On the measurement and classification of colour in studies of animal colour patterns

In studies of animal colouration it is no longer necessary to rely on subjective assessments of colour and conspicuousness, nor on methods which rely upon human vision. This is important because animals vary greatly in colour vision and colour is context-dependent. New methods make it practical to measure the colour spectrum of pattern elements (patches) of animals and their visual backgrounds for the conditions under which patch spectra reach the conspecific's, predator's or prey's eyes. These methods can be used in both terrestrial and aquatic habitats. A patch's colour is dependent not only upon its reflectance spectrum, but also upon the ambient light spectrum, the transmission properties of air or water, and the veiling light spectrum. These factors change with time of day, weather, season and microhabitat, so colours must be measured under the conditions prevalent when colour patterns are normally used. Methods of measuring, classifying and comparing colours are presented, as well as techniques for assessing the conspicuousness of colour patterns as a whole. Some implications of the effect of environmental light and vision are also discussed.     

On the design of a flexible stimulator for animal studies in auditory prostheses

The present paper describes the design of two stimulators (bench-top and portable) which can be used for animal studies in cochlear implants. The bench-top stimulator is controlled by a high-speed digital output board manufactured by National Instruments and is electrically isolated. The portable stimulator is controlled by a personal digital assistant (PDA) and is based on a custom interface board that communicates with the signal processor in the PDA through the secure digital IO (SDIO) slot. Both stimulators can provide 8 charge-balanced, bipolar channels of pulsatile and analog-like electrical stimulation, delivered simultaneously, interleaved or using a combination of both modes. Flexibility is provided into the construction of arbitrary, but charge-balanced, pulse shapes, which can be either symmetric or asymmetric.     

Fourier transform infrared microspectroscopy as a new tool for nematode studies

We report the results of a microspectroscopy study on the Fourier transform infrared (FT-IR) absorption spectra of Caenorhabditis elegans, collected from the different parts of a single intact specimen--pharynx, intestine and tail regions. The principal absorption bands were assigned to the molecular species present in C. elegans, with an excellent reproducibility for the pharynx spectrum. These results enabled us to explore if FT-IR microspectroscopy could offer a new tool for nematode identification. As an example, the discrimination among four well characterised nematode taxa is reported. The FT-IR results completely match those obtained by Blaxter and colleagues through molecular biology [Nature 392 (1998) 71].     

Cell electropermeabilization: a new tool for biochemical and pharmacological studies

Cell electropermeabilization is the transient permeabilization of the plasma membrane by means of short and intense electric pulses. Under optimized conditions, electropermeabilization is compatible with cell survival. It provides a direct access into the cytosol to ions, small molecules, exogenous drugs and macro-molecules. As cells remain functional, a large variety of cell biology questions can be addressed. Such ‘in situ biochemistry’ opens new possibilities beside the more classical studies dealing with unpermeabilized cells or subcellular extracts. Electropermeabilization also allows pharmacological studies with cells, cultured monolayers and in vivo tissues as well as the design of drug controlled-release systems.     

Comparative gene mapping: a valuable new tool for mammalian developmental studies

Technological advances in the 1970s encouraged the mapping of homologous gene loci in different mammalian species, including mouse and man. One hundred eighty-five homologous loci have now been mapped in these two species. Conservation of linkage is sufficient to identify substantial segments of the two genomes that have been left intact since their divergence from a common ancestor. The recognition of these conserved segments allows experimental manipulation of mouse chromosomes or chromosomal regions to produce models of human chromosomal anomalies of medical importance. Comparative gene mapping has been extended beyond mouse and man and the genomes of some species, including domestic cattle, appear to be more highly conserved relative to humans than the mouse. Such species may be particularly useful in providing models of human chromosomal anomalies that cannot be duplicated in laboratory mice.     

Solid-Phase Cross-Linking (SPCL): a new tool for protein structure studies

A wide range of chemical reagents are available to study the protein-protein interactions or protein structures. After reaction with such chemicals, covalently modified proteins are digested, resulting in shorter peptides that are analyzed by mass spectrometry (MS). Used especially when NMR of X-ray data are lacking, this methodology requires the identification of modified species carrying relevant information, among the unmodified peptides. To overcome the drawbacks of existing methods, we propose a more direct strategy relying on the synthesis of solid-supported cleavable monofunctional reagents and cross-linkers that react with proteins and that selectively release, after protein digestion and washings, the modified peptide fragments ready for MS analysis. Using this Solid-Phase Cross-Linking (SPCL) strategy, only modified sequences are analyzed and consistent data can be easily obtained since the signals of interest are not masked or suppressed by over-represented unmodified materials.     

The hidden code in genomics: a tool for gene discovery.

Among new insights coming from the completion of sequencing of the human genome, reported in Nature and Science, are clues of how evolution has increased the complexity of species, and in particular how the genetic code has enabled this process. It is clear that life has not only evolved by increasing the number of genes, but also by ingeniously evolving an efficient code for expressing diversity in the building blocks (i.e. the amino acids). The rules of nucleic acid base pairing and the classification of amino acids according to hydrophobicity/hydrophilicity relationships define a binary DNA code, which determines the general biophysical characteristics of proteins. Sense and antisense strands can encode protein segments having inverted and complementary hydropathy. The underlying binary code controls association and dissociation of proteins and presumably represents a primordial code that might have emerged in the early stages of self-organizing biochemical cycles. It is the purpose of this communication to provide a perspective of the code in the context of a binary language from its primordial origin to its present day format and to propose to use this code as a genomic mining tool.     

Extensive coloured identification of dog chromosomes to support karyotype studies: the colour code

The identification of individual dog chromosomes is problematic because the 38 pairs of autosomes are small and acrocentric. Here we describe the design and application of a FISH tool that enables definitive identification of each dog autosome in a normal karyotype, without relying on subjective interpretation of DAPI banding patterns. From a high-resolution physical map of the canine genome, we have chosen a panel of 80 canine chromosome-specific BAC clones. DNA from each clone is labeled with one of five different fluorochrome-conjugated nucleotides. By selecting one to three spatially separated BACs per chromosome, and labelling them with a distinctive combination of colours, each autosome can be identified objectively and orientated accurately, irrespective of the quality of DAPI chromosome banding. This tool, or part of it, can be used for any purpose where accurate identification of canine autosomes in a normal karyotype is essential. In this study, we demonstrate use of the 'colour code' for chromosome identification following CGH analysis of unbalanced genomic aberrations in a canine brain tumour. Our method is an improvement of an earlier procedure, featuring chromosome-specific BACs and sequential FISH hybridisations, as it enables simultaneous identification of all chromosomes in a single hybridisation.     

Toxicology and pathology considerations for the design of juvenile animal studies

Although exposure to drugs or toxicants can affect children and adults very differently, many compounds lack specific safety information for children. Studies in juvenile animals can help researchers assess pediatric patients' potential response to certain chemicals. Juvenile studies are highly sensitive to animal age, sex and species and must be planned with care to prevent misinterpretation of experimental data. The author reviews considerations for the design of these studies, focusing on toxicological and pathological aspects.     

I-SceI endonuclease: a new tool for DNA repair studies and genetic manipulations in streptomycetes

Actinomycetes are Gram-positive bacteria with a complex life cycle. They produce many pharmaceutically relevant secondary metabolites, including antibiotics and anticancer drugs. However, there is a limited number of biotechnological applications available as opposed to genetic model organisms like Bacillus subtilis or Escherichia coli. We report here a system for the functional expression of a synthetic gene encoding the I-SceI homing endonuclease in several streptomycetes. Using the synthetic sce(a) gene, we were able to create controlled genomic DNA double-strand breaks. A mutagenesis system, based on the homing endonuclease I-SceI, has been developed to construct targeted, non-polar, unmarked gene mutations in Streptomyces sp. Tü6071. In addition, we have shown that homologous recombination is a major pathway in streptomycetes to repair an I-SceI-generated DNA double-strand break. This novel I-SceI-based tool will be useful in fundamental studies on the repair mechanism of DNA double-strand breaks and for a variety of biotechnological applications.     

Thiessen polygons as a model for animal territory estimation

Thiessen polygons are often used to model territory characteristics. However, information about the quality of Thiessen polygon‐based estimates is currently lacking. We used published data to investigate the match between Thiessen polygons and mapped bird territories regarding territory size, shape and neighbourhood. Although territory sizes and the number of neighbours were strongly correlated between these two methods, both parameters were overestimated by the Thiessen polygons. Therefore, caution is required when Thiessen polygons are used as a model for absolute values and when the assumptions of Thiessen polygons, such as formation of discrete territories and a contiguous study area, are not met.     

Bean pod mottle virus: a new powerful tool for functional genomics studies in Pisum sativum

Pea (Pisum sativum L.) is an important legume worldwide. The importance of pea in arable rotations and nutritional value for both human and animal consumption have fostered sustained production and different studies to improve agronomic traits of interest. Moreover, complete sequencing of the pea genome is currently underway and will lead to the identification of a large number of genes potentially associated with important agronomic traits. Because stable genetic transformation is laborious for pea, virus‐induced gene silencing (VIGS) appears as a powerful alternative technology for determining the function of unknown genes. In this work, we present a rapid and efficient viral inoculation method using DNA infectious plasmids of Bean pod mottle virus (BPMV)‐derived VIGS vector. Six pea genotypes with important genes controlling biotic and/or abiotic stresses were found susceptible to BPMV carrying a GFP reporter gene and showed fluorescence in both shoots and roots. In a second step, we investigated 37 additional pea genotypes and found that 30 were susceptible to BPMV and only 7 were resistant. The capacity of BPMV to induce silencing of endogenes was investigated in the most susceptible genotype using two visual reporter genes: PsPDS and PsKORRIGAN1 (PsKOR1) encoding PHYTOENE DESATURASE and a 1,4‐β‐D‐glucanase, respectively. The features of the ‘one‐step’ BPMV‐derived VIGS vector include (i) the ease of rub‐inoculation, without any need for biolistic or agro‐inoculation procedures, (ii) simple cost‐effective procedure and (iii) noninterference of viral symptoms with silencing. These features make BPMV the most adapted VIGS vector in pea to make low‐ to high‐throughput VIGS studies.     

Lipolysis index: evaluation of a new tool for metabolic assessment in epidemiological studies on obesity

Lipid mobilization through adipocyte lipolysis is central for energy metabolism and is decreased in obesity. However, the factors of importance for lipolytic activity in the general population are not known. To further examine this we performed a cross-sectional study on teenagers and adults. We constructed and evaluated a simple index of lipolytic activity (ratio of fasting p-glycerol and body fat %) in population based samples in 316 teenagers (BMI 16-51 kg/m (2)) and 3,039 adults (BMI 16-70 kg/m (2)). In the adults, multiple regression analysis showed that waist and BMI but not age, plasma insulin, plasma noradrenaline or waist-to-hip ratio contributed independently and inversely to lipolytic activity (partial r=-0.37 and -0.28, respectively, p<0.0001). Together waist and BMI explained about 45% of the variability of lipolysis. Waist was a stronger factor than BMI in stepwise regression. The same analysis in teenagers showed that only BMI contributed independently and inversely to lipolytic activity (partial r=-0.90, p<0.0001) and explained about 55% of lipolysis variation. BMI had the strongest effect on lipolysis in lean teenagers. The results were the same for men and women. At all levels of lipolytic activity plasma fatty acid levels were elevated in obese subjects (p<0.0001). We conclude that during adolescence BMI is the major factor negatively influencing lipolytic activity, in particular among lean young subjects. In adulthood central fat accumulation together with increasing BMI decreases lipolysis. In spite of low lipolytic activity circulating fatty acid levels are increased in obesity, probably due to an adipose mass effect.     

Evaluation of spectrofluorometry as a tool for estimation in fed-batch fermentations

Native culture fluorescence was investigated as an additional source of information for predicting biomass and glucose concentrations in a fed-batch fermentation of Alcaligenes eutrophus. Partial least squares (PLS) regression and a feed forward neural network (FFNN) coupled with principle component analysis (PCA) were each used to model the kinetics of the fermentation. Data from three fermentations was combined to form a training set for model calibration and data from a fourth fermentation was used as the testing set. The fluorescent soft-sensors were compared with a previously developed feed forward neural network soft-sensor model which used oxygen uptake rate (OUR), carbon dioxide evolution rate (CER), aeration rate, feed rate, and fermentor volume to estimate biomass and glucose concentrations. The best model performance for predicting both biomass and glucose concentrations was achieved using the native fluorescence-based models. Real data predictions of the biomass concentration in the testing set were obtained using both the PLS and FFNN PCA modeling utilizing fluorescence measurements plus the rate of change of the fluorescence measurements. Accurate predictions of the glucose concentration in the testing set were obtained using the FFNN PCA modeling technique utilizing the rate of change of the fluorescence measurements. Substrate exhaustion was indicated qualitatively by a first-order PLS model utilizing the rate of change of fluorescence measurements. These results indicate that native culture fluorescence shows promise for providing additional valuable information to enhance predictive modeling which cannot be extracted from other easily acquired measurements.