Wiring of PQQ-dehydrogenases

The performance of pyrroloquinoline quinone (PQQ) dependent alcohol dehydrogenase (ADH) and two types of PQQ-glucose dehydrogenases in solution and when immobilized on the carbon paste electrodes modified with ferrocene derivatives is investigated. The immobilization of ADH consisting of PQQ and four hemes improves its stability up to 10 times. Both PQQ and heme moieties are involved in the electron transport from substrate to electrode. The ferrocene derivatives improve the electron transport 10-fold. Membrane-bound alcohol dehydrogenase from Gluconobacter sp. 33, intracellular soluble glucose dehydrogenase from Acinetobacter calcoaceticus L.M.D. 79.41 (s-GDH), and the membrane-bound enzyme (m-GDH) from Erwinia sp. 34-1 were purified and investigated. Soluble and membrane-bound PQQ-glucose dehydrogenases display different behavior during the immobilization on the modified carbon electrodes. The immobilization of s-GDH leads to a decrease in both stability and substrate specificity of the enzyme. This suggests that PQQ dissociates from the enzyme active center and operates as a free-diffusing mediator. The rate-limiting step of the process is likely the loading of PQQ onto the apo-enzyme. The immobilization of m-GDH leads to its substantial stabilization and improves the substrate specificity. The nature of m-GDH binding to the electrode surface is presumably similar to the binding to the cell membrane through its anchor-subunit. The enzyme operates as an enzyme and mediator complex.     

Membrane connectivity estimated by digital image analysis of HER2 immunohistochemistry is concordant with visual scoring and fluorescence in situ hybridization results: algorithm evaluation on breast cancer tissue microarrays

Because metaplastic carcinoma of the breast encompasses a great variety of histopathology, diagnostic challenges abound, especially within the realm of cytology. The authors compiled and studied an eight-case series comprised of metaplastic breast carcinomas and lesions initially suspicious cytologically for metaplastic carcinoma in order to assess the degree of cytologic-histologic correlation and to identify recurring problematic themes surrounding the cytology-based diagnosis of this neoplasm. The cytologic and histologic slides from eight cases suspicious for metaplastic breast carcinoma diagnosed by fine needle aspiration (FNA) were collected and analyzed through a seven-year retrospective search of case files at our institution. Based on cytologic characteristics, the cases were separated into three groups. Group 1 consisted of three cases presenting with poorly differentiated adenocarcinoma and squamoid components on FNA. Group 2 was composed of two cases that featured a monophasic, malignant ductal cell population on cytology, while the cytologic specimens for the third group of cases presented with a mesenchymal component with or without a malignant glandular constituent. Cytologic-histologic correlation was present in two of three cases demonstrating a mesenchymal component, and there was 100% sensitivity in the cytologic detection of those mesenchymal elements. However, in only one of three cases was there an accurate cytologic diagnosis of metaplastic carcinoma when squamoid changes were present on FNA. Both cases demonstrating only malignant glandular elements on cytologic specimens revealed an additional component of malignant squamous differentiation upon the examination of mastectomy-derived tissue. These results indicate that squamous-like changes identified on FNA should be interpreted with caution and that sampling error remains a problematic recurrence in cytology. Regardless, there appears to be promise concerning the accurate cytologic diagnosis of metaplastic carcinoma when the lesion is characterized by a mesenchymal component. A study implementing a larger case number is essential in determining the significance of these findings.     

Digital image analysis in pathology: benefits and obligation

Pathology has recently entered the era of personalized medicine. This brings new expectations for the accuracy and precision of tissue-based diagnosis, in particular, when quantification of histologic features and biomarker expression is required. While for many years traditional pathologic diagnosis has been regarded as ground truth, this concept is no longer sufficient in contemporary tissue-based biomarker research and clinical use. Another major change in pathology is brought by the advancement of virtual microscopy technology enabling digitization of microscopy slides and presenting new opportunities for digital image analysis. Computerized vision provides an immediate benefit of increased capacity (automation) and precision (reproducibility), but not necessarily the accuracy of the analysis. To achieve the benefit of accuracy, pathologists will have to assume an obligation of validation and quality assurance of the image analysis algorithms. Reference values are needed to measure and control the accuracy. Although pathologists' consensus values are commonly used to validate these tools, we argue that the ground truth can be best achieved by stereology methods, estimating the same variable as an algorithm is intended to do. Proper adoption of the new technology will require a new quantitative mentality in pathology. In order to see a complete and sharp picture of a disease, pathologists will need to learn to use both their analogue and digital eyes.     

Amperometric immunosensor for diagnosis of BLV infection

A new amperometric immunosensor for detection of antibodies against bovine leukemia protein (gp51) was designed. The detection of antibody-antigen complex formation was based on application of secondary antibodies labeled with horseradish peroxidase (HRP). Ferrocenecarboxylic acid (FCA) and N,N,N',N'-tetramethylbenzidine (TMB) were selected as suitable mediators for this immunosensor. Optimal conditions for amperometric detection were found. Sensitivity of created system was compared with the results of enzyme-linked immunosorbent assay (ELISA) and agar gel immunodiffusion (AGID) reaction, and was sufficient for detection of usual anti-gp51 antibody concentration present in the blood serum of BLV-infected cattle.     

Comparative kinetic study of D-glucose oxidation by ruthenium(III) compounds catalyzed by FAD-dependent glucose oxidase and PQQ-dependent glucose dehydrogenase

The comparative kinetic study of two glucose oxidizing enzymes, FAD-dependent glucose oxidase and PQQ-dependent glucose dehydrogenase, is presented in the artificial electron transfer mediator system based on ruthenium(III) compounds. It is demonstrated that FAD-dependent glucose oxidase and PQQ-dependent glucose dehydrogenase follow Michaelis kinetics in the D-glucose/ruthenium(III) system. PQQ-dependent glucose dehydrogenase is more active than FAD-dependent glucose oxidase in the process of D-glucose oxidation by ruthenium(III) compounds, this being due to the different catalytic mechanisms of these enzymes.     

Amperometric biosensors based on dehydrogenase/NAD and heterocyclic quinones

The electrocatalytic oxidation of NADH was evaluated from cyclic voltammetry with heterocyclic quinones dissolved in a water solution. The process of oxidation is not sensitive to the nature of the electrode surface and takes place on platinum, glassy carbon or carbon electrodes. NADH and glucose sensors constructed were investigated and operated at 0 mV vs. Ag/AgCl (in saturated KCl) using single potential step chronoamperometry.