Genomic and signalling pathway characterization of the NZM panel of melanoma cell lines: A valuable model for studying the impact of genetic diversity in melanoma

Melanoma is a disease associated with a very high mutation burden and thus the possibility of a diverse range of oncogenic mechanisms that allow it to evade therapeutic interventions and the immune system. Here, we describe the characterization of a panel of 102 cell lines from metastatic melanomas (the NZM lines), including using whole‐exome and RNA sequencing to analyse genetic variants and gene expression changes in a subset of this panel. Lines possessing all major melanoma genotypes were identified, and hierarchical clustering of gene expression profiles revealed four broad subgroups of cell lines. Immunogenotyping identified a range of HLA haplotypes as well as expression of neoantigens and cancer–testis antigens in the lines. Together, these characteristics make the NZM panel a valuable resource for cell‐based, immunological and xenograft studies to better understand the diversity of melanoma biology and the responses of melanoma to therapeutic interventions.     

The Mn4Ca photosynthetic water-oxidation catalyst studied by simultaneous X-ray spectroscopy and crystallography using an X-ray free-electron laser

The structure of photosystem II and the catalytic intermediate states of the Mn₄CaO₅ cluster involved in water oxidation have been studied intensively over the past several years. An understanding of the sequential chemistry of light absorption and the mechanism of water oxidation, however, requires a new approach beyond the conventional steady-state crystallography and X-ray spectroscopy at cryogenic temperatures. In this report, we present the preliminary progress using an X-ray free-electron laser to determine simultaneously the light-induced protein dynamics via crystallography and the local chemistry that occurs at the catalytic centre using X-ray spectroscopy under functional conditions at room temperature.     

Quantitative Modeling of GRK-Mediated β2AR Regulation

We developed a unified model of the GRK-mediated β2 adrenergic receptor (β2AR) regulation that simultaneously accounts for six different biochemical measurements of the system obtained over a wide range of agonist concentrations. Using a single deterministic model we accounted for (1) GRK phosphorylation in response to various full and partial agonists; (2) dephosphorylation of the GRK site on the β2AR; (3) β2AR internalization; (4) recycling of the β2AR post isoproterenol treatment; (5) β2AR desensitization; and (6) β2AR resensitization. Simulations of our model show that plasma membrane dephosphorylation and recycling of the phosphorylated receptor are necessary to adequately account for the measured dephosphorylation kinetics. We further used the model to predict the consequences of (1) modifying rates such as GRK phosphorylation of the receptor, arrestin binding and dissociation from the receptor, and receptor dephosphorylation that should reflect effects of knockdowns and overexpressions of these components; and (2) varying concentration and frequency of agonist stimulation “seen” by the β2AR to better mimic hormonal, neurophysiological and pharmacological stimulations of the β2AR. Exploring the consequences of rapid pulsatile agonist stimulation, we found that although resensitization was rapid, the β2AR system retained the memory of the previous stimuli and desensitized faster and much more strongly in response to subsequent stimuli. The latent memory that we predict is due to slower membrane dephosphorylation, which allows for progressive accumulation of phosphorylated receptor on the surface. This primes the receptor for faster arrestin binding on subsequent agonist activation leading to a greater extent of desensitization. In summary, the model is unique in accounting for the behavior of the β2AR system across multiple types of biochemical measurements using a single set of experimentally constrained parameters. It also provides insight into how the signaling machinery can retain memory of prior stimulation long after near complete resensitization has been achieved.     

Effect of ginger constituents and synthetic analogues on cyclooxygenase-2 enzyme in intact cells.

Seventeen pungent oleoresin principles of ginger (Zingiber officinale, Roscoe) and synthetic analogues were evaluated for inhibition of cyclooxygenase-2 (COX-2) enzyme activity in the intact cell. These compounds exhibited a concentration and structure dependent inhibition of the enzyme, with IC(50) values in the range of 1-25 microM. Ginger constituents, [8]-paradol and [8]-shogaol, as well as two synthetic analogues, 3-hydroxy-1-(4-hydroxy-3-methoxyphenyl)decane and 5-hydroxy-1-(4-hydroxy-3-methoxyphenyl)dodecane, showed strong inhibitory effects on COX-2 enzyme activity. The SAR analysis of these phenolic compounds revealed three important structural features that affect COX-2 inhibition: (i) lipophilicity of the alkyl side chain, (ii) substitution pattern of hydroxy and carbonyl groups on the side chain, and (iii) substitution pattern of hydroxy and methoxy groups on the aromatic moiety.     

Sterol and oxysterol synthases near the ciliary base activate the Hedgehog pathway

Vertebrate Hedgehog signals are transduced through the primary cilium, a specialized lipid microdomain that is required for Smoothened activation. Cilia-associated sterol and oxysterol lipids bind to Smoothened to activate the Hedgehog pathway, but how ciliary lipids are regulated is incompletely understood. Here we identified DHCR7, an enzyme that produces cholesterol, activates the Hedgehog pathway, and localizes near the ciliary base. We found that Hedgehog stimulation negatively regulates DHCR7 activity and removes DHCR7 from the ciliary microenvironment, suggesting that DHCR7 primes cilia for Hedgehog pathway activation. In contrast, we found that Hedgehog stimulation positively regulates the oxysterol synthase CYP7A1, which accumulates near the ciliary base and produces oxysterols that promote Hedgehog signaling in response to pathway activation. Our results reveal that enzymes involved in lipid biosynthesis in the ciliary microenvironment promote Hedgehog signaling, shedding light on how ciliary lipids are established and regulated to transduce Hedgehog signals.     

The release of DNA into the plasma of mice following hepatic cell death by apoptosis and necrosis

The goal of these investigations was to measure levels of DNA in the plasma of mice following administration of hepatotoxic agents to induce apoptotic or necrotic cell death and determine any differences in the release of this marker depending upon death pathway. For this purpose, the effects of varying doses of anti-Fas, acetaminophen (APAP) or carbon tetrachloride (CCl₄) were assessed in normal mice. Plasma DNA was measured fluorometrically by the dye PicoGreen while lactate dehydrogenase (LDH) and caspase 3, other molecules released with cell injury or death, were measured by enzymatic assays. Histology was used to assess the occurrence of apoptosis or necrosis. Results of these experiments indicate that increased blood DNA levels occurred with all three agents and were highest with anti-Fas and CCl₄; caspase 3 levels were much higher with anti-Fas than the other agents. Histological examination confirmed the predominance of apoptotic death with anti-Fas and necrotic death with APAP and CCl₄. These results indicate that increased blood DNA is common in hepatotoxic injury and is a feature of both apoptotic and necrotic death.     

Harvesting and seeding of stochastic populations: analysis and numerical approximation

Journal of Mathematical Biology - We study an ecosystem of interacting species that are influenced by random environmental fluctuations. At any point in time, we can either harvest or seed...