of the acetyl-CoA carboxylase PASTICCINO3 and the 3-hydroxy acyl-CoA dehydratase PASTICCINO2 revealed that VLCFAs are important for cell proliferation and tissue patterning. Here, we show that the immunophilin PASTICCINO1 (PAS1) is also required for VLCFA synthesis. Impairment of PAS1 function results in reduction of VLCFA levels that particularly affects the composition of sphingolipids, known to be important for cell polarity in animals. Moreover, PAS1 associates with several enzymes of the VLCFA elongase complex in the endoplasmic reticulum. The pas1 mutants are deficient in lateral root formation and are characterized by an abnormal patterning of the embryo apex, which leads to defective cotyledon organogenesis. Our data indicate that in both tissues, defective organogenesis is associated with the mistargeting of the auxin efflux carrier PIN FORMED1 in specific cells, resulting in local alteration of polar auxin distribution. Furthermore, we show that exogenous VLCFAs rescue lateral root organogenesis and polar auxin distribution, indicating their direct involvement in these processes. Based on these data, we propose that PAS1 acts as a molecular scaffold for the fatty acid elongase complex in the endoplasmic reticulum and that the resulting VLCFAs are required for polar auxin transport and tissue patterning during plant development.     

The PTEN phosphatase controls intestinal epithelial cell polarity and barrier function: role in colorectal cancer progression

Background

The PTEN phosphatase acts on phosphatidylinositol 3,4,5-triphosphates resulting from phosphatidylinositol 3-kinase (PI3K) activation. PTEN expression has been shown to be decreased in colorectal cancer. Little is known however as to the specific cellular role of PTEN in human intestinal epithelial cells. The aim of this study was to investigate the role of PTEN in human colorectal cancer cells.

Methodology/Principal Findings

Caco-2/15, HCT116 and CT26 cells were infected with recombinant lentiviruses expressing a shRNA specifically designed to knock-down PTEN. The impact of PTEN downregulation was analyzed on cell polarization and differentiation, intercellular junction integrity (expression of cell-cell adhesion proteins, barrier function), migration (wound assay), invasion (matrigel-coated transwells) and on tumor and metastasis formation in mice. Electron microscopy analysis showed that lentiviral infection of PTEN shRNA significantly inhibited Caco-2/15 cell polarization, functional differentiation and brush border development. A strong reduction in claudin 1, 3, 4 and 8 was also observed as well as a decrease in transepithelial resistance. Loss of PTEN expression increased the spreading, migration and invasion capacities of colorectal cancer cells in vitro. PTEN downregulation also increased tumor size following subcutaneous injection of colorectal cancer cells in nude mice. Finally, loss of PTEN expression in HCT116 and CT26, but not in Caco-2/15, led to an increase in their metastatic potential following tail-vein injections in mice.

Conclusions/Significance

Altogether, these results indicate that PTEN controls cellular polarity, establishment of cell-cell junctions, paracellular permeability, migration and tumorigenic/metastatic potential of human colorectal cancer cells.     

Fanconi DNA repair pathway is required for survival and long-term maintenance of neural progenitors

Although brain development abnormalities and brain cancer predisposition have been reported in some Fanconi patients, the possible role of Fanconi DNA repair pathway during neurogenesis is unclear. We thus addressed the role of fanca and fancg, which are involved in the activation of Fanconi pathway, in neural stem and progenitor cells during brain development and adult neurogenesis. Fanca(-/-) and fancg(-/-) mice presented with microcephalies and a decreased neuronal production in developing cortex and adult brain. Apoptosis of embryonic neural progenitors, but not that of postmitotic neurons, was increased in the neocortex of fanca(-/-) and fancg(-/-) mice and was correlated with chromosomal instability. In adult Fanconi mice, we showed a reduced proliferation of neural progenitor cells related to apoptosis and accentuated neural stem cells exhaustion with ageing. In addition, embryonic and adult Fanconi neural stem cells showed a reduced capacity to self-renew in vitro. Our study demonstrates a critical role for Fanconi pathway in neural stem and progenitor cells during developmental and adult neurogenesis.