Effect of removal of organic material on stable isotope ratios in skeletal carbonate from taxonomic groups with complex mineralogies.

RATIONALE: Stable oxygen and carbon isotope ratios are one of the most accurate ways of determining environmental changes in the past, which are used to predict future environmental change. Biogenic carbonates from marine organisms are the most common source of samples for stable isotope analysis. Before they are analyzed by mass spectrometry, any organic material is traditionally removed by one of three common pretreatment methods: roasting, bleaching, or with hydrogen peroxide at various strengths and durations. METHODS: This study compares δ18 O and δ13 C values in a control with no pretreatment with those from five different pretreatment methods using conventional acid digestion mass spectrometry. The objectives are to: assess the impact of the most common pretreatment methods on δ18 O and δ13 C values from (1) taxonomically underrepresented groups in previous studies, and (2) those that precipitate a wide range of biomineralogies, in the debate of whether to pretreat or not to pretreat. We analyzed the following biomineralogically complex temperate marine organisms from southern New Zealand: four species of bryozoans, four species of molluscs, two species of arthropods, and one species each of annelid, red alga, brachiopod, and echinoderm (test plates and spines treated separately). These species precipitate aragonite, High-, Intermediate-, and/or Low-Mg calcite (LMC) in their skeletons. We used linear mixed statistical models to compare the effects of the pretreatments and mineralogical composition on their δ18 O and δ13 C values. RESULTS: Roasting was the most effective pretreatment for the removal of organic matter and light H2 O2 the least, but the former had corresponding impacts on isotope ratios. δ18 O values were not directly affected by wt% MgCO3 , but they were significantly affected by the interaction of roasting and wt% MgCO3 . This same negative effect of roasting on species with higher wt% MgCO3 occurred in δ13 C values, but it was much more pronounced in δ18 O values. Both H2 O2 pretreatments significantly and negatively affected δ18 O values at higher wt% MgCO3 . Neither bleaching pretreatment significantly affected δ18 O values. δ13 C values were most negatively affected in skeletons with high wt% MgCO3 . There was also a strong negative roasting effect and more so at higher wt% MgCO3 . Bleaching and H2 O2 did not significantly affect δ13 C values. CONCLUSIONS: Based on these results, and when using skeletal carbonate of complex mineralogies, we recommend considering the abandonment of pretreatment of biogenic carbonate for stable isotope analysis due to confounded results from previous studies, difficulties with preparation, and/or the absence of significant effects of organic material on isotope ratios. If pretreatment is necessary, avoid roasting especially at higher temperatures and durations, use minimal bleaching, and in general avoid using High-Mg calcite species in O and C stable isotope studies. If bleaching is used, clearly indicate the concentration and duration of exposure.

Snake modulates constriction in response to prey's heartbeat.

Many species of snakes use constriction-the act of applying pressure via loops of their trunk-to subdue and kill their prey. Constriction is costly and snakes must therefore constrict their prey just long enough to ensure death. However, it remains unknown how snakes determine when their prey is dead. Here, we demonstrate that boas (Boa constrictor) have the remarkable ability to detect a heartbeat in their prey and, based on this signal, modify the pressure and duration of constriction accordingly. We monitored pressure generated by snakes as they struck and constricted warm cadaveric rats instrumented with a simulated heart. Snakes responded to the beating heart by constricting longer and with greater total pressure than when constricting rats with no heartbeat. When the heart was stopped midway through the constriction, snakes abandoned constriction shortly after the heartbeat ceased. Furthermore, snakes naive to live prey also responded to the simulated heart, suggesting that this behaviour is at least partly innate. These results are an example of how snakes integrate physiological cues from their prey to modulate a complex and ancient behavioural pattern.

CUDC-101, a multitargeted inhibitor of histone deacetylase, epidermal growth factor receptor, and human epidermal growth factor receptor 2, exerts potent anticancer activity.

Receptor tyrosine kinase inhibitors have recently become important therapeutics for a variety of cancers. However, due to the heterogeneous and dynamic nature of tumors, the effectiveness of these agents is often hindered by poor response rates and acquired drug resistance. To overcome these limitations, we created a novel small molecule, CUDC-101, which simultaneously inhibits histone deacetylase and the receptor kinases epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2) in cancer cells. Because of its integrated histone deacetylase inhibition, CUDC-101 synergistically blocked key regulators of EGFR/HER2 signaling pathways, also attenuating multiple compensatory pathways, such as AKT, HER3, and MET, which enable cancer cells to escape the effects of conventional EGFR/HER2 inhibitors. CUDC-101 displayed potent antiproliferative and proapoptotic activities against cultured and implanted tumor cells that are sensitive or resistant to several approved single-targeted drugs. Our results show that CUDC-101 has the potential to dramatically improve the treatment of heterogeneous and drug-resistant tumors that cannot be controlled with single-target agents. Further, they provide a framework to create individual small molecules that simultaneously antagonize multiple biochemically distinct oncogenic targets, suggesting a general paradigm to surpass conventional, single-target cancer therapeutics. Cancer Res; 70(9); 3647-56. (c)2010 AACR.

Discovery of 7-(4-(3-ethynylphenylamino)-7-methoxyquinazolin-6-yloxy)-N-hydroxyheptanamide (CUDc-101) as a potent multi-acting HDAC, EGFR, and HER2 inhibitor for the treatment of cancer.

By incorporating histone deacetylase (HDAC) inhibitory functionality into the pharmacophore of the epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2) inhibitors, we synthesized a novel series of compounds with potent, multiacting HDAC, EGFR, and HER2 inhibition and identified 7-(4-(3-ethynylphenylamino)-7-methoxyquinazolin-6-yloxy)-N-hydroxyheptanamide 8 (CUDC-101) as a drug candidate, which is now in clinical development. 8 displays potent in vitro inhibitory activity against HDAC, EGFR, and HER2 with an IC(50) of 4.4, 2.4, and 15.7 nM, respectively. In most tumor cell lines tested, 8 exhibits efficient antiproliferative activity with greater potency than vorinostat (SAHA), erlotinib, lapatinib, and combinations of vorinostat/erlotinib and vorinostat/lapatinib. In vivo, 8 promotes tumor regression or inhibition in various cancer xenograft models including nonsmall cell lung cancer (NSCLC), liver, breast, head and neck, colon, and pancreatic cancers. These results suggest that a single compound that simultaneously inhibits HDAC, EGFR, and HER2 may offer greater therapeutic benefits in cancer over single-acting agents through the interference with multiple pathways and potential synergy among HDAC and EGFR/HER2 inhibitors.

Targeting heat shock protein 90 with CUDC-305 overcomes erlotinib resistance in non-small cell lung cancer.

CUDC-305 is a heat shock protein 90 (HSP90) inhibitor of the novel imidazopyridine class. Here, we report its activities in non-small cell lung cancer (NSCLC) cell lines with gene deregulations conferring primary or secondary resistance to epidermal growth factor receptor (EGFR) inhibitors. We show that CUDC-305 binds strongly to HSP90 extracted from erlotinib-resistant NSCLC cells (IC50 70 nmol/L). This result correlates well with the potent antiproliferative activity in erlotinib-resistant NSCLC cell lines (IC50 120-700 nmol/L) reported previously. Furthermore, it exhibits durable inhibition of multiple oncoproteins and induction of apoptosis in erlotinib-resistant NSCLC cells. CUDC-305 potently inhibits tumor growth in subcutaneous xenograft models of H1975 and A549, which harbor EGFR T790M mutation or K-ras mutations conferring acquired and primary erlotinib resistance, respectively. In addition, CUDC-305 significantly prolongs animal survival in orthotopic lung tumor models of H1975 and A549, which may be partially attributed to its preferential exposure in lung tissue. Furthermore, CUDC-305 is able to extend animal survival in a brain metastatic model of H1975, further confirming its ability to cross the blood-brain barrier. Correlating with its effects in various tumor models, CUDC-305 induces degradation of receptor tyrosine kinases and downstream signaling molecules of the PI3K/AKT and RAF/MEK/ERK pathways simultaneously, with concurrent induction of apoptosis in vivo. In a combination study, CUDC-305 enhanced the antitumor activity of a standard-of-care agent in the H1975 tumor model. These results suggest that CUDC-305 holds promise for the treatment of NSCLC with primary or acquired resistance to EGFR inhibitor therapy.

CUDC-305, a novel synthetic HSP90 inhibitor with unique pharmacologic properties for cancer therapy.

PURPOSE: We designed and synthesized CUDC-305, an HSP90 inhibitor of the novel imidazopyridine class. Here, we report its unique pharmacologic properties and antitumor activities in a variety of tumor types. EXPERIMENTAL DESIGN: The potency of the compound was analyzed by fluorescence polarization competition binding assay. Its antiproliferative activities were assessed in 40 human cancer cell lines. Its pharmacologic properties and antitumor activities were evaluated in a variety of tumor xenograft models. RESULTS: CUDC-305 shows high affinity for HSP90alpha/beta (IC(50), approximately 100 nmol/L) and HSP90 complex derived from cancer cells (IC(50), 48.8 nmol/L). It displays potent antiproliferative activity against a broad range of cancer cell lines (mean IC(50), 220 nmol/L). CUDC-305 exhibits high oral bioavailability (96.0%) and selective retention in tumor (half-life, 20.4 hours) compared with normal tissues. Furthermore, CUDC-305 can cross blood-brain barrier and reach therapeutic levels in brain tissue. CUDC-305 exhibits dose-dependent antitumor activity in an s.c. xenograft model of U87MG glioblastoma and significantly prolongs animal survival in U87MG orthotopic model. CUDC-305 also displays potent antitumor activity in animal models of erlotinib-resistant non-small cell lung cancer and induces tumor regression in animal models of MDA-MB-468 breast cancer and MV4-11 acute myelogenous leukemia. Correlating with its efficacy in these various tumor models, CUDC-305 robustly inhibits multiple signaling pathways, including PI3K/AKT and RAF/MEK/ERK, and induces apoptosis. In combination studies, CUDC-305 enhances the antitumor activity of standard-of-care agents in breast and colorectal tumor models. CONCLUSION: CUDC-305 is a promising drug candidate for the treatment of a variety of cancers, including brain malignancies.

Quantification of diacylglycerol species from cellular extracts by electrospray ionization mass spectrometry using a linear regression algorithm.

Diacylglycerols (DAGs) play significant roles in both intermediate metabolism and signal transduction. These lipid species are second messengers involved in modulating a plethora of cellular processes. Evaluation of DAG species concentrations has been hampered by the lack of a reliable method for molecular species analysis within a complex mixture of cellular lipids. We describe a new method for quantitative analysis of DAG species from complex biological extracts based on positive mode electrospray ionization mass spectrometry without prior derivatization. Quantification is achieved using internal standards and calibration curves constructed by spiking cell extracts with different concentrations of DAG species containing various acyl chain lengths and degrees of unsaturation. The new mass spectral data processing algorithm incorporates a multiple linear regression model including a factor accountable for possible interactions between experimental preparations and the slope of the curve for the standards, allowing the examinations of the effects of sample origin conditions (such as cell types, phenotypes, etc.) and instrument variability on this slope. Internal standards provide a basis for quantification of 28 DAG molecular species detected in RAW 264.7 cells after stimulation of a G-protein coupled receptor with platelet activating factor. This method displays excellent reproducibility over the established range of concentrations with variations of < or =10% and is highly sensitive with a detection limit of 0.1-0.4 pmol/microL depending upon acyl chain composition. We have shown differential effects on various DAGs in response to a ligand which illustrates the importance of examining lipids at the molecular species level rather than as a single homogeneous entity.

Lipidomics: an analysis of cellular lipids by ESI-MS.

Recognition of the importance of lipid signaling in cellular function has led to rapid progress in the technology of lipid analysis. Measurements of lipid species changes are central to defining the networks of cell signaling (e.g., receptor activation by hormones or drugs) and lipids are involved in many biochemical and pathological processes. During the last several years our laboratory has focused on developing efficient methods for extraction of glycerophospholipids from biological systems and their detection and identification by mass spectrometry. We analyze phospholipid changes in mammalian cells as a result of a defined ligand stimulation strategy that supports the research questions of the consortium. The improvement of mass spectrometry techniques for phospholipid analysis combined with sophisticated computational methods developed in our group has facilitated simultaneous analysis of hundreds of phospholipid species in mammalian cells. This information is presented as Lipid Arrays (or more precisely as virtual arrays) and allows identification of temporal changes in membrane phospholipid species between two contrasting biological conditions (e.g., unstimulated basal vs. stimulated or as a contrast between normal and disease stages). Using the lipidomics approach, we are able to identify approximately 450 phospholipid species from total membrane extracts and qualitatively measure pattern response changes initiated by cell surface receptors. As such, this approach facilitates the elucidation of the metabolic changes induced by a perturbation in the cell and recognition of patterns of signaling.

LIPID arrays: new tools in the understanding of membrane dynamics and lipid signaling.

Phospholipids are the structural building blocks of the membrane bilayer, which retains and regulates intra-cellular content. In addition to creating a protective barrier around the cell, lipids modulate membrane trafficking and are themselves precursors of important intracellular signaling molecules. Identification and quantification of these molecular species is essential for a more complete understanding of cell signaling pathways, and more reliable and sensitive methods are needed for determining membrane phospholipid content. Recent improvements in electrospray ionization mass spectrometry have made possible the direct identification of more than 400 phospholipid species from biological extracts of a single cell type. Changes in the cellular concentration of diverse lipids can be determined by analysis of the mass spectra by statistical algorithms. In the future, lipid arrays will be integrated with other high-throughput profiling technologies, and computational lipidomics will expand our understanding of the molecular basis of cellular processes and diseases.

Computational lipidomics: a multiplexed analysis of dynamic changes in membrane lipid composition during signal transduction.

Recent successes in defining the roles of lipids in cell signaling have stimulated greater interest in these versatile biomolecules. Until recently, analysis of these molecules at the species level has required labor-intensive techniques. The development of electrospray ionization mass spectrometry (ESI-MS) has made possible the detection and identification of thermally labile biological molecules, such as phospholipids. The "soft" ionization does not cause extensive fragmentation, is highly sensitive, accurate, and reproducible. Thus, this method is well suited for analyzing a broad range of phospholipids without elaborate chromatographic separation. Evaluating the vast amounts of data resulting from these measurements is a rate-limiting step in the assessment of phospholipid composition, requiring the development and application of computational algorithms for mass spectrometry data. Here we describe computational lipidomics, a novel analytical technique, coupling mass spectrometry with statistical algorithms to facilitate the comprehensive analysis of hundreds of lipid species from cellular extracts. As a result, lipid arrays are generated to indicate qualitative changes that occur in lipid composition between experimental or disease states, similar to proteomic and genomic analyses. This review presents a methodological strategy for using ESI-MS combined with a high-power computational analysis to profile time-dependent changes in cellular phospholipids after the addition of an agonist or to evaluate changes promoted by pathophysiological processes. As an illustration, we describe the methods and approaches used to generate lipid arrays for The Alliance for Cellular Signaling (AfCS). These arrays are contributing to a more complete understanding of the participants of cellular signaling pathways after activation of cell surface receptors.