Analysis of eighteen antidepressants,four atypical antipsychotics and active metabolites in serum by liquid chromatography: a simple tool for therapeutic drug monitoring

Therapeutic drug monitoring necessitates efficient, fast and reliable analytical methods validated by external quality control. We therefore devised an isocratic reversed-phase HPLC method with ultraviolet detection and optimised this to quantify mirtazapine, reboxetine, moclobemide, venlafaxine, O-desmethylvenlafaxine, paroxetine, fluvoxamine, fluoxetine, norfluoxetine, sertraline, citalopram, amitriptyline, nortriptyline, imipramine, desipramine, doxepin, nordoxepin, clomipramine, norclomipramine, trimipramine, mianserine, maprotiline, normaprotiline, amisulpride, clozapine, norclozapine, quetiapine, risperidone and 9-OH-risperidone in human serum. After solid-phase extraction of the drugs and metabolites, the chromatographic separation was achieved on a Nucleosil 100-Protect 1 column with acetonitrile-potassium dihydrogenphosphate buffer as mobile phase. The method was validated for therapeutic and toxic serum ranges. A linear relationship (r>0.998) was obtained between the concentration and the detector signal. Recoveries were between 75 and 99% for the drugs and metabolites. The accuracy of the quality control samples, expressed as percent recovery, ranged from 91 to 118%; intra- and inter-assay-relative standard deviations were 0.9-10.2% and 0.9-9.7%, respectively. Additional external quality control is carried out since 3 years. This method is applicable to rapidly and effectively analyze serum or plasma samples for therapeutic drug monitoring of about 30 antidepressants and atypical antipsychotics.     

Estimating domain orientation of two human antibody IgG4 chimeras by crystallohydrodynamics

A modified crystallohydrodynamic approach introduced in 2001 is applied to two human IgG4 constructs from mouse IgG1. The constructs were point mutants of the chimeric antibody molecule cB72.3(4): cB72.3(4A), devoid of inter-chain disulfide bridging, and cB72.3(4P), which has full inter-chain bridging. As before, the known crystallographic structures for the Fab and Fc domains were combined with the measured translational frictional ratios to obtain an estimate for the apparent time-averaged hydration of the domains and hence for that of the intact molecule. The original approach was modified with the hydrated dimensions of the domains being applied, rather than the anhydrous crystallographic dimensions, for assessing the inter-domain orientations using the algorithms HYDROSUB and SOLPRO. Both chimeric IgG4 molecules were found to have open, rather than compact, structures, in agreement with the previous study on wild-type human IgG4. The insertion of a frictionless connector between the domains was necessary, however, for representing the cB72.3(4A) chimera. It therefore appears that the inter-chain disulfide bonds act as physical constraints in the cB72.3(4P) chimera, forcing the antibody domains together and producing a less elongated structure than that of cB72.3(4A). The open structures produced for the two IgG4 chimeras showed similarity to those structures identified for murine IgG1 and IgG2a molecules through X-ray crystallography.Presented at the conference for Advances in Analytical Ultracentrifugation and Hydrodynamics, 8–11 June 2002, Grenoble, France     

TRiC/CCT cooperates with different upstream chaperones in the folding of distinct protein classes

The role in protein folding of the eukaryotic chaperonin TRiC/CCT is only partially understood. Here, we show that a group of WD40 beta-propeller proteins in the yeast cytosol interact transiently with TRiC upon synthesis and require the chaperonin to reach their native state. TRiC cooperates in the folding of these proteins with the ribosome-associated heat shock protein (Hsp)70 chaperones Ssb1/2p. In contrast, newly synthesized actin and tubulins, the major known client proteins of TRiC, are independent of Ssb1/2p and instead use the co-chaperone GimC/prefoldin for efficient transfer to the chaperonin. GimC can replace Ssb1/2p in the folding of WD40 substrates such as Cdc55p, but combined deletion of SSB and GIM genes results in loss of viability. These findings expand the substrate range of the eukaryotic chaperonin by a structurally defined class of proteins and demonstrate an essential role for upstream chaperones in TRiC-assisted folding.     

Structure-activity relationships of novel anti-malarial agents: part 5. N-(4-acylamino-3-benzoylphenyl)-[5-(4-nitrophenyl)-2-furyl]acrylic acid amides

We have developed the [5-(4-nitrophenyl)-2-furyl]acrylic acid substituted benzophenone 4g as a novel lead for anti-malarial agents. Here, we demonstrated that the acyl residue at the 2-amino group of the benzophenone core structure has to be a phenylacetic acid substructure substituted in its para-position with methyl or other substituents of similar size. The trifluoromethyl substituted derivative displayed an IC(50) of 47 nM against the multi-drug resistant Plasmodium falciparum strain Dd2.     

3D parallel coordinate systems--a new data visualization method in the context of microscopy-based multicolor tissue cytometry.

BACKGROUND: Presentation of multiple interactions is of vital importance in the new field of cytomics. Quantitative analysis of multi- and polychromatic stained cells in tissue will serve as a basis for medical diagnosis and prediction of disease in forthcoming years. A major problem associated with huge interdependent data sets is visualization. Therefore, alternative and easy-to-handle strategies for data visualization as well as data meta-evaluation (population analysis, cross-correlation, co-expression analysis) were developed. METHODS: To facilitate human comprehension of complex data, 3D parallel coordinate systems have been developed and used in automated microscopy-based multicolor tissue cytometry (MMTC). Frozen sections of human skin were stained using the combination anti-CD45-PE, anti-CD14-APC, and SytoxGreen as well as the appropriate single and double negative controls. Stained sections were analyzed using automated confocal laser microscopy and semiquantitative MMTC-analysis with TissueQuest 2.0. The 3D parallel coordinate plots are generated from semiquantitative immunofluorescent data of single cells. The 2D and 3D parallel coordinate plots were produced by further processing using the Matlab environment (Mathworks, USA). RESULTS: Current techniques in data visualization primarily utilize scattergrams, where two parameters are plotted against each other on linear or logarithmic scales. However, data evaluation on cartesian x/y-scattergrams is, in general, only of limited value in multiparameter analysis. Dot plots suffer from serious problems, and in particular, do not meet the requirements of polychromatic high-context tissue cytometry of millions of cells. The 3D parallel coordinate plot replaces the vast amount of scattergrams that are usually needed for the cross-correlation analysis. As a result, the scientist is able to perform the data meta-evaluation by using one single plot. On the basis of 2D parallel coordinate systems, a density isosurface is created for representing the event population in an intuitive way. CONCLUSIONS: The proposed method opens new possibilities to represent and explore multidimensional data in the perspective of cytomics and other life sciences, e.g., DNA chip array technology. Current protocols in immunofluorescence permit simultaneous staining of up to 17 markers. Showing the cross-correlation between these markers requires 136 scattergrams, which is a prohibitively high number. The improved data visualization method allows the observation of such complex patterns in only one 3D plot and could take advantage of the latest developments in 3D imaging.     

Hygienisation and Nutrient Conservation of Sewage Sludge or Cattle Manure by Lactic Acid Fermentation

Manure from animal farms and sewage sludge contain pathogens and opportunistic organisms in various concentrations depending on the health of the herds and human sources. Other than for the presence of pathogens, these waste substances are excellent nutrient sources and constitute a preferred organic fertilizer. However, because of the pathogens, the risks of infection of animals or humans increase with the indiscriminate use of manure, especially liquid manure or sludge, for agriculture. This potential problem can increase with the global connectedness of animal herds fed imported feed grown on fields fertilized with local manures. This paper describes a simple, easy-to-use, low-tech hygienization method which conserves nutrients and does not require large investments in infrastructure. The proposed method uses the microbiotic shift during mesophilic fermentation of cow manure or sewage sludge during which gram-negative bacteria, enterococci and yeasts were inactivated below the detection limit of 3 log₁₀ cfu/g while lactobacilli increased up to a thousand fold. Pathogens like Salmonella, Listeria monocytogenes, Staphylococcus aureus, E. coli EHEC O:157 and vegetative Clostridium perfringens were inactivated within 3 days of fermentation. In addition, ECBO-viruses and eggs of Ascaris suum were inactivated within 7 and 56 days, respectively. Compared to the mass lost through composting (15–57%), the loss of mass during fermentation (< 2.45%) is very low and provides strong economic and ecological benefits for this process. This method might be an acceptable hygienization method for developed as well as undeveloped countries, and could play a key role in public and animal health while safely closing the nutrient cycle by reducing the necessity of using energy-inefficient inorganic fertilizer for crop production.     

Modeled Changes in Potential Grassland Productivity and in Grass-Fed Ruminant Livestock Density in Europe over 1961–2010

About 25% of European livestock intake is based on permanent and sown grasslands. To fulfill rising demand for animal products, an intensification of livestock production may lead to an increased consumption of crop and compound feeds. In order to preserve an economically and environmentally sustainable agriculture, a more forage based livestock alimentation may be an advantage. However, besides management, grassland productivity is highly vulnerable to climate (i.e., temperature, precipitation, CO₂ concentration), and spatial information about European grassland productivity in response to climate change is scarce. The process-based vegetation model ORCHIDEE-GM, containing an explicit representation of grassland management (i.e., herbage mowing and grazing), is used here to estimate changes in potential productivity and potential grass-fed ruminant livestock density across European grasslands over the period 1961–2010. Here “potential grass-fed ruminant livestock density” denotes the maximum density of livestock that can be supported by grassland productivity in each 25 km × 25 km grid cell. In reality, livestock density could be higher than potential (e.g., if additional feed is supplied to animals) or lower (e.g., in response to economic factors, pedo-climatic and biotic conditions ignored by the model, or policy decisions that can for instance reduce livestock numbers). When compared to agricultural statistics (Eurostat and FAOstat), ORCHIDEE-GM gave a good reproduction of the regional gradients of annual grassland productivity and ruminant livestock density. The model however tends to systematically overestimate the absolute values of productivity in most regions, suggesting that most grid cells remain below their potential grassland productivity due to possible nutrient and biotic limitations on plant growth. When ORCHIDEE-GM was run for the period 1961–2010 with variable climate and rising CO₂, an increase of potential annual production (over 3%) per decade was found: 97% of this increase was attributed to the rise in CO₂, -3% to climate trends and 15% to trends in nitrogen fertilization and deposition. When compared with statistical data, ORCHIDEE-GM captures well the observed phase of climate-driven interannual variability in grassland production well, whereas the magnitude of the interannual variability in modeled productivity is larger than the statistical data. Regional grass-fed livestock numbers can be reproduced by ORCHIDEE-GM based on its simple assumptions and parameterization about productivity being the only limiting factor to define the sustainable number of animals per unit area. Causes for regional model-data misfits are discussed, including uncertainties in farming practices (e.g., nitrogen fertilizer application, and mowing and grazing intensity) and in ruminant diet composition, as well as uncertainties in the statistical data and in model parameter values.