Angela J. Russell

Carbon Nanotube−Ionic Liquid Composite Sensors and Biosensors

A new composite electrode has been fabricated using multiwall carbon nanotubes (MWCNT) and the ionic liquid n-octylpyridinum hexafluorophosphate (OPFP). This electrode shows very attractive electrochemical performances compared to other conventional electrodes using graphite and mineral oil, notably improved sensitivity and stability. One major advantage of this electrode compared to other electrodes using carbon nanotubes and other ionic liquids is its extremely low capacitance and background currents. A 10% (w/w) loading of MWCNT was selected as the optimal composition based on voltammetric results, as well as the stability of the background response in solution. The new composite electrode showed good activity toward hydrogen peroxide and NADH, with the possibility of fabricating a sensitive biosensor for glucose and alcohol using glucose oxidase and alcohol dehydrogenase, respectively, by simply incorporating the specific enzyme within the composite matrix. The marked electrode stability and antifouling features toward NADH oxidation was much higher for this composite compared to a bare glassy carbon electrode. While a loading of 2% MWCNT showed very poor electrochemical behavior, a large enhancement was observed upon gentle heating to 70 degrees C, which gave a response similar to the optimum composition of 10%. The ease of preparation, low background current, high sensitivity, stability, and small loading of nanotubes using this composite can create new novel avenues and applications for fabricating robust sensors and biosensors for many important species.

DLL4-Notch Signaling Mediates Tumor Resistance to Anti-VEGF Therapy In Vivo

Resistance to VEGF inhibitors is emerging as a major clinical problem. Notch signaling has been implicated in tumor angiogenesis. Therefore, to investigate mechanisms of resistance to angiogenesis inhibitors, we transduced human glioblastoma cells with retroviruses encoding Notch delta-like ligand 4 (DLL4), grew them as tumor xenografts and then treated the murine hosts with the VEGF-A inhibitor bevacizumab. We found that DLL4-mediated tumor resistance to bevacizumab in vivo. The large vessels induced by DLL4-Notch signaling increased tumor blood supply and were insensitive to bevacizumab. However, blockade of Notch signaling by dibenzazepine, a γ-secretase inhibitor, disrupted the large vessels and abolished the tumor resistance. Multiple molecular mechanisms of resistance were shown, including decreased levels of hypoxia-induced VEGF and increased levels of the VEGF receptor VEGFR1 in the tumor stroma, decreased levels of VEGFR2 in large blood vessels, and reduced levels of VEGFR3 overall. DLL4-expressing tumors were also resistant to a VEGFR targeting multikinase inhibitor. We also observed activation of other pathways of tumor resistance driven by DLL4-Notch signaling, including the FGF2-FGFR and EphB4-EprinB2 pathways, the inhibition of which reversed tumor resistance partially. Taken together, our findings show the importance of classifying mechanisms involved in angiogenesis in tumors, and how combination therapy to block DLL4-Notch signaling may enhance the efficacy of VEGF inhibitors, particularly in DLL4-upregulated tumors, and thus provide a rational base for the development of novel strategies to overcome antiangiogenic resistance in the clinic.

Abrogation of E-cadherin-mediated cell-cell contact in mouse embryonic stem cells results in reversible LIF-independent self-renewal.

We have previously demonstrated that differentiation of embryonic stem (ES) cells is associated with downregulation of cell surface E‐cadherin. In this study, we assessed the function of E‐cadherin in mouse ES cell pluripotency and differentiation. We show that inhibition of E‐cadherin‐mediated cell–cell contact in ES cells using gene knockout (Ecad−/−), RNA interference (EcadRNAi), or a transhomodimerization‐inhibiting peptide (CHAVC) results in cellular proliferation and maintenance of an undifferentiated phenotype in fetal bovine serum‐supplemented medium in the absence of leukemia inhibitory factor (LIF). Re‐expression of E‐cadherin in Ecad−/−, EcadRNAi, and CHAVC‐treated ES cells restores cellular dependence to LIF supplementation. Although reversal of the LIF‐independent phenotype in Ecad−/− ES cells is dependent on the β‐catenin binding domain of E‐cadherin, we show that β‐catenin null (βcat−/−) ES cells also remain undifferentiated in the absence of LIF. This suggests that LIF‐independent self‐renewal of Ecad−/− ES cells is unlikely to be via β‐catenin signaling. Exposure of Ecad−/−, EcadRNAi, and CHAVC‐treated ES cells to the activin receptor‐like kinase inhibitor SB431542 led to differentiation of the cells, which could be prevented by re‐expression of E‐cadherin. To confirm the role of transforming growth factor β family signaling in the self‐renewal of Ecad−/− ES cells, we show that these cells maintain an undifferentiated phenotype when cultured in serum‐free medium supplemented with Activin A and Nodal, with fibroblast growth factor 2 required for cellular proliferation. We conclude that transhomodimerization of E‐cadherin protein is required for LIF‐dependent ES cell self‐renewal and that multiple self‐renewal signaling networks subsist in ES cells, with activity dependent upon the cellular context. STEM CELLS 2009;27:2069–2080

Hedgehog and Bmp Polarize Hematopoietic Stem Cell Emergence in the Zebrafish Dorsal Aorta

Hematopoietic stem cells (HSCs) are first detected in the floor of the embryonic dorsal aorta (DA), and we investigate the signals that induce the HSC program there. We show that while continued Hedgehog (Hh) signaling from the overlying midline structures maintains the arterial program characteristic of the DA roof, a ventral Bmp4 signal induces the blood stem cell program in the DA floor. This patterning of the DA by Hh and Bmp is the mirror image of that in the neural tube, with Hh favoring dorsal rather than ventral cell types, and Bmp favoring ventral rather than dorsal. With the majority of current data supporting a model whereby HSCs derive from arterial endothelium, our data identify the signal driving this conversion. These findings are important for the study of the production of HSCs from embryonic stem cells and establish a paradigm for the development of adult stem cells.

Optimization of 3,5-dimethylisoxazole derivatives as potent bromodomain ligands.

The bromodomain protein module, which binds to acetylated lysine, is emerging as an important epigenetic therapeutic target. We report the structure-guided optimization of 3,5-dimethylisoxazole derivatives to develop potent inhibitors of the BET (bromodomain and extra terminal domain) bromodomain family with good ligand efficiency. X-ray crystal structures of the most potent compounds reveal key interactions required for high affinity at BRD4(1). Cellular studies demonstrate that the phenol and acetate derivatives of the lead compounds showed strong antiproliferative effects on MV4;11 acute myeloid leukemia cells, as shown for other BET bromodomain inhibitors and genetic BRD4 knockdown, whereas the reported compounds showed no general cytotoxicity in other cancer cell lines tested.

Selective small molecule inhibitors of the potential breast cancer marker, human arylamine N-acetyltransferase 1, and its murine homologue, mouse arylamine N-acetyltransferase 2.

The identification, synthesis and evaluation of a series of rhodanine and thiazolidin-2,4-dione derivatives as selective inhibitors of human arylamine N-acetyltransferase 1 and mouse arylamine N-acetyltransferase 2 is described. The most potent inhibitors identified have submicromolar activity and inhibit both the recombinant proteins and human NAT1 in ZR-75 cell lysates in a competitive manner. (1)H NMR studies on purified mouse Nat2 demonstrate that the inhibitors bind within the putative active site of the enzyme.

Ammonium-Directed Oxidation of Cyclic Allylic and Homoallylic Amines

The ammonium-directed olefinic oxidation of a range of cyclic allylic and homoallylic amines has been investigated. Functionalization of a range of allylic 3-(N,N-dibenzylamino)cycloalk-1-enes with m-CPBA in the presence of Cl(3)CCO(2)H gives exclusively the corresponding syn-epoxide for the 5-membered ring (>99:1 dr), the anti-epoxide for the 8-membered ring (>99:1 dr), and predominantly the anti-epoxide for the 7-membered ring (94:6 dr). Oxidation of the homoallylic amines 3-(N-benzylamino)methylcyclohex-1-ene and 3-(N,N-dibenzylamino)methylcyclohex-1-ene gave, in both cases, the corresponding N-protected 1,2-anti-2,3-syn-3-aminomethylcyclohexane-1,2-diol with high levels of diastereoselectivity (>or=90:10 dr). The versatile synthetic intermediates resulting from these oxidation reactions are readily transformed into a range of amino diols.

Highly (E)-selective Wadsworth-Emmons reactions promoted by methylmagnesium bromide.

An experimentally simple protocol for the very highly (E)-selective Wadsworth-Emmons reaction [(E):(Z) selectivities in excess of 180:1 in some cases] of a range of straight-chain and branched aliphatic, substituted aromatic, and base-sensitive aldehydes via reaction with an alkyl diethylphosphonoacetate and MeMgBr is reported.

An oxidation and ring contraction approach to the synthesis of (+/-)-1-deoxynojirimycin and (+/-)-1-deoxyaltronojirimycin.

A reaction sequence involving the chemoselective olefinic oxidation of N(1)-benzyl-2,7-dihydro-1H-azepine with m-CPBA in the presence of HBF(4) and BnOH followed by ring contraction facilitates the stereoselective preparation of either of the epoxide diastereoisomers of (2RS,3SR)-N(1)-benzyl-2-chloromethyl-3-benzyloxy-4,5-epoxypiperidine by simple modification of the reaction conditions. Epoxide ring opening, functional group interconversion, and deprotection allow the synthesis of (+/-)-1-deoxynojirimycin and (+/-)-1-deoxyaltronojirimycin.

β-Fluoroamphetamines via the Stereoselective Synthesis of Benzylic Fluorides

A range of substituted aryl epoxides undergo efficient ring-opening hydrofluorination upon treatment with 0.33 equiv of BF(3) x OEt(2) in CH(2)Cl(2) at -20 degrees C to give the corresponding syn-fluorohydrins, consistent with a mechanism involving a stereoselective S(N)1-type epoxide ring-opening process. The benzylic fluoride products of these reactions are valuable templates for further elaboration, as demonstrated by the preparation of a range of aryl-substituted beta-fluoroamphetamines.