Random epigenetic modulation of CHO cells by repeated knockdown of DNA methyltransferases increases population diversity and enables sorting of cells with higher production capacities

Chinese hamster ovary (CHO) cells produce a large share of today's biopharmaceuticals. Still, the generation of satisfactory producer cell lines is a tedious undertaking. Recently, it was found that CHO cells, when exposed to new environmental conditions, modify their epigenome, suggesting that cells adapt their gene expression pattern to handle new challenges. The major aim of the present study was to employ artificially induced, random changes in the DNA-methylation pattern of CHO cells to diversify cell populations and consequently increase the finding of cell lines with improved cellular characteristics. To achieve this, DNA methyltransferases and/or the ten-eleven translocation enzymes were downregulated by RNA interference over a time span of ∼16 days. Methylation analysis of the resulting cell pools revealed that the knockdown of DNA methyltransferases was highly effective in randomly demethylating the genome. The same approach, when applied to stable CHO producer cells resulted in (a) an increased productivity diversity in the cell population, and (b) a higher number of outliers within the population, which resulted in higher specific productivity and titer in the sorted cells. These findings suggest that epigenetics play a previously underestimated, but actually important role in defining the overall cellular behavior of production clones.     

The geometry and genetics of hybridization

When divergent populations form hybrids, hybrid fitness can vary with genome composition, current environmental conditions, and the divergence history of the populations. We develop analytical predictions for hybrid fitness, which incorporate all three factors. The predictions are based on Fisher's geometric model, and apply to a wide range of population genetic parameter regimes and divergence conditions, including allopatry and parapatry, local adaptation, and drift. Results show that hybrid fitness can be decomposed into intrinsic effects of admixture and heterozygosity, and extrinsic effects of the (local) adaptedness of the parental lines. Effect sizes are determined by a handful of geometric distances, which have a simple biological interpretation. These distances also reflect the mode and amount of divergence, such that there is convergence toward a characteristic pattern of intrinsic isolation. We next connect our results to the quantitative genetics of line crosses in variable or patchy environments. This means that the geometrical distances can be estimated from cross data, and provides a simple interpretation of the “composite effects.” Finally, we develop extensions to the model, involving selectively induced disequilibria, and variable phenotypic dominance. The geometry of fitness landscapes provides a unifying framework for understanding speciation, and wider patterns of hybrid fitness.     

Characterization at nucleotide resolution of the homogeneously staining region sites of insertion in two cancer cell lines

The mechanisms of formation of intrachromosomal amplifications in tumours are still poorly understood. By using quantitative polymerase chain reaction, DNA sequencing, chromosome walking, in situ hybridization on metaphase chromosomes and whole-genome analysis, we studied two cancer cell lines containing an MYC oncogene amplification with acquired copies ectopically inserted in rearranged chromosomes 17. These intrachromosomal amplifications result from the integration of extrachromosomal DNA molecules. Replication stress could explain the formation of the double-strand breaks involved in their insertion and in the rearrangements of the targeted chromosomes. The sequences of the junctions indicate that homologous recombination was not involved in their formation and support a non-homologous end-joining process. The replication stress-inducible common fragile sites present in the amplicons may have driven the intrachromosomal amplifications. Mechanisms associating break-fusion-bridge cycles and/or chromosome fragmentation may have led to the formation of the uncovered complex structures. To our knowledge, this is the first characterization of an intrachromosomal amplification site at nucleotide resolution.     

Ectomycorrhizal fungal diversity in Quercus ilex Mediterranean woodlands: variation among sites and over soil depth profiles in hyphal exploration types, species richness and community composition

Understanding the factors underlying the distribution of biodiversity is a challenging issue in ecology. Here, we examined the distribution patterns of ectomycorrhizal fungal diversity across the soil profile in three Quercus ilex forests. Contact exploration type strongly dominated at all sites, but was most prevalent in the upper, organic-rich soil layers. At each site, three quarters of the ectomycorrhizal tips and 59 % of taxa were restricted to the ten first centimeters of the soil profile. The relative abundance of the dominant family Russulaceae increased with increasing soil depth. Species composition varied significantly among sites, with most species being rare. Species that occurred in only one of the three sites accounted for 78.9 % of all species, and 57.3 % of species were represented by a single ECM root tip. Our results suggest that (i) rare species at both local and regional scales contribute to the highly diverse fungal assemblages in Mediterranean forests and (ii) multi-sites studies including the whole soil profile are needed to provide comprehensive overviews of the taxonomic and functional diversities of ectomycorrhizal communities.     

Differential proliferation rates generate patterns of mechanical tension that orient tissue growth

Orientation of cell divisions is a key mechanism of tissue morphogenesis. In the growing Drosophila wing imaginal disc epithelium, most of the cell divisions in the central wing pouch are oriented along the proximal–distal (P–D) axis by the Dachsous‐Fat‐Dachs planar polarity pathway. However, cells at the periphery of the wing pouch instead tend to orient their divisions perpendicular to the P–D axis despite strong Dachs polarization. Here, we show that these circumferential divisions are oriented by circumferential mechanical forces that influence cell shapes and thus orient the mitotic spindle. We propose that this circumferential pattern of force is not generated locally by polarized constriction of individual epithelial cells. Instead, these forces emerge as a global tension pattern that appears to originate from differential rates of cell proliferation within the wing pouch. Accordingly, we show that localized overgrowth is sufficient to induce neighbouring cell stretching and reorientation of cell division. Our results suggest that patterned rates of cell proliferation can influence tissue mechanics and thus determine the orientation of cell divisions and tissue shape.     

Antibacterial and leishmanicidal activities of temporin-SHd,a 17-residue long membrane-damaging peptide

Temporins are a family of short antimicrobial peptides (8–17 residues) that mostly show potent activity against Gram-positive bacteria. Herein, we demonstrate that temporin-SHd, a 17-residue peptide with a net charge of +2 (FLPAALAGIGGILGKLF_amide), expressed a broad spectrum of antimicrobial activity. This peptide displayed potent antibacterial activities against Gram-negative and Gram-positive bacteria, including multi-drug resistant Staphylococcus aureus strains, as well as antiparasitic activity against promastigote and the intracellular stage (amastigote) of Leishmania infantum, at concentration not toxic for the macrophages. Temporin-SHd that is structured in a non-amphipathic α-helix in anionic membrane-mimetic environments, strongly and selectively perturbs anionic bilayer membranes by interacting with the polar head groups and acyl region of the phospholipids, with formation of regions of two coexisting phases: one phase rich in peptide and the other lipid-rich. The disruption of lipid packing within the bilayer may lead to the formation of transient pores and membrane permeation/disruption once a threshold peptide accumulation is reached. To our knowledge, Temporin-SHd represents the first known 17-residue long temporin expressing such broad spectrum of antimicrobial activity including members of the trypanosomatidae family. Additionally, since only a few shorter members (13 residues) of the temporin family are known to display antileishmanial activity (temporins-TA, -TB and -SHa), SHd is an interesting tool to analyze the antiparasitic mechanism of action of temporins.     

Membrane-bound mucin modular domains: From structure to function

Mucins belong to a heterogeneous family of large O-glycoproteins composed of a long peptidic chain called apomucin on which are linked hundreds of oligosaccharidic chains. Among mucins, membrane-bound mucins are modular proteins and have a structural organization usually containing Pro/Thr/Ser-rich O-glycosylated domains (PTS), EGF-like and SEA domains. Via these modular domains, the membrane-bound mucins participate in cell signalling and cell interaction with their environment in normal and pathological conditions. Moreover, the recent knowledge of these domains and their biological activities led to the development of new therapeutic approaches involving mucins. In this review, we show 3D structures of EGF and SEA domains. We also describe the functional features of the evolutionary conserved domains of membrane-bound mucins and discuss consequences of splice events.