LPA-induced mutually exclusive subcellular localization of active RhoA and Arp2 mRNA revealed by sequential FRET and FISH

We previously demonstrated that mRNAs for the subunits of the Arp2/3 complex localize to protrusions in fibroblasts (Mingle et al. in J Cell Sci 118:2425–2433, 2005). However, the signaling pathway that regulates Arp2/3 complex mRNA localization remains unknown. In this study we have identified lysophosphatidic acid (LPA) as a potent inducer of Arp2 mRNA localization to protrusions in fibroblasts via the RhoA-ROCK pathway. As RhoA is known to be activated locally in the cells, we sought to understand how spatial activation of Rho affects Arp2 mRNA localization. By sequentially performing fluorescence resonance energy transfer (FRET) and fluorescence in situ hybridization (FISH), we have visualized active RhoA and Arp2 mRNA in the same cells. Upon LPA stimulation, approximately two times more cells than those in the serum-free medium showed mutually exclusive localization of active RhoA and Arp2 mRNA. These results demonstrate the importance of localized activation of Rho in Arp2 mRNA localization and provide new insights as to how Rho regulates Arp2/3 complex mRNA localization. To our best knowledge, this is the first report in which FRET and FISH are combined to detect localized protein activity and mRNA in the same cells. This method should be easily adopted for the detection of other fluorescence protein based biosensors and DNA/RNA in the same cells.     

Estimation of the effective yield properties of human trabecular bone using nonlinear micro-finite element analyses

Micro-finite element (\(\upmu \)FE) analyses are often used to determine the apparent mechanical properties of trabecular bone volumes. Yet, these apparent properties depend strongly on the applied boundary conditions (BCs) for the limited size of volumes that can be obtained from human bones. To attenuate the influence of the BCs, we computed the yield properties of samples loaded via a surrounding layer of trabecular bone (“embedded configuration”). Thirteen cubic volumes (10.6 mm side length) were collected from \(\upmu \)CT reconstructions of human vertebrae and femora and converted into \(\upmu \)FE models. An isotropic elasto-plastic material model was chosen for bone tissue, and nonlinear \(\upmu \)FE analyses of six uniaxial, shear, and multi-axial load cases were simulated to determine the yield properties of a subregion (5.3 mm side length) of each volume. Three BCs were tested. Kinematic uniform BCs (KUBCs: each boundary node is constrained with uniform displacements) and periodicity-compatible mixed uniform BCs (PMUBCs: each boundary node is constrained with a uniform combination of displacements and tractions mimicking the periodic BCs for an orthotropic material) were directly applied to the subregions, while the embedded configuration was achieved by applying PMUBCs on the larger volumes instead. Yield stresses and strains, and element damage at yield were finally compared across BCs. Our findings indicate that yield strains do not depend on the BCs. However, KUBCs significantly overestimate yield stresses obtained in the embedded configuration (+43.1 ± 27.9%). PMUBCs underestimate (?10.0 ± 11.2%), but not significantly, yield stresses in the embedded situation. Similarly, KUBCs lead to higher damage levels than PMUBCs (+51.0 ± 16.9%) and embedded configurations (+48.4 ± 15.0%). PMUBCs are better suited for reproducing the loading conditions in subregions of the trabecular bone and deliver a fair estimation of their effective (asymptotic) yield properties.     

Discovery of 2-oxopiperazine dengue inhibitors by scaffold morphing of a phenotypic high-throughput screening hit

A series of 2-oxopiperazine derivatives were designed from the pyrrolopiperazinone cell-based screening hit 4 as a dengue virus inhibitor. Systematic investigation of the structure-activity relationship (SAR) around the piperazinone ring led to the identification of compound (S)-29, which exhibited potent anti-dengue activity in the cell-based assay across all four dengue serotypes with EC₅₀ < 0.1 μM. Cross-resistant analysis confirmed that the virus NS4B protein remained the target of the new oxopiperazine analogs obtained via scaffold morphing from the HTS hit 4.     

Gastric gelatinase B/matrix metalloproteinase-9 is rapidly increased in Helicobacter felis-induced gastritis

It has previously been shown that matrix metalloproteinase-9 (MMP-9) levels, originating from macrophages, are considerably increased in human Helicobacter pylori-associated gastritis. Here, the early kinetics of the MMP-9 response resulting from Helicobacter infection in C57BL/6 and MMP-9 knock-out mice using the murine Helicobacter felis model were examined. H. felis infection induced severe gastritis in the murine stomach at just 2 weeks after infection. Before gastritis, an increase was observed in MMP-9-positive cells detected by immunohistochemistry in the basal lamina propria. This finding was corroborated by gelatin zymography of stomach samples. As the gastritis increased so did the concentration of MMP-9 and the incidence of gastric MMP-9-positive cells, their location corresponding to that of macrophages. In contrast, systemic levels of MMP-9 remained unchanged. When MMP-9-deficient mice were infected with H. felis, no significant difference in gastritis development was detected compared with disease development in wild-type animals. We conclude that MMP-9 production is an early event in the response to gastric Helicobacter infection, a feature that may favor the recruitment of immune cells early during infection. At later stages, however, the increased levels of MMP-9 may damage the integrity of the stomach mucosa.     

Identification of biologically active chemokine isoforms from ascitic fluid and elevated levels of CCL18/pulmonary and activation-regulated chemokine in ovarian carcinoma

Chemokines are important in leukocyte homeostasis, inflammation, angiogenesis, and metastasis. Here, the molecular diversity of chemokines present in ovarian carcinoma was studied by purifying the proteins to homogeneity from ascitic fluid. Biologically active intact CCL2 and processed CXCL8, CCL3, and CCL18 isoforms were recovered. CCL7 and CCL20 were also purified, but their levels were 10-fold lower compared with CXCL8, CCL2, and CCL3 and even 100-fold lower than the amounts of CCL18 isolated. In ascitic fluids from patients with ovarian carcinoma (n = 12), significantly higher levels of CXCL8 and CCL18 (2.0 versus 0.7 ng/ml (p = 0.01) and 120 versus 44 ng/ml (p = 0.0002), respectively) were detected compared with those in nonovarian carcinoma patients (n = 12). In contrast to CXCL8, CCL18 was not inducible in carcinoma cell lines. Immunostaining showed CCL18 expression in tumor-infiltrating cells with monocyte/macrophage morphology but not in the ovarian carcinoma cells. Our data demonstrate that biochemically heterogenous but biologically active forms of several chemokines are present at different concentrations in ovarian carcinoma ascitic fluid. This points to a delicate balance of chemokines in epithelial ovarian cancer and to a potentially major role for CXCL8 and CCL18 in this tumor.     

Identification of a pathway for the utilization of the Amadori product fructoselysine in Escherichia coli

Escherichia coli was found to grow on fructoselysine as an energetic substrate at a rate of about one-third of that observed with glucose. Extracts of cells grown on fructoselysine catalyzed in the presence of ATP the phosphorylation of fructoselysine and a delayed formation of glucose 6-phosphate from this substrate. Data base searches allowed us to identify an operon containing a putative kinase (YhfQ) belonging to the PfkB/ ribokinase family, a putative deglycase (YhfN), homologous to the isomerase domain of glucosamine-6-phosphate synthase, and a putative cationic amino acid transporter (YhfM). The proteins encoded by YhfQ and YhfN were overexpressed in E. coli, purified, and shown to catalyze the ATP-dependent phosphorylation of fructoselysine to a product identified as fructoselysine 6-phosphate by 31P NMR (YhfQ), and the reversible conversion of fructoselysine 6-phosphate and water to lysine and glucose 6-phosphate (YhfN). The K(m) of the kinase for fructoselysine amounted to 18 microm, and the K(m) of the deglycase for fructoselysine 6-phosphate, to 0.4 mm. A value of 0.15 m was found for the equilibrium constant of the deglycase reaction. The kinase and the deglycase were both induced when E. coli was grown on fructoselysine and then reached activities sufficient to account for the rate of fructoselysine utilization.     

Spatial segregation between immatures and adults in a pelagic seabird suggests age‐related competition

Individual competitiveness conditions access to resources when they are limited. Immature individuals that are less skilled than adults have to adapt their foraging strategies to survive. Among strategies to reduce competition, spatial segregation has been widely demonstrated. However, the use of spatial segregation by immatures to limit intra‐specific competition with adults has rarely been tested. In this study, we investigated and compared habitat preferences and distributions of free‐ranging immature and breeding adult northern gannets Morus bassanus in order to determine whether they compete for similar habitats during the year, and if this results in a spatial segregation between birds of different age groups. Based on > 66 000 km of aerial surveys conducted in the North‐East Atlantic Ocean during winter and summer 2012, habitats selected by immatures and adult birds were modelled independently, linking gannet density to a set of oceanographic and physiographic predictors. Their large‐scale seasonal distribution was then predicted. We found that gannets displayed a strong season‐dependent competition between immatures and adults, as a consequence of immatures and adults using similar habitats in both summer and winter. During summer, when adults are constrained by reproduction, both groups were spatially highly segregated despite similar habitat preferences (thermal fronts), with youngest individuals selecting habitats out of range of central‐place foragers, highlighting intra‐specific competition. Contrastingly during winter, when reproductive constraints disappear, immature and adult distributions largely overlapped. Our study provides new insights into the role played by age, foraging experience and reproductive constraints on the distribution of marine predators. More specifically, these results highlight in seabirds how the youngest fraction mitigates, through spatial segregation, the competition with experienced adults, and suggest a progressive strategy along the maturation process.     

Stacking three late blight resistance genes from wild species directly into African highland potato varieties confers complete field resistance to local blight races

Considered responsible for one million deaths in Ireland and widespread famine in the European continent during the 1840s, late blight, caused by Phytophthora infestans, remains the most devastating disease of potato (Solanum tuberosum L.) with about 15%–30% annual yield loss in sub‐Saharan Africa, affecting mainly smallholder farmers. We show here that the transfer of three resistance (R) genes from wild relatives [RB, Rpi‐blb2 from Solanum bulbocastanum and Rpi‐vnt1.1 from S. venturii] into potato provided complete resistance in the field over several seasons. We observed that the stacking of the three R genes produced a high frequency of transgenic events with resistance to late blight. In the field, 13 resistant transgenic events with the 3R‐gene stack from the potato varieties ‘Desiree’ and ‘Victoria’ grew normally without showing pathogen damage and without any fungicide spray, whereas their non‐transgenic equivalent varieties were rapidly killed. Characteristics of the local pathogen population suggest that the resistance to late blight may be long‐lasting because it has low diversity, and essentially consists of the single lineage, 2_A1, which expresses the cognate avirulence effector genes. Yields of two transgenic events from ‘Desiree’ and ‘Victoria’ grown without fungicide to reflect small‐scale farm holders were estimated to be 29 and 45 t/ha respectively. This represents a three to four‐fold increase over the national average. Thus, these late blight resistant potato varieties, which are the farmers’ preferred varieties, could be rapidly adopted and bring significant income to smallholder farmers in sub‐Saharan Africa.     

To What Extent is Primate Second Molar Enamel Occlusal Morphology Shaped by the Enamel-Dentine Junction?

The form of two hard tissues of the mammalian tooth, dentine and enamel, is the result of a combination of the phylogenetic inheritance of dental traits and the adaptive selection of these traits during evolution. Recent decades have been significant in unveiling developmental processes controlling tooth morphogenesis, dental variation and the origination of dental novelties. The enamel-dentine junction constitutes a precursor for the morphology of the outer enamel surface through growth of the enamel cap which may go along with the addition of original features. The relative contribution of these two tooth components to morphological variation and their respective response to natural selection is a major issue in paleoanthropology. This study will determine how much enamel morphology relies on the form of the enamel-dentine junction. The outer occlusal enamel surface and the enamel-dentine junction surface of 76 primate second upper molars are represented by polygonal meshes and investigated using tridimensional topometrical analysis. Quantitative criteria (elevation, inclination, orientation, curvature and occlusal patch count) are introduced to show that the enamel-dentine junction significantly constrains the topographical properties of the outer enamel surface. Our results show a significant correlation for elevation, orientation, inclination, curvature and occlusal complexity between the outer enamel surface and the enamel dentine junction for all studied primate taxa with the exception of four modern humans for curvature (p<0.05). Moreover, we show that, for all selected topometrical parameters apart from occlusal patch count, the recorded correlations significantly decrease along with enamel thickening in our sample. While preserving tooth integrity by providing resistance to wear and fractures, the variation of enamel thickness may modify the curvature present at the occlusal enamel surface in relation to enamel-dentine junction, potentially modifying dental functionalities such as blunt versus sharp dental tools. In terms of natural selection, there is a balance between increasing tooth resistance and maintaining efficient dental tools. In this sense the enamel cap acts as a functional buffer for the molar occlusal pattern. In primates, results suggest a primary emergence of dental novelties on the enamel-dentine junction and a secondary transposition of these novelties with no or minor modifications of dental functionalities by the enamel cap. Whereas enamel crenations have been reported by previous studies, our analysis do not support the presence of enamel tubercles without dentine relief nuclei. As is, the enamel cap is, at most, a secondary source of morphological novelty.