BOX elements modulate gene expression in Streptococcus pneumoniae: impact on the fine-tuning of competence development

More than 100 BOX elements are randomly distributed in intergenic regions of the pneumococcal genome. Here we demonstrate that these elements can affect expression of neighboring genes and present evidence that they are mobile. Together, our findings show that BOX elements enhance genetic diversity and genomic plasticity in Streptococcus pneumoniae.     

Phagocytosis of N-acetyl-D-glucosamine particles, a Th1 adjuvant, by RAW 264.7 cells results in MAPK activation and TNF-alpha, but not IL-10, production

A practical and highly effective Th1 adjuvant should induce Th1 cytokines (IL-12, IL-18, and TNF-alpha) but not the Th2 cytokine IL-10, an inhibitor of Th1 responses. In this study, phagocytosis of N-acetyl-d-glucosamine polymer (chitin) particles by RAW 264.7 cells, a murine macrophage-like cell line, resulted in phosphorylation of MAPK (p38, Erk 1/2, and JNK), and production of relatively high levels of TNF-alpha and COX-2 with increased PGE(2) release. Similar results were observed in response to oligonucleotides with CpG motifs, mycobacterial components and endotoxin. However, these bacterial components also induced a large amount of IL-10. Chitin particles, in contrast, failed to induce detectable levels of IL-10, although the production of high levels of PGE(2) and TNF-alpha and the activation of MAPK's are potentially positive signals for IL-10 production. Thus, our results indicate that chitin particles act as a unique Th1 adjuvant for macrophages without inducing increased production of IL-10.     

A beta1,4-galactosyltransferase is required for convergent extension movements in zebrafish

Our understanding of how complex carbohydrates function during embryonic development is still very limited, primarily due to the large number of glycosyltransferases now known to be involved in their synthesis. To overcome these limitations, we have taken advantage of the zebrafish system to analyze the function of complex carbohydrates during development by down-regulating the expression of specific glycosyltransferases. Herein, we report the identification of the zebrafish ortholog of mammalian beta1,4-galactosyltransferase I, beta4GalT1, and its requirement for proper convergent extension movements during gastrulation. beta4GalT1 is expressed in the oocyte and throughout the embryo during the first 24 h of development. Knockdown of zebrafish beta4GalT1 by two independent morpholino oligonucleotides results in embryos with a truncated anterior-posterior axis, as well as elongated somites and moderate defects in the patterning of the head mesenchyme. Co-injection of zebrafish beta4GalT1 mRNA returns galactosyltransferase activity to control levels and rescues the defects produced by morpholino oligonucleotides. In situ hybridizations of various molecular markers reveal that the axial mesoderm of epiboly stage embryos is abnormally widened in beta4GalT1 morphants, indicative of abnormal convergent extension. Consistent with this, the rate of anterior-posterior axis elongation is reduced relative to control-injected embryos, similar to that seen in known convergent extension mutants. Among the many potential substrates for beta4GalT1 is laminin, a principle component of the extracellular matrix that supports cell movements such as those that occur during convergent extension. Previous in vitro studies have shown that the galactosylation status of laminin directly influences its ability to support cell spreading and migration. In this regard, laminin isolated from beta4GalT1 morphant embryos is poorly galactosylated, which may contribute to defective cell migration during convergent extension movements. This work demonstrates that zebrafish can be used to identify critical developmental roles for specific glycosyltransferases that would not be obvious otherwise, such as an absolute requirement for beta4GalT1 during convergent extension movements.     

The iron/lead transporter superfamily of Fe/Pb2+ uptake systems

Oxidase-dependent ferrous iron uptake transporters of the OFeT family and lead uptake transporters of the PbrT family comprise the iron/lead transporter (ILT) superfamily (transporter classification No. 9.A.10). All sequenced homologues of the ILT superfamily were multiply aligned, and conserved motifs, including fully conserved acidic residues in putative transmembrane segments (TMSs) 1 and 4, previously implicated in heavy metal binding, were identified. Topological analyses confirmed the presence of 7 conserved TMSs in a 3 + 3 + 1 arrangement where the two 3 TMS elements are internally repeated. Phylogenetic analyses revealed the presence of several sequence divergent clusters of orthologous proteins that group roughly according to the phylogenes of the organisms of origin. The results serve to characterize and provide evolutionary insight into a novel superfamily of heavy metal uptake transporters.     

Spatial distribution of tissue level properties in a human femoral cortical bone

The mechanical properties of cortical bone are determined by a combination bone tissue composition, and structure at several hierarchical length scales. In this study the spatial distribution of tissue level properties within a human femoral shaft has been investigated. Cylindrically shaped samples (diameter: 4.4mm, N=56) were prepared from cortical regions along the entire length (20-85% of the total femur length), and around the periphery (anterior, medial, posterior and lateral quadrants). The samples were analyzed using scanning acoustic microscopy (SAM) at 50MHz and synchrotron radiation micro computed tomography (SRμCT). For all samples the average cortical porosity (Ct.Po), tissue elastic coefficients (c(ij)) and the average tissue degree of mineralization (DMB) were determined. The smallest coefficient of variation was observed for DMB (1.8%), followed by BV/TV (5.4%), c(ij) (8.2-45.5%), and Ct.Po (47.5%). Different variations with respect to the anatomical position were found for DMB, Ct.Po and c(ij). These data address the anatomical variations in anisotropic elastic properties and link them to tissue mineralization and porosity, which are important input parameters for numerical multi-scale bone models.     

Identification of Mutations in TMEM5 and ISPD as a Cause of Severe Cobblestone Lissencephaly

Cobblestone lissencephaly is a peculiar brain malformation with characteristic radiological anomalies. It is defined as cortical dysplasia that results when neuroglial overmigration into the arachnoid space forms an extracortical layer that produces agyria and/or a “cobblestone” brain surface and ventricular enlargement. Cobblestone lissencephaly is pathognomonic of a continuum of autosomal-recessive diseases characterized by cerebral, ocular, and muscular deficits. These include Walker-Warburg syndrome, muscle-eye-brain disease, and Fukuyama muscular dystrophy. Mutations in POMT1, POMT2, POMGNT1, LARGE, FKTN, and FKRP identified these diseases as alpha-dystroglycanopathies. Our exhaustive screening of these six genes, in a cohort of 90 fetal cases, led to the identification of a mutation in only 53% of the families, suggesting that other genes might also be involved. We therefore decided to perform a genome-wide study in two multiplex families. This allowed us to identify two additional genes: TMEM5 and ISPD. Because TMEM has a glycosyltransferase domain and ISPD has an isoprenoid synthase domain characteristic of nucleotide diP-sugar transferases, these two proteins are thought to be involved in the glycosylation of dystroglycan. Further screening of 40 families with cobblestone lissencephaly identified nonsense and frameshift mutations in another four unrelated cases for each gene, increasing the mutational rate to 64% in our cohort. All these cases displayed a severe phenotype of cobblestone lissencephaly A. TMEM5 mutations were frequently associated with gonadal dysgenesis and neural tube defects, and ISPD mutations were frequently associated with brain vascular anomalies.     

X-Ray Phase Nanotomography Resolves the 3D Human Bone Ultrastructure

Bone strength and failure are increasingly thought to be due to ultrastructural properties, such as the morphology of the lacuno-canalicular network, the collagen fiber orientation and the mineralization on the nanoscale. However, these properties have not been studied in 3D so far. Here we report the investigation of the human bone ultrastructure with X-ray phase nanotomography, which now provides the required sensitivity, spatial resolution and field of view. The 3D organization of the lacuno-canalicular network is studied in detail over several cells in osteonal and interstitial tissue. Nanoscale density variations are revealed and show that the cement line separating these tissues is hypermineralized. Finally, we show that the collagen fibers are organized as a twisted plywood structure in 3D.     

Low expression of Beclin 1, associated with high Bcl-xL, predicts a malignant phenotype and poor prognosis of gastric cancer

Recent studies have suggested that dysregulation of autophagy plays a pivotal role in tumorigenesis. Here, we determined the prognostic value of autophagy-related protein Beclin 1 in gastric cancer. A total of 153 primary gastric cancer patients were subjected to analysis of Beclin 1 expression and survival prognosis. Among them, 68 patients were assigned randomly and used as a training set to generate a cutoff score for Beclin 1 expression by receive operating characteristic (ROC) curve analysis. The ROC-generated cutoff score was subjected to analyze the association of Beclin 1 with clinical characteristics and patient outcome. In a testing set (n = 85) and overall patients (n = 153), both univariate and multivariate analysis found that low expression of Beclin 1 predicted adverse overall survival and progression-free survival for gastric cancer patients. Furthermore, in each stage of gastric cancer patients, Beclin 1 expression was a prognostic indicator in patients with stage II, III and IV. Importantly, a reverse relationship between Beclin 1 and Bcl-xL expression was demonstrated. In patients of elevated Bcl-xL expression, a subset with lower Beclin 1 expression displayed an inferior overall survival and progression-free survival than those with higher Beclin 1 expression. Thus, our data demonstrated that low expression of Beclin 1, associated with high Bcl-xL, played as an independent biomarker, contributing to a more aggressive cancer cell phenotype and poor prognosis for gastric tumor.