Assessing the instability of the isoelectric focusing patterns of erythropoietin in urine

IEF can be used to differentiate human urinary erythropoietin (uEPO), recombinant human erythropoietin or epoetin (rEPO) and darbepoetin (novel erythropoiesis stimulating protein (NESP)). This is the basis of the method currently used to detect misuse of rEPO and NESP by elite athletes. Recently, an unknown activity has been attributed to some urine samples (denominated 'unstable' urine by the World Anti-Doping Agency; WADA). This activity has shown to give rise to artefactual profiles for both rEPO and NESP when incubated with such urine and, thus, raised concerns with respect to doping control. We have evaluated which charges produce the characteristic IEF profiles of uEPO, rEPO and NESP and how these profiles respond to distinct enzymatic reactions. From sialidase digestions it became evident that only uEPO contains charges different from sialic acid, and a comparison of all substances after complete de-N-glycosylation localized these charges in the carbohydrate moiety. Partial desialylation, or digestion with arylsulfatase from Helix pomatia yielded profiles for recombinants species similar to those observed for unstable urine samples. The contributions from our studies to the anti-doping problem include: (i) protocols that may corroborate the potential misuse of rEPO or NESP based on the particular enzymatic activity of an arylsulfatase preparation, or a broad-specificity sialidase; (ii) assurance that the instability observed in some urine samples may only result from false-negatives, but not from false-positive testing; and (iii) a simple remedy to prevent an unstable urine from altering the IEF profile by adding selective competitive substrates.     

Halloysite Clay Nanotubes for Resveratrol Delivery to Cancer Cells

Halloysite is natural aluminosilicate clay with hollow tubular structure which allows loading with low soluble drugs using their saturated solutions in organic solvents. Resveratrol, a polyphenol known for having antioxidant and antineoplastic properties, is loaded inside these clay nanotubes lumens. Release time of 48 h is demonstrated. Spectroscopic and ζ‐potential measurements are used to study the drug loading/release and for monitoring the nanotube layer‐by‐layer (LbL) coating with polyelectrolytes for further release control. Resveratrol‐loaded clay nanotubes are added to breast cell cultures for toxicity tests. Halloysite functionalization with LbL polyelectrolyte multilayers remarkably decrease nanotube self‐toxicity. MTT measurements performed with a neoplastic cell lines model system (MCF‐7) as function of the resveratrol‐loaded nanotubes concentration and incubation time indicate that drug‐loaded halloysite strongly increase of cytotoxicity leading to cell apoptosis.

     

Hydroxamate represents a versatile zinc binding group for the development of new carbonic anhydrase inhibitors

Hydroxamates (R-CONHOH) have been scarcely investigated as carbonic anhydrase (CA, EC 4.2.1.1) inhibitors (CAIs). An inhibition/structural study of PhCONHOH is reported against all human isoforms. Comparing aliphatic (R = Me and CF(3)) and aromatic (R = Ph) hydroxamates as CAIs, we prove that CONHOH is a versatile zinc binding group. Depending on the nature of the R moiety, it can adopt different coordination modes to the catalytic ion within the CA active site.     

Reparameterization of the scalar potential in the two Higgs doublet model

In the two Higgs doublet model with no additional symmetries in the scalar sector (different from the gauge and Lorentz symmetries), it is customary to reparameterize the model by rotating the scalar doublets so that one of the vacuum expectation values vanishes. It is well known that the Yukawa sector of the model is unaffected by such a transformation. Notwithstanding this, since the Higgs potential must also be transformed, it is necessary to show that such a sector is also unaltered in its physical content. We demonstrate that the physical content of the potential is invariant even when the charge conjugation symmetry is demanded. PACS 12.60.Fr; 11.30.Er; 11.15.Ex; 11.30.Hv     

Biosensors for effective environmental and agrifood protection and commercialization: from research to market

Biosensors are projected to find many applications due to their high selectivity and sensitivity, rapid reaction, economy and ease of handling in field measurements. Even though biosensors for a wide range of environmental pollutants have been extensively reported in the literature, the decision to develop a suitable biosensing system that can be approved by a regulatory perspective for environmental applications is fraught with technical issues. These issues mainly concern the biological recognition element, the physico-chemical transducer and the interfaces between the biological and the physical components, but also aspects of fluidics, electronics, and software for data processing. This article reviews methods together with a process to move biosensor technology from research laboratories to market, focusing as a case in point on challenges and possible opportunities in the development of photosynthetic-based biosensors for environmental applications.     

A matrix-assisted laser desorption/ionization mass spectrometry approach to the lipid A from Mesorhizobium loti

The isolation, purification and analysis of the lipid A obtained from Mesorhizobium loti Ayac 1 BII strain is presented. Analysis of the carbohydrate moiety after acid hydrolysis by high-pH anion-exchange chromatography with pulse amperometric detection (HPAEC-PAD) showed the presence of glucosamine and galacturonic acid as the only sugar components. Gas chromatographic (GC) and GC/mass spectrometric (MS) analysis of the fatty acids revealed the presence of 3-OH-C12:0; 3-OH-C13:0; 3-OH-C20:0 and 27-OH-C28:0 among the major hydroxylated species. In addition, C16:0, C17:0, C18:0 and C 20:0 were shown as main saturated fatty acids. Different polyacylated species were evidenced by thin layer chromatography of lipid A, allowing the purification of two fractions. Ultraviolet matrix-assisted laser desorption/ionization time-of-flight (UV-MALDI-TOF) MS analysis with different matrices, in the positive- and negative-ion mode, was performed. The fast moving component revealed the presence of hexa-acylated species, varying in the fatty acid composition. Species containing three 3-OH fatty acids and a 27-OH-C28:0 fatty acid were observed. Individual ions within this family differ by +/-14 mass units. The slow moving component was enriched mainly in penta-acylated species. Among them, three subgroups were detected: the major one compatible with the sugar core bearing two 3-OH 20:0 fatty acids, a 3-OH 13:0 or a 3-OH 12:0 fatty acid, a 27-OH 28:0 fatty acid and one saturated fatty acid. Each signal differs in a C18:0 acyl unit from the corresponding hexa-acylated family. On the other hand, a subgroup bearing one 3-OH 20:0 fatty acid, one 27-OH 28:0 fatty acid and two non-polar fatty acids was shown. A minor subgroup compatible with structures containing two hydroxylated and three non-polar fatty acids was also detected. The results obtained showed that nor-harmane was an excellent matrix for charged lipid A structural studies in both, positive and negative ion modes.     

Quantum study of the active sites of the γ alumina surface (II): QM/MM (LSCF) approach to water, hydrogen disulfide and carbon monoxide adsorption

The mixed quantum mechanics/molecular mechanics (QM/MM) local self consistent field (LSCF) method is applied to study the adsorption of water, hydrogen sulfide, and carbon monoxide molecules on γ alumina surfaces. The effect of the long-range contributions included in the LSCF adsorption/dissociation energies are compared to cluster results. For the carbon monoxide, the long-range contributions do not change the adsorption energies in comparison with the cluster approach. In opposition, the long-range contributions lower the adsorption and dissociation energies of water and hydrogen disulfide. Cautions to be taken on the application of the LSCF method to γ alumina are also discussed.     

Salt-induced Charge Separation in Photoinduced Electron Transfer Reactions. The Effect of Ion Size*

The effect of added salts on the efficiency for photoinduced charge separation in two typical electron acceptor (A)/electron donor (D) systems was studied by the technique of laser flash photolysis. We investigate the exciplex-forming pyrene/p-dicyanobenzene (Py/DCB) and pyrene/N,N-dimethylaniline (Py/DMA) systems in ethyl acetate. The salts selected for this study are tetrabutylammonium chloride, tetrahexylammonium chloride, lithium perchlorate, sodium perchlorate, tetrabutylammonium perchlorate, sodium tetraphenylborate and tetrabutylammonium tetraphenyl-borate. In most cases, the salts quench the emission of the exciplexes with rate constants near the diffusional rate limit in ethyl acetate. An apparent red shift of the fluorescence maximum of the exciplexes with increasing salt concentration is also generally observed. Laser flash photolysis experiments showed that in the absence of the salts both A/D systems yield exclusively the triplet excited state of the polyaromatic. However, in the presence of many of the electrolytes studied, induced free radical ion formation is observed. The experimental efficiencies for induced charge separation (n) depend on the A/D system and on the nature of the salt. The measured n values vary between 0 and 0.5. The most striking variation corresponds to the lithium and sodium perchlorates. These salts are almost totally inefficient in quenching the Py/DCB exciplex, while they quench and induce charge separation from the Py/DMA exciplex with a high yield. The effect of the different salts on both exciplexes may be rationalized by using the concept of the soft/hard character of the interacting ions.     

Optimization of photodynamic therapy response by survivin gene knockdown in human metastatic breast cancer T47D cells

Photodynamic therapy (PDT) leads to the generation of cytotoxic oxygen species that appears to stimulate several different signaling pathways, some of which lead to cell death, whereas others mediate cell survival. In this context, we observed that PDT mediated by methyl-5-aminolevulinic acid as the photosensitizer resulted in over-expression of survivin, a member of the inhibitor of apoptosis (IAP) family that correlates inversely with patient prognosis. The role of survivin in resistance to anti-cancer therapies has become an area of intensive investigation. In this study, we demonstrate a specific role for survivin in modulating PDT-mediated apoptotic response. In our experimental system, we use a DNA vector-based siRNA, which targets exon-1 of the human survivin mRNA (pSil_1) to silence survivin expression. Metastatic T47D cells treated with both pSil_1 and PDT exhibited increased apoptotic indexes and cytotoxicity when compared to single-agent treated cells. The treatment resulted in increased PARP and caspase-3 cleavage, a decrease in the Bcl-2/Bak ratio and no participation of heat shock proteins. In contrast, the overexpression of survivin by a survivin-expressed vector increased cell viability and reduced cell death in breast cancer cells treated with PDT. Therefore, our data suggest that combining PDT with a survivin inhibitor may attribute to a more favorable clinical outcome than the use of single-modality PDT.     

A monoclinic form of dendocarbin A: a borderline case of one‐dimensional isostructural polymorphism

The title compound, dendocarbin A [systematic name: (1R,5aS,9aS,9bR)‐1‐hydroxy‐6,6,9a‐trimethyldodecahydronaphtho[1,2‐c]furan‐3‐one], C₁₅H₂₂O₃, is a sesquiterpene lactone isolated from Drimys winteri var chilensis. The monoclinic phase described herein displays an identical molecular structure to the orthorhombic phase that we reported previously [Paz Robles et al. (2014). Acta Cryst. C 70 , 1007–1010], while varying significantly in chain pitch, and can thus be considered as a borderline case of one‐dimensional isostructural polymorphism.