Harnessing functional plasticity of enzymes: a fluorogenic probe for imaging 17beta-HSD10 dehydrogenase, an enzyme involved in Alzheimer's and Parkinson's diseases

In this paper, we describe the development of a fluorogenic substrate for 17beta-hydroxysteroid-dehydrogenase type 10 (17beta-HSD10), which is a multifunctional metabolic enzyme fulfilling several metabolic roles (beta-oxidation of fatty acids, catabolism of isoleucine, and metabolism of steroids). In recent years, it has emerged as an important stress and pathological marker in neurons and glial cells (expression down-regulation in Parkinson's disease, up-regulation and association with beta-amyloid peptide in Alzheimer's disease). Through the iterative molecular design and chemical synthesis described herein, compound 1 was developed, which possesses all required properties for a selective optical reporter substrate: alcohol-ketone optical switching, the ability to function as a good enzyme substrate (expressed in kinetic parameters), cell permeability, and cell retention. Probe 1 provides a blue-to-green/yellow bright switch and enables non-invasive, real-time imaging of 17beta-HSD10 in live human cells. The selectivity of reporter 1 was established by the quantitative correlation of metabolic activity to protein expression in human kidney cell line HEK-293T.     

Wiring of glucose oxidase with graphene nanoribbons: an electrochemical third generation glucose biosensor

A reagentless third generation electrochemical glucose biosensor was fabricated based on wiring the template enzyme glucose oxidase (GOx) with graphene nanoribbons (GN) in order to create direct electron transfer between the co-factor (flavin adenine dinucleotide, FAD) and the electrode. The strategy involved: (i) isolation of the apo-enzyme by separating it from its co-enzyme; (ii) preparation of graphene nanoribbons (GN) by oxidative unzipping of multi-walled carbon nanotubes; (iii) adsorptive immobilization of GNs on the surface of a screen printed carbon electrode (SPCE); (iv) covalent attachment of FAD to the nanoribbons; (v) recombination of the apo-enzyme with the covalently bound FAD to the holoenzyme; and (vi) stabilization of the bio-layer with a thin membrane of Nafion. The biosensor (referred to as GN/FAD/apo-GOx/Nafion/SPCE) is operated at a potential of +0.475 V vs Ag/AgCl/{3 M KCl} in flow-injection mode with an oxygen-free phosphate buffer (pH 7.5) acting as a carrier. The signals are linearly proportional to the concentration of glucose in the range from 50 to 2000 mg?L^?1 with a detection limit of 20 mg?L^?1. The repeatability (10 measurements, at 1000 mg?L^?1 glucose) is ±1.4% and the reproducibility (5 sensors, 1000 mg?L^?1 glucose) is ±1.8%. The biosensor was applied to the determination of glucose in human serum.

Open image in new window

Graphical abstract Wiring of the apo-enzyme of glucose oxidase (apo-GOx) with graphene nanoribbons (GN) bound to FAD at a screen-printed carbon electrode (SPCE). Cyclic voltammetric and amperometric responses to various glucose concentrations.

     

Exponential attractors for a class of reaction-diffusion problems with time delays

We consider a reaction-diffusion system subject to homogeneous Neumann boundary conditions on a given bounded domain. The reaction term depends on the population densities as well as on their past histories in a very general way. This class of systems is widely used in population dynamics modelling. Due to its generality, the longtime behavior of the solutions can display a certain complexity. Here we prove a qualitative result which can be considered as a common denominator of a large family of specific models. More precisely, we demonstrate the existence of an exponential attractor, provided that a bounded invariant region exists and the past history decays exponentially fast. This result will be achieved by means of a suitable adaptation of the l-trajectory method coming back to the seminal paper of Málek and Nečas. The first author was partially supported by the Italian PRIN 2006 research project Problemi a frontiera libera, transizioni di fase e modelli di isteresi. The second author was supported by the research project MŠM 0021620839 and by the project LC06052 (Jindřich Nečas Center for Mathematical Modeling).     

Critical evaluation of a new method for quantitative determination of minerals in solid samples

The new method for the Chemical Quantitative Mineral Analysis (CQMA) is presented and critically evaluated. The percentages of minerals are calculated of the elemental bulk chemical analysis using identified minerals and their crystallochemical formulas. For this calculation an optimization procedure is used. CQMA enables also the feedback calculation of the bulk chemical analysis from CQMA calculated mineral contents. Subsequent comparison of the feedback calculated bulk chemical analysis and actual bulk chemical analysis of the same sample is used for critical evaluation of the input data. If necessary the input data are completed and/or reanalyzed and used for repeated calculation to achieve better match between the feedback calculated and actual chemical analyses, which consequently leads to more accurate mineral analysis. Moreover the feedback bulk chemical analysis can be also separately calculated from any mineral analyses determined e.g. by XRD, FTIR and/or optical method. Six German rock reference samples representing a very wide range of minerals and chemical composition of materials were selected and their mineral analyses carried out by CQMA and Rietveld X‐ray diffraction methods. The results were compared with reference mineral analyses and critically evaluated.     

Investigation of the Qx–Qy Equilibrium in a Metal‐Free Phthalocyanine

Phthalocyanines (Pcs) have attracted a lot of interest as small molecules for organic electronics. However, some excited‐state properties of metal‐free phthalocyanines, as for example, the dynamics of the transition between the nondegenerate Q_x and Q_y states in a metal‐free phthalocyanine, have not been fully established. This effect results in a blue‐shifted shoulder with low intensity in the Pc fluorescence spectrum. This shoulder was suggested to be related to emission from the more energetic Q_y state. By using ultrafast femtosecond transient absorption, we have found a clear equilibrium between the Q_x and Q_y state of metal‐free phthalocyanines in solution.     

Remarks on the uniqueness of second order ODEs

We are concerned with the uniqueness problem for solutions to the second order ODE of the form x″+f(x, t) = 0, subject to appropriate initial conditions, under the sole assumption that f is non-decreasing with respect to x, for each t fixed. We show that there is non-uniqueness in general; on the other hand, several types of reasonable additional assumptions make the problem uniquely solvable.     

Organoplatinum(II) complexes containing chelating or bridging bis(N-heterocyclic carbene) ligands: formation of a platinum(II) carbonate complex by aerial CO2 fixation

The preparation of two new bis(N-heterocyclic carbene) platinum(II) complexes, in which NHC rings are joined by a CH(2) linker group, is described. While, the chelate complex [PtMe(2)(bis-NHC1)], 1, was formed with large tert-butyl wingtips, the iso-propyl N-substituent analogue favors formation of the cluster complex [Pt(2)Me(4)(μ-SMe(2))(μ-bis-NHC2)](2)(μ-Ag(2)Br(2)), 2, in which two binuclear platinum(II) complexes are linked together by an Ag(2)Br(2) unit. The chelating platinum complex 1 undergoes aerial CO(2) fixation and forms platinum(II) carbonate complex [Pt(CO(3))(bis-NHC1)], 3.     

Dependence of adenine isolation efficiency on the chain length evidenced using paramagnetic particles and voltammetry measurements

The main aim of this work was to study the dependence of oligoadenine isolation efficiency on the chain length by using paramagnetic particles covered by homo-deoxythymidines ((dT)25) with subsequent detection by adsorptive transfer technique coupled with square wave voltammetry. For this purpose, the oligonucleotides of the length A5, A10, A15, A20, A25, A30, A35, A40 and poly(A) in various concentrations were chosen. We determined that the isolation efficiency defined as “isolated oligonucleotide concentration”/“given oligonucleotide concentration” was about 55% on average. Sequence A25 demonstrated the best binding onto microparticles surface.     

Lessons from the first international proficiency test for the detection of spores from the honey bee pathogen Paenibacillus larvae

We report on the first proficiency test in the context of honey bee disease testing on a broad international scale. Honey samples were distributed to 12 participating laboratories for isolation and identification of spores of the etiological agent of American foulbrood, Paenibacillus larvae. Of the 11 laboratories responding to this proficiency testing event, only 6 (54%) provided data that were in full agreement with the previously determined status of the honey samples; eight (72%) laboratories fell within the threshold for qualification as defined a priori in this proficiency test (=level of agreement of at least 90%). Some lessons can now be drawn from the organizational point of view and will certainly improve new initiatives to be taken.     

Automated nucleic acids isolation using paramagnetic microparticles coupled with electrochemical detection

Easy, efficient and low demanding separation of mRNA from biological material is needed to study gene expression and to use in chip technologies. It is common knowledge that each mRNA molecule contains sequence of 25 adenines. This feature can be used for binding mRNA on the surface of the particles coated by thymine chains. The present work reports on suggesting and optimizing of mRNA separation and detection from biological material via paramagnetic microparticles coupled with electrochemical detection. Primarily we optimized cyclic and square wave voltammetric conditions to detect poly(A), which was used as standard to mimic behaviour of mRNA. Under the optimized square wave voltammetric conditions (frequency 280 Hz, accumulation time 200 s, supporting electrolyte and its temperature: acetate buffer 4.6 and 35 °C) we estimated detection limit down to 1 ng of poly(A) per ml. To enhance effectiveness and repeatability of isolation of nucleic acid automated approach for rinsing and hybridizing was proposed. We optimized the whole procedure and experimental conditions. Using automated way of isolation and under optimized conditions the yield of poly(A) (isolated concentration of poly(A)/given concentration of poly(A)*100) was approximately 75%. The suggested and optimized method for poly(A) isolation and detection was utilized for the analysis of brain tissues of patients with traumatic brain injury. The total amount of isolated mRNA varied from 40 to 760 g of mRNA per g of brain tissue. The isolation of mRNA from six samples per run was not longer than 2.5 h. Moreover, we applied the optimized procedure on fully automated pipetting instrument to isolate mRNA. The instrument was successfully tested on the analysis of extracts from roots of maize plants treated with cadmium(II) ions.