Michael Adsetts Edberg Hansen

Ecology, Inhibitory Activity, and Morphogenesis of a Marine Antagonistic Bacterium Belonging to the Roseobacter Clade

ABSTRACT Roseobacter strain 27-4 has been isolated from a turbot larval rearing unit and is capable of reducing mortality in turbot egg yolk sac larvae. Here, we demonstrate that the supernatant of Roseobacter 27-4 is lethal to the larval pathogens Vibrio anguillarum and Vibrio splendidus in a buffer system and inhibited their growth in marine broth. Liquid chromatography (LC) with both UV spectral detection and high-resolution mass spectrometry (HR-MS) identified the known antibacterial compound thiotropocin or its closely related precursor tropodithietic acid in the bioactive fractions. Antibacterial activity correlated with the appearance of a brownish pigment and was only formed in marine broth under static growth conditions. A thick biofilm of multicellular star-shaped aggregated cells formed at the air-liquid interface under static growth conditions. Here, the bioactive compound was the base peak in the LC-UV chromatograms of the extracts where it constituted 15% of the total peak area. Aerated conditions results in 10-fold-higher cell yield, however, cultures were nonpigmented, did not produce antibacterial activity, and grew as single cells. Production of antibacterial compounds may be quorum regulated, and we identified the acylated homoserine lactone (3-hydroxy-decanoyl homoserine lactone) from cultures of Roseobacter 27-4 using LC-HR-MS. The signal molecule was primarily detected in stagnant cultures. Roseobacter 27-4 grew between 10 and 30°C but died rapidly at 37°C. Also, the antibacterial compounds was sensitive to heat and was inactivated at 37°C in less than 2 days and at 25°C in 8 days. Using Roseobacter 27-4 as a probiotic culture will require that is be established in stagnant or adhered conditions and, due to the temperature sensitivity of the active compound, constant production must be ensured.

Real-Time Tracking of Moving Objects with an Active Camera

This article is concerned with the design and implementation of a system for real-time monocular tracking of a moving object using the two degrees of freedom of a camera platform. Figure-ground segregation is based on motion without making anya prioriassumptions about the object form. Using only the first spatiotemporal image derivatives, substraction of the normal optical flow induced by camera motion yields the object image motion. Closed-loop control is achieved by combining a stationary Kalman estimator with an optimal Linear Quadratic Regulator. The implementation on a pipeline architecture enables a servo rate of 25 Hz. We study the effects of time-recursive filtering and fixed-point arithmetic in image processing and we test the performance of the control algorithm on controlled motion of objects.

Effect of Anaplerotic Fluxes and Amino Acid Availability on Hepatic Lipoapoptosis

To identify metabolic pathways involved in hepatic lipoapoptosis, metabolic flux analysis using [U-13C5]glutamine as an isotopic tracer was applied to quantify phenotypic changes in H4IIEC3 hepatoma cells treated with either palmitate alone (PA-cells) or both palmitate and oleate in combination (PA/OA-cells). Our results indicate that palmitate inhibited glycolysis and lactate dehydrogenase fluxes while activating citric acid cycle (CAC) flux and glutamine uptake. This decoupling of glycolysis and CAC fluxes occurred during the period following palmitate exposure but preceding the onset of apoptosis. Oleate co-treatment restored most fluxes to their control levels, resulting in steatotic lipid accumulation while preventing apoptosis. In addition, palmitate strongly increased the cytosolic NAD+/NADH ratio, whereas oleate co-treatment had the opposite effect on cellular redox. We next examined the influence of amino acids on these free fatty acid-induced phenotypic changes. Increased medium amino acids enhanced reactive oxygen species (ROS) generation and apoptosis in PA-cells but not in PA/OA-cells. Overloading the medium with non-essential amino acids induced apoptosis, but essential amino acid overloading partially ameliorated apoptosis. Glutamate was the most effective single amino acid in promoting ROS. Amino acid overloading also increased cellular palmitoyl-ceramide; however, ceramide synthesis inhibitors had no effect on measurable indicators of apoptosis. Our results indicate that free fatty acid-induced ROS generation and apoptosis are accompanied by the decoupling of glycolysis and CAC fluxes leading to abnormal cytosolic redox states. Amino acids play a modulatory role in these processes via a mechanism that does not involve ceramide accumulation.

Production of mycotoxins by Aspergillus lentulus and other medically important and closely related species in section Fumigati

The production of mycotoxins and other secondary metabolites have been studied by LC-DAD-MS from six species in Aspergillus section Fumigati. This includes the three new species Aspergillus lentulus, A. novofumigatus and A. fumigatiaffinis as well as A. fumigatus, Neosartoria fisheri and N. pseudofisheri. A major finding was detection of gliotoxin from N. pseudofisheri, a species not previously reported to produce this mycotoxin. Gliotoxin was also detected from A. fumigatus together with fumagillin, fumigaclavine C, fumitremorgin C, fumiquinazolines, trypacidin, methyl-sulochrin, TR-2, verruculogen, helvolic acid and pyripyropenes. Major compounds from A. lentulus were cyclopiazonic acid, terrein, neosartorin, auranthine and pyripyropenes A, E and O. Thus in the present study A. fumigatus and A. lentulus did not produce any of the same metabolites except for pyripyropenes. The fact that A. lentulus apparently does not produce gliotoxin supports the idea that other compounds than gliotoxin might play an important role in the effective invasiveness of A. lentulus. An overall comparison of secondary metabolite production by strains of the six species was achieved by analysis of fungal extracts by direct injection mass spectrometry and cluster analysis. Separate groupings were seen for all the six species even though only one isolate was included in this study for the two species A. novofumigatus and A. fumigatiaffinis.

Identification of Novel Compounds Inhibiting Chikungunya Virus-Induced Cell Death by High Throughput Screening of a Kinase Inhibitor Library

Chikungunya virus (CHIKV) is a mosquito-borne arthrogenic alphavirus that causes acute febrile illness in humans accompanied by joint pains and in many cases, persistent arthralgia lasting weeks to years. The re-emergence of CHIKV has resulted in numerous outbreaks in the eastern hemisphere, and threatens to expand in the foreseeable future. Unfortunately, no effective treatment is currently available. The present study reports the use of resazurin in a cell-based high-throughput assay, and an image-based high-content assay to identify and characterize inhibitors of CHIKV-infection in vitro. CHIKV is a highly cytopathic virus that rapidly kills infected cells. Thus, cell viability of HuH-7 cells infected with CHIKV in the presence of compounds was determined by measuring metabolic reduction of resazurin to identify inhibitors of CHIKV-associated cell death. A kinase inhibitor library of 4,000 compounds was screened against CHIKV infection of HuH-7 cells using the resazurin reduction assay, and the cell toxicity was also measured in non-infected cells. Seventy-two compounds showing ≥50% inhibition property against CHIKV at 10 µM were selected as primary hits. Four compounds having a benzofuran core scaffold (CND0335, CND0364, CND0366 and CND0415), one pyrrolopyridine (CND0545) and one thiazol-carboxamide (CND3514) inhibited CHIKV-associated cell death in a dose-dependent manner, with EC50 values between 2.2 µM and 7.1 µM. Based on image analysis, these 6 hit compounds did not inhibit CHIKV replication in the host cell. However, CHIKV-infected cells manifested less prominent apoptotic blebs typical of CHIKV cytopathic effect compared with the control infection. Moreover, treatment with these compounds reduced viral titers in the medium of CHIKV-infected cells by up to 100-fold. In conclusion, this cell-based high-throughput screening assay using resazurin, combined with the image-based high content assay approach identified compounds against CHIKV having a novel antiviral activity - inhibition of virus-induced CPE - likely by targeting kinases involved in apoptosis.

Automated and Unbiased Image Analyses as Tools in Phenotypic Classification of Small-Spored Alternaria spp.

ABSTRACT For more than 25 years, controversy has surrounded the characterization and differentiation of small-spored Alternaria spp. And, therefore, the application of names of several species that are involved in the pathology of diseases related to host-specific toxin production. The name A. alternata often has been broadly applied to various morphologically and chemically distinct groups of isolates from different hosts. The purpose of this study was to develop and evaluate automated and unbiased image analysis systems that will analyze different phenotypic characters and facilitate testing and application of the morphological species concept in Alternaria taxonomy. Host-specific toxin-producing Alternaria isolates assigned to five morpho-species were compared with representative isolates of morphologically distinct A. alternata. Combined results of growth rates at different temperatures, colony morphology, and metabolite profiles were found to be useful in characterization and differentiation of small-spored Alternaria spp. when standardized conditions are applied and representative isolates employed for comparison.

An image-based algorithm for precise and accurate high throughput assessment of drug activity against the human parasite Trypanosoma cruzi.

We present a customized high content (image-based) and high throughput screening algorithm for the quantification of Trypanosoma cruzi infection in host cells. Based solely on DNA staining and single-channel images, the algorithm precisely segments and identifies the nuclei and cytoplasm of mammalian host cells as well as the intracellular parasites infecting the cells. The algorithm outputs statistical parameters including the total number of cells, number of infected cells and the total number of parasites per image, the average number of parasites per infected cell, and the infection ratio (defined as the number of infected cells divided by the total number of cells). Accurate and precise estimation of these parameters allow for both quantification of compound activity against parasites, as well as the compound cytotoxicity, thus eliminating the need for an additional toxicity-assay, hereby reducing screening costs significantly. We validate the performance of the algorithm using two known drugs against T.cruzi: Benznidazole and Nifurtimox. Also, we have checked the performance of the cell detection with manual inspection of the images. Finally, from the titration of the two compounds, we confirm that the algorithm provides the expected half maximal effective concentration (EC50) of the anti-T. cruzi activity.

Biologically inspired calibration-free adaptive saccade control of a binocular camera-head

Abstract. This paper describes fast and accurate calibration-free adaptive saccade control of a four-degrees-of-freedom binocular camera-head by means of Dynamic Cell Structures (DCS). The approach has been inspired by biology because primates face a similar problem and there is strong evidence that they have solved it in a similar way, i.e., by error feedback learning of an inverse model. Yet the emphasis of this article is not on detailed biological modeling but on how incremental growth of our artificial neural network model up to a prespecified precision results in very small networks suitable for real-time saccade control. Error-feedback-based training of this network proceeds in two phases. In the first phase we use a crude model of the cameras and the kinematics of the head to learn the topology of the input manifold together with a rough approximation of the control function off-line. In contrast to, for example, Kohonen-type adaptation rules, the distribution of neural units minimizes the control error and does not merely mimic the input probability density. In the second phase, the operating phase, the linear output units of the network continue to adapt on-line. Besides our TRC binocular camera-head we use a Datacube image processing system and a Stäubli R90 robot arm for automated training in the second phase. It will be demonstrated that the controller successfully corrects errors in the model and rapidly adapts to changing parameters.

High Content Screening of a Kinase-Focused Library Reveals Compounds Broadly-Active against Dengue Viruses

Dengue virus is a mosquito-borne flavivirus that has a large impact in global health. It is considered as one of the medically important arboviruses, and developing a preventive or therapeutic solution remains a top priority in the medical and scientific community. Drug discovery programs for potential dengue antivirals have increased dramatically over the last decade, largely in part to the introduction of high-throughput assays. In this study, we have developed an image-based dengue high-throughput/high-content assay (HT/HCA) using an innovative computer vision approach to screen a kinase-focused library for anti-dengue compounds. Using this dengue HT/HCA, we identified a group of compounds with a 4-(1-aminoethyl)-N-methylthiazol-2-amine as a common core structure that inhibits dengue viral infection in a human liver-derived cell line (Huh-7.5 cells). Compounds CND1201, CND1203 and CND1243 exhibited strong antiviral activities against all four dengue serotypes. Plaque reduction and time-of-addition assays suggests that these compounds interfere with the late stage of viral infection cycle. These findings demonstrate that our image-based dengue HT/HCA is a reliable tool that can be used to screen various chemical libraries for potential dengue antiviral candidates.

A comparison of dimension reduction methods with application to multi-spectral images of sand used in concrete

This paper presents a comparison of dimension reduction methods based on a novel machine vision application for estimating moisture content in sand used to make concrete. For the application in question it is very important to know the moisture content of the sand so as to ensure good-quality concrete. In order to achieve a continuous in-line approach for the concrete mixing, digital image analysis is used. Multi-spectral images, consisting of nine spectral bands in the visible and near infrared (NIR) range, were acquired. Each image consists of approximately 9 million pixels. Five different sand types were examined with 20–60 images for each type. To reduce the amount of data, features were extracted from the multi-spectral images; the features were summary statistics on single bands and pairs of bands as well as morphological summaries. The number of features (2,016) is high in relation to the number of observations and, therefore, dimension reductive methods are needed. Furthermore, speed, which is an important consideration, is aided by the use of a small number of variables. On top of that, fewer dimensions tend to give more robust results. Two traditional statistical methods for dimension reduction (forward selection and principal components) combined with ordinary least squares and one sophisticated chemometrics algorithm (genetic algorithm-partial least squares) are compared to the recently proposed least angle regression-elastic net (LARS-EN) model selection method.