Fluorescence Quenching as a Tool to Investigate Quinolone Antibiotic Interactions with Bacterial Protein OmpF

The outer membrane porin OmpF is an important protein for the uptake of antibiotics through the outer membrane of gram-negative bacteria; however, the possible binding sites involved in this uptake are still not recognized. Determination, at the molecular level, of the possible sites of antibiotic interaction is very important, not only to understand their mechanism of action but also to unravel bacterial resistance. Due to the intrinsic OmpF fluorescence, attributed mainly to its tryptophans (Trp²¹⁴, Trp⁶¹), quenching experiments were used to assess the site(s) of interaction of some quinolone antibiotics. OmpF was reconstituted in different organized structures, and the fluorescence quenching results, in the presence of two quenching agents, acrylamide and iodide, certified that acrylamide quenches Trp⁶¹ and iodide Trp²¹⁴. Similar data, obtained in presence of the quinolones, revealed distinct behaviors for these antibiotics, with nalidixic acid interacting near Trp²¹⁴ and moxifloxacin near Trp⁶¹. These studies, based on straightforward and quick procedures, show the existence of conformational changes in the protein in order to adapt to the different organized structures and to interact with the quinolones. The extent of reorganization of the protein in the presence of the different quinolones allowed an estimate on the sites of protein/quinolone interaction.     

Cnidarian Primary Cell Culture as a Tool to Investigate the Effect of Thermal Stress at Cellular Level

In the context of global change, symbiotic cnidarians are largely affected by seawater temperature elevation leading to symbiosis breakdown. This process, also called bleaching, is triggered by the dysfunction of the symbiont photosystems causing an oxidative stress and cell death to both symbiont and host cells. In our study, we wanted to elucidate the intrinsic capacity of isolated animal cells to deal with thermal stress in the absence of symbiont. In that aim, we have characterized an animal primary cell culture form regenerating tentacles of the temperate sea anemone Anemonia viridis. We first compared the potential of whole tissue tentacle or separated epidermal or gastrodermal monolayers as tissue sources to settle animal cell cultures. Interestingly, only isolated cells extracted from whole tentacles allowed establishing a viable and proliferative primary cell culture throughout 31 days. The analysis of the expression of tissue-specific and pluripotency markers defined cultivated cells as differentiated cells with gastrodermal origin. The characterization of the animal primary cell culture allowed us to submit the obtained gastrodermal cells to hyperthermal stress (+?5 and +?8 °C) during 1 and 7 days. Though cell viability was not affected at both hyperthermal stress conditions, cell growth drastically decreased. In addition, only a +?8 °C hyperthermia induced a transient increase of antioxidant defences at 1 day but no ubiquitin or carbonylation protein damages. These results demonstrated an intrinsic resistance of cnidarian gastrodermal cells to hyperthermal stress and then confirmed the role of symbionts in the hyperthermia sensitivity leading to bleaching.     

19F Nuclear Magnetic Resonance as a Tool To Investigate Microbial Degradation of Fluorophenols to Fluorocatechols and Fluoromuconates

A method was developed to study the biodegradation and oxidative biodehalogenation of fluorinated phenols by ¹⁹F nuclear magnetic resonance (NMR). Characterization of the ¹⁹F NMR spectra of metabolite profiles of a series of fluorophenols, converted by purified phenol hydroxylase, catechol 1,2-dioxygenase, and/or by the yeast-like fungus Exophiala jeanselmei, provided possibilities for identification of the ¹⁹F NMR chemical shift values of fluorinated catechol and muconate metabolites. As an example, the ¹⁹F NMR method thus defined was used to characterize the time-dependent metabolite profiles of various halophenols in either cell extracts or in incubations with whole cells of E. jeanselmei. The results obtained for these two systems are similar, except for the level of muconates observed. Altogether, the results of the present study describe a ¹⁹F NMR method which provides an efficient tool for elucidating the metabolic pathways for conversion of fluorine-containing phenols by microorganisms, with special emphasis on possibilities for biodehalogenation and detection of the type of fluorocatechols and fluoromuconates involved. In addition, the method provides possibilities for studying metabolic pathways in vivo in whole cells.     

Principal Components Analysis as a Tool to Summarise Biotransformation Data: Influence on Cells of Solvent Type and Phase Ratio

The effect of some solvents, present in different amounts, upon whole cells of Rhodococcus erythropolis DCL14 carrying out the biotransformation of (-)-carveol to (-)-carvone was studied. The solvents tested were ethyl butyrate, n-hexane, cyclohexane, iso-octane, n-dodecane, dimethyl sulfoxide, bis(2-ethylhexyl) phthalate and FC-70. The volumes of each solvent corresponded to organic:aqueous phase ratios of 0.0005, 0.0025, 0.005, 0.025 and 0.2. To assess any potential solvent protection towards substrate toxicity, assays were carried out at two initial carveol concentrations (15 and 50 mM). Carvone accumulation was followed by gas chromatography. Cell viability, several aspects of cell morphology and the ability to form clusters were monitored by fluorescence microscopy. Principal components analysis (PCA) was used as a tool to explain the differences in the observations of the multidimensional data set obtained from the multiple conditions. PCA using the different volumes of each solvent as variable suggests that the variability of the observations can be summarised in six components which represent 79.4% of the variance of the data. Conversely, using cell and solvent data to perform the PCA, 97.1% of the variance of the data can be summarised in three components, the first two capturing 91.0% of the information. These components seem to represent solvent toxicity and a protective effect of the solvent from carveol toxicity.     

Principal Components Analysis as a Tool to Summarise Biotransformation Data: Influence on Cells of Solvent Type and Phase Ratio

The effect of some solvents, present in different amounts, upon whole cells of Rhodococcus erythropolis DCL14 carrying out the biotransformation of (?)-carveol to (?)-carvone was studied. The solvents tested were ethyl butyrate, n-hexane, cyclohexane, iso-octane, n-dodecane, dimethyl sulfoxide, bis(2-ethylhexyl) phthalate and FC-70. The volumes of each solvent corresponded to organic:aqueous phase ratios of 0.0005, 0.0025, 0.005, 0.025 and 0.2. To assess any potential solvent protection towards substrate toxicity, assays were carried out at two initial carveol concentrations (15 and 50 mM). Carvone accumulation was followed by gas chromatography. Cell viability, several aspects of cell morphology and the ability to form clusters were monitored by fluorescence microscopy. Principal components analysis (PCA) was used as a tool to explain the differences in the observations of the multidimensional data set obtained from the multiple conditions. PCA using the different volumes of each solvent as variable suggests that the variability of the observations can be summarised in six components which represent 79.4% of the variance of the data. Conversely, using cell and solvent data to perform the PCA, 97.1% of the variance of the data can be summarised in three components, the first two capturing 91.0% of the information. These components seem to represent solvent toxicity and a protective effect of the solvent from carveol toxicity.     

Self-Organising Maps and Correlation Analysis as a Tool to Explore Patterns in Excitation-Emission Matrix Data Sets and to Discriminate Dissolved Organic Matter Fluorescence Components

Dissolved organic matter (DOM) is a complex mixture of organic compounds, ubiquitous in marine and freshwater systems. Fluorescence spectroscopy, by means of Excitation-Emission Matrices (EEM), has become an indispensable tool to study DOM sources, transport and fate in aquatic ecosystems. However the statistical treatment of large and heterogeneous EEM data sets still represents an important challenge for biogeochemists. Recently, Self-Organising Maps (SOM) has been proposed as a tool to explore patterns in large EEM data sets. SOM is a pattern recognition method which clusterizes and reduces the dimensionality of input EEMs without relying on any assumption about the data structure. In this paper, we show how SOM, coupled with a correlation analysis of the component planes, can be used both to explore patterns among samples, as well as to identify individual fluorescence components. We analysed a large and heterogeneous EEM data set, including samples from a river catchment collected under a range of hydrological conditions, along a 60-km downstream gradient, and under the influence of different degrees of anthropogenic impact. According to our results, chemical industry effluents appeared to have unique and distinctive spectral characteristics. On the other hand, river samples collected under flash flood conditions showed homogeneous EEM shapes. The correlation analysis of the component planes suggested the presence of four fluorescence components, consistent with DOM components previously described in the literature. A remarkable strength of this methodology was that outlier samples appeared naturally integrated in the analysis. We conclude that SOM coupled with a correlation analysis procedure is a promising tool for studying large and heterogeneous EEM data sets.     

Strain Analysis as a Tool for Functional Morphology

Strain gauge analysis can provide tremendous benefits to functionalmorphologists by accurately assessing the performance of a particularhard tissue structure in the mechanicalloading environmentsin which it functions. This method replaces estimated calculationsof skeletal stresses or strains with empirical measurements,and thus allows explicit testing of predictions derived fromtheoretical mechanical analyses or models. This direct measurementis particularly useful and important when the assumptions ofmechanical models are violated in the biological world, suchas when the geometry of the element of interest is complex and/orwhen the loads applied to a structure are either poorly knownof dynamically changing. In many, perhaps most, systems of interestto functional morphologists, the assumptions of simple shapeand well-defined loading necessary for most theoretical analysesare untenable; in these situations, strain gauge analysis canprovide considerable crucial information not obtainable anyother way. Although there are important limitations to the useof this approach, it can be applied to a variety of kinds offunctional analyses and may supply information about skeletalperformance that can contribute to meaningful functional interpretationsof morphological design.     

Multi-Variate EEG Analysis as a Novel Tool to Examine Brain Responses to Naturalistic Music Stimuli

Note onsets in music are acoustic landmarks providing auditory cues that underlie the perception of more complex phenomena such as beat, rhythm, and meter. For naturalistic ongoing sounds a detailed view on the neural representation of onset structure is hard to obtain, since, typically, stimulus-related EEG signatures are derived by averaging a high number of identical stimulus presentations. Here, we propose a novel multivariate regression-based method extracting onset-related brain responses from the ongoing EEG. We analyse EEG recordings of nine subjects who passively listened to stimuli from various sound categories encompassing simple tone sequences, full-length romantic piano pieces and natural (non-music) soundscapes. The regression approach reduces the 61-channel EEG to one time course optimally reflecting note onsets. The neural signatures derived by this procedure indeed resemble canonical onset-related ERPs, such as the N1-P2 complex. This EEG projection was then utilized to determine the Cortico-Acoustic Correlation (CACor), a measure of synchronization between EEG signal and stimulus. We demonstrate that a significant CACor (i) can be detected in an individual listener''s EEG of a single presentation of a full-length complex naturalistic music stimulus, and (ii) it co-varies with the stimuli’s average magnitudes of sharpness, spectral centroid, and rhythmic complexity. In particular, the subset of stimuli eliciting a strong CACor also produces strongly coordinated tension ratings obtained from an independent listener group in a separate behavioral experiment. Thus musical features that lead to a marked physiological reflection of tone onsets also contribute to perceived tension in music.     

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g. the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.     

Histone H1 as a Reporter Protein to Investigate Glycation in Bacteria

Nonenzymatic glycosylation (glycation) of proteins is a multistage chemical process starting as a condensation reaction between reducing sugars and primary amino groups (mainly from the side chains of Lis and Arg) and ending up with formation of complex heterocyclic compounds called advanced glycation end products (AGEs). For a long time, glycation has been attributed to the long-lived eukaryotes (including in humans) only. In a recent study, we showed that glycation also occurs in bacteria. The present study aims to prove that bacterial cytoplasm contains soluble glycating compounds. To this end, Lis/Arg-rich histone H1 isolated from rat liver was treated with deproteinized Escherichia coli cytoplasm through a dialysis membrane. This treatment leads to accumulation of AGEs as well as to a remarkable degradation of the reporter protein on storage at 4°C. Our results indicate also that glycation can be inhibited by acetylsalicylic acid (aspirin), thiamine (vitamin B1), and pyridoxine (vitamin B6).     

Hazard Analysis of Critical Control Points Assessment as a Tool to Respond to Emerging Infectious Disease Outbreaks

Highly pathogenic avian influenza virus (HPAI) strain H5N1 has had direct and indirect economic impacts arising from direct mortality and control programmes in over 50 countries reporting poultry outbreaks. HPAI H5N1 is now reported as the most widespread and expensive zoonotic disease recorded and continues to pose a global health threat. The aim of this research was to assess the potential of utilising Hazard Analysis of Critical Control Points (HACCP) assessments in providing a framework for a rapid response to emerging infectious disease outbreaks. This novel approach applies a scientific process, widely used in food production systems, to assess risks related to a specific emerging health threat within a known zoonotic disease hotspot. We conducted a HACCP assessment for HPAI viruses within Vietnam’s domestic poultry trade and relate our findings to the existing literature. Our HACCP assessment identified poultry flock isolation, transportation, slaughter, preparation and consumption as critical control points for Vietnam’s domestic poultry trade. Introduction of the preventative measures highlighted through this HACCP evaluation would reduce the risks posed by HPAI viruses and pressure on the national economy. We conclude that this HACCP assessment provides compelling evidence for the future potential that HACCP analyses could play in initiating a rapid response to emerging infectious diseases.     

Restriction Endonuclease Analysis of Highly Repetitive DNA as a Phylogenetic Tool

Multiple band patterns of DNA repeats in the 20–500-nucleotide range can be detected by digesting genomic DNA with short—cutting restriction endonucleases, followed by end labeling of the restriction fragments and fractionation in nondenaturing polyacrylamide gels. We call such band patterns obtained from genomic DNA ``taxonprints' (Fedorov et al. 1992). Here we show that taxonprints for the taxonomic groups studied (mammals, reptiles, fish, insects—altogether more than 50 species) have the following properties: (1) All individuals from the same species have identical taxonprints. (2) Taxonprint bands can be subdivided into those specific for a single species and those specific for groups of closely related species, genera, and even families. (3) Each restriction endonuclease produces unique band patterns; thus, five to ten restriction enzymes (about 100 bands) may be sufficient for a statistical treatment of phylogenetic relationships based on polymorphisms of restriction endinuclease sites. We demonstrate that taxonprint analysis allows one to distinguish closely related species and to establish the degree of similarity among species and among genera. These characteristics make taxonprint analysis a valuable tool for taxonomic and phylogenetic studies. Received: 10 February 1997 / Accepted: 10 March 1997     

The Insect Galleria mellonella as a Powerful Infection Model to Investigate Bacterial Pathogenesis

The study of bacterial virulence often requires a suitable animal model. Mammalian models of infection are costly and may raise ethical issues. The use of insects as infection models provides a valuable alternative. Compared to other non-vertebrate model hosts such as nematodes, insects have a relatively advanced system of antimicrobial defenses and are thus more likely to produce information relevant to the mammalian infection process. Like mammals, insects possess a complex innate immune system¹. Cells in the hemolymph are capable of phagocytosing or encapsulating microbial invaders, and humoral responses include the inducible production of lysozyme and small antibacterial peptides^2,3. In addition, analogies are found between the epithelial cells of insect larval midguts and intestinal cells of mammalian digestive systems. Finally, several basic components essential for the bacterial infection process such as cell adhesion, resistance to antimicrobial peptides, tissue degradation and adaptation to oxidative stress are likely to be important in both insects and mammals¹. Thus, insects are polyvalent tools for the identification and characterization of microbial virulence factors involved in mammalian infections.Larvae of the greater wax moth Galleria mellonella have been shown to provide a useful insight into the pathogenesis of a wide range of microbial infections including mammalian fungal (Fusarium oxysporum, Aspergillus fumigatus, Candida albicans) and bacterial pathogens, such as Staphylococcus aureus, Proteus vulgaris, Serratia marcescens Pseudomonas aeruginosa, Listeria monocytogenes or Enterococcus faecalis^4-7. Regardless of the bacterial species, results obtained with Galleria larvae infected by direct injection through the cuticle consistently correlate with those of similar mammalian studies: bacterial strains that are attenuated in mammalian models demonstrate lower virulence in Galleria, and strains causing severe human infections are also highly virulent in the Galleria model^8-11. Oral infection of Galleria is much less used and additional compounds, like specific toxins, are needed to reach mortality.G. mellonella larvae present several technical advantages: they are relatively large (last instar larvae before pupation are about 2 cm long and weight 250 mg), thus enabling the injection of defined doses of bacteria; they can be reared at various temperatures (20 °C to 30 °C) and infection studies can be conducted between 15 °C to above 37 °C^12,13, allowing experiments that mimic a mammalian environment. In addition, insect rearing is easy and relatively cheap. Infection of the larvae allows monitoring bacterial virulence by several means, including calculation of LD₅₀¹⁴, measurement of bacterial survival^15,16 and examination of the infection process¹⁷. Here, we describe the rearing of the insects, covering all life stages of G. mellonella. We provide a detailed protocol of infection by two routes of inoculation: oral and intra haemocoelic. The bacterial model used in this protocol is Bacillus cereus, a Gram positive pathogen implicated in gastrointestinal as well as in other severe local or systemic opportunistic infections^18,19.