CREB3L1 Is a Metastasis Suppressor That Represses Expression of Genes Regulating Metastasis,Invasion, and Angiogenesis

The unfolded protein response (UPR) is activated in response to hypoxia-induced stress such as in the tumor microenvironment. This study examined the role of CREB3L1 (cyclic AMP [cAMP]-responsive element-binding protein 3-like protein 1), a member of the UPR, in breast cancer development and metastasis. Initial experiments identified the loss of CREB3L1 expression in metastatic breast cancer cell lines compared to low-metastasis or nonmetastatic cell lines. When metastatic cells were transfected with CREB3L1, they demonstrated reduced invasion and migration in vitro, as well as a significantly decreased ability to survive under nonadherent or hypoxic conditions. Interestingly, in an in vivo rat mammary tumor model, not only did CREB3L1-expressing cells fail to form metastases compared to CREB3L1 null cells but regression of the primary tumors was seen in 70% of the animals as a result of impaired angiogenesis. Microarray and chromatin immunoprecipitation with microarray technology (ChIP on Chip) analyses identified changes in the expression of many genes involved in cancer development and metastasis, including a decrease in those involved in angiogenesis. These data suggest that CREB3L1 plays an important role in suppressing tumorigenesis and that loss of expression is required for the development of a metastatic phenotype.     

A novel epidermal growth factor receptor-signaling platform and its targeted translation in pancreatic cancer

Epidermal growth factor (EGF)-induced EGFR tyrosine kinase receptor activation in cancer cell survival responses has become a strategic molecular-targeting clinical therapeutic intent, but the failures of these targeted approaches in the clinical setting demand alternate strategies. Here, we uncover a novel neuraminidase-1 (Neu1) and matrix metalloproteinase-9 (MMP-9) cross-talk in alliance with GPCR neuromedin B, which is essential for EGF-induced receptor activation and cellular signaling. Neu1 and MMP-9 form a complex with EGFR on the cell surface. Tamiflu (oseltamivir phosphate), anti-Neu1 antibodies, broad range MMP inhibitor galardin (GM6001), neuromedin B GPCR specific antagonist BIM-23127, the selective inhibitor of whole heterotrimeric G-protein complex BIM-46174 and MMP-9 specific inhibitor dose-dependently inhibited Neu1 activity associated with EGF stimulated 3T3–hEGFR cells. Tamiflu, anti-Neu1 antibodies and MMP9i attenuated EGFR phosphorylation associated with EGF-stimulated cells. Preclinical data provide the proof-of-evidence for a therapeutic targeting of Neu1 with Tamiflu in impeding human pancreatic cancer growth and metastatic spread in heterotopic xenografts of eGFP-MiaPaCa-2 tumors growing in RAGxCγ double mutant mice. Tamiflu-treated cohort exhibited a reduction of phosphorylation of EGFR-Tyr1173, Stat1-Tyr701, Akt-Thr308, PDGFRα-Tyr754 and NFκBp65-Ser311 but an increase in phospho-Smad2-Ser465/467 and -VEGFR2-Tyr1175 in the tumor lysates from the xenografts of human eGFP-MiaPaCa-2 tumor-bearing mice. The findings identify a novel promising alternate therapeutic treatment of human pancreatic cancer.     

Attenuated PLD1 association and signalling at the H452Y polymorphic form of the 5-HT2A receptor

The 5-HT_2A receptor (5-HT_2AR) is implicated in psychotropic changes within the central nervous system (CNS). A number of polymorphisms have been reported in the 5-HT_2AR gene; one of these results in a non-synonymous change, H452Y, in the carboxy-terminal tail of the receptor protein. The minor allele (9% occurrence) has been statistically associated with CNS dysfunction such as impaired memory processing and resistance to neuroleptic treatment in schizophrenic patients. We investigated the impact of H452Y mutation of the 5-HT_2AR expressed in COS7 cells on distinctly coupled intracellular signalling pathways from the receptor, focusing on the heterotrimeric G protein-independent phospholipase D (PLD) pathway, compared to the conventional Gq/11-linked phospholipase C (PLC) pathway. The H452Y mutation selectively attenuated PLD signalling, which as in the wild-type receptor, was mediated by a molecular complex involving PLD1 docked to the receptor's carboxy-terminal tail domain. Co-immunoprecipitation and GST-fusion protein experiments revealed that the H452Y mutation selectively reduced PLD1 binding to the receptor. Experiments with blocking peptides to mimic short sections of the 5-HT_2AR tail sequence revealed that the peptide spanning residue 452 strongly reduced PLD but not PLC responses of the receptor. Similar observations were made when assessing both PLD responses and PLD-dependent cellular proliferation elicited by activation of 5-HT_2ARs natively expressed in MCF-7 cells. Overall these findings indicate that the H452Y polymorphic variant of the 5-HT_2AR displays selective disruption of its PLD signalling pathway. This may potentially play a role in the CNS dysfunction associated with the H452Y allele of the 5-HT_2AR.     

The Effect of the PB2 Mutation 627K on Highly Pathogenic H5N1 Avian Influenza Virus Is Dependent on the Virus Lineage

Clade 2.2 Eurasian-lineage H5N1 highly pathogenic avian influenza viruses (HPAIVs) were first detected in Qinghai Lake, China, in 2005 and subsequently spread through Asia, Europe, and Africa. Importantly, these viruses carried a lysine at amino acid position 627 of the PB2 protein (PB2 627K), a known mammalian adaptation motif. Previous avian influenza virus isolates have carried glutamic acid in this position (PB2 627E), commonly described to restrict virus polymerase function in the mammalian host. We sought to examine the effect of PB2 627K on viral maintenance in the avian reservoir. Viruses constructed by reverse genetics were engineered to contain converse PB2 627K/E mutations in a Eurasian H5N1 virus (A/turkey/Turkey/5/2005 [Ty/05]) and, for comparison, a historical pre-Asian H5N1 HPAIV that naturally bears PB2 627E (A/turkey/England/50-92/1991 [50-92]). The 50-92 PB2 627K was genetically unstable during virus propagation, resulting in reversion to PB2 627E or the accumulation of the additional mutation PB2 628R and/or a synonymous mutation from an A to a G nucleotide at nucleotide position 1869 (PB2 A1869G). Intriguingly, PB2 628R and/or A1869G appeared to improve the genetic stability of 50-92 PB2 627K. However, the replication of 50-92 PB2 627K in conjunction with these stabilizing mutations was significantly restricted in experimentally infected chickens, where reversion to PB2 627E occurred. In contrast, no significant effects on viral fitness were observed for Ty/05 PB2 627E or 627K in in vitro or in vivo experiments. Our observations suggest that PB2 627K is supported in Eurasian-lineage viruses; in contrast, PB2 627K carries a significant fitness cost in the historical pre-Asian 50-92 virus.     

Planar cell polarity proteins differentially regulate extracellular matrix organization and assembly during zebrafish gastrulation

Zebrafish gastrulation cell movements occur in the context of dynamic changes in extracellular matrix (ECM) organization and require the concerted action of planar cell polarity (PCP) proteins that regulate cell elongation and mediolateral alignment. Data obtained using Xenopus laevis gastrulae have shown that integrin–fibronectin interactions underlie the formation of polarized cell protrusions necessary for PCP and have implicated PCP proteins themselves as regulators of ECM. By contrast, the relationship between establishment of PCP and ECM assembly/remodeling during zebrafish gastrulation is unclear. We previously showed that zebrafish embryos carrying a null mutation in the four-pass transmembrane PCP protein vang-like 2 (vangl2) exhibit increased matrix metalloproteinase activity and decreased immunolabeling of fibronectin. These data implicated for the first time a core PCP protein in the regulation of pericellular proteolysis of ECM substrates and raised the question of whether other zebrafish PCP proteins also impact ECM organization. In Drosophila melanogaster, the cytoplasmic PCP protein Prickle binds Van Gogh and regulates its function. Here we report that similar to vangl2, loss of zebrafish prickle1a decreases fibronectin protein levels in gastrula embryos. We further show that Prickle1a physically binds Vangl2 and regulates both the subcellular distribution and total protein level of Vangl2. These data suggest that the ability of Prickle1a to impact fibronectin organization is at least partly due to effects on Vangl2. In contrast to loss of either Vangl2 or Prickle1a function, we find that glypican4 (a Wnt co-receptor) and frizzled7 mutant gastrula embryos with disrupted non-canonical Wnt signaling exhibit the opposite phenotype, namely increased fibronectin assembly. Our data show that glypican4 mutants do not have decreased proteolysis of ECM substrates, but instead have increased cell surface cadherin protein expression and increased intercellular adhesion. These data indicate that Wnt/Glypican4/Frizzled signaling regulates ECM assembly through effects on cadherin-mediated cell cohesion. Together, our results demonstrate that zebrafish Vangl2/Prickle1a and non-canonical Wnt/Frizzled signaling have opposing effects on ECM organization underlying PCP and gastrulation cell movements.     

Energy,ecology and the distribution of microbial life

Mechanisms that govern the coexistence of multiple biological species have been studied intensively by ecologists since the turn of the nineteenth century. Microbial ecologists in the meantime have faced many fundamental challenges, such as the lack of an ecologically coherent species definition, lack of adequate methods for evaluating population sizes and community composition in nature, and enormous taxonomic and functional diversity. The accessibility of powerful, culture-independent molecular microbiology methods offers an opportunity to close the gap between microbial science and the main stream of ecological theory, with the promise of new insights and tools needed to meet the grand challenges humans face as planetary engineers and galactic explorers. We focus specifically on resources related to energy metabolism because of their direct links to elemental cycling in the Earth''s history, engineering applications and astrobiology. To what extent does the availability of energy resources structure microbial communities in nature? Our recent work on sulfur- and iron-oxidizing autotrophs suggests that apparently subtle variations in the concentration ratios of external electron donors and acceptors select for different microbial populations. We show that quantitative knowledge of microbial energy niches (population-specific patterns of energy resource use) can be used to predict variations in the abundance of specific taxa in microbial communities. Furthermore, we propose that resource ratio theory applied to micro-organisms will provide a useful framework for identifying how environmental communities are organized in space and time.     

Combined molecular MRI and immuno-spin-trapping for in vivo detection of free radicals in orthotopic mouse GL261 gliomas

Free radicals play a major role in gliomas. By combining immuno-spin-trapping (IST) and molecular magnetic resonance imaging (mMRI), in vivo levels of free radicals were detected within mice bearing orthotopic GL261 gliomas. The nitrone spin trap DMPO (5,5-dimethyl pyrroline N-oxide) was administered prior to injection of an anti-DMPO probe (anti-DMPO antibody covalently bound to a bovine serum albumin (BSA)–Gd (gadolinium)-DTPA (diethylene triamine penta acetic acid)–biotin MRI contrast agent) to trap tumor-associated free radicals. mMRI detected the presence of anti-DMPO adducts by either a significant sustained increase (p < 0.001) in MR signal intensity or a significant decrease (p < 0.001) in T₁ relaxation, measured as %T₁ change. In vitro assessment of the anti-DMPO probe indicated a significant decrease (p < 0.0001) in T₁ relaxation in GL261 cells that were oxidatively stressed with hydrogen peroxide, compared to controls. The biotin moiety of the anti-DMPO probe was targeted with fluorescently-labeled streptavidin to locate the anti-DMPO probe in excised brain tissues. As a negative control a non-specific IgG antibody covalently bound to the albumin–Gd-DTPA–biotin construct was used. DMPO adducts were also confirmed in tumor tissue from animals administered DMPO, compared to non-tumor brain tissue. GL261 gliomas were found to have significantly increased malondialdehyde (MDA) protein adducts (p < 0.001) and 3-nitrotyrosine (3-NT) (p < 0.05) compared to normal mouse brain tissue, indicating increased oxidized lipids and proteins, respectively. Co-localization of the anti-DMPO probe with either 3-NT or 4-hydroxynonenal was also observed. This is the first report regarding the detection of in vivo levels of free radicals from a glioma model.     

Modeling Social Transmission Dynamics of Unhealthy Behaviors for Evaluating Prevention and Treatment Interventions on Childhood Obesity

Research evidence indicates that obesity has spread through social networks, but lever points for interventions based on overlapping networks are not well studied. The objective of our research was to construct and parameterize a system dynamics model of the social transmission of behaviors through adult and youth influence in order to explore hypotheses and identify plausible lever points for future childhood obesity intervention research. Our objectives were: (1) to assess the sensitivity of childhood overweight and obesity prevalence to peer and adult social transmission rates, and (2) to test the effect of combinations of prevention and treatment interventions on the prevalence of childhood overweight and obesity. To address the first objective, we conducted two-way sensitivity analyses of adult-to-child and child-to-child social transmission in relation to childhood overweight and obesity prevalence. For the second objective, alternative combinations of prevention and treatment interventions were tested by varying model parameters of social transmission and weight loss behavior rates. Our results indicated child overweight and obesity prevalence might be slightly more sensitive to the same relative change in the adult-to-child compared to the child-to-child social transmission rate. In our simulations, alternatives with treatment alone, compared to prevention alone, reduced the prevalence of childhood overweight and obesity more after 10 years (1.2–1.8% and 0.2–1.0% greater reduction when targeted at children and adults respectively). Also, as the impact of adult interventions on children was increased, the rank of six alternatives that included adults became better (i.e., resulting in lower 10 year childhood overweight and obesity prevalence) than alternatives that only involved children. The findings imply that social transmission dynamics should be considered when designing both prevention and treatment intervention approaches. Finally, targeting adults may be more efficient, and research should strengthen and expand adult-focused interventions that have a high residual impact on children.