Short communication: Evaluation of an automated in-house hematology analyzer for bovine blood

The objective of this study was to evaluate the suitability of the V-Sight hematology analyzer (A. Menarini Pharma GmbH, Vienna, Austria) for bovine blood by a comparison with a reference device (Advia 2120i, Siemens AG, Erlangen, Germany). In total, 97 blood samples were obtained from 75 dairy cows. Analyzed parameters included counts of white blood cells (WBC), lymphocytes, monocytes, granulocytes, red blood cells (RBC), and platelets (PLT), as well as hemoglobin concentration (HGB), hematocrit (HCT), mean corpuscular volume, mean corpuscular hemoglobin (MCH), MCH concentration (MCHC), mean platelet volume (MPV), and plateletcrit (PCT). Based on Passing-Bablok regression, the V-Sight provided accurate and precise results for MCH and MCHC only. The PCT results were comparable to the reference method, but precision was inconclusive. Significant proportional differences were detected for monocytes, granulocytes, HCT, and PLT. For all other analytes, significant proportional and systemic differences were observed. The WBC and lymphocyte results from the V-Sight were characterized by poor accuracy, poor precision, and a high number of false positive outliers. Bland-Altman analysis indicated negative biases for all WBC parameters, the erythrocyte indices, and PLT. Positive biases were observed for RBC, HGB, HCT, MPV, and PCT. Correlation coefficients of >0.9 between the V-Sight and the reference method were found only for RBC, HGB, HCT, and MPV. Intraassay precision of the V-Sight analyzer was acceptable (coefficient of variation <5%) for granulocytes, the erythrocyte indices, and MPV. It was unacceptable (coefficient of variation ≥5%) for WBC, lymphocytes, monocytes, as well as RBC, and inconclusive for HGB, HCT, PLT, and PCT. Sensitivity was high for all RBC counts and indices as well as PLT, but low for monocytes, granulocytes, and MPV. Specificity was high for monocytes and granulocytes, but low for RBC, HCT, MCH, and MCHC. With accurate and precise results for only 2 out of 13 parameters, the V-Sight cannot be recommended for analysis of bovine blood.     

Wind energy potential in southern Sweden-Example of planning methodology

This study presents a planning model for Swedish activities in the field of wind power. Models and results of calculations of the land-based wind energy potential with the Wind Atlas Analysis and Application Programme, WA^sP, are described and analysed in a geographical information systems (GIS) called the ArcView^® GIS system. One county in southern Sweden was chosen as a case study to present the methods used. The results from that case study indicate a great wind energy potential but there are nevertheless many factors limiting that potential. The study calls for a further development of planning tools in the field. Important areas for the future are the development of knowledge in market issues, wind power technology, environmental issues, and public opinion on constructing wind turbines.     

Nitroreductase-Mediated Release of Inhibitors of Lysine-Specific Demethylase 1 (LSD1) from Prodrugs in Transfected Acute Myeloid Leukaemia Cells

Lysine-specific demethylase 1 (LSD1) has evolved as a promising therapeutic target for cancer treatment, especially in acute myeloid leukaemia (AML). To approach the challenge of site-specific LSD1 inhibition, we developed an enzyme-prodrug system with the bacterial nitroreductase NfsB (NTR) that was expressed in the virally transfected AML cell line THP1-NTR⁺. The cellular activity of the NTR was proven with a new luminescent NTR probe. We synthesised a diverse set of nitroaromatic prodrugs that by design do not affect LSD1 and are reduced by the NTR to release an active LSD1 inhibitor. The emerging side products were differentially analysed using negative controls, thereby revealing cytotoxic effects. The 2-nitroimidazolyl prodrug of a potent LSD1 inhibitor emerged as one of the best prodrug candidates with a pronounced selectivity window between wild-type and transfected THP1 cells. Our prodrugs are selectively activated and release the LSD1 inhibitor locally, proving their suitability for future targeting approaches.     

Pressure cell for investigations of solid-liquid interfaces by neutron reflectivity

We describe an apparatus for measuring scattering length density and structure of molecular layers at planar solid-liquid interfaces under high hydrostatic pressure conditions. The device is designed for in situ characterizations utilizing neutron reflectometry in the pressure range 0.1-100 MPa at temperatures between 5 and 60?°C. The pressure cell is constructed such that stratified molecular layers on crystalline substrates of silicon, quartz, or sapphire with a surface area of 28 cm(2) can be investigated against noncorrosive liquid phases. The large substrate surface area enables reflectivity to be measured down to 10(-5) (without background correction) and thus facilitates determination of the scattering length density profile across the interface as a function of applied load. Our current interest is on the stability of oligolamellar lipid coatings on silicon surfaces against aqueous phases as a function of applied hydrostatic pressure and temperature but the device can also be employed to probe the structure of any other solid-liquid interface.     

Rational Structure-Based Design of Fluorescent Probes for Amyloid Folds

Amyloid fibrils are pathological hallmarks of various human diseases, including Parkinson's, Alzheimer's, amyotrophic lateral sclerosis (ALS or motor neurone disease), and prion diseases. Treatment of the amyloid diseases are hindered, among other factors, by timely detection and therefore, early detection of the amyloid fibrils would be beneficial for treatment against these disorders. Here, a small molecular fluorescent probe is reported that selectively recognize the fibrillar form of amyloid beta(1–42), α-synuclein, and HET-s(218–289) protein over their monomeric conformation. The rational design of the reporters relies on the well-known cross-β-sheet repetition motif, the key structural feature of amyloids.     

Discovery of Affinity-Based Probes for Btk Occupancy Assays

Bruton's tyrosine kinase (Btk) is an attractive target for the treatment of a wide array of B-cell malignancies and autoimmune diseases. Small-molecule covalent irreversible Btk inhibitors targeting Cys481 have been developed for the treatment of such diseases. In clinical trials, probe molecules are required in occupancy studies to measure the level of engagement of the protein by these covalent irreversible inhibitors. The result of this pharmacodynamic (PD) activity provides guidance for appropriate dosage selection to optimize inhibition of the drug target and correlation of target inhibition with disease treatment efficacy. This information is crucial for successful evaluation of drug candidates in clinical trials. Based on the pyridine carboxamide scaffold of a novel solvent-accessible pocket (SAP) series of covalent irreversible Btk inhibitors, we successfully developed a potent and selective affinity-based biotinylated probe 12 (2-[(4-{4-[5-(1-{5-[(3aS,4S,6aR)-2-oxo-hexahydro-1H-thieno[3,4-d]imidazol-4-yl]pentanamido}-3,6,9,12-tetraoxapentadecan-15-amido)pentanoyl]piperazine-1-carbonyl}phenyl)amino]-6-[1-(prop-2-enoyl)piperidin-4-yl]pyridine-3-carboxamide). Compound 12 has been used in Btk occupancy assays for preclinical studies to determine the therapeutic efficacy of Btk inhibition in two mouse lupus models driven by TLR7 activation and type I interferon.     

Development of Biarylalkyl Carboxylic Acid Amides with Improved Anti-schistosomal Activity

The parasitic disease schistosomiasis is the cause of more than 200 000 human deaths per year. Although the disease is treatable, there is one major shortcoming: praziquantel has been the only drug used to combat these parasites since 1977. The risk of the emergence of resistant schistosomes is known to be increasing, as a reduced sensitivity of these parasites toward praziquantel has been observed. We developed a new class of substances, which are derived from inhibitors of human aldose reductase, and which showed promising activity against Schistosoma mansoni couples in vitro. Further optimisation of the compounds led to an increase in anti-schistosomal activity with observed phenotypes such as reduced egg production, vitality, and motility as well as tegumental damage and gut dilatation. Here, we performed structure–activity relationship studies on the carboxylic acid moiety of biarylalkyl carboxylic acids. Out of 82 carboxylic acid amides, we identified 10 compounds that are active against S. mansoni at 25 μm . The best five compounds showed an anti-schistosomal activity up to 10 μm and induced severe phenotypes. Cytotoxicity tests in human cell lines showed that two derivatives had no cytotoxicity at 50 or 100 μm . These compounds are promising candidates for further optimisation toward the new anti-schistosomal agents.