Analysis of cyanobacterial toxins (anatoxin-a, cylindrospermopsin, microcystin-LR) by capillary electrophoresis

Capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC) were applied to the simultaneous separation of cyanobacterial toxins (anatoxin-a, microcystin-LR, cylindrospermopsin). The analytical performance data of both methods, optimized for the three toxins, were similar with a precision of migration times smaller than 0.8 RSD% and a detection limit in the range of 1-4 microg/mL, using spectrophotometric detection at 230 nm. Both methods were applied to an analysis of cyanotoxins in water bloom samples and crude cyanobacterial extracts. The results obtained indicate that, for complex matrices, the sequential application of CZE and MEKC is necessary. It is recommended to use both CE techniques for the analysis of the same sample in order to confirm the results by an orthogonal approach.     

Preparation and structural studies on the Bu2Sn(IV) complexes with aromatic mono- and dicarboxylic acids containing hetero {N} donor atom

Nine complexes of ^tBu₂Sn(IV)²⁺ were obtained in the solid state with ligands containing -COOH group(s) and aromatic {N} donor atom. The binding sites of the ligands were identified by FT-IR spectroscopic measurements. It was found that in most cases the -COO⁻ groups are co-ordinated in monodentate manner. Nevertheless, in some of our complexes, the -COO⁻ group forms bridges between two central {Sn} atoms resulting in the formation of an oligomeric structure, a motif that is characteristic only to the nicotinate compound. These pieces of information and the rationalisation of the experimental ¹¹⁹Sn Mössbauer nuclear quadrupole splittings, Δ, - according to the point charge model formalism - support the formation of octahedral (O_h) or trigonal bipyramidal (TBP) molecular structures. The X-ray diffraction analysis of one complex obtained as single crystal revealed the distortion of the TBP geometry towards square pyramidal (SP) one. This was rationalised by PM3 molecular modelling of the ^tBu₂Sn(pdc) complex. In the asymmetric unit, the two chemically similar but symmetry independent molecules form pseudo-dimers, in which the Sn?Sn separation amounts to ca. 6.4 Å. The crystal lattice is stabilised by C-H?O hydrogen bonding between individual molecules.     

Biological UV dosimeters in the assessment of the biological hazard from environmental radiation

To determine the impact of environmental UV radiation, biological dosimeters that weight directly the incident UV components of sunlight have been developed, improved and evaluated in the frame of the BIODOS project. Four DNA-based biological dosimeters ((i) phage T7, (ii) uracil thin layer, (iii) spore dosimeter and (iv) DLR-biofilm) have been assessed from the viewpoint of their biological relevance, spectral response and quantification of their biological effectiveness. The biological dosimeters have been validated by comparing their readings with weighted spectroradiometer data, by comparison with other biological doses, as well as with the determined amounts of DNA UV photoproducts. The data presented here demonstrate that the biological dosimeters are potentially reliable field dosimeters for measuring the integrated biologically effective irradiance for DNA damage.     

Submillimeter wave generation by second harmonic operation of tunable gyrotrons

The gyrotrons developed at the University of Sydney are tunable sources of millimetre and submillimetre radiation. The generation of submillimetre wavelengths requires operation at the second harmonic of the electron cyclotron frequency, or higher. Our latest gyrotron, GYROTRON IVA, has successfully achieved second harmonic operation and obtained frequencies up to 590 GHz (=0.51 mm). The experimental results and conditions for second harmonic operation will be presented. The design of a new gyrotron, GYROTRON V, which is especially optimised for the second harmonic, will also be included.     

Comparative study of recent wide-pore materials of different stationary phase morphology, applied for the reversed-phase analysis of recombinant monoclonal antibodies

Various recent wide-pore reversed-phase stationary phases were studied for the analysis of intact monoclonal antibodies (mAbs) of 150 kDa and their fragments possessing sizes between 25 and 50 kDa. Different types of column technology were evaluated, namely, a prototype silica-based inorganic monolith containing mesopores of ～250 Å and macropores of ～?1.1 μm, a column packed with 3.6 μm wide-pore core-shell particles possessing a wide pore size distribution with an average around 200 Å and a column packed with fully porous 1.7 μm particles having pore size of ～300 Å. The performance of these wide-pore materials was compared with that of a poly(styrene–divinyl benzene) organic monolithic column, with a macropore size of approximately 1 μm but without mesopores (stagnant pores). A systematic investigation was carried out using model IgG1 and IgG2 mAbs, namely rituximab, panitumumab, and bevacizumab. Firstly, the recoveries of intact and reduced mAbs were compared on the two monolithic phases, and it appeared that adsorption was less pronounced on the organic monolith, probably due to the difference in chemistry (C18 versus phenyl) and the absence of mesopores (stagnant zones). Secondly, the kinetic performance was investigated in gradient elution mode for all columns. For this purpose, peak capacities per meter as well as peak capacities per time unit and per pressure unit (PPT) were calculated at various flow rates, to compare performance of columns with different dimensions. In terms of peak capacity per meter, the core-shell 3.6 μm and fully porous 1.7 μm columns outperformed the two monolithic phases, at a temperature of 60 °C. However, when considering the PPT values, the core-shell 3.6 μm column remained the best phase while the prototype silica-based monoliths became very interesting, mostly due to a very high permeability compared with the organic monolith. Therefore, these core-shell and silica-based monolith provided the fastest achievable separation. Finally, at the maximal working temperature of each column, the core-shell 3.6 μm column was far better than the other one, because it is the only one stable up to 90 °C. Lastly, the loading capacity was also measured on these four different phases. It appeared that the organic monolith was the less interesting and rapidly overloaded, due to the absence of mesopores. On the other hand, the loading capacity of prototype silica-based monolith was indeed reasonable.     

Comparison of various silica‐based monoliths for the analysis of large biomolecules†

In the present study, three types of silica‐based monoliths, i.e. the first and second generations of commercial silica monolithic columns and a wide‐pore prototype monolith were compared for the analysis of large biomolecules. These molecules possess molecular weights between 1 and 66 kDa. The gradient kinetic performance of the first‐generation monolith was lower than that of the second generation, for large biomolecules (>14 kDa) but very close with smaller ones (1.3–5.8 kDa). In contrast, the wide‐pore prototype column was particularly attractive with proteins larger than 19 kDa (higher peak capacity). Among these three columns, the selectivity and retention remained quite similar but a possible larger number of accessible and charged residual silanols was noticed on the wide‐pore prototype material, which led to unpredicted small changes in selectivity and slightly broader peaks than expected. The peak shapes attained with the addition of 0.1% formic acid in the mobile phase remained acceptable for MS coupling, particularly for biomolecules of less than 6 kDa. It was found that one of the major issues with all of these silica‐based monoliths is the possible poor recovery of large biomolecules (principally with monoclonal antibody fragments of more than 25 kDa).     

Capillary zone electrophoresis of proteins applying ionic liquids for dynamic coating and as background electrolyte component

The use of ionic liquids in capillary electrophoresis, either as coating material or as components of the background electrolyte needs systematic standardization to set up optimal conditions. Excellent separation of the proteins was achieved using 1-ethyl-3-methylimidazolium tetrafluoroborate ([emim][BF₄]) or 1-butyl-3-methylimidazolium tetrafluoroborate ([bmim][BF₄]) ionic liquids using the properly made ionic-liquid–water binary mixtures for the experiments. The binary mixture has a distinctly stable and well perceptible low pH, which depends on the concentration of the ionic liquid, and on the preparation time of the mixture. Optimal conditions for the electrophoretic separation were obtained upon a multivariate analysis of the experimental parameters (applied voltage, migration time, concentration, and type of the ionic liquid). The standardized condition provides a low electroendosmotic flow toward the anode, which, however, did not hinder the proteins to migrate toward the cathode. The migration of cytochrome c, lysozyme, myoglobin, trypsin, and apo-transferrin at a pH around 2, far below the isoelectric points of the proteins, showed RSD values of the migration times less than 7.5% and less than 6.5% when using [emim][BF₄] or [bmim][BF₄], respectively, either in run-to-run or day-to-day experiments. The determination of the extent of the EOF is not possible with the commonly used EOF markers, due to interaction with the ionic-liquid constituents. The interaction of the ionic liquids with the proteins influences the migration order in zone electrophoresis. This method has been applied successfully for the analyses of real biological samples such as proteins from egg whites and human tears.     

A New Oxygen Containing Pyclen-Type Ligand as a Manganese(II) Binder for MRI and 52Mn PET Applications: Equilibrium,Kinetic, Relaxometric,Structural and Radiochemical Studies

A new pyclen-3,9-diacetate derivative ligand (H₂3,9-OPC2A) was synthesized possessing an etheric O-atom opposite to the pyridine ring, to improve the dissociation kinetics of its Mn(II) complex (pyclen = 3,6,9,15-tetraazabicyclo(9.3.1)pentadeca-1(15),11,13-triene). The new ligand is less basic than the N-containing analogue (H₂3,9-PC2A) due to the non-protonable O-atom. In spite of its lower basicity, the conditional stability of the [Mn(3,9-OPC2A)] (pMn = −log(Mn(II)), c_L = c_Mn(II) = 0.01 mM. pH = 7.4) remains unaffected (pMn = 8.69), compared to the [Mn(3,9-PC2A)] (pMn = 8.64). The [Mn(3,9-OPC2A)] possesses one water molecule, having a lower exchange rate with bulk solvents (k_ex²⁹⁸ = 5.3 ± 0.4 × 10⁷ s⁻¹) than [Mn(3,9-PC2A)] (k_ex²⁹⁸ = 1.26 × 10⁸ s⁻¹). These mild differences are rationalized by density-functional theory (DFT) calculations. The acid assisted dissociation of [Mn(3,9-OPC2A)] is considerably slower (k₁ = 2.81 ± 0.07 M⁻¹ s⁻¹) than that of the complexes of diacetates or bisamides of various 12-membered macrocycles and the parent H₂3,9-PC2A. The [Mn(3,9-OPC2A)] is inert in rat/human serum as confirmed by ⁵²Mn labeling (nM range), as well as by relaxometry (mM range). However, a 600-fold excess of EDTA (pH = 7.4) or a mixture of essential metal ions, propagated some transchelation/transmetalation in 7 days. The H₂3,9-OPC2A is labeled efficiently with ⁵²Mn at elevated temperatures, yet at 37 °C the parent H₂3,9-PC2A performs slightly better. Ultimately, the H₂3,9-OPC2A shows advantageous features for further ligand designs for bifunctional chelators.     

A minimization version of a directed subgraph homeomorphism problem

We consider a special case of the directed subgraph homeomorphism or topological minor problem, where the host graph has a specific regular structure. Given an acyclic directed pattern graph, we are looking for a host graph of minimal height which still allows for an embedding. This problem has applications in compiler design for certain coarse-grain reconfigurable architectures. In this application domain, the task is to simultaneously schedule, bind and route a so-called data-flow graph, where vertices represent operations and arcs stand for data dependencies between the operations, given an orthogonal grid structure of reconfigurable processing elements (PEs) that have restricted communication abilities. We show that the problem of simultaneously scheduling, binding and routing is NP-complete by describing a logic engine reduction from NAE-3-SAT. This result holds even when the input graph is a directed tree with maximum indegree two. We also give a |V|^3/2-approximation algorithm. J. A. Brenner’s research supported by the DFG Research Center Matheon “Mathematics for key technologies”. J. C. van der Veen’s research supported by DFG Focus Program 1148, “Reconfigurable Architectures”, Grants FE 407/8-1 and FE 407/8-2.