A high-precision wound healing assay based on photosensitized culture substrates.

Quantitative assessment of cell migration in vitro is often required in fundamental and applied research from different biomedical areas including wound repair, tumor metastasis or developmental biology. A collection of assays has been established throughout the years like the most widely used scratch assay or the so-called barrier assay. It is the principle of these assays to introduce a lesion into an otherwise confluent monolayer in order to study the migration of cells from the periphery into this artificial wound and determine the migration rate from the time necessary for wound closure.

Development of Specialized Microelectrode Arrays with Local Electroporation Functionality.

When a cell or tissue is exposed to a pulsed electric field (100-1000 V/cm), the cellular membrane permeabilizes for biomolecules that cannot pass an intact cellular membrane. During this electropermeabilization (EP), plasmid deoxyribonucleic acid sequences encoding therapeutic or regulatory genes can enter the cell, which is called gene electrotransfer (GET). GET using micro-/nano technology provides higher spatial resolution and operates with lower voltage amplitudes compared to conventional bulk EP.

Label-free impedance measurements to unravel biomolecular interactions involved in G protein-coupled receptor signaling.

G protein-coupled receptors (GPCRs) are among the most heavily addressed drug targets in medicinal chemistry and pharmacology. The screening for new agonists or antagonists has been largely based on genetically engineered cells overexpressing the receptor to study binding of ligands directly or via intracellular signaling events downstream of receptor activation. These approaches are often invasive in nature, need to be conducted as endpoint assays, require isotope- or fluorophore-labeling and significant genetic manipulation.

Asymmetric Synthesis of ,-Heterobicyclic Octanes and (-)-Geissman-Waiss Lactone.

A short, asymmetric synthesis of tetrahydro-2-furo[3,2-]pyrrole derivatives and (-)-Geissman-Waiss lactone starting from -cyclohexadiene epoxide is described. Pivotal transformations in the developed synthetic procedure include asymmetric epoxide ring opening to install the requisite 1,5 stereocenters and oxidative lactonization/lactamization sequences. This route provides a streamlined synthetic pathway toward necine alkaloids.

PTER-assay: Combined Impedimetric Detection of Permeability (P) and Resistance (TER) of Barrier-Forming Cell Layers.

Epithelial and endothelial barrier function is typically studied in vitro by growing the cells of interest on permeable supports that are sandwiched between two fluid compartments. This setup mimics the physiological situation with the cell layer as the diffusion barrier at the interface between two chemically distinct fluids. Routinely, the barrier function is quantitatively described by two key parameters: (i) the transepithelial or transendothelial electrical resistance (TER) as a measure of the permeability for small inorganic ions and (ii) the permeability coefficient (P) as a descriptor of the permeability for molecular tracers.

Label-free profiling of cell dynamics: A sequence of impedance-based assays to estimate tumor cell invasiveness in vitro.

Dynamic properties of cancer cells, most notably their ability to migrate, have been correlated successfully with their invasive nature in vivo. To establish a stronger experimental basis for such a correlation we subjected five different cancer cell lines of well-defined metastatic potential to a sequence of three independent assays reporting on three different aspects of cell dynamics, namely (1) the kinetics of cell spreading, (2) cell shape fluctuations, and (3) cell migration. The sequentially applied assays correspond to different measuring modes of the well-established ECIS technique that is based on non-invasive and label-free impedance readings of planar gold-film electrodes that serve as the growth substrate for the cells under study.

Synthesis of 2,4,6-Trisubstituted Pyridines by Oxidative Eosin Y Photoredox Catalysis.

Eosin Y, an organic dye, was activated as a photoredox catalyst in the presence of molecular oxygen using visible light and, when it was used in the reaction of aryl ketones and benzyl amines, afforded good yields (52-87%) of 2,4,6-triarylpyridines (21 examples) at ambient temperature. The aryl groups at the 2- and 6-positions are derived from ketones, while benzyl amine plays the dual role of providing an aryl functionality at the 4-position of pyridine as well as being a nitrogen donor.

Draft Genome Sequence of Pyrodictium occultum PL19T, a Marine Hyperthermophilic Species of Archaea That Grows Optimally at 105°C.

We report here the draft genome sequence of Pyrodictium occultum PL19(T), a marine hyperthermophilic archaeon. The genome provides insights into molecular and cellular adaptation mechanisms to life in extreme environments and the evolution of early organisms on Earth.

Evaluation of a Push-Pull Approach for Aedes aegypti (L.) Using a Novel Dispensing System for Spatial Repellents in the Laboratory and in a Semi-Field Environment.

The increase in insecticide resistant mosquito populations necessitates the exploration of novel vector control intervention measures. Push-pull strategies for insect control have been successful when used in integrated crop pest management. Through the combinatory use of deterring and attracting stimuli, the abundance of insect pests can be changed in a given area.

Composite collective decision-making.

Individual animals are adept at making decisions and have cognitive abilities, such as memory, which allow them to hone their decisions. Social animals can also share information. This allows social animals to make adaptive group-level decisions.

Enantioselective synthesis of 4-heterosubstituted cyclopentenones.

Racemic 4-hydroxycyclopentenone, readily derived from furfuryl alcohol, can be transformed via its O-Boc derivative to 4-acyloxy, 4-aryloxy-, 4-amino-, or 4-thio-substituted cyclopentenones with high enantioselectivity by palladium-catalyzed kinetic resolution via nucleophilic allylic substitutions. Applying this methodology, a short formal synthesis of ent-noraristeromycin was readily accomplished.

Laboratory evaluation techniques to investigate the spatial potential of repellents for push and pull mosquito control systems.

A protocol has been developed for the indoor evaluation of candidate spatial repellents intended for use in push and pull systems. Single treatments (catnip oil, 1-methylpiperazine, and homopiperazine) and a mixture of catnip oil and homopiperazine were tested with yellow-fever mosquitoes (Aedes aegypti) in Y-tube olfactometers to determine 1) if these compounds inhibited mosquito host-seeking at short distances and 2) if results obtained in olfactometer tests can be correlated with a larger scale set-up, that is, a room test. All test materials significantly decreased the ability of mosquitoes to find host odors (from a human finger) by up to 96.

Macroporous silicon chips for laterally resolved, multi-parametric analysis of epithelial barrier function.

This study describes a novel assay to visualize the macromolecular permeability of epithelial and endothelial cell layers with subcellular lateral resolution. Defects within the cell layer and details about the permeation route of the migrating solute are revealed. The assay is based on silicon chips with densely packed, highly ordered, dead-ended pores of μm-diameters on one side.

Impedance analysis of adherent cells after in situ electroporation: non-invasive monitoring during intracellular manipulations.

In this study adherent animal cells were grown to confluence on circular gold-film electrodes of 250 μm diameter that had been deposited on the surface of a regular culture dish. The impedance of the cell-covered electrode was measured at designated frequencies to monitor the behavior of the cells with time. This approach is referred to as electric cell-substrate impedance sensing or short ECIS in the literature.

A novel test cage with an air ventilation system as an alternative to conventional cages for the efficacy testing of mosquito repellents.

We have developed a novel test cage and improved method for the evaluation of mosquito repellents. The method is compatible with the United States Environmental Protection Agency, 2000 draft OPPTS 810.3700 Product Performance Test Guidelines for Testing of Insect Repellents.

Reduction in ribosomal protein synthesis is sufficient to explain major effects on ribosome production after short-term TOR inactivation in Saccharomyces cerevisiae.

Ribosome synthesis depends on nutrient availability, sensed by the target of rapamycin (TOR) signaling pathway in eukaryotes. TOR inactivation affects ribosome biogenesis at the level of rRNA gene transcription, expression of ribosomal proteins (r-proteins) and biogenesis factors, preribosome processing, and transport. Here, we demonstrate that upon TOR inactivation, levels of newly synthesized ribosomal subunits drop drastically before the integrity of the RNA polymerase I apparatus is severely impaired but in good correlation with a sharp decrease in r-protein production.

Archaea in artificial environments: their presence in global spacecraft clean rooms and impact on planetary protection.

The presence and role of Archaea in artificial, human-controlled environments is still unclear. The search for Archaea has been focused on natural biotopes where they have been found in overwhelming numbers, and with amazing properties. However, they are considered as one of the major group of microorganisms that might be able to survive a space flight, or even to thrive on other planets.

Methanocaldococcus villosus sp. nov., a heavily flagellated archaeon that adheres to surfaces and forms cell-cell contacts.

A novel chemolithoautotrophic, hyperthermophilic methanogen was isolated from a submarine hydrothermal system at the Kolbeinsey Ridge, north of Iceland. Based on its 16S rRNA gene sequence, the strain belongs to the order Methanococcales within the genus Methanocaldococcus, with approximately 95 % sequence similarity to Methanocaldococcus jannaschii as its closest relative. Cells of the novel organism stained Gram-negative and appeared as regular to irregular cocci possessing more than 50 polar flagella.

Cultivation of anaerobic and facultatively anaerobic bacteria from spacecraft-associated clean rooms.

In the course of this biodiversity study, the cultivable microbial community of European spacecraft-associated clean rooms and the Herschel Space Observatory located therein were analyzed during routine assembly operations. Here, we focused on microorganisms capable of growing without oxygen. Anaerobes play a significant role in planetary protection considerations since extraterrestrial environments like Mars probably do not provide enough oxygen for fully aerobic microbial growth.

Physarum polymalic acid hydrolase: Recombinant expression and enzyme activation.

As a platform for syntheses of nanoconjugates in antitumor drug delivery, polymalic acid together with its tailoring specific exohydrolase is purified from plasmodium cultures of the slime mold Physarum polycephalum, a member of the phylum myxomycota. Polymalic acid hydrolase is expressed in an inactive form that functions as a molecular adapter for polymalic acid trafficking within the plasmodium and is activated only during secretion. Activation follows specific protein tyrosine phosphorylation and dissociation from plasma membranes.

Actively transcribed rRNA genes in S. cerevisiae are organized in a specialized chromatin associated with the high-mobility group protein Hmo1 and are largely devoid of histone molecules.

Synthesis of ribosomal RNAs (rRNAs) is the major transcriptional event in proliferating cells. In eukaryotes, ribosomal DNA (rDNA) is transcribed by RNA polymerase I from a multicopy locus coexisting in at least two different chromatin states. This heterogeneity of rDNA chromatin has been an obstacle to defining its molecular composition.

Transcription factor E is a part of transcription elongation complexes.

A homologue of the N-terminal domain of the alpha subunit of the general eukaryotic transcription factor TFE is encoded in the genomes of all sequenced archaea, but the position of archaeal TFE in transcription complexes has not yet been defined. We show here that TFE binds nonspecifically to single-stranded DNA, and photochemical cross-linking revealed TFE binding to a preformed open transcription bubble. In preinitiation complexes, the N-terminal part of TFE containing a winged helix-turn-helix motif is cross-linked specifically to DNA of the nontemplate DNA strand at positions -9 and -11.

Construction of adenovirus type 5 early region 1 and 4 virus mutants.

This chapter describes a novel strategy that simplifies the generation and production of adenovirus type 5 (Ad5) mutants carrying defined mutations in early transcription units 1 (E1) and 4 (E4). The strategy involves three recombinant plasmids containing E1 (pE1-1235), E4 (pE4-1155), or the wild-type genome that lacks a portion of E3 (pH5pg4100). To generate recombinant viruses, mutations are first introduced into pE1- and/or pE4-transfer plasmids by site-directed mutagenesis.

Association of the metabolic syndrome with early coronary disease in families with frequent myocardial infarction.

This study examined the extent to which the metabolic syndrome (MS) augments the risk for major cardiovascular events in healthy patients with a strong genetic background for coronary artery disease (CAD). In a prospective cohort study, we examined 1,316 patients without previously diagnosed CAD or diabetes mellitus. Patients were participants of the Regensburg Myocardial Infarction Family Study, in which > or = 2 family members had severe CAD and 1 had myocardial infarction (MI) at < 60 years of age.

Pir proteins of Saccharomyces cerevisiae are attached to beta-1,3-glucan by a new protein-carbohydrate linkage.

A family of covalently linked cell wall proteins of Saccharomyces cerevisiae, called Pir proteins, are characterized by up to 10 conserved repeating units. Ccw5/Pir4p contains only one complete repeating sequence and its deletion caused a release of the protein into the medium. The exchange of each of three glutamines (Gln69, Gln74, Gln76) as well as one aspartic acid (Asp72) within the repeating unit leads to a loss of the protein from the cell wall.