Bioanalysis of new designer drugs

Since the late 1990s the illicit drug market has undergone considerable change: along with the traditional drugs of abuse that still dominate, more than 100 psychotropic substances designed to bypass controlled substances legislation have appeared and led to intoxications and fatalities. Starting from the huge class of phenylalkylamines, containing many subgroups, the spectrum of structures has grown from tryptamines, piperazines, phenylcyclohexyl derivates and pyrrolidinophenones to synthetic cannabinoids and the first synthetic cocaine. Due to the small prevalence and high number of unknown substances, the detection of new designer drugs is a challenge for clinical and forensic toxicologists. Standard screening procedures might fail because a recently discovered or yet unknown substance has not been incorporated in the library used. Nevertheless, many metabolism studies, case reports, screening methods and substance-profiling papers concentrating on single compounds have been published. This review provides an overview of the developed bioanalytical and analytical methods, the matrices used, sample-preparation procedures, concentration of analytes in case of intoxication and also gives a résumé of immunoassay experiences. Additionally, six screening methods for biological matrices with a larger spectrum of analytes are described in more detail.

In vivo analysis of uropod function during physiological T cell trafficking

Migrating lymphocytes acquire a polarized phenotype with a leading and a trailing edge, or uropod. Although in vitro experiments in cell lines or activated primary cell cultures have established that Rho-p160 coiled-coil kinase (ROCK)-myosin II-mediated uropod contractility is required for integrin de-adhesion on two-dimensional surfaces and nuclear propulsion through narrow pores in three-dimensional matrices, less is known about the role of these two events during the recirculation of primary, nonactivated lymphocytes. Using pharmacological antagonists of ROCK and myosin II, we report that inhibition of uropod contractility blocked integrin-independent mouse T cell migration through narrow, but not large, pores in vitro. T cell crawling on chemokine-coated endothelial cells under shear was severely impaired by ROCK inhibition, whereas transendothelial migration was only reduced through endothelial cells with high, but not low, barrier properties. Using three-dimensional thick-tissue imaging and dynamic two-photon microscopy of T cell motility in lymphoid tissue, we demonstrated a significant role for uropod contractility in intraluminal crawling and transendothelial migration through lymph node, but not bone marrow, endothelial cells. Finally, we demonstrated that ICAM-1, but not anatomical constraints or integrin-independent interactions, reduced parenchymal motility of inhibitor-treated T cells within the dense lymphoid microenvironment, thus assigning context-dependent roles for uropod contraction during lymphocyte recirculation.

Functionalisation of PLLA nanofiber scaffolds using a possible cooperative effect between collagen type I and BMP-2: impact on colonization and bone formation in vivo

The reconstruction of large bone defects after injury or tumor resection often requires the use of bone substitution. Artificial scaffolds based on synthetic biomaterials can overcome disadvantages of autologous bone grafts, like limited availability and donor side morbidity. Among them, scaffolds based on nanofibers offer great advantages. They mimic the extracellular matrix, can be used as a carrier for growth factors and allow the differentiation of human mesenchymal stem cells. Differentiation is triggered by a series of signaling processes, including integrin and bone morphogenetic protein (BMP), which act in a cooperative manner. The aim of this study was to analyze whether these processes can be remodeled in artificial poly-(l)-lactide acid (PLLA) based nanofiber scaffolds in vivo. Electrospun matrices composed of PLLA-collagen type I or BMP-2 incorporated PLLA-collagen type I were implanted in calvarial critical size defects in rats. Cranial CT-scans were taken 4, 8 and 12 weeks after implantation. Specimens obtained after euthanasia were processed for histology and immunostainings on osteocalcin, BMP-2 and Smad5. After implantation the scaffolds were inhomogeneously colonized and cells were only present in wrinkle- or channel-like structures. Ossification was detected only in focal areas of the scaffold. This was independent of whether BMP-2 was incorporated in the scaffold. However, cells that migrated into the scaffold showed an increased ratio of osteocalcin and Smad5 positive cells compared to empty defects. Furthermore, in case of BMP-2 incorporated PLLA-collagen type I scaffolds, 4 weeks after implantation approximately 40 % of the cells stained positive for BMP-2 indicating an autocrine process of the ingrown cells. These findings indicate that a cooperative effect between BMP-2 and collagen type I can be transferred to PLLA nanofibers and furthermore, that this effect is active in vivo. However, this had no effect on bone formation. The reason for this seems to be an unbalanced colonization of the scaffolds with cells, due to insufficient pore size.

Determination of free and total valproic acid in human plasma by capillary electrophoresis with contactless conductivity detection

A new approach for the determination of free and total valproic acid in small samples of 140 μL human plasma based on capillary electrophoresis with contactless conductivity detection is proposed. A dispersive liquid-liquid microextraction technique was employed in order to remove biological matrices prior to instrumental analysis. The free valproic acid was determined by isolating free valproic acid from protein-bound valproic acid by ultrafiltration under centrifugation of 100 μL sample. The filtrate was acidified to turn valproic acid into its protonated neutral form and then extracted. The determination of total valproic acid was carried out by acidifying 40 μL untreated plasma to release the protein-bound valproic acid prior to extraction. A solution consisting of 10 mM histidine, 10 mM 3-(N-morpholino)propanesulfonic acid and 10 μM hexadecyltrimethylammonium bromide of pH 6.5 was used as background electrolyte for the electrophoretic separation. The method showed good linearity in the range of 0.4-300 μg/mL with a correlation coefficient of 0.9996. The limit of detection was 0.08 μg/mL, and the reproducibility of the peak area was excellent (RSD=0.7-3.5%, n=3, for the concentration range from 1 to 150 μg/mL). The results for the free and total valproic acid concentration in human plasma were found to be comparable to those obtained with a standard immunoassay. The corresponding correlation coefficients were 0.9847 for free and 0.9521 for total valproic acid.

Anisotropically oriented electrospun matrices with an imprinted periodic micropattern: a new scaffold for engineered muscle constructs

Engineered muscle constructs provide a promising perspective on the regeneration or substitution of irreversibly damaged skeletal muscle. However, the highly ordered structure of native muscle tissue necessitates special consideration during scaffold development. Multiple approaches to the design of anisotropically structured substrates with grooved micropatterns or parallel-aligned fibres have previously been undertaken. In this study we report the guidance effect of a scaffold that combines both approaches, oriented fibres and a grooved topography. By electrospinning onto a topographically structured collector, matrices of parallel-oriented poly(ε-caprolactone) fibres with an imprinted wavy topography of 90 µm periodicity were produced. Matrices of randomly oriented fibres or parallel-oriented fibres without micropatterns served as controls. As previously shown, un-patterned, parallel-oriented substrates induced myotube orientation that is parallel to fibre direction. Interestingly, pattern addition induced an orientation of myotubes at an angle of 24° (statistical median) relative to fibre orientation. Myotube length was significantly increased on aligned micropatterned substrates in comparison to that on aligned substrates without pattern (436 ± 245 µm versus 365 ± 212 µm; p < 0.05). We report an innovative, yet simple, design to produce micropatterned electrospun scaffolds that induce an unexpected myotube orientation and an increase in myotube length.

Enzymatic formation of modular cell-instructive fibrin analogs for tissue engineering

The molecular engineering of cell-instructive artificial extracellular matrices is a powerful means to control cell behavior and enable complex processes of tissue formation and regeneration. This work reports on a novel method to produce such smart biomaterials by recapitulating the crosslinking chemistry and the biomolecular characteristics of the biopolymer fibrin in a synthetic analog. We use activated coagulation transglutaminase factor XIIIa for site-specific coupling of cell adhesion ligands and engineered growth factor proteins to multiarm poly(ethylene glycol) macromers that simultaneously form proteolytically sensitive hydrogel networks in the same enzyme-catalyzed reaction. Growth factor proteins are quantitatively incorporated and released upon cell-derived proteolytic degradation of the gels. Primary stromal cells can invade and proteolytically remodel these networks both in an in vitro and in vivo setting. The synthetic ease and potential to engineer their physicochemical and bioactive characteristics makes these hybrid networks true alternatives for fibrin as provisional drug delivery platforms in tissue engineering.

Data-driven estimates of the number of clusters in multivariate time series

An important problem in unsupervised data clustering is how to determine the number of clusters. Here we investigate how this can be achieved in an automated way by using interrelation matrices of multivariate time series. Two nonparametric and purely data driven algorithms are expounded and compared. The first exploits the eigenvalue spectra of surrogate data, while the second employs the eigenvector components of the interrelation matrix. Compared to the first algorithm, the second approach is computationally faster and not limited to linear interrelation measures.

Multi-parametric classification of Alzheimer's disease and mild cognitive impairment: the impact of quantitative magnetization transfer MR imaging

Multi-parametric and quantitative magnetic resonance imaging (MRI) techniques have come into the focus of interest, both as a research and diagnostic modality for the evaluation of patients suffering from mild cognitive decline and overt dementia. In this study we address the question, if disease related quantitative magnetization transfer effects (qMT) within the intra- and extracellular matrices of the hippocampus may aid in the differentiation between clinically diagnosed patients with Alzheimer disease (AD), patients with mild cognitive impairment (MCI) and healthy controls. We evaluated 22 patients with AD (n=12) and MCI (n=10) and 22 healthy elderly (n=12) and younger (n=10) controls with multi-parametric MRI. Neuropsychological testing was performed in patients and elderly controls (n=34). In order to quantify the qMT effects, the absorption spectrum was sampled at relevant off-resonance frequencies. The qMT-parameters were calculated according to a two-pool spin-bath model including the T1- and T2 relaxation parameters of the free pool, determined in separate experiments. Histograms (fixed bin-size) of the normalized qMT-parameter values (z-scores) within the anterior and posterior hippocampus (hippocampal head and body) were subjected to a fuzzy-c-means classification algorithm with downstreamed PCA projection. The within-cluster sums of point-to-centroid distances were used to examine the effects of qMT- and diffusion anisotropy parameters on the discrimination of healthy volunteers, patients with Alzheimer and MCIs. The qMT-parameters T2(r) (T2 of the restricted pool) and F (fractional pool size) differentiated between the three groups (control, MCI and AD) in the anterior hippocampus. In our cohort, the MT ratio, as proposed in previous reports, did not differentiate between MCI and AD or healthy controls and MCI, but between healthy controls and AD.

Fenofibrate metabolism in the cynomolgus monkey using ultraperformance liquid chromatography-quadrupole time-of-flight mass spectrometry-based metabolomics

Fenofibrate, widely used for the treatment of dyslipidemia, activates the nuclear receptor, peroxisome proliferator-activated receptor alpha. However, liver toxicity, including liver cancer, occurs in rodents treated with fibrate drugs. Marked species differences occur in response to fibrate drugs, especially between rodents and humans, the latter of which are resistant to fibrate-induced cancer. Fenofibrate metabolism, which also shows species differences, has not been fully determined in humans and surrogate primates. In the present study, the metabolism of fenofibrate was investigated in cynomolgus monkeys by ultraperformance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOFMS)-based metabolomics. Urine samples were collected before and after oral doses of fenofibrate. The samples were analyzed in both positive-ion and negative-ion modes by UPLC-QTOFMS, and after data deconvolution, the resulting data matrices were subjected to multivariate data analysis. Pattern recognition was performed on the retention time, mass/charge ratio, and other metabolite-related variables. Synthesized or purchased authentic compounds were used for metabolite identification and structure elucidation by liquid chromatographytandem mass spectrometry. Several metabolites were identified, including fenofibric acid, reduced fenofibric acid, fenofibric acid ester glucuronide, reduced fenofibric acid ester glucuronide, and compound X. Another two metabolites (compound B and compound AR), not previously reported in other species, were characterized in cynomolgus monkeys. More importantly, previously unknown metabolites, fenofibric acid taurine conjugate and reduced fenofibric acid taurine conjugate were identified, revealing a previously unrecognized conjugation pathway for fenofibrate.

Comparative study of four retentive anchor systems for implant supported overdentures--retention force changes

OBJECTIVES: Wear of attachments leads to a loss of retention and potentially reduces the function of complete dentures. This study evaluated the retention force changes of different prefabricated attachment systems for implant-supported overdentures to estimate the wear constancy and applicability in clinical practice. METHODS: Four prefabricated attachment systems were tested [Group SG: retentive ball attachment (Straumann, Switzerland) with gold matrix, Group ST: retentive ball attachment (Straumann, Switzerland) with titanium spring matrix, Group IB: UNOR i-Ball with Ecco matrix (UNOR, Switzerland) and Group IMZ: IMZ-TwinPlus ball attachment with gold matrix (DENTSPLY Friadent, Germany)]. Ten samples of each system were subjected to 10,000 insertion-separation cycles. RESULTS: Results showed that all types of attachments showed wear, which led to a loss of retention force after an initial increase at the beginning of the wear simulation. Attachments with a plastic retention insert or gold matrices underwent the smallest changes in retention force. The titanium spring system showed the largest changes in retention force and a greater variation between the different cycles and specimen. This behaviour is probably caused by a large fitting tolerance of the titanium spring. CONCLUSIONS: Attachment systems which possess a male and female component of different material composition are preferable. They show smaller changes in the retention force. For retention force increase and wear compensation, an attachment system should be adjustable.

Parameter-free extraction of the functional network topology from intracranial EEG recordings: a time-resolved study of graph properties in focal onset seizures

Rationale: Focal onset epileptic seizures are due to abnormal interactions between distributed brain areas. By estimating the cross-correlation matrix of multi-site intra-cerebral EEG recordings (iEEG), one can quantify these interactions. To assess the topology of the underlying functional network, the binary connectivity matrix has to be derived from the cross-correlation matrix by use of a threshold. Classically, a unique threshold is used that constrains the topology [1]. Our method aims to set the threshold in a data-driven way by separating genuine from random cross-correlation. We compare our approach to the fixed threshold method and study the dynamics of the functional topology. Methods: We investigate the iEEG of patients suffering from focal onset seizures who underwent evaluation for the possibility of surgery. The equal-time cross-correlation matrices are evaluated using a sliding time window. We then compare 3 approaches assessing the corresponding binary networks. For each time window: * Our parameter-free method derives from the cross-correlation strength matrix (CCS)[2]. It aims at disentangling genuine from random correlations (due to finite length and varying frequency content of the signals). In practice, a threshold is evaluated for each pair of channels independently, in a data-driven way. * The fixed mean degree (FMD) uses a unique threshold on the whole connectivity matrix so as to ensure a user defined mean degree. * The varying mean degree (VMD) uses the mean degree of the CCS network to set a unique threshold for the entire connectivity matrix. * Finally, the connectivity (c), connectedness (given by k, the number of disconnected sub-networks), mean global and local efficiencies (Eg, El, resp.) are computed from FMD, CCS, VMD, and their corresponding random and lattice networks. Results: Compared to FMD and VMD, CCS networks present: *topologies that are different in terms of c, k, Eg and El. *from the pre-ictal to the ictal and then post-ictal period, topological features time courses that are more stable within a period, and more contrasted from one period to the next. For CCS, pre-ictal connectivity is low, increases to a high level during the seizure, then decreases at offset. k shows a ‘‘U-curve’’ underlining the synchronization of all electrodes during the seizure. Eg and El time courses fluctuate between the corresponding random and lattice networks values in a reproducible manner. Conclusions: The definition of a data-driven threshold provides new insights into the topology of the epileptic functional networks.

On the use of textural features for writer identification in old handwritten music scores

Writer identification consists in determining the writer of a piece of handwriting from a set of writers. In this paper we present a system for writer identification in old handwritten music scores which uses only music notation to determine the author. The steps of the proposed system are the following. First of all, the music sheet is preprocessed for obtaining a music score without the staff lines. Afterwards, four different methods for generating texture images from music symbols are applied. Every approach uses a different spatial variation when combining the music symbols to generate the textures. Finally, Gabor filters and Grey-scale Co-ocurrence matrices are used to obtain the features. The classification is performed using a k-NN classifier based on Euclidean distance. The proposed method has been tested on a database of old music scores from the 17th to 19th centuries, achieving encouraging identification rates.

A computer-aided diagnostic system to characterize CT focal liver lesions: design and optimization of a neural network classifier

In this paper, a computer-aided diagnostic (CAD) system for the classification of hepatic lesions from computed tomography (CT) images is presented. Regions of interest (ROIs) taken from nonenhanced CT images of normal liver, hepatic cysts, hemangiomas, and hepatocellular carcinomas have been used as input to the system. The proposed system consists of two modules: the feature extraction and the classification modules. The feature extraction module calculates the average gray level and 48 texture characteristics, which are derived from the spatial gray-level co-occurrence matrices, obtained from the ROIs. The classifier module consists of three sequentially placed feed-forward neural networks (NNs). The first NN classifies into normal or pathological liver regions. The pathological liver regions are characterized by the second NN as cyst or "other disease." The third NN classifies "other disease" into hemangioma or hepatocellular carcinoma. Three feature selection techniques have been applied to each individual NN: the sequential forward selection, the sequential floating forward selection, and a genetic algorithm for feature selection. The comparative study of the above dimensionality reduction methods shows that genetic algorithms result in lower dimension feature vectors and improved classification performance.