LRP5 Signaling in Osteosarcomagenesis: a Cautionary Tale of Translation from Cell Lines to Tumors

Previous reports document expression of low-density lipoprotein receptor-related protein 5 (LRP5) in osteosarcoma (OS) tissue. Expression of this Wnt receptor correlated with metastatic disease and poor disease-free survival. Forced expression of dominant-negative LRP5 (dnLRP5), which lacks the membrane binding domain of the native protein and therefore functions as a soluble receptor-sponge for Wnt ligands, reduced in vitro cellular invasion and in vivo xenograft tumor growth for osteosarcoma cell lines. Here, we use a genetically engineered mouse model of osteosarcomagenesis with and without expression of dnLRP5 to assess to what degree tumorigenesis is affected and whether Wnt/β-catenin signaling is circumvented or maintained. Each cohort of mice developed osteosarcoma at a similar ultimate prevalence, but after a slightly increased latency in those also expressing dnLRP5. On histology, there was no difference between groups, despite previous reports that the dnLRP5 osteosarcoma cells specifically undergo a mesenchymal-to-epithelial transition in vitro. Finally, immunohistochemistry showed the presence of cytosolic and nuclear β-catenin and nuclear Cyclin D1, markers consistent with preserved Wnt/β-catenin signaling despite constitutive blockade of the cell surface receipt of Wnt signaling ligand. These data suggest that canonical Wnt signaling plays a role in OS progression and that while blockade of singular nodes in signaling pathways can have dramatic effects on individual cell lines, real tumors readily evade such focused attacks.     

Ligand binding to anti-cancer target CD44 investigated by molecular simulations

CD44 is a cell-surface glycoprotein and receptor for hyaluronan, one of the major components of the tumor extracellular matrix. There is evidence that the interaction between CD44 and hyaluronan promotes breast cancer metastasis. Recently, the molecule F-19848A was shown to inhibit hyaluronan binding to receptor CD44 in a cell-based assay. In this study, we investigated the mechanism and energetics of F-19848A binding to CD44 using molecular simulation. Using the molecular mechanics/Poisson Boltzmann surface area (MM-PBSA) method, we obtained the binding free energy and inhibition constant of the complex. The van der Waals (vdW) interaction and the extended portion of F-19848A play key roles in the binding affinity. We screened natural products from a traditional Chinese medicine database to search for CD44 inhibitors. From combining pharmaceutical requirements with docking and molecular dynamics simulations, we found ten compounds that are potentially better or equal to the F-19848A ligand at binding to CD44 receptor. Therefore, we have identified new candidates of CD44 inhibitors, based on molecular simulation, which may be effective small molecules for the therapy of breast cancer.     

Nucleotide composition string selection in HIV-1 subtyping using whole genomes

MOTIVATION: The availability of the whole genomic sequences of HIV-1 viruses provides an excellent resource for studying the HIV-1 phylogenies using all the genetic materials. However, such huge volumes of data create computational challenges in both memory consumption and CPU usage. RESULTS: We propose the complete composition vector representation for an HIV-1 strain, and a string scoring method to extract the nucleotide composition strings that contain the richest evolutionary information for phylogenetic analysis. In this way, a large-scale whole genome phylogenetic analysis for thousands of strains can be done both efficiently and effectively. By using 42 carefully curated strains as references, we apply our method to subtype 1156 HIV-1 strains (10.5 million nucleotides in total), which include 825 pure subtype strains and 331 recombinants. Our results show that our nucleotide composition string selection scheme is computationally efficient, and is able to define both pure subtypes and recombinant forms for HIV-1 strains using the 5000 top ranked nucleotide strings. AVAILABILITY: The Java executable and the HIV-1 datasets are accessible through 'http://www.cs.ualberta.ca/~ghlin/src/WebTools/hiv.php. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.     

Inhibitory effect of 2-arylbenzofurans from Erythrina addisoniae on protein tyrosine phosphatase-1B

Bioassay-guided fractionation of an EtOAc-soluble extract of the stem bark of Erythrina addisoniae (Leguminosae), using an in vitro PTP1B inhibitory assay, resulted in the isolation of three new (1-3) and three known (4-6) 2-arylbenzofuran derivatives. The new compounds were identified as 2-[2',4'-dihydroxy-3'-(3-methylbut-2-enyl)phenyl]-6-hydroxybenzofuran (1), 2-[2'-methoxy-4'-hydroxy-5'-(3-methylbut-2-enyl)phenyl]-6-hydroxybenzofuran (2), and 2-(2'-methoxy-4'-hydroxyphenyl)-5-(3-methylbut-2-enyl)-6-hydroxybenzofuran (3). The new 2-arylbenzofurans 1-3 inhibited PTP1B activity with IC(50) values ranging from 13.6+/-1.1 to 17.5+/-1.2 microM in vitro assay. On the basis of the data obtained, 2-arylbenzofurans with prenyl group may be considered as a new class of PTP1B inhibitors.     

Effect of refining on pulp surface charge accessible to polydadmac and FTIR characteristic bands of high yield kraft fibres

Studies were carried out to determine the surface charge and total charge of refined softwood high yield kraft pulps at different levels by titration methods. The results showed that the surface charge was highly dependent on the degree of refining. However, the total fibre charge was not affected by refining. The change of the fibre surface charge during refining monitored using ATR-FTIR characteristic bands was within 1700-1300 cm(-1). Reliable PLS1 calibration models could be established to correlate the FTIR spectral data and the surface charge of the refined pulps.     

The effects of Bacillus subtilis on Caenorhabditis elegans fitness after heat stress

Microbes can provide their hosts with protection from biotic and abiotic factors. While many studies have examined how certain bacteria can increase host lifespan, fewer studies have examined how host reproduction can be altered. The nematode Caenorhabditis elegans has been a particularly useful model system to examine how bacteria affect the fitness of their hosts under different contexts. Here, we examine how the bacterium Bacillus subtilis, compared to the standard C. elegans lab diet, Escherichia coli, affects C. elegans survival and reproduction after experiencing a period of intense heat stress. We find that under standard conditions, nematodes reared on B. subtilis produce fewer offspring than when reared on E. coli.However, despite greater mortality rates on B. subtilis after heat shock, young adult nematodes produced more offspring after heat shock when fed B. subtilis compared to E. coli. Because offspring production is necessary for host population growth and evolution, the reproductive advantage conferred by B. subtilis supersedes the survival advantage of E. coli. Furthermore, we found that nematodes must be reared on B. subtilis (particularly at the early stages of development) and not merely be exposed to the bacterium during heat shock, to obtain the reproductive benefits provided by B. subtilis. Taken together, our findings lend insight into the importance of environmental context and interaction timing in shaping the protective benefits conferred by a microbe toward its host.     

Identification and genetic characterization of the Pseudomonas aeruginosaleuB gene encoding 3-isopropylmalate dehydrogenase

The Pseudomonas aeruginosa leuB gene, encoding 3-isopropylmalate dehydrogenase, was identified upstream of asd, encoding aspartate-β-semialdehyde dehydrogenase. Genetic analysis indicated that leuB is identical to the previously mapped gene defined by the leu-10 allele. The chromosomal leuB locus was inactivated by gene replacement. The insertions had no adverse effect on expression of the downstream asd gene but resulted in leucine auxotrophy. The leuB gene encodes a protein containing 360 amino acids (with a molecular weight of 39153), which was expressed in Escherichia coli as a M, 42000 protein. The results suggested that, in contrast to the situation in other bacteria (E. coli, Salmonella typhimurium and Bacillus subtilis) the P. aeruginosa leuB gene is physically separated from the genes encoding the other enzymes of the isopropylmalate pathway.     

Molecular basis for interaction between Icap1 alpha PTB domain and beta 1 integrin.

Icap1 alpha is a 200-amino acid protein that binds to the COOH-terminal 13 amino acids ((786)AVTTVVNPKYEGK(798)) of the integrin beta(1) subunit. Alanine scanning mutagenesis of this region revealed that Val(787), Val(790), and (792)NPKY(795) are critical for Icap1 alpha binding. The NPXY motif is a known binding substrate for phosphotyrosine binding (PTB) domain proteins. The sequences of Icap1 alpha, residues 58--200, and the beta(1) integrin, residues 786-797, were aligned to the available PTB-peptide structures to generate a high quality structural model. Site-directed mutagenesis showed that Leu(135), Ile(138), and Ile(139) of Icap1 alpha, residues predicted by the model to be in close proximity to (792)NPKY(795), and Leu(82) and Tyr(144), residues expected to form a hydrophobic pocket near Val(787), are required for the Icap1 alpha-beta(1) integrin interaction. These findings indicate that Icap1 alpha is a PTB domain protein, which recognizes the NPXY motif of beta(1) integrin. Furthermore, our date suggest that an interaction between Val(787) and the hydrophobic pocket created by Leu(82) and Tyr(144) of Icap1 alpha forms the basis for the specificity of Icap1 alpha for the beta(1) integrin subunit.