Long chain ceramides and very long chain ceramides have opposite effects on human breast and colon cancer cell growth

Ceramides are known to be key players in intracellular signaling and are involved in apoptosis, cell senescence, proliferation, cell growth and differentiation. They are synthesized by ceramide synthases (CerS). So far, six different mammalian CerS (CerS1-6) have been described. Recently, we demonstrated that human breast cancer tissue displays increased activity of CerS2, 4, and 6, together with enhanced generation of their products, ceramides C(16:0), C(24:0), and C(24:1). Moreover, these increases were significantly associated with tumor dignity. To clarify the impact of this observation, we manipulated cellular ceramide levels by overexpressing ceramide synthases 2, 4 or 6 in MCF-7 (breast cancer) and HCT-116 (colon cancer) cells, respectively. Overexpression of ceramide synthases 4 and 6 elevated generation of short chain ceramides C(16:0), C(18:0) and C(20:0), while overexpression of ceramide synthase 2 had no effect on ceramide production in vivo, presumably due to limited substrate availability, because external addition of very long chain acyl-CoAs resulted in a significant upregulation of very long chain ceramides. We also demonstrated that upregulation of CerS4 and 6 led to the inhibition of cell proliferation and induction of apoptosis, whereas upregulation of CerS2 increased cell proliferation. On the basis of our data, we propose that a disequilibrium between ceramides of various chain length is crucial for cancer progression, while normal cells require an equilibrium between very long and long chain ceramides for normal physiology.     

The role of barriers and gradients in differentiation processes of pyrgulinid microgastropods of Lake Ohrid

Ancient Lake Ohrid is characterized by vertical (bathymetrical) zones within the lake, presumably promoting allopatric speciation due to barriers or parapatric speciation along gradients. Examples within the lake include the belt of Chara algae as well as the shell zone, both presumably impeding migrations of benthic invertebrates. Three potential cases of vertical differentiation leading to distinct depth forms have been reported for the gastropod subfamily Pyrgulinae (Caenogastropoda: Hydrobiidae): Ginaia munda ssp., Macedopyrgula spp. and Ochridopyrgula macedonica ssp. Based on DNA data of the COI gene from a total of 145 specimens, this article aims at investigating the vertical differentiation within these depth forms and thus patterns of speciation in Lake Ohrid. An initial morphometric analysis showed a clear correlation of shell shape and collecting depth for Ginaia munda ssp. and Macedopyrgula spp. This morphological trend is largely reflected in the genetic structure of the respective taxa. The data presented here indicate the existence of strong gradients of abiotic and biotic factors in Lake Ohrid rather than distinct barriers. Therefore, parapatric speciation may be the predominant form of differentiation of benthic invertebrates in the lake. Incomplete lineage sorting, hybridization and phenotypic plasticity possibly caused by epigenetic mechanisms are discussed as possible reasons for the incongruence between geno- and phenotype observed in few specimens of Ginaia munda ssp. and Macedopyrgula spp. For the third taxon, Ochridopyrgula macedonica ssp., morphometric and genetic analyses revealed only weak support for the previously proposed depth forms. However, a horizontal differentiation of lake and spring populations was revealed instead, and parapatric and allopatric differentiations are discussed in this taxon.     

Mollusc biodiversity in a European ancient lake system: lakes Prespa and Mikri Prespa in the Balkans

The spatial distribution of (endemic) biodiversity in ancient and potentially ancient lakes in Europe is poorly understood. Examples include Lakes Prespa and Mikri Prespa in the Central Balkans. Utilizing information of the most species-rich taxon in these lakes, the Mollusca, we therefore attempt to statistically assess and visualize the spatial distribution of biodiversity, to analyse biogeographical patterns, and to carry out a conservation assessment. We estimate that at least 40 (sub)species (29 gastropod and 11 bivalve taxa) occur in the lakes. For both lakes combined, 37.5% of the mollusc taxa are endemic. In general, the mollusc richness in Lake Mikri Prespa is lower than in Lake Prespa and less heterogeneously distributed. The highest species richness can be seen on the western and south eastern shores of Lake Prespa. Based on the presence/absence of genera, a minimum spanning tree analysis supports the sister lake relationship of both lakes, which, in turn, are most closely related to lakes in the western Balkans and not to nearby Lake Ohrid. The IUCN red list assessment revealed (A) a tendency towards mollusc faunal change, (B) a contemporary decline and potential loss of mollusc diversity, and (C) that all endemic species are of conservation concern.     

Root Suberin Forms an Extracellular Barrier That Affects Water Relations and Mineral Nutrition in Arabidopsis

Though central to our understanding of how roots perform their vital function of scavenging water and solutes from the soil, no direct genetic evidence currently exists to support the foundational model that suberin acts to form a chemical barrier limiting the extracellular, or apoplastic, transport of water and solutes in plant roots. Using the newly characterized enhanced suberin1 (esb1) mutant, we established a connection in Arabidopsis thaliana between suberin in the root and both water movement through the plant and solute accumulation in the shoot. Esb1 mutants, characterized by increased root suberin, were found to have reduced day time transpiration rates and increased water-use efficiency during their vegetative growth period. Furthermore, these changes in suberin and water transport were associated with decreases in the accumulation of Ca, Mn, and Zn and increases in the accumulation of Na, S, K, As, Se, and Mo in the shoot. Here, we present direct genetic evidence establishing that suberin in the roots plays a critical role in controlling both water and mineral ion uptake and transport to the leaves. The changes observed in the elemental accumulation in leaves are also interpreted as evidence that a significant component of the radial root transport of Ca, Mn, and Zn occurs in the apoplast.     

Short-term activation induces multifunctional dendritic cells that generate potent antitumor T-cell responses in vivo

Dendritic cell (DC) vaccines have emerged as a promising strategy to induce antitumoral cytotoxic T cells for the immunotherapy of cancer. The maturation state of DC is of critical importance for the success of vaccination, but the most effective mode of maturation is still a matter of debate. Whereas immature DC carry the risk of inducing tolerance, extensive stimulation of DC may lead to DC unresponsiveness and exhaustion. In this study, we investigated how short-term versus long-term DC activation with a Toll-like receptor 9 agonist influences DC phenotype and function. Murine DC were generated in the presence of the hematopoietic factor Flt3L (FL-DC) to obtain both myeloid and plasmacytoid DC subsets. Short activation of FL-DC for as little as 4 h induced fully functional DC that rapidly secreted IL-12p70 and IFN-α, expressed high levels of costimulatory and MHC molecules and efficiently presented antigen to CD4 and CD8 T cells. Furthermore, short-term activated FL-DC overcame immune suppression by regulatory T cells and acquired high migratory potential toward the chemokine CCL21 necessary for DC recruitment to lymph nodes. In addition, vaccination with short-term activated DC induced a strong cytotoxic T-cell response in vivo and led to the eradication of tumors. Thus, short-term activation of DC generates fully functional DC for tumor immunotherapy. These results may guide the design of new protocols for DC generation in order to develop more efficient DC-based tumor vaccines.     

Receptor Density Is Key to the Alpha2/Beta Interferon Differential Activities

Multiple type I interferons (IFN-α/β) elicit Jak/Stat activation, rapid gene induction, and pleiotropic effects, such as differentiation, antiviral protection, and blocks in proliferation, which are dependent on the IFN subtype and the cellular context. To date, ligand- and receptor-specific molecular determinants underlying IFN-α/β differential activities or potencies have been well characterized. To analyze cellular determinants that impact subtype-specific potency, human fibrosarcoma U5A-derived clones, exhibiting a gradient of IFN sensitivity by virtue of increasing receptor levels, were monitored for Jak/Stat signaling, gene induction, cell cycle lengthening, and apoptosis. In cells with scarce receptors, IFN-β was more potent than IFN-α2 in antiproliferative activity, while the two subtypes were equipotent in all other readouts. Conversely, in cells with abundant receptors, IFN-α2 matched or even surpassed IFN-β in all readouts tested. Our results suggest that the differential activities of the IFN subtypes are dictated not only by the intrinsic ligand/receptor binding kinetics but also by the density of cell surface receptor components.A persistent question in the field of helically bundled cytokines concerns the molecular basis of intracellular signal activation following binding to cognate cell surface receptors. Typically, cytokine-induced dimerization of the receptor subunits is thought to trigger catalytic transactivation of the associated Jak tyrosine kinases. Phosphorylation of critical receptor tyrosine motifs by the activated Jak proteins allows recruitment and activation of downstream Stat effectors (25, 34). A clear distinction can be made between the short homodimeric Jak2-activating receptors, such as the growth hormone or the erythropoietin receptors, and the more complex heteromeric receptors. Among these latter is the type I interferon (IFN) receptor, a prototypic class 2 receptor, made of two subunits, each associated with a different Jak enzyme (29). IFNAR2 contains extracellularly two fibronectin III domains forming a well-defined cytokine binding module. The cytoplasmic region of IFNAR2 is 250 amino acids long, interacts with Jak1, and contains two principal Tyr-based Stat recruitment motifs (24, 35). IFNAR1 is made of a large ectodomain of four fibronectin III domains, not all involved in ligand binding, and a 100-amino-acid-long cytoplasmic region complexed with Tyk2 and subjected to ligand-induced ubiquitination driving receptor proteolysis (13, 14).A large array of IFNs (over a dozen α subtypes and one β subtype) bind to this ubiquitously expressed receptor complex to induce rapid gene expression programs that elicit measurable antiviral responses and cell growth inhibition as well as cell context-specific functional changes (4, 31). Several studies have reported on differential activities of type I IFNs, but no unique function has ever been attributed to a given subtype (see references in reference 29). Thus, a differential can be defined as a lack of correlation between two specific activities. For instance, depending on the cell system, IFN-α2 and IFN-β can exhibit equivalent antiproliferative potency or over a 100-fold difference in antiproliferative potency and nearly equipotency in antiviral activity. Since no overt differences are observed in the structure or stoichiometry of the ligand-receptor complex formed with different subtypes, the concordant view points to the way each IFN subtype engages the available receptors. Indeed, kinetic measurements of the interaction of IFN-α2 and IFN-β with receptor ectodomains have shown substantial differences. IFNAR2 represents the high-affinity subunit, toward which IFN-α2 exhibits nanomolar binding affinity and IFN-β exhibits ∼100 pM binding affinity. Conversely, IFNAR1 is the low-affinity subunit, toward which IFN-α2 exhibits micromolar affinity and IFN-β ∼50 nM affinity (19, 22). The contribution of the individual and combined affinities on ternary complex formation by either IFN subtype have been thoroughly studied (10, 26). However, how these dynamic parameters influence receptor function and translate into activation of Jak, recruitment of Stats and additional effectors, gene induction, and bioactivities remains ill defined.Rather than focusing on ligand and receptor determinants, here we investigated the relationship between receptor subunit levels and IFN-α2 versus IFN-β signaling and functional outcomes (IFN-α2/β differential potencies). Since we previously showed that no simple relationship between receptor levels and Jak/Stat signaling can be inferred by comparing different cell types (18), we have used a reductionist approach in a single cell type, from which we have engineered and studied clones expressing low or abundant receptor levels. We show that the density of receptors at the cell surface represents a critical determinant of the level of differential activity exhibited by two IFN subtypes.     

Assessing changes in macrophyte assemblages with salinity in non-riverine wetlands: A Bayesian approach

Bayesian modeling techniques (which accounted for imperfect detection) were used to assess changes in macrophyte assemblages in 58 wetlands along a typical salinity gradient in Western Victoria, Australia. By incorporating detectability into our predictions, an unbiased estimate was made of the relationship between salinity and both individual species occupancy and the expected number of species. When compared to the freshest wetlands, macrophyte species number was predicted to decrease by as much as 60–70% at conductivities of around 6.0 mS cm⁻¹ (11% seawater), a value often considered the upper salinity tolerance for many freshwater aquatic plants. The model also predicted a 40–50% drop in species number at conductivities of around 1.5 mS cm⁻¹ (3% seawater). It was also found that 25 out of 76 freshwater species were unlikely to occur at conductivities above 1.0 mS cm⁻¹. Consequently, secondary salinisation of fresh non-riverine wetlands is highly likely to markedly and negatively impact upon non-riverine wetland macrophyte assemblages.