Serum metabolomics using ultra performance liquid chromatography coupled to mass spectrometry in lactating dairy cows following a single dose of sporidesmin.

INTRODUCTION: Photosensitization is a common clinical sign in cows suffering from liver damage caused by the mycotoxin sporidesmin. This disease, called facial eczema (FE), is of major importance in New Zealand. Current techniques for diagnosing animals with subclinical sporidesmin-induced liver damage (i.e. without photosensitization) are nonspecific. In addition, little is known of the mechanisms involved in sporidesmin resistance, nor the early effects seen following low-dose sporidesmin intoxication. OBJECTIVE: The objective of this study was to identify individual metabolites or metabolic profiles that could be used as serum markers for early stage FE in lactating cows. METHODS: Results are presented from a 59-day sporidesmin challenge in Friesian-cross dairy cows. Serum metabolite profiles were obtained using reversed phase ultra-performance liquid chromatography (UPLC) electrospray ionization mass spectrometry (MS) and UPLC tandem MS. Multivariate and time series analyses were used to assess the data. RESULTS: Statistical analysis, both with and without the temporal component, could distinguish the profiles of animals with clinical signs from the others, but not those affected subclinically. An increase in the concentrations of a combination of taurine- and glycine-conjugated secondary bile acids (BAs) was the most likely cause of the separation. This is the first time that MS methods have been applied to FE and that bile acids changes have been detected in cattle exposed to sporidesmin. CONCLUSIONS: It is well known that BA concentrations increase during cholestasis due to damage to bile ducts and leakage of the bile. This is the first study to investigate metabolomic changes in serum following a sporidesmin challenge. Further work to establish the significance of the elevation of individual BAs concentrations in the serum of early-stage sporidesmin-poisoned cows is necessary.

G-Quadruplex Supramolecular Assemblies in Photochemical Upconversion.

Parallel, tetramolecular G-quadruplex (G4) DNA possessing TINA monomer, (R)-1-O-[4-(1-pyrenylethynyl)phenylmethyl]glycerol, were synthesised and evaluated in complexes with tris(2,2'-bipyridine)ruthenium(II), [Ru(bpy)3 ](2+) , and the Zn(2+) derivative of 5,10,15,20-tetrakis-(1-methyl-4-pyridyl)-21 H,23H-porphine, ZnTMpyP4. UV/Vis, fluorescence, and circular dichroism (CD) spectroscopy showed that the use of G4-DNA as a template resulted in the effective communication between the ligands and the TINA molecule that was covalently attached to the 5'-end and between T and dG at the 5'-end of the dTG4 T sequence. Only one G4-DNA possessing the TINA molecule at the 5'-end of the dTG4 T sequence was able to yield a green-to-blue photochemical upconversion (PUC, λem =420 nm) in the presence of [Ru(bpy)3 ](2+) upon excitation at 500 nm. Different DNA secondary structures can thus be used in DNA-based assemblies for PUC and the way of attachment of chromophores to DNA plays a pivotal role for the creation of a photosynthetic centre.

DNA-Based Assemblies for Photochemical Upconversion.

In the present study DNA was used as a scaffold for the supramolecular assembly of organic chromophores for photochemical upconversion (PUC). Initially, a green-to-blue PUC was observed using free chromophores in solution: tris(2,2'-bipyridine)ruthenium(II), [Ru(bpy)3](2+), which acts as a long-wavelength absorber (λex = 500 nm), and an in situ energy donor to an acceptor (R)-1-O-[4-(1-pyrenylethynyl)phenylmethyl]glycerol (PEPy or TINA monomer), which acts as an annihilator and short-wavelength photoemitter (λem = 420 nm). Then, DNA duplexes possessing TINA monomers were synthesized, and complexes with [Ru(bpy)3](2+) were investigated. In contrast to the dynamic interactions of [Ru(bpy)3](2+) with TINA monomer free in solution, ground-state complex formation was the predominant mechanism of interaction between [Ru(bpy)3](2+) and DNA duplexes bearing two TINA monomers at the 5' ends as shown by fluorescence, circular dichroism (CD), and UV-vis spectroscopy studies. The use of TINA-modified DNAs led to PUC occurring at concentrations significantly lower than that for free chromophores in solution: 2.5 µM [Ru(bpy)3](2+) and 5.0 µM TINA-modified duplex in the DNA-based systems in aqueous buffer versus 46 µM [Ru(bpy)3](2+) and 4.6 mM TINA monomer for the free donor and acceptor in DCM, respectively. Providing vast capabilities of DNA in the development of novel photonic systems as a result of the controllable organization of various chromophores, this study opens a new perspective for the development of DNA-based light-harvesting systems using PUC.

Synthesis of ß-pyrrolic-modified porphyrins and their incorporation into DNA.

A synthetic methodology for the synthesis of various ß-pyrrolic-functionalised porphyrins and their covalent attachment to 2'-deoxyuridine and DNA is described. Palladium(0)-catalysed Sonogashira and copper(I)-catalysed Huisgen 1,3-dipolar cycloaddition reactions were used to insert porphyrins into the structure of 2'-deoxyuridine and DNA. Insertion of a porphyrin into the middle of single-stranded CT oligonucleotides possessing a 5'-terminal run of four cytosines was shown to trigger the formation of pH- and temperature-dependent i-motif structures. Porphyrin insertion also led to the aggregation of single-stranded purine-pyrimidine sequences, which could be dissociated by heating at 90 °C for 5 min. Parallel triplexes and anti-parallel duplexes were formed in the presence of the appropriate complementary strand(s). Depending on the modification, porphyrins were placed in the major and minor grooves of duplexes and were used as bulged intercalating insertions in duplexes and triplexes. In general, the thermal stabilisation of parallel triplexes possessing porphyrin-modified triplex-forming oligonucleotide (TFO) strands was observed, whereas anti-parallel duplexes were destabilised. These results are compared and discussed on the basis of the results of molecular modelling calculations.

Spectroscopic and computational study of ß-ethynylphenylene substituted zinc and free-base porphyrins.

A series of tetraphenylporphyrins appended at the ß-pyrrolic position with an ethynylphenylene- or ethynylpyridine-substituent have been subjected to spectroscopic and density functional theory (DFT) analyses. The mean absolute deviation between corresponding experimental and DFT-derived vibrational spectra is up to 10.2 cm(-1), suggesting that the DFT B3LYP/6-31G(d) method provides an accurate model of the ß-substituted porphyrin systems. The configuration interactions that give rise to prominent electronic absorptions have been calculated using time-dependant DFT (TD-DFT) and have been rationalized with reference to the energy and topology of DFT calculated molecular orbitals. As the electron withdrawing capacity of the ß-substituent increases the LUMO orbital gains appreciable amplitude over the substituent moiety and is stabilised. This represents a departure from the assumptions underpinning the Gouterman four-orbital model, resulting in atypical electronic absorption spectra. This phenomenon is also manifested in the enhancement patterns of the resonance Raman spectra insofar as B-band excitation engenders an enhancement of substituent based modes. These observations demonstrate that the ß-substituent exerts an appreciable electronic influence on the porphyrin π-electron system and provides a means of introducing charge-transfer character to prominent electronic transitions.