Determination of 8-hydroxy-2'-deoxyguanosine in untreated urine by capillary electrophoresis with UV detection

A capillary electrophoresis method with UV detection was developed for the determination of 8-hydroxy-2'-deoxyguanosine (8-OHdG) in untreated urine samples. The calibration graph for 8-OHdG in urine is linear in the concentration range 10-500 mg/l. and the detection limit is 5 mg/l (17 microM). 8-OHdG was determined in urine from oncological patients treated by radiation therapy. Its concentrations relative to creatinine were found to be in the range 10-47 microg 8-OHdG/l mg creatinine (4-19 micromol 8-OHdG/mmol creatinine). The overall time of the analysis of a urine sample was less than 15 min.     

Photoinduced phase transitions and helix untwisting in the SmC* phase of a novel cinnamoyl-based liquid crystal

A photoinduced phase transition and helix untwisting in a new liquid crystal forming the SmC* phase were studied in detail. The compound consists of a cinnamoyl photosensitive fragment with C?=?C double bond capable of photoisomerisation and photocycloaddition. It was shown that ultraviolet (UV) irradiation (365 nm) induces an extreme decrease in phase transitions temperatures (SmC*–SmA*, SmA*–N*, N*–I). Vertically aligned samples in the SmC* phase cause selective light reflection in the visible spectral range. The light action results in a noticeable helix untwisting that causes a shift in the selective light reflection peak to the long-wavelength spectral region. The temperature dependence of spontaneous polarisation P _s was measured and it was found that UV irradiation induces a decrease in the values of P _s. Photo-optical phenomena taking place in the liquid crystal are attributed to the formation of photoproducts having low anisometry, which disrupts mesophases.     

Zinc-phosphate nanoparticles with reversibly attached TNF-α analogs: an interesting concept for potential use in active immunotherapy

The authors’ intention was to prepare nanometer-sized zinc-phosphate nanoparticles that would be capable of binding histidine-rich TNF-α analogs onto their surface via a coordinative bond. Zinc-phosphate nanoparticles with a size of around 60 nm were prepared by a wet precipitation method and characterized using SEM, EDX, XRD, and DLS. First, BSA was bound as a testing protein, afterward two TNF-α analogs with decreased activity were bound to the described nanoparticles. The efficiency of binding and the existence of coordinative bond were confirmed with SDS-PAGE analysis. During binding, particle storage, and release experiments, the prepared TNF-α analogs retained their biological activity—hence the epitopes necessary for formation of antibodies stayed intact. The particle size did not change within a period of 2 weeks. No significant agglomeration was observed, the particles could be quickly dispersed in ultrasound. The present nanoparticles and the general approach of coordinative binding are widely applicable for natural and engineered histidine-rich proteins. The nanoparticles bearing appropriate TNF-α analogs could also be potentially used for active immunotherapy to tackle the chronic inflammatory diseases associated with pathogenically elevated levels of TNF-α.     

Benchmarking of Density Functionals for the Description of Optical Properties of Newly Synthesized π-Conjugated TADF Blue Emitters

Computational modeling of the optical characteristics of organic molecules with potential for thermally activated delayed fluorescence (TADF) may assist markedly the development of more efficient emitting materials for organic light-emitting diodes. Recent theoretical studies in this area employ mostly methods from density functional theory (DFT). In order to obtain accurate predictions within this approach, the choice of a proper functional is crucial. In the current study, we focus on testing the performance of a set of DFT functionals for estimation of the excitation and emission energy and the excited singlet-triplet energy gap of three newly synthesized compounds with capacity for TADF. The emitters are designed specifically to enable charge transfer by π-electron conjugation, at the same time possessing high-energy excited triplet states. The functionals chosen for testing are from various groups ranging from gradient-corrected through global hybrids to range-separated ones. The results show that the monitored optical properties are especially sensitive to how the long-range part of the exchange energy is treated within the functional. The accurate functional should also be able to provide well balanced distribution of the π-electrons among the molecular fragments. Global hybrids with moderate (less than 0.4) share of exact exchange (B3LYP, PBE0) and the meta-GGA HSE06 are outlined as the best performing methods for the systems under study. They can predict all important optical parameters correctly, both qualitatively and quantitatively.     

Histidines in affinity tags and surface clusters for immobilized metal-ion affinity chromatography of trimeric tumor necrosis factor alpha.

In order to achieve efficient IMAC (immobilized metal-ion affinity chromatography) purification of tumor necrosis factor alpha (TNF-alpha) and its analogs by a common chromatographic procedure, we tested four histidine-rich affinity tags attached to the N-termini of the trimeric TNF-alpha molecule. Using low cultivation temperature and appropriate protease deficient E. coli strains, it was possible to obtain intact, full-length proteins with NHis2Xa and HisArg tags, which could be purified to over 95% purity in a single step. However, in comparison to model proteins bearing a surface histidine cluster, accumulation of the histidine-tagged proteins in E. coli was significantly reduced, even in protease deficient strains. In addition, the histidine tagged TNF-alpha proteins never displayed good chromatographic behavior, which was otherwise easily achieved with model proteins. Although the most popular hexa-histidine tag is generally recognized as very convenient for single step isolation of monomeric proteins, our results with trimeric TNF-alpha indicate that oligomeric proteins may require further optimization of the tag, with respect to its length, composition, and location. Histidines, relatively rigidly inserted in the structure, as in our model proteins, display superior chromatographic characteristics vis a vis flexible tags with the same total number of histidines.     

Spin exchange interaction through phenylene-ethynylene bridge in diradicals based on iminonitroxide and nitronylnitroxide radical derivatives. 1. Experimental investigation of the through-bond spin exchange coupling

A series of bis-iminonitroxide diradical derivatives of different lengths and geometry have been prepared that incorporate a conjugated phenylene-ethynylene bridge as a rigid spacer. This paper describes the synthesis of these new components and their main characterizations. An unexpected singlet ground state and substituent effects on the singlet-triplet gap have been found for substituted "m-phenylene"-based diradicals. The effects of the pi-conjugation on the intramolecular through-bond spin coupling have been investigated by changing the length of the spacer within linear derivatives. The EPR studies demonstrate the intramolecular magnetic coupling between the radical spins within all compounds. This result is very attractive and unusual, given the large distance between the radicals from 15 A in the dimer to 36 A in the pentamer.     

Characterization of Carotenoid Triplet States in the Light-Harvesting Complex B800–850from the Purple Bacterium Rubrivivax gelationsus

The carotenoid triplet states in the light-harvesting complex B800–850from purple bacterium Rubrivivax gelatinosus were characterized by absorption-detected magnetic resonance in zero magnetic field (ADMR) spectroscopy. Detailed HPLC analysis of carotenoids from B800–850demonstrated the presence of several carotenoids bound to the complex: the major ones are hydroxyspheroidene and spheroidene (together 80%), followed by neurosporene and hydroxyneurosporene (7%), spheroidenone and hydroxyspheroidenone (7.5%) and two other minor carotenoids that could be 3,4-dihydrospheroidenone and 3,4-dihydrohydroxyspheroidenone (5.5%). Three triplet states originating from carotenoids present in the B800–850were observed. The identical T-S spectra recorded at selectively chosen 2|E| transitions of carotenoids indicated that all these triplet states can be attributed to three different populations of one carotenoid family, probably to spheroidene and to hydroxyspheroidene, with different out-of-plane distortions of their polyene chain due to a different protein environment. Triplet states of the neurosporene and the spheroidenone families are probably not observed because of the low signal amplitude.     

Chemometric approach in quantification of structural identity/similarity of proteins in biopharmaceuticals

We present a chemometrics study in which we show the identity or degree of similarity of 3D protein structures of various G-CSF (Granulocyte Colony-Stimulating Factor) isolates. The G-CSF isolates share the same amino acid sequence, but the preparation was carried out by somehow diverse technologies. The comparison of 3D structures was made on the basis of 2D NMR NOESY (Nuclear Overhauser Enhancement Spectroscopy) spectra of proteins. In searching for the most appropriate criteria to determine the identity or degree of similarity of selected spectral regions of different isolates, two methods for quantitative evaluation of identity/similarity were used. The first method compares all peaks in the two investigated protein spectral regions; the extent of peaks that overlap is determined. The second method includes spectral invariants originating from graph theory. The criteria of identity/similarity were calculated from graphs, derived from a collection of up to 200 peaks of investigated 2D NMR spectral region. The peaks were linked into a graph according to the sequential nearest neighborhoods. According to the first method all peaks were relevant, considering that spectral noise was previously removed; the largest similarity was found between the protein of a commercially available G-CSF drug and one of the three new isolates produced in the laboratory. The second method indicated that the pairwise similarity of the three new isolates is larger than the similarity of any of the new isolates with the commercially available drug. This is an expected result taking into account that the new isolates are produced by the same technology, while the commercial product has additives for long-term storage that could not be completely compensated. The proposed measure of similarity may help the developers of biosimilar products to optimize the controllable parameters of the production technology and eventually to argue the identity of the new isolate in comparison with the originator commercial product.