Glycoconjugated Metallohelices have Improved Nuclear Delivery and Suppress Tumour Growth In Vivo

Monosaccharides are added to the hydrophilic face of a self‐assembled asymmetric Fe^II metallohelix, using CuAAC chemistry. The sixteen resulting architectures are water‐stable and optically pure, and exhibit improved antiproliferative selectivity against colon cancer cells (HCT116 p53^+/+) with respect to the non‐cancerous ARPE‐19 cell line. While the most selective compound is a glucose‐appended enantiomer, its cellular entry is not mainly glucose transporter‐mediated. Glucose conjugation nevertheless increases nuclear delivery ca 2.5‐fold, and a non‐destructive interaction with DNA is indicated. Addition of the glucose units affects the binding orientation of the metallohelix to naked DNA, but does not substantially alter the overall affinity. In a mouse model, the glucose conjugated compound was far better tolerated, and tumour growth delays for the parent compound (2.6 d) were improved to 4.3 d; performance as good as cisplatin but with the advantage of no weight loss in the subjects.     

Determination of ethyl glucuronide in human serum by capillary zone electrophoresis and an immunoassay

Ethyl glucuronide (EtG) is a marker of recent alcohol consumption. For the optimization of the analysis of EtG by CZE with indirect absorbance detection, the use of capillaries with permanent and dynamic wall coatings, the composition of the BGE, and various sample preparation procedures, including dilution with water, ultrafiltration, protein precipitation, and SPE, were investigated. Two validated screening assays for the determination of EtG in human serum, a CZE‐based approach and an enzyme immunoassay (EIA), are described. The CZE assay uses a coated capillary, 2,4‐dimethylglutaric acid as an internal standard, and a pH 4.65 BGE comprising 9 mM nicotinic acid, ε‐aminocaproic acid and 10% v/v ACN. Proteins are removed via precipitation with ACN prior to analysis and the LOQ is 0.50 mg/L. The EIA is based upon commercial reagents which are promoted for the determination of urinary EtG. Krebs–Ringer solution containing 5% BSA is used as a calibration matrix. All samples are ultrafiltered prior to analysis of the ultrafiltrate on a Mira Plus analyzer. Assay calibration ranged between 0 and 2 mg/L and the upper reference limit was determined to be 0.05 mg/L. Both assays proved to be suitable for the analysis of samples from different individuals. For EtG levels above 0.50 mg/L, good agreement was observed for the comparison of the results of the two methods.     

Effect of Two Different UVA Doses on the Rabbit Cornea and Lens

The aim of the present paper was to examine the irradiation effect of two doses of UVA rays (365 nm) on the rabbit cornea and lens. Corneas of anesthetized adult albino rabbits were irradiated with UVA rays for 5 days (daily dose 1.01 J cm⁻² in one group of rabbits and daily dose 2.02 J cm⁻² in the second group of animals). The third day after the last irradiation, the rabbits were killed, and their eyes were employed for spectrophotometrical, biochemical and immunohistochemical investigations. Normal eyes served as controls. Absorption spectra of the whole corneal centers were recorded over the UV–VIS (visible) spectral range. Levels of antioxidant and prooxidant enzymes, nitric oxide synthases and nitric oxide (indirectly measured as nitrate concentration) were investigated in the cornea. Malondialdehyde, a byproduct of lipid peroxidation, was examined in the cornea and lens. The results show that the staining for endothelial nitric oxide synthase was more pronounced in corneas irradiated with the higher UVA dose. Otherwise, UVA rays at either dose did not significantly change corneal light absorption properties and did not cause statistically significant metabolic changes in the cornea or lens. In conclusion, UVA rays at the employed doses did not evoke harmful effects in the cornea or lens.     

Supercritical fluid extraction as a tool for isolation of monoterpenes from coniferous needles and walnut-tree leaves

Several monoterpenes, i.e., (+)-α-pinene, (−)-camphene, sabinene, (−)-β-pinene, myrcene, R-(+)-limonene, (−)-bornylacetate, (−)-trans-caryophyllene and a-humulene were identified and determined by gas chromatography-mass spectrometry (GC-MS) in needles of Pinaceae (Picea abies, P. omorika, P. pungens, P. Breweriana, Pinus nigra, P. mungo turra, P. black, P. sylvestris, Abies pinsapo, A. holophylla, A. Bronmuelleris, A. alba, Larix Kaempferi L. decidua) and tree-leaves of Juglandaceae (Juglans regia, J. nigra, J. sieboldiana var. Cordiformis) families. Supercritical fluid extraction (SFE) was found to be very useful for their isolation at optimised conditions (needles/leaves: pressure 20/30 MPa, temperature 80/130°C, time of extraction 60/60 min, modifier chloroform/chloroform). Their seasonal distribution, evaluation of differences in concentrations and relative amounts in different trees and their varieties grown in different localities were evaluated.      

Characterization of the volatile fraction emitted by Pinus spp. by one- and two-dimensional chromatographic techniques with mass spectrometric detection

The chemical composition of the needles of P. pinea, P. pinaster, P. halepensis, P. nigra, P. brutia, P. patula, P. radiata, P. taeda, P. elliotti, P. kesiya, P. sylvestris and P. eldarica was investigated. Headspace solid-phase microextraction and steam distillation extraction were used to collect the volatile fractions. Samples were analyzed using one-dimensional gas chromatography (1D-GC) and comprehensive two-dimensional gas chromatography (GC × GC) associated with a quadrupole and a time-of-flight mass detectors. Results showed that the analytical capabilities of 1D-GC are partially limited by the separation power of the columns. The higher sensibility and the absence of peak skewing of the time-of-flight mass analyzer, with the use of automated peak finding and deconvolution algorithms, allowed for the detection of trace components with qualitative full spectra and the extraction of true mass spectra from coeluting compounds, promoting their reliable identification and thus significantly improving results obtained by 1D-GC/MS, when using a quadrupole mass analyzer. The use of GC × GC resulted in enhanced separation efficiency and increased signal to noise ratio (sensitivity) of the analytes, maximizing mass spectra quality and improving compound detection and identification. This work shows the use of 1D-GC/ToFMS for the analysis of pine needles volatiles, achieving the detection of 177 compounds, that is more than twice the number previously identified by standard 1D-GC/MS. The analysis by GC × GC for the same sample allowed the detection of 212 compounds. The enantioselective GC × GC analysis performed for all the Pinus spp. under study achieved the detection of 422 different compounds. Cross-over phenomena according to operational conditions are highlighted and discussed.     

Analysis of anthocyanin pigments in Lonicera (Caerulea) extracts using chromatographic fractionation followed by microcolumn liquid chromatography-mass spectrometry

Anthocyanins from the fruit Lonicera caerulea L. var. kamtschatica (blueberry honeysuckle, Caprifoliaceae) were studied via (semi)preparative chromatographic fractionation followed by MS and μLC/MS analysis. The extraction procedure was optimized with respect to analytical purposes as well as its potential use for the preparation of nutraceuticals. The highest yield of anthocyanins was obtained using acidified methanol as the extraction medium. A comparable total anthocyanin content was obtained using a mixture of methanol and acetone. However, when Lonicera anthocyanins were in contact with acetone, a condensation reaction occurred to a large extent and related 5-methylpyranoanthocyanins were found. The effect of other extraction media, including ethanol as a "green" solvent, is also discussed. The potential of two fractionation procedures for extract purification differing in their chromatographic selectivity and scale was studied (i.e. using a Sephadex LH-20 gel column and a reversed phase). Fractions obtained by both procedures were used for a detailed analysis. MS and μLC/MS(2) methods were used for monitoring anthocyanin and 5-methylpyranoderivatives content as well as identifying less common and more complex dyes (dimer of cyanidin-3-hexoside, cyanidin-ethyl-catechin-hexosides, etc.). These more complex dyes are most likely formed during fruit treatment.     

Evaluation of comprehensive two-dimensional gas chromatography coupled to time-of-flight mass spectrometry for the diagnosis of inherited metabolic disorders using an automated data processing strategy

Organic acidurias are a large group of inherited metabolic disorders (IMDs), commonly diagnosed by GC-MS analysis of organic acids in urine after acidic extraction and trimethylsilylation. In this study, a GC×GC-ToF-MS method has been optimized for the analysis of pathological metabolites in urine. An automated data processing strategy based on the use of mass spectra and GC retention times for the target search and quantification of pathological metabolites has been developed. Using this procedure, each unknown sample is automatically examined for the presence of markers of several diseases at the same time. The method has been applied for the analysis of 6 challenging proficiency testing samples from patients with IMDs (thymidine phosphorylase deficiency, mevalonic aciduria, hawkinsinuria, aromatic l-amino acid decarboxylase deficiency, propionic acidemia and medium-chain acyl-CoA dehydrogenase deficiency). Using the GC×GC-ToF-MS method, we were able to determine complete sets of markers for all the IMDs. The quality of the mass spectral matches for the pathological markers was higher than 800 (out of 1000).     

Dynamic behaviors of a delay differential equation model of plankton allelopathy

In this paper, we consider a modified delay differential equation model of the growth of n-species of plankton having competitive and allelopathic effects on each other. We first obtain the sufficient conditions which guarantee the permanence of the system. As a corollary, for periodic case, we obtain a set of delay-dependent condition which ensures the existence of at least one positive periodic solution of the system. After that, by means of a suitable Lyapunov functional, sufficient conditions are derived for the global attractivity of the system. For the two-dimensional case, under some suitable assumptions, we prove that one of the components will be driven to extinction while the other will stabilize at a certain solution of a logistic equation. Examples show the feasibility of the main results.     

Statistical methods for comparing comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometry results: metabolomic analysis of mouse tissue extracts

The potential for the utilization of GC x GC-time-of-flight (TOF) MS for high-resolution metabolomics studies is discussed, with the implementation of some statistical comparisons for biomarker detection. Metabolite profiles from NZO obese mice versus BL/6 control mice are compared and contrasted using a number of chromatogram comparison routines, including direct chromatogram comparisons, chromatogram subtraction and averaging routines, as well as a method for generating relative weighted peak surface difference chromatograms, and a more conventional Student's t-test statistical approach.