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In situ imaging of microorganisms in geologic material.

In order to fully delineate the interactions of microorganisms with geological substrates, unequivocal identification of intact microbial cells within geologic samples is required without the disruption of either the rock texture or the relationship of the microorganisms to the mineral fabric. To achieve this objective we developed a protocol that enables the visualization of intact microbial cells in petrographic thin sections, avoids detaching the cells from their host mineral surfaces and avoids microbial contamination during the lapidary process. Propidium iodide and POPO-3, nucleic acid stains that specifically target double-stranded DNA and RNA were utilized for in situ visualization of cells in surface and subsurface basalts from northeastern Idaho. Additionally, examination of samples incubated with acetic acid-UL-14C via phosphor imagining facilitated the in situ visualization of 14C labeled biomass. Biomass observed was low (<10(7) cells/g). These observations indicate that the microbial distribution in these rocks exhibits a high degree of spatial heterogeneity at the sub-centimeter scale.

Bacteria↗

A note on using a laser-based technique for recording of behaviour and location of free-ranging animals.

We developed a precise, remote (up to 300m) observation system to record animal location and behaviour that requires no animal handling or disruption of the normal environment. Our system, combining a survey laser and a laptop, also allows recording of observed animal behaviour from seconds to hours, with accuracy of 1m or better. Up to one individual per second can be located, which supports data collection of large numbers of animals not possible with other methods. The laser system was used to track a halter-broken heifer led in an arc beginning and ending about 50m from the laser with a maximum distance of about 150m. We recorded the location of the heifer at 35 points along the arc using the laser, a global positioning system (GPS), and a nylon tape. There was an average linear difference of 1.16m (S.D. 0.63) between the laser data and the GPS data. The laser was potentially more accurate than GPS for this application because the laser averaged only 0.21m (S.D. 0.24) linear difference from the tape. Tests of the laser to relocate points in the field to within 0.20m and 0.1 degrees, averaged 0.42m (S.D. 0.29) from the original points. Our technique allows precise location of behaviour and navigation to grazed sites, potentially revealing how animals interact with the resources they exploit and showing the effect of landscape spatial heterogeneity on foraging and habitat use patterns.

Journal Article↗

Membrane inlet mass spectrometric measurement of O2 and CO2 gradients in cultures of Lactobacillus paracasei and a developing Cheddar cheese ecosystem.

Membrane inlet mass spectrometry was used to measure O2 and CO2 as depth profiles in stab cultures of 0.1% agar Man Rogosa Sharpe medium inoculated with Lactobacillus paracasei CI3. Diffusion of CO2 from the central column of growth into the medium was observed to show lower concentrations where bacteria were absent. CO2 profiles developed in a manner similar to those in Cheddar cheese and O2 was undetectable at similar depths. Gases were analysed in Cheddar cheese over a maturation period of 200 d. O2 was detectable to depths of 13, 6 and 2.5 mm on days 2, 9 and 15, respectively, but then became undetectable at depths of 2.5-3 mm. CO2 concentrations measured within the cheese increased 10-fold from day 2 to day 200 to reach a value of around 15 mM. The progress of measured CO2 concentration over time at a given depth in cheese shows a hyperbolic type increase. Coefficient of regression values increase with depth to a maximum value of R2 = 0.93. In both systems, reductions and increases in CO2 were due to the absence or presence of bacterial growth, respectively. Confocal scanning laser and scanning electron microscopy was used to show spatial heterogeneity of microcolonies within the cheese ecosystem. This information can potentially be used as a non-sensory evaluation of cheese maturity status. Measurement of gases in a cheese ecosystem provides the first description of mass spectrometry being used to monitor the processes of microbial gaseous exchange with respect to O2 and CO2 in a cheese ecosystem.

Carbon Dioxide↗

Natural attenuation of chlorinated solvents at Area 6, Dover Air Force Base: characterization of microbial community structure.

A polyphasic approach based on cultivation and direct recovery of 16S rRNA gene sequences was utilized for microbial characterization of an aquifer contaminated with chlorinated ethenes. This work was conducted in order to support the evaluation of natural attenuation of chlorinated ethenes in groundwater at Area 6 at Dover Air Force Base (Dover, DE). Results from these studies demonstrated the aquifer contained relatively low biomass (e.g. direct microscopic counts of < 10(7) bacteria/g of sediment) comprised of a physiologically diverse group of microorganisms including iron reducers, acetogens, sulfate reducers, denitrifiers, aerobic and anaerobic heterotrophs. Laboratory microcosms prepared with authentic sediment and groundwater provided direct microbiological evidence that the mineralization of vinyl chloride and cis-dichloroethene as well as each step in the complete reductive dechlorination of tetracloroethene to ethene can occur in the Area 6 aquifer. Enrichment cultures capable of the oxidative degradation of cis-1,2-dichloroethene (cis-DCE) and vinyl chloride (VC) were obtained from groundwater across the aquifer demonstrating the possible importance of direct, non-cometabolic oxidation of cis-DCE and VC in natural attenuation. Culture-independent analyses based upon recovery of 16S rRNA gene sequences revealed the presence of anaerobic organisms distributed primarily between two major bacterial divisions: the delta subdivision of the Proteobacteria and low-G + C gram positive. Recovery of sequences affiliated with phylogenetic groups containing known anaerobic-halorespiring organisms such as Desulfitobacterium, Dehalobacter, and certain groups of iron reducers provided qualitative support for a role of reductive dechlorination processes in the aquifer. This molecular data is suggestive of a functional linkage between the microbiology of the site and the apparent natural attenuation process. The presence and distribution of microorganisms were found to be consistent with a microbially driven attenuation of chlorinated ethenes within the aquifer and in accord with a conceptual model of aquifer geochemistry which suggest that both reductive and oxidative mechanisms are involved in heterogeneous, spatially distributed processes across the aquifer.

Biodegradation, Environmental↗

Spatio-logical processes in intracellular signalling.

Classical models of intracellular signalling describe how small changes in a cell's external environment can bring about major changes in cellular activity. Recent findings from experimental biology indicate that many intracellular signalling systems show a high level of spatial organisation. This permits the modification, by protein kinase or protein phosphatase action, of specific subsets of intracellular proteins - an attribute that is not addressed in classical signalling models. Here we use ideas and concepts from computer science to describe the information processing nature of intracellular signalling pathways and the impact of spatial heterogeneity of their components (e.g. protein kinases and protein phosphatases) on signalling activity. We argue that it is useful to view the signalling ecology as a vast parallel distributed processing network of agents operating in heterogeneous microenvironments, and we conclude with an overview of the mathematical and semantic methodologies that might help clarify this analogy between biological and computational systems.

Models, Biological↗

Differentiation of cerebral tumors using multi-section echo planar MR perfusion imaging.

OBJECTIVE: We have investigated the performance of magnetic resonance (MR) perfusion imaging to differentiate between astrocytomas grade II, grade III and glioblastomas in a prospective study. MATERIALS AND METHODS: In 33 patients with suspected supratentorial primary cerebral tumors we performed multi-section Echo Planar MR perfusion imaging. Regional cerebral blood volume (rCBV) maps were calculated and the maximum rCBV was determined from the entire lesion. This value was divided by the mean rCBV value from the contralateral side, which provided the rCBV index used in this study. The rCBV index was correlated with the histological tumor classification after stereotactic biopsy (n=7) or open resection (n=26). RESULTS: The maximum rCBV index was 1.2+/-0.8 for grade II astrocytomas (n=3), 4.0+/-1.2 for grade III astrocytomas (n=13), and 10.3+/-3.3 for glioblastomas (n=17). The difference between grade III astrocytomas and glioblastomas was highly significant (P<0.001). DISCUSSION AND CONCLUSION: The rCBV index measured with multi-section Echo Planar MR perfusion is capable of differentiating grade III astrocytomas from glioblastomas. It serves as an additional parameter to establish a diagnosis in cases where it is not possible to clearly differentiate between these types of tumors on the basis of conventional MR imaging. MR perfusion imaging also provides information about spatial heterogeneities within a tumor which might improve diagnostic performance. This technology may also be of interest for follow-up examinations after histological diagnosis and further treatment.

Adult↗

Studies of porous media by thermally polarized gas NMR: current status.

Three examples of thermally polarized gas NMR performed at New Mexico Resonance are presented to demonstrate its unique advantages in porous media studies. 1) In-vivo animal lung imaging by Kuethe et al., in which useful quality 3D images of rat lungs were obtained in 30 min. It is conjectured that comparable human lung images would take much less time to make, possibly by the ratio of body weights, a factor of several hundred. 2) The success of the lung imaging suggested other porous media as candidates for thermally polarized gas NMR. Caprihan and coworkers obtained excellent images from partially sintered ceramics and Vycor glass. Since then, Beyea has developed the technique of spatially resolved BET curves for ceramics and other nanoporous solids. In this way, surface area, pore size, and porosity, averaged over an image voxel, can be spatially resolved. This greatly aids in the characterization of such materials, especially with regards to spatial heterogeneities. 3) Finally, we describe Codd's propagator experiments on propane gas flowing through a packed bed of 300 microm beads. In order to increase signal-to-noise ratio, the flowing gas was pressurized to 170 kPa. Excellent quality propagators showing the discrete nature of the bead pack were obtained. This type of information is not available in comparable liquid studies because most spins will not diffuse far enough to sample the walls in the time available.

Animals↗

Age-related differences in NMDA/metabotropic glutamate receptor binding in rat substantia nigra.

Both N-methyl-D-aspartate (NMDA) and quisqualate/AMPA-insensitive metabotropic glutamate (mGlu) receptors mediate glutamate neurotransmission in substantia nigra (SN). In this work, NMDA and mGlu receptor sites in substantia nigra pars compacta (SNC) and pars reticulata were autoradiographically mapped in rat brains using specific binding of (+)3H-MK801 or 3H-glutamate, with saturating concentrations of NMDA, AMPA and quisqualate. In brains of both adult and postnatal day 15 (PN15) male rats, prepared at subjective mid-day of a 12h light/12h dark (12h L/12h D) cycle, specific binding at NMDA and mGlu sites in substantia nigra was pronounced when compared with control binding. The (+)3H-MK801 binding in adults was spatially heterogeneous. Overall binding density in pars compacta was higher relative to binding density in pars reticulata with a mean percent change (Deltaxmacr;%) of 32%. Within the pars reticulata but not pars compacta, there were rostro-caudal differences with considerably denser binding in the posterior compared with the anterior pars reticulata (Deltaxmacr;%=108%). PN15 rats showed a less pronounced heterogeneity in pars compacta versus pars reticulata binding, (Deltaxmacr;%=27%), and less rostro-caudal differentiation in (+)3H-MK801 binding density throughout pars reticulata (Deltaxmacr;%=46%). 3H-glutamate binding in both adult and PN15 rats was less dense overall than (+)3H-MK801 binding. In adults, there was no difference in binding density between pars compacta and pars reticulata (Deltaxmacr;%=0.4%), but there were marked heterogeneities when binding was compared between anterior versus posterior pars compacta (Deltaxmacr;%=29%), and anterior versus posterior pars reticulata (Deltaxmacr;%=25%). This rostro-caudal heterogeneity in 3H-glutamate binding density was also present in PN15 pars compacta (Deltaxmacr;%=45%) but not in pars reticulata. Our findings mirror similar anterior/posterior heterogeneities in the GABAergic system in adult and PN15 male rats and may reflect a developmental change in both the structure and anticonvulsant/proconvulsant properties of substantia nigra pars reticulata (SNR) with age.

Aging↗

Gene regulation by patterned electrical activity during neural and skeletal muscle development.

Patterned neural activity modifies central synapses during development and the physiological properties of skeletal muscle by selectively repressing or stimulating transcription of distinct genes. The effects of neural activity are mostly mediated by calcium. Of particular interest are the cellular mechanisms that may be used to sense and convert changes in calcium into specific alterations in gene expression. Recent studies have addressed the importance of spatial heterogeneity or of temporal changes in calcium levels for the regulation of gene expression.

Action Potentials↗

Magnetic resonance imaging in cancer research.

Non-invasive assessment of antineoplastic response and correlation of the location, magnitude and duration of transgene expression in vivo would be particularly useful for evaluating cancer gene therapy protocols. This review presents selected examples of how magnetic resonance (MR) has been used to assess therapeutic efficacy by non-invasive quantitation of cell kill, to detect a therapeutic response prior to a change in tumour volume and to detect spatial heterogeneity of the tumour response and quantitate transgene expression. In addition, applications of the use of bioluminescence imaging (BLI) for the evaluation of treatment efficacy and in vivo transgene expression are also presented. These examples provide an overview of areas in which imaging of animal tumour models can contribute towards improving the evaluation of experimental therapeutic agents.

Animals↗

Genetic differentiation of neutral markers and quantitative traits in predominantly selfing metapopulations: confronting theory and experiments with Arabidopsis thaliana.

The comparison of the genetic differentiation of quantitative traits (QST) and molecular markers (FST) can inform on the strength and spatial heterogeneity of selection in natural populations, provided that markers behave neutrally. However, selection may influence the behaviour of markers in selfing species with strong linkage disequilibria among loci, therefore invalidating this test of detection of selection. We address this issue by monitoring the genetic differentiation of five microsatellite loci (FST) and nine quantitative traits (QST) in experimental metapopulations of the predominantly selfing species Arabidopsis thaliana, that evolved during eight generations. Metapopulations differed with respect to population size and selection heterogeneity. In large populations, the genetic differentiation of neutral microsatellites was much larger under heterogeneous selection than under uniform selection. Using simulations, we show that this influence of selection heterogeneity on FST can be attributable to initial linkage disequilibria among loci, creating stronger genetic differentiation of QTL than expected under a simple additive model with no initial linkage. We found no significant differences between FST and QST regardless of selection heterogeneity, despite a demonstrated effect of selection on QST values. Additional data are required to validate the role of mating system and linkage disequilibria in the joint evolution of neutral and selected genetic differentiation, but our results suggest that FST/QST comparisons can be conservative tests to detect selection in selfing species.

Arabidopsis↗

Long-term dynamics of natural populations of Schistosoma mansoni among Rattus rattus in patchy environment.

Dynamics of natural populations of Schistosoma mansoni were studied during 8 consecutive years among Rattus rattus populations from 8 transmission sites of the marshy forest focus of Guadeloupe (French West Indies). The schistosome population is over-dispersed (k = 0.119) within the murine hosts and ecological factors linked to the patchy environment may be responsible for such aggregated distribution. Analysis of the spatio-temporal variations in prevalences, intensities and abundances showed limited variations of the infection during the 8 years at the level of the whole parasite population but great spatial heterogeneity at the level of local schistosome populations. Inter-populational genetic variability linked to the degree of adaptation of this human parasite to the murine host may explain differences in transmission dynamics between the local populations of S. mansoni.

Animals↗

The evaluation of potential global morbidity attributable to intestinal nematode infections.

This paper presents a method of estimating the potential global morbidity due to human intestinal nematode infections (Ascaris lumbricoides, Trichuris trichiura and hookworms), based on the observed prevalence of infection. The method relies on the observed relationships between prevalence and intensity of infection, and between worm burden and potential morbidity. This approach is shown to be sensitive to the precision of the original prevalence estimates and, in particular, to the degree of spatial heterogeneity in levels of infection. The estimates presented here indicate that several tens of millions of children are likely to suffer developmental consequences from infection, and suggest that the global disease burden of geohelminthiasis may be significantly greater than was supposed.

Adolescent↗

Characterization of interlayer Cs+ in clay samples using secondary ion mass spectrometry with laser sample modification.

Ultraviolet laser irradiation was used to greatly enhance the secondary ion mass spectrometry (SIMS) detection of Cs(+) adsorbed to soil consisting of clay and quartz. Imaging SIMS showed that the enhancement of the Cs(+) signal was spatially heterogeneous: the intensity of the Cs(+) peak was increased by factors up to 100 for some particles but not at all for others. Analysis of standard clay samples exposed to Cs(+) showed a variable response to laser irradiation depending on the type of clay analyzed. The Cs(+) abundance was significantly enhanced when Cs(+)-exposed montmorillonite was irradiated and then analyzed using SIMS, which contrasted with the behavior of Cs(+)-exposed kaolinite, which displayed no Cs(+) enhancement. Exposed illitic clays displayed modest enhancement of Cs(+) upon laser irradiation, intermediate between that of kaolinite and montmorillonite. The results for Cs(+) were rationalized in terms of adsorption to interlayer sites within the montmorillonite, which is an expandable phyllosilicate. In these locations, Cs(+) was not initially detectable using SIMS. Upon irradiation, Cs(+) was thermally redistributed, which enabled detection using SIMS. Since neither the illite nor the kaolinite is an expandable clay, adsorption to inner-layer sites does not occur, and either modest or no laser enhancement of the Cs(+) signal is observed. Laser irradiation also produced unexpected enhancement of Ti(+) from illite and kaolinite clays that contained small quantities of Ti, which indicates the presence of microscopic titanium oxide phases in the clay materials.

Journal Article↗

Parallel chemical dosing of subcellular targets.

To characterize the role of spatially heterogeneous signaling in cellular function, methods are required for differentially exposing distinct regions of individual cells to externally applied reagents. Although a range of standard approaches exists for generating localized chemical gradients in culture, including puffer pipet spritzing and photolytic release of caged effectors, each is limited in key respects. Here, we report development of a cell-dosing strategy that addresses these limitations, providing the means to create steep gradients of any aqueous-miscible compound at essentially unlimited numbers of sites in parallel. In this approach, cells are cultured on a micrometer-thick polymer membrane that serves as a barrier between two stacked laminar-flow channels: one containing the cell culture and the other serving as a reagent flow cell. By focusing a pulsed laser beam onto one or more selected membrane positions, micrometer-diameter pores can be ablated upstream of desired cellular targets. Nascent pores thus serve as ports of entry into the culture environment for reagent streams capable of modifying subcellular features at positions potentially hundreds of micrometers from ablation sites. Importantly, individual reagent streams also can be rapidly eliminated by photo-cross-linking a protein plug over a selected pore. This versatile strategy for dynamically reshaping the chemical microenvironments in which cells reside should be useful in a variety of cell biology applications, ranging from neurotrophic modulation of neurite pathfinding to stimulation of cellular networks.

Cell Differentiation↗

Kinetic chain lengths in highly cross-linked networks formed by the photoinitiated polymerization of divinyl monomers: a gel permeation chromatography investigation.

Highly cross-linked networks formed by the photoinitiated polymerization of multifunctional monomers are finding application in the field of biomaterials because of their chemical versatility, reaction control, and ability to polymerize under physiological conditions. Typically, degradation is introduced into these networks via the cross-links and leads to the release of nondegradable but water-soluble kinetic chains formed during the chain polymerization process. In this study, gel permeation chromatography (GPC) was used to characterize kinetic chain length distributions in highly cross-linked systems that are being developed for orthopedic applications. By polymerizing divinyl monomers to various conversions and subsequently degrading them, we investigated the aspects of network structural evolution related to kinetic chain formation. In general, the average kinetic chain length increased with conversion until the onset of autodeceleration, when the kinetic chains decreased in length as the propagation reaction became diffusion-controlled. The distribution of kinetic chains also changed when different initiation conditions (i.e., initiator concentration and incident light intensity) were used, and a decrease in the kinetic chain lengths was observed at higher initiation rates. Finally, kinetic chain lengths were examined as a function of depth in thick samples polymerized with different light intensities and with a photobleaching initiator. Light attenuation through the sample led to different initiation rates as a function of depth and, consequently, spatial heterogeneity in the network structure as measured by the distributions of kinetic chains.

Butadienes↗

Plutonium partitioning to colloidal and particulate matter in an acidic, sandy sediment: implications for remediation alternatives and plutonium migration.

Plutonium partitioning within a bulk, freshwater sediment and to specific size fractions of the sediment was determined. Fission track analysis was used to observe the spatial heterogeneity of fissile isotopes of Pu and U in the sediment. For the bulk sediment, a six-step sequential extraction scheme was used to elucidate Pu partitioning. Although no direct Pu speciation is obtained from our sequential extraction scheme, this study demonstrates that the partitioning information obtained is more useful for evaluating ex-situ remediation treatments than information obtained from complete digestion and analysis of the bulk sediment. The majority of 238Pu and 239 + 240Pu appear to be partitioned in the oxidizable fraction, suggesting that Pu is primarily associated with organic matter in the bulk sediment or may exist as a discrete, oxidizable phase. By varying filter pore size used to separate the sequential extraction leachate solution from the remaining solid phase, a fraction of 238Pu associated with colloidal material was observed, and chemical evidence suggests that this colloidal material is relatively refractory. Pu partitioning to various size fractions of the bulk sediment was also compared to the percent organic carbon present in those size fractions. Interestingly, little correlation was observed between the percentage of organic carbon and concentrations of Pu isotopes in the various size fractions, although differences were observed in the distributions of 238Pu versus 239 + 240Pu in the size fractions. These results suggest that other sediment phases may also be important for Pu partitioning. Our observations are described in the context of feasibility of various remediation options.

Colloids↗

Formation of ferrihydrite and associated iron corrosion products in permeable reactive barriers of zero-valent iron.

Ferrihydrite, which is known to form in the presence of oxygen and to be stabilized by the adsorption of Si, PO4 and SO4, is ubiquitous in the fine-grained fractions of permeable reactive barrier (PRB) samples from the U.S. Coast Guard Support Center (Elizabeth City, NC) and the Denver Federal Center (Lakewood, CO) studied by high-resolution transmission electron microscopy and selected area electron diffraction. The concurrent energy-dispersive X-ray data indicate a strong association between ferrihydrite and metals such as Si, Ca, and Cr. Magnetite, green rust 1, aragonite, calcite, mackinawite, greigite and lepidocrocite were also present, indicative of a geochemical environment that is temporally and spatially heterogeneous. Whereas magnetite, which is known to form due to anaerobic Fe0 corrosion, passivates the Fe0 surface, ferrihydrite precipitation occurs away from the immediate Fe0 surface, forming small (<0.1 microm) discrete clusters. Consequently, Fe0-PRBs may remain effective for a longer period of time in slightly oxidized groundwater systems where ferrihydrite formation occurs compared to oxygen-depleted systems where magnetite passivation occurs. The ubiquitous presence of ferrihydrite suggests that the use of Fe0-PRBs may be extended to applications that require contaminant adsorption rather than, or in addition to, redox-promoted contaminant degradation.

Adsorption↗