Global analysis of osteosarcoma lipidomes reveal altered lipid profiles in metastatic versus nonmetastatic cells.

Osteosarcoma (OS) is the most common form of primary bone cancer in humans. The early detection and subsequent control of metastasis has been challenging in OS. Lipids are important constituents of cells that maintain structural integrity that can be converted into lipid-signaling molecules and are reprogrammed in cancerous states. Here, we investigate the global lipidomic differences in metastatic (143B) and nonmetastatic (HOS) human OS cells as compared with normal fetal osteoblast cells (FOB) using lipidomics. We detect 15 distinct lipid classes in all three cell lines that included over 1,000 lipid species across various classes including phospholipids, sphingolipids and ceramides, glycolipids, and cholesterol. We identify a key class of lipids, diacylglycerols, which are overexpressed in metastatic OS cells as compared with their nonmetastatic or nontumorigenic counterparts. As a proof of concept, we show that blocking diacylglycerol synthesis reduces cellular viability and reduces cell migration in metastatic OS cells. Thus, the differentially regulated lipids identified in this study might aid in biomarker discovery, and the synthesis and metabolism of specific lipids could serve as future targets for therapeutic development.

Development of Poly Unsaturated Fatty Acid Derivatives of Aspirin for Inhibition of Platelet Function.

The inhibition of platelet aggregation is key to preventing conditions such as myocardial infarction and ischemic stroke. Aspirin is the most widely used drug to inhibit platelet aggregation. Aspirin absorption can be improved further to increase its permeability across biologic membranes via esterification or converting the carboxylic acid to an anhydride. There are several reports indicating that ω-3 and ω-6 fatty acids such as linoleic acid, eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) separately inhibit platelet aggregation. Herein, we synthesize anhydride conjugates of aspirin with linoleic acid, EPA, and DHA to form aspirin anhydrides that are expected to have higher permeability across cellular membranes. These aspirin-fatty acid anhydrides inhibited platelet aggregation in washed human platelets and platelet-rich plasma in a dose-dependent manner. In particular, the aspirin-DHA anhydride displayed similar effectiveness to aspirin. Platelet aggregation studies conducted in the presence of various platelet agonists indicated that the aspirin-lipid conjugates act through inhibition of the cyclooxygenase (COX)-thromboxane synthase (TXAS) pathway. Hence, we performed detailed biochemical studies using purified COX-1 as well as TXAS stabilized in nanoscale lipid bilayers of nanodiscs to confirm results from the platelet aggregation studies. We show that although all of the aspirin conjugates act through the COX-TXAS pathway by inhibiting COX-1, the parent fatty acids do not act via this pathway. Finally, we studied the hydrolysis of these compounds in buffer and human plasma, and we demonstrate that all of the aspirin-fatty acid conjugates hydrolyze to the parent molecules aspirin and fatty acid in a controlled manner.

Direct Capture of Functional Proteins from Mammalian Plasma Membranes into Nanodiscs.

Mammalian plasma membrane proteins make up the largest class of drug targets yet are difficult to study in a cell free system because of their intransigent nature. Herein, we perform direct encapsulation of plasma membrane proteins derived from mammalian cells into a functional nanodisc library. Peptide fingerprinting was used to analyze the proteome of the incorporated proteins in nanodiscs and to further demonstrate that the lipid composition of the nanodiscs directly affects the class of protein that is incorporated. Furthermore, the functionality of the incorporated membrane proteome was evaluated by measuring the activity of membrane proteins: Na(+)/K(+)-ATPase and receptor tyrosine kinases. This work is the first report of the successful establishment and characterization of a cell free functional library of mammalian membrane proteins into nanodiscs.

Cerium(III) Nitrate Containing Electrospun Wound Dressing for Mitigating Burn Severity.

Thermal injuries pose a risk for service members in prolonged field care (PFC) situations or to civilians in levels of lower care. Without access to prompt surgical intervention and treatment, potentially salvageable tissues are compromised, resulting in increases in both wound size and depth. Immediate debridement of necrotic tissue enhances survivability and mitigates the risks of burn shock, multiple organ failure, and infection. However, due to the difficulty of surgical removal of the burn eschar in PFC situations and lower levels of care, it is of utmost importance to develop alternative methods for burn stabilization. Studies have indicated that cerium(III) nitrate may be used to prolong the time before surgical intervention is required. The objective of this study was to incorporate cerium(III) nitrate into an electrospun dressing that could provide burst release. Select dosages of cerium(III) nitrate were dissolved with either pure solvent or polyethylene oxide (PEO) for coaxial or traditional electrospinning set-ups, respectively. The solutions were coaxially electrospun onto a rotating mandrel, resulting in a combined nonwoven mesh, and then compared to traditionally spun solutions. Dressings were evaluated for topography, morphology, and porosity using scanning electron microscopy and helium pycnometry. Additionally, cerium(III) loading efficiency, release rates, and cytocompatibility were evaluated in both static and dynamic environments. Imaging showed randomly aligned polymer nanofibers with fiber diameters of 1161 ± 210 nm and 1090 ± 250 nm for traditionally and coaxially spun PEO/cerium(III) nitrate dressings, respectively. Assay results indicated that the electrospun dressings contained cerium(III) nitrate properties, with the coaxially spun dressings containing 33% more cerium(III) nitrate than their traditionally spun counterparts. Finally, release studies revealed that PEO-based dressings released the entirety of their contents within the first hour with no detrimental cytocompatibility effects for coaxially-spun dressings. The study herein shows the successful incorporation of cerium(III) nitrate into an electrospun dressing.

Antitumorigenic Properties of Omega-3 Endocannabinoid Epoxides.

Accumulating studies have linked inflammation to tumor progression. Dietary omega-3 fatty acids, such as docosahexaenoic acid (DHA), have been shown to suppress tumor growth through their conversion to epoxide metabolites. Alternatively, DHA is converted enzymatically into docosahexaenoylethanolamide (DHEA), an endocannabinoid with antiproliferative activity. Recently, we reported a novel class of anti-inflammatory DHEA-epoxide derivative called epoxydocospentaenoic-ethanolamide (EDP-EA) that contain both ethanolamide and epoxide moieties. Herein, we study the antitumorigenic properties of EDP-EAs in an osteosarcoma (OS) model. First, we show ∼80% increase in EDP-EAs in metastatic versus normal lungs of mice. We found significant differences in the apoptotic and antimigratory potencies of the different EDP-EA regioisomers, which were partially mediated through cannabinoid receptor 1 (CB1). Next, we synthesized derivatives of the most pro-apoptotic regioisomer. These derivatives had reduced hydrolytic susceptibility to fatty acid amide hydrolase (FAAH) and increased CB1-selective binding. Collectively, we report a novel class of EDP-EAs that exhibit antiangiogenic, antitumorigenic, and antimigratory properties in OS.

Comparative proteomic investigation of metastatic and non-metastatic osteosarcoma cells of human and canine origin.

Osteosarcoma is the most common bone cancer in dogs and people. In order to improve clinical outcomes, it is necessary to identify proteins that are differentially expressed by metastatic cells. Membrane bound proteins are responsible for multiple pro-metastatic functions. Therefore characterizing the differential expression of membranous proteins between metastatic and non-metastatic clonal variants will allow the discovery of druggable targets and consequently improve treatment methodology. The objective of this investigation was to systemically identify the membrane-associated proteomics of metastatic and non-metastatic variants of human and canine origin. Two clonal variants of divergent in vivo metastatic potential from human and canine origins were used. The plasma membranes were isolated and peptide fingerprinting was used to identify differentially expressed proteins. Selected proteins were further validated using western blotting, flow cytometry, confocal microscopy and immunohistochemistry. Over 500 proteins were identified for each cell line with nearly 40% of the proteins differentially regulated. Conserved between both species, metastatic variants demonstrated significant differences in expression of membrane proteins that are responsible for pro-metastatic functions. Additionally, CD147, CD44 and vimentin were validated using various biochemical techniques. Taken together, through a comparative proteomic approach we have identified several differentially expressed cell membrane proteins that will help in the development of future therapeutics.

Recent advances in nanodisc technology for membrane protein studies (2012-2017).

Historically, the main barrier to membrane protein investigations has been the tendency of membrane proteins to aggregate (due to their hydrophobic nature), in aqueous solution as well as on surfaces. The introduction of biomembrane mimetics has since stimulated momentum in the field. One such mimetic, the nanodisc (ND) system, has proved to be an exceptional system for solubilizing membrane proteins. Herein, we critically evaluate the advantages and imperfections of employing nanodiscs in biophysical and biochemical studies. Specifically, we examine the techniques that have been modified to study membrane proteins in nanodiscs. Techniques discussed here include fluorescence microscopy, solution-state/solid-state nuclear magnetic resonance, electron microscopy, small-angle X-ray scattering, and several mass spectroscopy methods. Newer techniques such as SPR, charge-sensitive optical detection, and scintillation proximity assays are also reviewed. Lastly, we cover how nanodiscs are advancing nanotechnology through nanoplasmonic biosensing, lipoprotein-nanoplatelets, and sortase-mediated labeling of nanodiscs.

Functional role of the conserved i-helix residue I346 in CYP5A1-Nanodiscs.

Thromboxane synthase (CYP5A1) is a non-classical cytochrome P450 (CYP) expressed in human platelets that mediates vascular homeostasis by producing thromboxane A2 (TXA2) through the isomerization of prostaglandin H2 (PGH2). A homology alignment of CYP5A1 with human CYPs indicates that a highly conserved I-helix threonine residue is occupied by an isoleucine at position 346 in CYP5A1. We find that reverse-engineering CYP5A1 to contain either threonine or serine in this position dramatically increases TXA2 formation. Interestingly, the levels of malondialdehyde (MDA), a homolytic fragmentation product of PGH2 formed via a pathway independent of TXA2 formation, remain constant. Furthermore, spectral analysis using two PGH2 substrate analogs supports the observed activity changes in the hydroxyl-containing mutants. The more constrained active site of the I346T mutant displays altered PGH2 substrate analog binding properties. Together these studies provide new mechanistic insights into CYP5A1 mediated isomerization of PGH2 with respect to a critical active site residue.

Synthesis of most polyene natural product motifs using just 12 building blocks and one coupling reaction.

The inherent modularity of polypeptides, oligonucleotides and oligosaccharides has been harnessed to achieve generalized synthesis platforms. Importantly, like these other targets, most small-molecule natural products are biosynthesized via iterative coupling of bifunctional building blocks. This suggests that many small molecules also possess inherent modularity commensurate with systematic building block-based construction. Supporting this hypothesis, here we report that the polyene motifs found in >75% of all known polyene natural products can be synthesized using just 12 building blocks and one coupling reaction. Using the same general retrosynthetic algorithm and reaction conditions, this platform enabled both the synthesis of a wide range of polyene frameworks that covered all of this natural-product chemical space and the first total syntheses of the polyene natural products asnipyrone B, physarigin A and neurosporaxanthin b-D-glucopyranoside. Collectively, these results suggest the potential for a more generalized approach to making small molecules in the laboratory.