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Distribution of a genetically-engineered Escherichia coli population introduced into soil.

The spatial localization of the cells and the DNA of a genetically-engineered Escherichia coli population introduced into soil was investigated. Inoculated soils were size fractioned and bacterial numbers and E. coli EL1003 specific chromosomal DNA target sequences were enumerated in each fraction using plate-counting and MPN-PCR, respectively. Different numbers of either indigenous or introduced bacteria were found in each fraction indicating that their distribution in the soil was non-uniform. The distributions of the indigenous bacteria and the E. coli cells within the size fractions were significantly different: the E. coli population was mainly associated with the dispersible clay fraction (79.0%) from which only 10.7% of the indigenous bacteria were recovered. The distribution of the E. coli target DNA sequences was in agreement with the location of the cells. The different distribution of the two populations is likely to restrict genetic interactions. These results are relevant to potential interactions between native soil microflora and populations introduced into soil for competitive purposes.

Bacteria↗

Visualization of Helicobacter species within the murine cecal mucosa using specific fluorescence in situ hybridization.

BACKGROUND: Members of the genus Helicobacter have been associated with colitis development in a number of immunodeficient animal models. While it is known that these organisms can initiate colitis development, the location and spatial distribution of these bacteria within the intestinal tract is currently unknown. In this study, we developed and optimized fluorescence in situ hybridization (FISH) probes specifically for Helicobacter species. MATERIALS AND METHODS: Based on 16S-RNA gene alignments, two probes specific for the entire family Helicobacteraceae and two probes specific for Helicobacter ganmani and Helicobacter hepaticus were designed. Evaluation of these probes was determined using ATCC reference strains and cecum samples from ten IL-10 knockout mice. The presence of Helicobacter species was determined using FISH and verified using PCR-DGGE and microscopic examination of silver stained sections. RESULTS: Analysis of the ATCC reference strains revealed that the probes HEL274/HEL717 were specific for the family Helicobacteraceae, while HEP642 was specific for H. hepaticus and GAN1237 for H. ganmani. Using these probes, a pattern of spatial localization of the two different Helicobacter species was observed in the cecum tissues of IL-10 knockout mice. This consistently showed that H. ganmani was localized to the lower regions and H. hepaticus to the mid-upper regions of the crypts. CONCLUSION: We have developed FISH probes specific for the family Helicobacteraceae as well as two individual Helicobacter species. This study will allow the future use of the FISH to better understand host-pathogen interactions in vitro.

Animals↗

Cochlear or nerve stimulation.

To stimulate through electrical stimulation the auditory nerve excitation patterns produced by a normal ear, it is necessary to control both the spatial and the timing aspects of the neural firing pattern. While the spatial localization, the "place" aspect of stimulation can probably be achieved adequately with either scala tympani or direct eighth nerve electrodes, only the eighth nerve electrodes have the potential to reproduce the detailed timing patterns of the normal nerve. While such complete control of the stimulation pattern is probably necessary to produce "natural" auditory percepts, it may well not be necessary for the conveying of the information in speech because of the plasticity and pattern recognition capabilities of the brain. Present encouraging results as open speech comprehension with scala tympani electrode arrays suggest that, in many cases at least, the cruder stimulation pattern control will suffice.

Cochlear Implants↗

CD 95 mediated apoptosis in embryogenesis: implication in tooth development.

INTRODUCTION: Understanding of apoptotic mechanisms involved in tissue shaping is of particular interest because of possible targeted modulation of the development of organ structures such as teeth. Research of CD 95 mediated apoptosis has been focused particularly on cell death in the immune system and related disorders. However, CD 95 mediated apoptosis is also involved in embryogenesis of many organs as the kidney, the lung, the intestine and tissue networks such as the nervous system. DESIGN: Narrative review. RESULTS: This review briefly summarizes the current knowledge of CD 95 mediated apoptosis in embryogenesis with possible implication in tooth development. CD 95 receptor and CD 95 ligand are found at early stages of tooth development. The data suggest some positive correlations with dental apoptosis distribution, particularly in the primary enamel knot where apoptosis occurs during elimination of this structure. CD 95 deficient (lpr) adult mouse tooth phenotype, however, did not show any alterations in final tooth pattern and morphology. CONCLUSION: To date studies of apoptotic machinery during tooth development show spatial localization of many of the components together with precise and localized timing of cell death. There is still much to be learned about the regulation and importance of apoptosis in tooth development. Nevertheless, the involvement of apoptotic regulatory mechanisms interplaying with other molecules participates to the cellular cross-talk in developing tissues, which opens possible targeted modulations as suggested, e.g. for future molecular dentistry.

Animals↗

Imaging epileptiform discharges in slices of piriform cortex with voltage-sensitive fluorescent dyes.

Voltage imaging techniques were used to investigate epileptiform discharges in brain slices containing piriform cortex (PC). These experiments pinpointed the site of discharge onset in the endopiriform nucleus (En). Under some conditions, discharge onset also occurred simultaneously in adjoining neocortex. With slightly suprathreshold electrical stimulation, discharge generation was a two-stage process in which onset was preceded by a sustained spatially localized depolarization denoted as plateau activity. Plateau activity was seen away from the onset site, in a border region between En and layer III of PC. A similar two-stage sequence was seen for slices taken from a variety of planes, using two different interictal models as well as an ictal model. Plateau activity was found to be necessary for the generation of both kinds of discharge. Synaptic transmission at the site of onset was found to be required for the generation of interictal-like discharges, but ictal-like discharges were different in that they could still be generated when synaptic transmission at this site was impaired. These studies identify specialized regions with potentially important roles in epileptogenesis and help to elucidate the neuronal circuitry that can produce epileptiform activity.

Animals↗

Two-color two-photon excitation of fluorescence.

We report the observation of two-photon excitation of an organic fluorophore with two different wavelengths, a phenomenon we refer to as two-color two-photon (2C2P) excitation. Ultraviolet emission of p-terphenyl at 340 nm was observed when the sample was illuminated with both 375 and 750 nm pulses from a picosecond dye laser. The emission of p-terphenyl was about 100-fold and more than 1000-fold less for illumination at only 375 or 750 nm, respectively. Observation of the 2C2P signal required temporal and spatial overlap of the 375 and 750 nm pulses. The amplitude of the signal depended on the polarization of each beam. 2C2P excitation can have applications in fluorescence microscopy and elsewhere when spatially localized excitation is desirable.

Fluorescence Polarization↗

A STEM approach to the study of the proteinaceous architecture of a ribosome.

We used a high-resolution STEM to attempt to recognize the spatial localization of the macromolecular components of ribosomes isolated from Tetrahymena pyriformis. Images obtained either by the annular dark field detector or by the ratio-contrast mode (Z-contrast) show a ribosome as constituted of rounded dark areas, the number and size of which are compatible with those of the protein molecules recognized by biochemistry. Images of different ribosomes, when similar orientations are observed, exhibit a roughly constant pattern of the dark areas. A set of images of a tilted ribosome allows to recognize that some masses are in fact two more superimposed components. We propose that the dark areas represent essentially the proteinaceous architecture of the ribosome.

Animals↗

Fluctuations in intracellular calcium concentration and their effect on tonic tension in canine cardiac Purkinje fibres.

Ca2+-activated aequorin luminescence and tension were measured in dog Purkinje fibres during twitches and during the increase in resting force produced by exposure of the fibres to a low-Na+ solution after 3 min without external K+. Over the restricted range which could be examined, the relation between tension and 'mean' aequorin luminescence (luminescence filtered at 0.2 Hz) was approximately linear during the development and maintenance of contracture. For a given level of force, the mean aequorin luminescence during contracture was up to 20 times greater than the peak luminescence during the twitch. Noise analysis of aequorin luminescence and tension during contracture indicated the presence of periodic fluctuations, with a predominant frequency in the range 1-4 Hz. Ryanodine (1 microM) or caffeine (10 mM) abolished the fluctuations in luminescence and tension and made the relation between tension and mean aequorin luminescence much steeper. A mathematical model, the key feature of which is periodicity in the asynchronous occurrence of spatially localized regions of relatively high [Ca2+], reproduces the experimental data derived from contractures. From the model analysis, we infer that tonic tension is produced by recruitment of increasing numbers of regions of high [Ca2+], rather than by homogeneous graded activation. These results indicate that during contracture or 'tonic tension', intracellular [Ca2+] is not at steady state, but rather undergoes large, asynchronous spatio-temporal fluctuations. Thus the assumptions that intracellular [Ca2+] is at steady state or homogeneous during tonic tension are not valid.

Action Potentials↗

Propagating potassium and chloride conductances during activation and fertilization of the egg of the frog, Rana pipiens.

Fertilization or artificial activation of the frog egg (Rana pipiens) elicits a positive-going shift in membrane potential which results from an opening of Cl- and K+ channels in the egg membrane. We examined the spatial localization of the currents produced by the opening of these channels, using large patch electrodes (tip diameters 3-10 microns). We also used small patch electrodes (tip diameters about 1 micron) to study the single K+ channel currents. After activation, with the patch electrode at any position on the egg surface, we observed a transient current, with a main peak lasting several seconds. This activation current occurred after a variable delay of 0-3 min following the rise of the activation potential. With 10% Ringer solution in the bath and pipette, the current was usually outward, although it sometimes had an inward component. With one patch electrode on the animal surface of the egg and another patch electrode on the vegetal surface, we observed that the activation current propagated over the egg surface. In experiments where the egg was activated by applying a hyperpolarizing pulse, the response in the animal half preceded that in the vegetal half by an average of about 1 min. The amplitude of the peak outward current was similar for animal and vegetal recordings (1-2 mA/cm2). Tetraethylammonium (11 mM) in the patch pipette blocked most of the outward component of the activation current and revealed an underlying inward component. The inward component of the activation current was carried by Cl-, since it could be reversed by raising the Cl- concentration in the pipette. The Cl- component of the activation current propagated over the egg surface, with timing similar to that of the total current. The average amplitude of the peak Cl- current was six or more times larger at the animal than the vegetal surface. Fertilization caused a current to propagate from the animal to the vegetal surface, like the current observed during activation. With a small patch electrode, single channel currents of the K+ component of the activation current could be seen. The probability that the channels were open increased at more positive potentials. The single channel conductance was estimated to be 25 pS, and the reversal potential to be -150 mV. Single Cl- channel currents have not yet been seen. Activation or fertilization of the frog egg resulted in a wave-like opening of Cl- and K+ channels, which spread from the animal to the vegetal half of the egg.(ABSTRACT TRUNCATED AT 400 WORDS)

Action Potentials↗

Human perceptual learning in identifying the oblique orientation: retinotopy, orientation specificity and monocularity.

1. Human perceptual learning in discrimination of the oblique orientation was studied using psychophysical methods. Subjects were trained daily to improve their ability to identify the orientation of a circular 2.5 deg diameter unidimensional noise field. Dramatic improvements in sensitivity to contour orientation occurred over a period of 15-20 days. The improved performance persisted for several months. Improvement was more evident between daily sessions than within sessions. This was partly due to fatigue interfering with the learning effect. Moreover, a consolidation period seemed to be required. 2. Improvement was restricted to the position of the stimulus being trained. This position dependency of the learning effect proved very precise. After training at a specific stimulus position, merely displacing the stimulus to an adjacent position caused a marked increase in thresholds. 3. No transfer of the training effect was observed between orientations. Following a shift of 90 deg away from the trained orientation, performance fell, even below the initial level. 4. We observed complete to almost complete transfer between the two eyes. 5. Our results suggest plastic changes at a level of the visual processing stream where input from both eyes has come together, but where generalization for spatial localization and orientation has not yet occurred.

Discrimination, Psychological↗

Spectroscopic time-resolved diffuse reflectance and transmittance measurements of the female breast at different interfiber distances.

The first, to our knowledge, in-vivo broadband spectroscopic characterization of breast tissue using different interfiber distances as well as transmittance measurements is presented. Absorption and scattering properties are measured on six healthy subjects, using time-resolved diffuse spectroscopy and an inverse model based on the diffusion equation. Wavelength-tunable picosecond-pulse lasers and time-correlated single-photon counting detection are employed, enabling fully spectroscopic measurements in the range 610 to 1040 nm. Characterization of the absorption and reduced scattering coefficients of breast tissue is made with the aim of investigating individual variations, as well as variations due to different measurement geometries. Diffuse reflectance measurements at different interfiber distances (2, 3, and 4 cm) are performed, as well as measurements in transmittance mode, meaning that different sampling volumes are involved. The results show a large variation in the absorption and scattering properties depending on the subject, correlating mainly with the water versus lipid content of the breast. Intrasubject variations, due to different interfiber distances or transmittance modes, correlate with the known structures of the breast, but these variations are small compared to the subject-to-subject variation. The intrasubject variations are larger for the scattering data than the absorption data; this is consistent with different spatial localization of the measurements of these parameters, which is explained by the photon migration theory.

Adult↗

Toward the discrimination of early melanoma from common and dysplastic nevus using fiber optic diffuse reflectance spectroscopy.

We describe a study of the discrimination of early melanoma from common and dysplastic nevus using fiber optic diffuse reflectance spectroscopy. Diffuse reflectance spectra in the wavelength range 550 to 1000 nm are obtained using 400-microm core multimode fibers arranged in a six-illumination-around-one-collection geometry with a single fiber-fiber spacing of 470 microm. Spectra are collected at specific locations on 120 pigmented lesions selected by clinicians as possible melanoma, including 64 histopathologically diagnosed as melanoma. These locations are carried through to the histopathological diagnosis, permitting a spatially localized comparison with the corresponding spectrum. The variations in spectra between groups of lesions with different diagnoses are examined and reduced to features suitable for discriminant analysis. A classifier distinguishing between benign and malignant lesions performs with sensitivity/specificity of between 6469% and 7278%. Classifiers between pairs of the group common nevus, dysplastic nevus, in situ melanoma, and invasive melanoma show better or similar performance than the benign/malignant classifier, and analysis provides evidence that different spectral features are needed for each pair of groups. This indicates that multiple discriminant systems are likely to be required to distinguish between melanoma and similar lesions.

Algorithms↗

Peak velocity determination using fast Fourier velocity encoding with minimal spatial encoding.

For quantitative peak velocity determination, a technique was developed that uses Fourier velocity encoding (FVE) for the fast acquisition of images of velocity with no spatial encoding other than slice selection. The technique produces images of velocity versus temporal frequency. In applications where the quantity of interest is the peak velocity and in-plane spatial localization is not required, high SNR images are produced with reduced sensitivity to errors due to slice thickness and motion. The technique was validated using steady and pulsatile flow in a straight tube, and compared to both phase contrast measurements and numerical models using steady flow in a 50% and a 75% cosinusoidal stenosis phantom. Results show that for slices as large as 2 cm and/or undergoing periodic motion, FVE can accurately measure the peak velocity in cases where a distribution of velocities exist.

Blood Flow Velocity↗

Proton magnetic resonance spectroscopy in the brain: report of AAPM MR Task Group #9.

AAPM Magnetic Resonance Task Group #9 on proton magnetic resonance spectroscopy (MRS) in the brain was formed to provide a reference document for acquiring and processing proton (1H) MRS acquired from brain tissue. MRS is becoming a common adjunct to magnetic resonance imaging (MRI), especially for the differential diagnosis of tumors in the brain. Even though MR imaging is an offshoot of MR spectroscopy, clinical medical physicists familiar with MRI may not be familiar with many of the common practical issues regarding MRS. Numerous research laboratories perform in vivo MRS on other magnetic nuclei, such as 31P, 13C, and 19F. However, most commercial MR scanners are generally only capable of spectroscopy using the signals from protons. Therefore this paper is of limited scope, giving an overview of technical issues that are important to clinical proton MRS, discussing some common clinical MRS problems, and suggesting how they might be resolved. Some fundamental issues covered in this paper are common to many forms of magnetic resonance spectroscopy and are written as an introduction for the reader to these methods. These topics include shimming, eddy currents, spatial localization, solvent saturation, and post-processing methods. The document also provides an extensive review of the literature to guide the practicing medical physicist to resources that may be useful for dealing with issues not covered in the current article.

Artifacts↗

Acoustic field variability induced by time evolving internal wave fields

A space- and time-dependent internal wave model was developed for a shallow water area on the New Jersey continental shelf and combined with a propagation algorithm to perform numerical simulations of acoustic field variability. This data-constrained environmental model links the oceanographic field, dominated by internal waves, to the random sound speed distribution that drives acoustic field fluctuations in this region. Working with a suite of environmental measurements along a 42-km track, a parameter set was developed that characterized the influence of the internal wave field on sound speed perturbations in the water column. The acoustic propagation environment was reconstructed from this set in conjunction with bottom parameters extracted by use of acoustic inversion techniques. The resulting space- and time-varying sound speed field was synthesized from an internal wave field composed of both a spatially diffuse (linear) contribution and a spatially localized (nonlinear) component, the latter consisting of solitary waves propagating with the internal tide. Acoustic simulation results at 224 and 400 Hz were obtained from a solution to an elastic parabolic equation and are presented as examples of propagation through this evolving environment. Modal decomposition of the acoustic field received at a vertical line array was used to clarify the effects of both internal wave contributions to the complex structure of the received signals.

Journal Article↗

Atmospheric scattering for varying degrees of saturation and turbulent intermittency.

Atmospheric turbulence is inherently inhomogeneous and intermittent. Short periods of high activity are embedded in longer periods of relative calm. Local spatial and temporal changes in sound speed associated with this intermittency increase the likelihood of measuring large values of scattered acoustic signals. Previous work successfully predicted the probability density functions (pdf's) of fully saturated, scattered signals measured within an acoustic shadow zone [Wilson et al., J. Acoust. Soc. Am. 99, 3393-3400 (1996)]. The more general case of incompletely saturated scattering is considered in this paper; using the Rice-Nagakami distribution a theory is developed. The predicted intensity pdf has two free parameters: one to describe the degree of intermittency and a second for the degree of saturation. For validation purposes, outdoor propagation measurements were made over a flat, hard ground at ranges of 146-283 m and at frequencies of 50-540 Hz. The saturation parameter was determined from the acoustic data and also estimated from the turbulence conditions. The degree of saturation increased with frequency, and measured intensity pdf's were found to be in excellent agreement with the theory.

Journal Article↗

Speech processing in the auditory system. I: The representation of speech sounds in the responses of the auditory nerve.

In a previous paper the speech evoked spatio-temporal response patterns recorded in large populations of auditory-nerve fibers in the cat were examined [M.I. Miller and M.B. Sachs, J. Acoust. Soc. Am. 74, 502-517 (1983)]. The distribution of the relative phases of synchronized activity emerges as an important response feature reflecting the stimulus spectral parameters. Specifically, each strong low-order harmonic of the stimulus (less than or equal to 1.5-2 kHz) dominates the synchrony of a relatively broad segment of fibers near its corresponding characteristic frequency (CF) location in a pattern which mirrors the underlying traveling wave component. Each such fiber segment can be roughly subdivided into two regions: (1) a region basal to the point of resonance of the harmonic where the fiber PST histograms accumulate only small delays (or phase shifts) relative to each other reflecting the fast speed of propagation of the traveling wave, and (2) a region at or very near the point of resonance where the responses exhibit drastic relative phase shifts owing to the sudden slow down of the traveling wave and the consequent rapid accumulation of phase shifts. These rapid phase shifts thus manifest themselves as steep and localized spatial discontinuities in an otherwise relatively uniform instantaneous pattern of activity across the fiber array, all occurring at the CF locations corresponding to the low-order harmonics of the stimulus.

Acoustic Stimulation↗

Speech processing in the auditory system. II: Lateral inhibition and the central processing of speech evoked activity in the auditory nerve.

A biologically realistic model of a uniform lateral inhibitory network (LIN) is shown capable of extracting from the complex spatio-temporal firing patterns of the cat's auditory nerve the formants and low-order harmonics of synthetic voiced speech stimuli. The model provides a realistic mechanism to utilize the temporal aspects of the firing and thus supports the hypothesis that the neural coding of complex sounds in terms of average rates can be supplemented by the information coded in the synchronous firing. At low levels of intensity the LIN can sharpen the average rate profiles. At moderate and high levels the LIN uses the cues available in the distribution of phases of the synchronous activity which exhibit rapid relative phase shifts at specific characteristic frequency (CF) locations (corresponding to the frequencies of the low-order harmonics in the stimulus). These temporal phase shifts manifest themselves at the input of the LIN as steep and localized spatial discontinuities in the instantaneous pattern of activity across the fiber array. The LIN enhances its output from these spatially steep input regions while suppressing its output from spatially smooth input regions (where little phase shifts occur). In this manner the LIN recreates from the response patterns a representation of the stimulus spectrum using the temporal cues as spatial markers of the stimulus components rather than as absolute measures of their frequencies. Similar results are obtained with various lateral inhibitory topologies, e.g., recurrent versus nonrecurrent, single versus double layer, and linear versus nonlinear.

Animals↗