Conversion of embryonic stem cells into neuroectodermal precursors in adherent monoculture

Mouse embryonic stem (ES) cells are competent for production of all fetal and adult cell types. However, the utility of ES cells as a developmental model or as a source of defined cell populations for pharmaceutical screening or transplantation is compromised because their differentiation in vitro is poorly controlled. Specification of primary lineages is not understood and consequently differentiation protocols are empirical, yielding variable and heterogeneous outcomes. Here we report that neither multicellular aggregation nor coculture is necessary for ES cells to commit efficiently to a neural fate. In adherent monoculture, elimination of inductive signals for alternative fates is sufficient for ES cells to develop into neural precursors. This process is not a simple default pathway, however, but requires autocrine fibroblast growth factor (FGF). Using flow cytometry quantitation and recording of individual colonies, we establish that the bulk of ES cells undergo neural conversion. The neural precursors can be purified to homogeneity by fluorescence activated cell sorting (FACS) or drug selection. This system provides a platform for defining the molecular machinery of neural commitment and optimizing the efficiency of neuronal and glial cell production from pluripotent mammalian stem cells.     

Enteric nervous system progenitors are coordinately controlled by the G protein-coupled receptor EDNRB and the receptor tyrosine kinase RET

The enteric nervous system (ENS) in vertebrates is derived mainly from vagal neural crest cells that enter the foregut and colonize the entire wall of the gastrointestinal tract. Failure to completely colonize the gut results in the absence of enteric ganglia (Hirschsprung's disease). Two signaling systems mediated by RET and EDNRB have been identified as critical players in enteric neurogenesis. We demonstrate that interaction between these signaling pathways controls ENS development throughout the intestine. Activation of EDNRB specifically enhances the effect of RET signaling on the proliferation of uncommitted ENS progenitors. In addition, we reveal novel antagonistic roles of these pathways on the migration of ENS progenitors. Protein kinase A is a key component of the molecular mechanisms that integrate signaling by the two receptors. Our data provide strong evidence that the coordinate and balanced interaction between receptor tyrosine kinases and G protein-coupled receptors controls the development of the nervous system in mammals.     

Neuron and glia generating progenitors of the mammalian enteric nervous system isolated from foetal and postnatal gut cultures

Cultures of dissociated foetal and postnatal mouse gut gave rise to neurosphere-like bodies, which contained large numbers of mature neurons and glial cells. In addition to differentiated cells, neurosphere-like bodies included proliferating progenitors which, when cultured at clonal densities, gave rise to colonies containing many of the neuronal subtypes and glial cells present in the mammalian enteric nervous system. These progenitors were also capable of colonising wild-type and aganglionic gut in organ culture and had the potential to generate differentiated progeny that localised within the intrinsic ganglionic plexus. Similar progenitors were also derived from the normoganglionic small intestine of mice with colonic aganglionosis. Our findings establish the feasibility of expanding and isolating early progenitors of the enteric nervous system based on their ability to form distinct neurogenic and gliogenic structures in culture. Furthermore, these experiments provide the rationale for the development of novel approaches to the treatment of congenital megacolon (Hirschsprung's disease) based on the colonisation of the aganglionic gut with progenitors derived from normoganglionic bowel segments.     

The simultaneous determination of 1-aminocyclopropane-1-carboxylic acid and cyclopropane-1,1-dicarboxylic acid in Lycopersicum esculentum by high-performance liquid chromatography--electrospray tandem mass spectrometry

Varying concentrations of cyclopropane-1,1-dicarboxylic acid (CDA), an inhibitor of 1-aminocyclopropane-1-carboxylic acid oxidase, added to the solid culture medium of tomato nodal shoot segments resulted in a reduction in the level of endogenous ethylene according to the concentration of inhibitor applied. Following treatment with inhibitor, plants were homogenised and the concentrations of CDA and of 1-aminocyclopropane-1-carboxylic acid (ACC) were measured simultaneously in the resulting juice using an HPLC-ESI/MS-MS method. The levels of CDA and ACC measured in the plant tissues were associated with the concentration of inhibitor added to the solid medium. The HPLC-ESI/MS-MS method described produced limits of detection of 0.8 pmol for ACC and of 4 pmol for CDA.     

MITOCHONDRIAL PHYLOGEOGRAPHY OF THE LAND SNAIL ALBINARIA IN CRETE: LONG-TERM GEOLOGICAL AND SHORT-TERM VICARIANCE EFFECTS

The land snail genus Albinaria exhibits an extreme degree of morphological differentiation in Greece, especially in the island of Crete. Twenty-six representatives of 17 nominal species and a suspected hybrid were examined by sequence analysis of a PCR-amplified mitochondrial DNA fragment of the large rRNA subunit gene. Maximum parsimony and neighbor-joining phylogenetic analyses demonstrate a complex pattern of speciation and differentiation and suggest that Albinaria species from Crete belong to at least three distinct monophyletic groups, which, however, are not monophyletic with reference to the genus as a whole. There is considerable variation of genetic distance within and among “species” and groups. The revealed phylogenetic relations do not correlate well with current taxonomy, but exhibit biogeographical coherence. Certain small- and large-scale vicariance events can be traced, although dispersal and parapatric speciation may also be present. Our analysis suggests that there was an early and rapid differentiation of Albinaria groups across the whole of the range followed by local speciation events within confined geographical areas.     

Salmonella InvG forms a ring-like multimer that requires the InvH lipoprotein for outer membrane localization

Salmonella species translocate virulence effector proteins from the bacterial cytoplasm into mammalian host cells by means of a type III secretion apparatus, encoded by the pathogenicity island-1 (SPI-1). Little is known about the assembly and structure of this secretion apparatus, but the InvG protein is essential and could be an outer membrane secretion channel for the effector proteins. We observed that in recombinant Escherichia coli , the yield of InvG was enhanced by co-expression of InvH, and showed that mutation of invH decreased the level of InvG in wild-type Salmonella typhimurium . In E. coli , InvG alone was able to form an SDS-resistant multimer, but InvG localization to the outer membrane was dependent upon InvH, a lipoprotein itself located in the outer membrane, and no other SPI-1 specific protein. InvG targeted to the outer membrane by InvH became accessible to extracellular protease. InvG and InvH did not, however, appear to form a stable complex. Electron microscopy of InvG membrane protein purified from E. coli revealed that it forms an oligomeric ring-like structure with inner and outer diameters, 7 nm and 15 nm respectively.     

Network Reconstruction Based on Proteomic Data and Prior Knowledge of Protein Connectivity Using Graph Theory

Modeling of signal transduction pathways is instrumental for understanding cells’ function. People have been tackling modeling of signaling pathways in order to accurately represent the signaling events inside cells’ biochemical microenvironment in a way meaningful for scientists in a biological field. In this article, we propose a method to interrogate such pathways in order to produce cell-specific signaling models. We integrate available prior knowledge of protein connectivity, in a form of a Prior Knowledge Network (PKN) with phosphoproteomic data to construct predictive models of the protein connectivity of the interrogated cell type. Several computational methodologies focusing on pathways’ logic modeling using optimization formulations or machine learning algorithms have been published on this front over the past few years. Here, we introduce a light and fast approach that uses a breadth-first traversal of the graph to identify the shortest pathways and score proteins in the PKN, fitting the dependencies extracted from the experimental design. The pathways are then combined through a heuristic formulation to produce a final topology handling inconsistencies between the PKN and the experimental scenarios. Our results show that the algorithm we developed is efficient and accurate for the construction of medium and large scale signaling networks. We demonstrate the applicability of the proposed approach by interrogating a manually curated interaction graph model of EGF/TNFA stimulation against made up experimental data. To avoid the possibility of erroneous predictions, we performed a cross-validation analysis. Finally, we validate that the introduced approach generates predictive topologies, comparable to the ILP formulation. Overall, an efficient approach based on graph theory is presented herein to interrogate protein–protein interaction networks and to provide meaningful biological insights.     

Efficacy of Lapatinib in Therapy-Resistant HER2-Positive Circulating Tumor Cells in Metastatic Breast Cancer

Background

To evaluate the efficacy of lapatinib, a dual EGFR and HER2 tyrosine kinase inhibitor, in therapy-resistant HER2-positive CTCs in metastatic breast cancer (MBC).

Patients and Methods

Patients with MBC and HER2-positive CTCs despite disease stabilization or response to prior therapy, received lapatinib 1500 mg daily in monthly cycles, till disease progression or CTC increase. CTC monitoring was performed by immunofluorescent microscopy using cytospins of peripheral blood mononuclear cells (PBMCs) double stained for HER2 or EGFR and cytokeratin.

Results

A total of 120 cycles were administered in 22 patients; median age was 62.5 years, 15 (68.2%) patients were post-menopausal and 20 (90.1%) had HER2-negative primary tumors. At the end of the second course, HER2-positive CTC counts decreased in 76.2% of patients; the median number of HER2-positive CTCs/patient also declined significantly (p = 0.013), however the decrease was significant only among patients presenting disease stabilization (p = 0.018) but not among those with disease progression during lapatinib treatment. No objective responses were observed. All CTC-positive patients harbored EGFR-positive CTCs on progression compared to 62.5% at baseline (p = 0.054). The ratio of EGFR-positive CTCs/total CTCs detected in all patients increased from 17.1% at baseline to 37.6% on progression, whereas the mean percentage of HER2-negative CTCs/patient increased from 2.4% to 30.6% (p = 0.03).

Conclusions

The above results indicate that lapatinib is effective in decreasing HER2-positive CTCs in patients with MBC irrespectively of the HER2 status of the primary tumor and imply the feasibility of monitoring the molecular changes on CTCs during treatment with targeted agents.

Trial Registration

Clinical trial.gov NCT00694252     

Crystal structure of alkaline phosphatase from the Antarctic bacterium TAB5

Alkaline phosphatases (APs) are non-specific phosphohydrolases that are widely used in molecular biology and diagnostics. We describe the structure of the cold active alkaline phosphatase from the Antarctic bacterium TAB5 (TAP). The fold and the active site geometry are conserved with the other AP structures, where the monomer has a large central beta-sheet enclosed by alpha-helices. The dimer interface of TAP is relatively small, and only a single loop from each monomer replaces the typical crown domain. The structure also has typical cold-adapted features; lack of disulfide bridges, low number of salt-bridges, and a loose dimer interface that completely lacks charged interactions. The dimer interface is more hydrophobic than that of the Escherichia coli AP and the interactions have tendency to pair with backbone atoms, which we propose to result from the cold adaptation of TAP. The structure contains two additional magnesium ions outside of the active site, which we believe to be involved in substrate binding as well as contributing to the local stability. The M4 site stabilises an interaction that anchors the substrate-coordinating R148. The M5 metal-binding site is in a region that stabilises metal coordination in the active site. In other APs the M5 binding area is supported by extensive salt-bridge stabilisation, as well as positively charged patches around the active site. We propose that these charges, and the TAP M5 binding, influence the release of the product phosphate and thus might influence the rate-determining step of the enzyme.