Interactions of halichondrin B and eribulin with tubulin

Compounds that modulate microtubule dynamics include highly effective anticancer drugs, leading to continuing efforts to identify new agents and improve the activity of established ones. Here, we demonstrate that [(3)H]-labeled halichondrin B (HB), a complex, sponge-derived natural product, is bound to and dissociated from tubulin rapidly at one binding site per αβ-heterodimer, with an apparent K(d) of 0.31 μM. We found no HB-induced aggregation of tubulin by high-performance liquid chromatography, even following column equilibration with HB. Binding of [(3)H]HB was competitively inhibited by a newly approved clinical agent, the truncated HB analogue eribulin (apparent K(i), 0.80 μM) and noncompetitively by dolastatin 10 and vincristine (apparent K(i)'s, 0.35 and 5.4 μM, respectively). Our earlier studies demonstrated that HB inhibits nucleotide exchange on β-tubulin, and this, together with the results presented here, indicated the HB site is located on β-tubulin. Using molecular dynamics simulations, we determined complementary conformations of HB and β-tubulin that delineated in atomic detail binding interactions of HB with only β-tubulin, with no involvement of the α-subunit in the binding interaction. Moreover, the HB model served as a template for an eribulin binding model that furthered our understanding of the properties of eribulin as a drug. Overall, these results established a mechanistic basis for the antimitotic activity of the halichondrin class of compounds.     

CYANOS: a data management system for natural product drug discovery efforts using cultured microorganisms

A software package, termed "CYANOS", has been developed to facilitate the data management and mining for natural product drug discovery efforts. This system allows for the management of data associated with field collections, culture conditions, harvests, extractions, chemical separations, and biological evaluation. This software utilizes a MySQL database for data storage, which allows for reporting and data mining via third party tools. In addition, a Web-based interface was constructed to allow for multiuser access from a variety of desktop platforms. The code for this system is freely available and has been released under the Illinois Open Source license.     

Development and validation of a rapid method for microcystins in fish and comparing LC-MS/MS results with ELISA

Microcystins (MCs) are the most common cyanotoxins found worldwide in freshwater, brackish, and marine environments. The rapid and accurate analysis of MCs and nodularin (Nod-R) in fish tissue is important for determining occurrence, following trends, and monitoring exposure for risk assessment and other purposes. The aim of this study was to develop a streamlined and reliable sample preparation method for eight MCs (MC-RR, MC-YR, MC-LR, MC-WR, MC-LA, MC-LY, MC-LW, and MC-LF) and Nod-R in fish, and conduct a validation of the new method using liquid chromatography–tandem mass spectrometry (LC-MS/MS) for analysis and compare the results with a commercial enzyme-linked immunosorbent assay (ELISA) kit. Different sample preparation methods were compared, and a simple extraction protocol with acidified acetonitrile/water (3:1) followed by hexane partitioning cleanup was found to be most effective. Thorough validation of the final method was conducted, and 90–115% recoveries were achieved for all analytes except for MC-RR, which gave 130% average recovery (isotopically labeled internal standards were unavailable to correct for possible biases). The use of electrospray ionization in the negative mode gave few interferences and minimal matrix effects in the LC-MS/MS analysis overall. Precision was typically 10–20% RSD among multiple days in experiments, detection limits were <10 ng/g in the fish tissue (catfish, basa, and swai filets), and no false-positives or false-negatives occurred in blind analyses of many spiked samples. The ELISA was unable to distinguish between MCs but was found to correctly assess the presence or absence of MCs and Nod-R in the blind-fortified fish tissues. The capability of these approaches to measure covalently bound MCs was not assessed.     

Forcing operators on MTL‐algebras

We study the forcing operators on MTL‐algebras, an algebraic notion inspired by the Kripke semantics of the monoidal t ‐norm based logic (MTL). At logical level, they provide the notion of the forcing value of an MTL‐formula. We characterize the forcing operators in terms of some MTL‐algebras morphisms. From this result we derive the equality of the forcing value and the truth value of an MTL‐formula (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Wettability control and patterning of PDMS using UV-ozone and water immersion

We demonstrate a simple method to tune and pattern the wettability of polydimethylsiloxane (PDMS) to generate microfluidic mimics of heterogeneous porous media. This technique allows one to tailor the capillary forces at different regions within the PDMS channel to mimic multi-phase flow in oil reservoirs. In this method, UV-ozone treatment is utilized to oxidize and hydrophilize the surface of PDMS. To maintain a stable surface wettability, the oxidized surfaces are immersed in water. Additionally, the use of a photomask makes it convenient to pattern the wettability in the porous media. A one-dimensional diffusive reaction model is established to understand the UV-ozone oxidation as well as hydrophobic recovery of oxidized PDMS surfaces. The modeling results show that during UV-ozone, surface oxidation dominates over diffusion of low-molecular-weight (LMW) species. However, the diffusivity of LMW species plays an important role in wettability control of PDMS surfaces.     

Dissociation and fractionation of heavy and light chains from IgG monoclonal antibodies

A basic method for dissociation and fractionation of monoclonal IgG heavy and light chain is described. It employs less noxious and hazardous reagents than the classical mercaptoethanol/propionic acid process and replaces size exclusion chromatography with cation exchange on a monolith to improve productivity. Significant scope remains to refine the conditions. The method can be applied to other disulfide bonded proteins with significant affinity for cation exchangers.     

Electrokinetic properties of wavellite and its floatability with cationic and anionic collectors

The reverse apatite flotation with fatty acids has been widely used for the reduction of phosphorus content of magmatic origin iron ores. However, the occurrence of phosphorus intensely disseminated as secondary minerals such as wavellite renders the anionic reverse flotation a challenge. Zeta potential measurements and microflotation tests of wavellite with the use of anionic and cationic collectors were carried out in this work. The wavellite's IEP value was achieved at pH 4.5. Below the IEP value, the surface positively charged sites are made up of aluminum ions. The species H(+), Al(OH)(2)(+), Al(OH)(2+), Al(3+), OH(-), H(2)PO(4)(-), HPO(4)(2-), and PO(4)(3-) play a role in the protonation and deprotonation reactions that will determine the wavellite-solution interface properties. The highest values of wavellite's floatability under basic pH conditions were achieved in the presence of cationic collectors (1 × 10(-4) mol L(-1)). The formation of surface complexes and the precipitation of insoluble salt of aluminum onto wavellite surface seems to be the most likely hypothesis for the chemical nature interactions between amines and wavellite. The surface formation of aluminum oleate on the wavellite's surface seems to be the most probable hypothesis for the adsorption mechanism and the resultant high floatability of wavellite between pH 7.5 and pH 10.0 in the presence of sodium oleate (1 × 10(-4) mol L(-1)). The results showed that the cationic reverse flotation of secondary phosphates is a promising route to reduce the phosphorus content of iron ores from deposits that underwent a supergene enrichment process, since wavellite floatability in the alkaline pH range, using amine as collector, was not significantly affected by the presence of corn starch.     

Molybdenum site structure of Escherichia coli YedY, a novel bacterial oxidoreductase

We report a structural characterization using X-ray absorption spectroscopy of the molybdenum site of Escherichia coli YedY, a novel oxidoreductase related to be the sulfite oxidase family of molybdenum enzymes. We find that the enzyme can exist in Mo(V) and Mo(IV) oxidation states but cannot be readily oxidized to the Mo(VI) form. Mo(V) YedY has molybdenum coordination similar to that of sulfite oxidase, with one Mo═O at 1.71 ?, three Mo-S at 2.39 ?, and one Mo-OH at 2.09 ?, which elongates to 2.20 ? upon reduction to Mo(IV), indicating Mo-OH(2) coordination. The Mo(V) enzyme also possesses a long Mo-O coordination at 2.64 ?, which may be due to oxygen coordination by Asn-45 O(δ), with Mo-O(δ) approximately trans to the Mo═O group. A comparison with sulfite oxidase indicates that YedY possesses a much more uniform Mo-S coordination, with a maximum permitted deviation of less than 0.05 ?. Our results indicate that the YedY active site shows considerable similarity to but also important differences from that of reduced forms of sulfite oxidase.     

The 1(5)Π(g) state of C2

We report ab initio spectroscopic constants for the recently identified 1(5)Π(g) state of C(2) [P. Bornhauser, Y. Sych, G. Knopp, T. Gerber, and P. P. Radi, J. Chem. Phys. 134, 044302 (2011)]. The calculations are performed at the multi-reference configuration interaction level of theory with Davidson's correction using aug-cc-pV6Z basis sets and include core-valence correlation and relativistic corrections obtained with quadruple-zeta bases. Such treatment accurately reproduces the experimentally observed constants of the a(3)Π(u) and other states. Thus, we expect our calculated ω(e) value for the 1(5)Π(g) state to be within a few cm(-1), and rotational constants to be within 0.1% of experiment. Agreement with available spectroscopic data is excellent, with the calculations strongly suggesting that the 1(5)Π(g) vibrational level observed by Bornhauser et al. is v = 0.