Identification of Odorant-Receptor Interactions by Global Mapping of the Human Odorome

The human olfactory system recognizes a broad spectrum of odorants using approximately 400 different olfactory receptors (hORs). Although significant improvements of heterologous expression systems used to study interactions between ORs and odorant molecules have been made, screening the olfactory repertoire of hORs remains a tremendous challenge. We therefore developed a chemical systems level approach based on protein-protein association network to investigate novel hOR-odorant relationships. Using this new approach, we proposed and validated new bioactivities for odorant molecules and OR2W1, OR51E1 and OR5P3. As it remains largely unknown how human perception of odorants influence or prevent diseases, we also developed an odorant-protein matrix to explore global relationships between chemicals, biological targets and disease susceptibilities. We successfully experimentally demonstrated interactions between odorants and the cannabinoid receptor 1 (CB1) and the peroxisome proliferator-activated receptor gamma (PPARγ). Overall, these results illustrate the potential of integrative systems chemical biology to explore the impact of odorant molecules on human health, i.e. human odorome.     

Cancer Cells Differentially Activate and Thrive on De Novo Lipid Synthesis Pathways in a Low-Lipid Environment

Increased lipogenesis is a hallmark of a wide variety of cancers and is under intense investigation as potential antineoplastic target. Although brisk lipogenesis is observed in the presence of exogenous lipids, evidence is mounting that these lipids may adversely affect the efficacy of inhibitors of lipogenic pathways. Therefore, to fully exploit the therapeutic potential of lipid synthesis inhibitors, a better understanding of the interrelationship between de novo lipid synthesis and exogenous lipids and their respective role in cancer cell proliferation and therapeutic response to lipogenesis inhibitors is of critical importance. Here, we show that the proliferation of various cancer cell lines (PC3M, HepG2, HOP62 and T24) is attenuated when cultured in lipid-reduced conditions in a cell line-dependent manner, with PC3M being the least affected. Interestingly, all cell lines - lipogenic (PC3M, HepG2, HOP62) as well as non-lipogenic (T24) - raised their lipogenic activity in these conditions, albeit to a different degree. Cells that attained the highest lipogenic activity under these conditions were best able to cope with lipid reduction in term of proliferative capacity. Supplementation of the medium with very low density lipoproteins, free fatty acids and cholesterol reversed this activation, indicating that the mere lack of lipids is sufficient to activate de novo lipogenesis in cancer cells. Consequently, cancer cells grown in lipid-reduced conditions became more dependent on de novo lipid synthesis pathways and were more sensitive to inhibitors of lipogenic pathways, like Soraphen A and Simvastatin. Collectively, these data indicate that limitation of access to exogenous lipids, as may occur in intact tumors, activates de novo lipogenesis is cancer cells, helps them to thrive under these conditions and makes them more vulnerable to lipogenesis inhibitors. These observations have important implications for the design of new antineoplastic strategies targeting the cancer cell''s lipid metabolism.     

Improving biophysical properties of a bispecific antibody scaffold to aid developability

While the concept of Quality-by-Design is addressed at the upstream and downstream process development stages, we questioned whether there are advantages to addressing the issues of biologics quality early in the design of the molecule based on fundamental biophysical characterization, and thereby reduce complexities in the product development stages. Although limited number of bispecific therapeutics are in clinic, these developments have been plagued with difficulty in producing materials of sufficient quality and quantity for both preclinical and clinical studies. The engineered heterodimeric Fc is an industry-wide favorite scaffold for the design of bispecific protein therapeutics because of its structural, and potentially pharmacokinetic, similarity to the natural antibody. Development of molecules based on this concept, however, is challenged by the presence of potential homodimer contamination and stability loss relative to the natural Fc. We engineered a heterodimeric Fc with high heterodimeric specificity that also retains natural Fc-like biophysical properties, and demonstrate here that use of engineered Fc domains that mirror the natural system translates into an efficient and robust upstream stable cell line selection process as a first step toward a more developable therapeutic.     

A novel gene, phcA from Pseudomonas syringae induces programmed cell death in the filamentous fungus Neurospora crassa

The phytopathogen Pseudomonas syringae competes with other epiphytic organisms, such as filamentous fungi, for resources. Here we characterize a gene in P. syringae pv. syringae B728a and P. syringae pv. tomato DC3000, termed phcA , that has homology to a filamentous fungal gene called het-c . phcA is conserved in many P. syringae strains, but is absent in one of the major clades, which includes the P. syringae pathovar phaseolicola. In the filamentous fungus Neurospora crassa , HET-C regulates a conserved programmed cell death pathway called heterokaryon incompatibility (HI). Ectopic expression of phcA in N. crassa induced HI and cell death that was dependent on the presence of a functional het-c pin-c haplotype. Further, by co-immunoprecipitation experiments, a heterocomplex between N. crassa HET-C1 and PhcA was associated with phcA- induced HI . P. syringae was able to attach and extensively colonize N. crassa hyphae, while an Escherichia coli control showed no association with the fungus. We further show that the P. syringae is able to use N. crassa as a sole nutrient source. Our results suggest that P. syringae has the potential to utilize phcA to acquire nutrients from fungi in nutrient-limited environments like the phyllosphere by the novel mechanism of HI induction.     

Integrins in mammary development

Integrins are ubiquitously expressed major cell surface receptors for extracellular matrix. Integrin interaction with their extracellular ligands triggers activation of the intracellular signaling pathways that control cell shape, motility, proliferation, survival, cell-type-specific gene expression. In this review, we summarize recent studies analyzing contribution of integrins to the control of the mammary morphogenesis and differentiation, function and maintenance of mammary stem and progenitor cells and resume the data from mouse models revealing the contribution of the integrin-mediated signaling to mammary tumorigenesis.     

A functional screen for genes involved in Xenopus pronephros development

In Xenopus, the pronephros is the functional larval kidney and consists of two identifiable components; the glomus, the pronephric tubules, which can be divided into four separate segments, based on marker gene expression. The simplicity of this organ, coupled with the fact that it displays the same basic organization and function as more complex mesonephros and metanephros, makes this an attractive model to study vertebrate kidney formation. In this study, we have performed a functional screen specifically to identify genes involved in pronephros development in Xenopus. Gain-of-function screens are performed by injecting mRNA pools made from a non-redundant X. tropicalis full-length plasmid cDNA library into X. laevis eggs, followed by sib-selection to identify the single clone that caused abnormal phenotypes in the pronephros. Out of 768 egg and gastrula stage cDNA clones, 31 genes, approximately 4% of the screened clones, affected pronephric marker expression examined by whole mount in situ hybridization or antibody staining. Most of the positive clones had clear expression patterns in pronephros and predicted/established functions highly likely to be involved in developmental processes. In order to carry out a more detailed study, we selected Sox7, Cpeb3, P53csv, Mecr and Dnajc15, which had highly specific expression patterns in the pronephric region. The over-expression of these five selected clones indicated that they caused pronephric abnormalities with different temporal and spatial effects. These results suggest that our strategy to identify novel genes involved in pronephros development was highly successful, and that this strategy is effective for the identification of novel genes involved in late developmental events.     

Immunoregulatory protein B7‐H3 promotes growth and decreases sensitivity to therapy in metastatic melanoma cells

B7‐H3 (CD276) belongs to the B7 family of immunoregulatory proteins and has been implicated in cancer progression and metastasis. In this study, we found that metastatic melanoma cells with knockdown expression of B7‐H3 showed modest decrease in proliferation and glycolytic capacity and were more sensitive to dacarbazine (DTIC) chemotherapy and small‐molecule inhibitors targeting MAP kinase (MAPK) and AKT/mTOR pathways: vemurafenib (PLX4032; BRAF inhibitor), binimetinib (MEK‐162; MEK inhibitor), everolimus (RAD001; mTOR inhibitor), and triciribidine (API‐2; AKT inhibitor). Similar effects were observed in melanoma cells in the presence of an inhibitory B7‐H3 monoclonal antibody, while the opposite was seen in B7‐H3‐overexpressing cells. Further, combining B7‐H3 inhibition with small‐molecule inhibitors resulted in significantly increased antiproliferative effect in melanoma cells, as well as in BRAF^V600E mutated cell lines derived from patient biopsies. Our findings indicate that targeting B7‐H3 may be a novel alternative to improve current therapy of metastatic melanoma.