Diatoms are unicellular phytoplankton accounting for approximately 40% of global marine primary productivity [1], yet the molecular mechanisms underlying their ecological success are largely unexplored. We use a functional-genomics approach in the marine diatom Phaeodactylum tricornutum to characterize a novel protein belonging to the widely conserved YqeH subfamily [2] of GTP-binding proteins thought to play a role in ribosome biogenesis [3], sporulation [4], and nitric oxide (NO) generation [5]. Transgenic diatoms overexpressing this gene, designated PtNOA, displayed higher NO production, reduced growth, impaired photosynthetic efficiency, and a reduced ability to adhere to surfaces. A fused YFP-PtNOA protein was plastid localized, distinguishing it from a mitochondria-localized plant ortholog. PtNOA was upregulated in response to the diatom-derived unsaturated aldehyde 2E,4E/Z-decadienal (DD), a molecule previously shown to regulate intercellular signaling, stress surveillance [6], and defense against grazers [7]. Overexpressing cell lines were hypersensitive to sublethal levels of this aldehyde, manifested by altered expression of superoxide dismutase and metacaspases, key components of stress and death pathways [8, 9]. NOA-like sequences were found in diverse oceanic regions, suggesting that a novel NO-based system operates in diatoms and may be widespread in phytoplankton, providing a biological context for NO in the upper ocean [10].

Angiogenesis and inflammation are important therapeutic targets in non-small cell lung cancer (NSCLC). It is well known that proteolysis mediated by matrix metalloproteinases (MMPs) promotes angiogenesis and inflammation in the tumor microenvironment. Here, the effects of the MMP-inhibitor TIMP-2 on NSCLC inflammation and angiogenesis were evaluated in TIMP-2 deficient (timp2−/−) mice injected subcutaneously with Lewis-Lung carcinoma cells and compared with the effects on tumors in wild-type (WT) mice. TIMP-2-deficient mice demonstrated increased tumor growth, enhanced expression of angiogenic marker αv β3 in tumor and endothelial cells and significantly higher serum-VEGF-A levels. Tumor-bearing-timp2−/− mice showed a significant number of inflammatory cells in their tumors, upregulation of inflammation mediators, NF-κB and Annexin A1, as well as higher levels of serum IL-6. Phenotypic analysis revealed an increase in myeloid-derived-suppressor-cell (MDSC) cells (CD11b+, Gr-1+) that co-expressed VEGF-R1+ and elevated MMP activation present in tumors and spleens from timp2−/− mice. Furthermore, TIMP-2-deficient tumors up-regulated expression of the immunosuppressing genes controlling MDSC growth, IL-10, IL-13, IL-11, and CCL-5/RANTES, as well as decreased IFN-γ and increased CD40L. Moreover, forced TIMP-2 expression in human lung-adenocarcinoma A-549 resulted in a significant reduction of MDSCs recruited into tumors, as well as suppression of angiogenesis and tumor growth. The increase in MDSCs has been linked to cancer immunosuppression and angiogenesis. Therefore, this study supports TIMP-2 as a negative regulator of MDSCs with important implications for the immunotherapy and /or anti-angiogenic treatment of NSCLC.

As part of their strategy to infect the globally important coccolithophore, Emiliania huxleyi (Lohmann) W.W. Hay & H.P. Mohler, Coccolithoviruses trigger and regulate the host's programmed cell death (PCD) machinery during lytic infection. The induction and recruitment of host metacaspases, specialized, ancestral death proteases that facilitate viral lysis, suggests they may be important subcellular determinants to infection. We examined the "basal" levels and patterns of caspase activity and metacaspase expression in exponentially growing resistant and sensitive E. huxleyi strains and linked them with susceptibility to E. huxleyi virus 1 (EhV1). Resistant E. huxleyi strains were consistently characterized by low caspase specific activity and a relatively simple metacaspase expression profile. In contrast, sensitive E. huxleyi strains had markedly elevated caspase specific activity and consistently expressed more diverse metacaspase proteins. Using pooled data sets from triplicate experiments, we observed statistically significant linear correlations between infectivity, caspase activity, and metacaspase expression, with each strain forming distinct clusters, within a gradient in viral susceptibility. At the same time, we observed positive correlations between the expression of a subset of metacaspase proteins and lower susceptibility, suggestive of potential protective roles. Our findings implicate the importance of subtle differences in the basal physiological regulation of the PCD machinery to viral resistance or sensitivity and cell fate.

A novel recombinant protein vaccine for human schistosomiasis caused by Schistosoma mansoni is under development. The Sm-TSP-2 schistosomiasis vaccine is comprised of a 9 kDa recombinant protein corresponding to the extracellular domain of a unique S. mansoni tetraspanin. Here, we describe the cloning and the expression of the external loop of Sm-TSP-2 recombinant protein secreted by Pichia Pink the process development at 20L scale fermentation, and the two-steps purification, which resulted in a protein recovery yield of 31% and a protein purity of 97%. The developed processes are suitable for the production of purified protein for subsequent formulation and Phase 1 clinical studies.