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The sequencing platform and workflow strongly influence microbial community analyses through potential errors at each step. Effective diagnostics and experimental controls are needed to validate data and improve reproducibility. This cross-laboratory study evaluates sources of variability and error at three main steps of a standardized amplicon sequencing workflow (DNA extraction, polymerase chain reaction [PCR], and sequencing) using Oxford Nanopore MinION to analyze agricultural soils and a simple mock community. Variability in sequence results occurs at each step in the workflow with PCR errors and differences in library size greatly influencing diversity estimates. Common bioinformatic diagnostics and the mock community are ineffective at detecting PCR abnormalities. This work outlines several diagnostic checks and techniques to account for sequencing depth and ensure accuracy and reproducibility in soil community analyses. These diagnostics and the inclusion of a reference soil can help ensure data validity and facilitate the comparison of multiple sequencing runs within and between laboratories., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Background: Soil microbial communities are difficult to measure and critical to soil processes. The bulk soil microbiome is highly diverse and spatially heterogeneous, which can make it difficult to detect and monitor the responses of microbial communities to differences or changes in management, such as different crop rotations in agricultural research. Sampling a subset of actively growing microbes should promote monitoring how soil microbial communities respond to management by reducing the variation contributed by high microbial spatial and temporal heterogeneity and less active microbes. We tested an in-growth bag method using sterilized soil in root-excluding mesh, "sterile sentinels," for the capacity to differentiate between crop rotations. We assessed the utility of different incubation times and compared colonized sentinels to concurrently sampled bulk soils for the statistical power to differentiate microbial community composition in low and high diversity crop rotations. We paired this method with Oxford Nanopore MinION sequencing to assess sterile sentinels as a standardized, fast turn-around monitoring method., Results: Compared to bulk soil, sentinels provided greater statistical power to distinguish between crop rotations for bacterial communities and equivalent power for fungal communities. The incubation time did not affect the statistical power to detect treatment differences in community composition, although longer incubation time increased total biomass. Bulk and sentinel soil samples contained shared and unique microbial taxa that were differentially abundant between crop rotations., Conclusions: Overall, compared to bulk soils, the sentinels captured taxa with copiotrophic or ruderal traits, and plant-associated taxa. The sentinels show promise as a sensitive, scalable method to monitor soil microbial communities and provide information complementary to traditional soil sampling., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Crop rotation is an important management tactic that farmers use to manage crop production and reduce pests and diseases. Long-term crop rotations may select groups of microbes that form beneficial or pathogenic associations with the following crops, which could explain observed crop yield differences with different crop sequences. To test this hypothesis, we used two locations each with four long-term (12-14-year), replicated, rotation treatments: continuous corn (CCC), corn/corn/soybean (SCC), corn/soybean (CSC), and soybean/corn (SCS). Afterwards, soybean was planted, and yield and soil health indicators, bulk soil microbiome, and soybean root-associated microbiome were assessed. Soybean yields, as well as soil protein, and POXC as soil health indicators were higher following CCC than in the other three treatments at both locations. A bacterial taxon in family JG30-KF-AS9 was enriched in CCC, whereas Microvirga, Rhodomicrobium, and Micromonosporaceae were enriched in SCS. Several ascomycetes explain lowered yield as soybean pathogens in SCS. Surprisingly, Tumularia, Pyrenochaetopsis and Schizothecium were enriched in soybean roots after CCC, suggesting corn pathogens colonizing soybean roots as nonpathogens. Our finding of associations between soil health indicators related to microbiomes and soybean yield has wide-ranging implications, opening the possibility of manipulating microbiomes to improve crop yield potential.

Arbuscular mycorrhizal (AM) fungi are considered to be a key group of soil organisms for assessments of soil biological properties and developing relationships among crop production management practices, soil properties, crop performance, and ecosystem services. In a field study of cover crop treatments established during the transition from small grains to corn (Zea mays L), we assessed multiple measures of AM fungal responses to the management treatments: soil propagule numbers, biomass via lipid biomarkers, and root colonization extent. Our objectives were to determine response variables that reliably distinguished cover crop treatments and formed consistent relationships with grain yield, plant biomass, and mineral nutrient concentrations of the following corn crop. The number of soil AM fungal propagules and amount of the NLFA biomarker C16:1cis11 measured on fall-collected soils most consistently and significantly responded to fall cover crop treatments. Neither of these measures of soil inoculum potential was strongly related to measures of crop performance. The PLFA biomarker C16:1cis11 was marginally responsive to cover crop but did not strongly relate to crop performance parameters. Corn root colonization by AM fungi was not significantly affected by cover crop treatment, but significant negative relationships were found between root colonization and grain N concentration and plant biomass at maturity. In contrast, a significant positive relationship between root colonization and plant N concentration at the 6-leaf stage was found. Understanding the relative effectiveness and limitations of AM fungal response variables will inform their application in field studies of agricultural management practices.

Excessive crop residue returned to the soil hinders farm operations, but residue removal can affect soil quality. In contrast, cover cropping can return additional residue to the soil and improve soils and environmental quality compared with no cover cropping. Residue and cover crop impacts on soil surface greenhouses gas (GHG) emissions are undetermined and site specific. Thus, the present study was conducted to investigate the impacts of corn ( L.) residue management and cover cropping on GHG fluxes. The fluxes were measured from 2013 to 2015 using static chamber under corn and soybean [ (L.) Merr.] rotation initiated in 2000 at Brookings, SD. Treatments included two residue management levels (residue returned [RR] and residue not returned [RNR]) and two cover cropping (cover crops [CC] and no cover crops [NCC]). Results showed that RR under corn and soybean phases significantly reduced cumulative CO fluxes (2681.3 kg ha in corn and 2419.8 kg ha in soybeans) compared with RNR (3331.0 kg ha in corn and 2755.0 kg ha in soybeans) in 2013. The RR emitted significantly less cumulative NO fluxes than RNR from both the phases in 2013 and 2014, but not in 2015. The CC treatment had significantly lower cumulative NO fluxes than the NCC for corn and soybean phases in 2013 and 2014. We conclude that crop residue retention and cover cropping can mitigate the GHG emissions compared with residue removal and no cover cropping., (Copyright © by the American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America, Inc.)

To evaluate crop rotation effects on maize seedling performance and its associated microbiome, maize plants were grown in the greenhouse in soils preceded by either maize, pea, soybean or sunflower. Soils originated from a replicated field experiment evaluating different four-year rotation combinations. In the greenhouse, a stressor was introduced by soil infestation with western corn rootworm (WCR) or Fusarium graminearum. Under non-infested conditions, maize seedlings grown in soils preceded by sunflower or pea had greater vigor. Stress with WCR or F. graminearum resulted in significant root damage. WCR root damage was equivalent for seedlings regardless of soil provenance; whereas F. graminearum root damage was significantly lower in maize grown in soils preceded by sunflower. Infestation with WCR affected specific microbial taxa (Acinetobacter, Smaragdicoccus, Aeromicrobium, Actinomucor). Similarly, F. graminearum affected fungal endophytes including Trichoderma and Endogone. In contrast to the biological stressors, rotation sequence had a greater effect on rhizosphere microbiome composition, with larger effects observed for fungi compared to bacteria. In particular, relative abundance of Glomeromycota was significantly higher in soils preceded by sunflower or maize. Defining the microbial players involved in crop rotational effects in maize will promote selection and adoption of favorable crop rotation sequences.

Killifish (Fundulus heteroclitus) are euryhaline teleosts that are widely used in environmental and toxicological studies, and they are tolerant to arsenic, in part due to very low assimilation of arsenic from the environment. The mechanism of arsenic uptake by the intestine, a major route of arsenic uptake in humans is unknown. Thus, the goal of this study was to determine if aquaglyceroporins (AQPs), which transport water and other small molecules including arsenite across cell membranes, are expressed in the killifish intestine, and whether AQP expression is affected by osmotic stress. Through RT-PCR and sequence analysis of PCR amplicons, we demonstrated that the intestine expresses kfAQP3a and kfAQP3b, two previously identified variants, and also identified a novel variant of killifish AQP3 (kfAQP3c) in the intestine. The variants likely represent alternate splice forms. A BLAST search of the F. heteroclitus reference genome revealed that the AQP3 gene resides on a single locus, while an alignment of the AQP3 sequence among 384 individuals from eight population ranging from Rhode Island to North Carolina revealed that its coding sequence was remarkably conserved with no fixed polymorphism residing in the region that distinguishes these variants. We further demonstrate that the novel variant transports arsenite into HEK293T cells. Whereas kfAQP3a, which does not transport arsenite, was expressed in both freshwater (FW) and saltwater (SW) acclimated fish, kfAQP3b, an arsenic transporter, was expressed only in FW acclimated fish, and kfAQP3c was expressed only in SW acclimated fish. Thus, we have identified a novel, putative splice variant of kfAQP3, kfAQP3c, which transports arsenic and is expressed only in SW acclimated fish., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The nutrients found in prey and nonprey foods, and relative digestibility of these foods, has a major influence on diet selection by omnivorous insects. Many insects have developed symbiotic relationships with gut bacteria to help with extracting nutrition from nonprey diets. Gryllus pennsylvanicus (Burmeister) (Orthoptera: Gryllidae) was assigned to one of two treatment groups, antibiotic-treated and nonantibiotic-treated, and consumption of seeds (nonprey) and eggs (prey) were measured. Male crickets administered antibiotics consumed more seeds and greater seed weight, while antibiotic-fed female crickets consumed fewer seeds and less seed weight, relative to the untreated male and female crickets, respectively. Both male and female antibiotic-treated crickets consumed similar weight of eggs as nonantibiotic-treated male and female crickets, respectively. These results provide evidence that gut symbionts influence diet selection of male and female G. pennsylvanicus differently. This sex-specific dietary selection may be because of the fact that male and female crickets have different nutritional requirements.

The first human stereotactic surgery based on intracerebral landmarks and Cartesian coordinates was performed in 1947. With this followed the publication of a number of stereotactic frames and atlases. The intercommissural line joining the anterior and posterior commissures was to define stereotactic coordinate systems used in movement disorders and other functional neurosurgical procedures. Initially the target for Parkinson disease was the globus pallidus internus (GPi), but many investigators soon turned to the thalamus or parts of the subthalamus, but not the subthalamic nucleus. Microelectrode recording was introduced in 1961. With the apparent clinical efficacy of L-DOPA in 1965 interest in stereotactic surgery for Parkinson disease declined. The failure of prolonged, consistent pharmacologic management of bradykinesia and tremor, the side effects of dyskinesias, and the fading therapeutic success of medical treatment of movement disorders led to a resurgence of interest in the surgical management of movement disorders. With advances in understanding of the functional anatomy of the corticobasal ganglia circuit, advances in brain imaging, more sophisticated electrophysiologic recordings, and the use of deep brain stimulation as a reversible lesion, stereotactic surgery returned as a viable option for the treatment of movement disorders. The posterior medial part of the globus pallidus, ventral intermediate nucleus of the thalamus, and the subthalamus, its nuclei and pathways, are sites for interrupting pathophysiologic circuits. Not only has this been applied to movement disorders, but to epilepsy, chronic pain, and behavioral disorders., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Background: The storability of distillers wet grains (DWG) influences the economic, energetic, and carbon balances of fuel ethanol production, yet there are limited published data on the deterioration of DWG following its production. We used biogenic CO(2) production to assess the aerobic stability of DWG incubated at three temperatures (12 °C, 22 °C, 32 °C) and compared CO(2) production over time to the appearance of mold and changes in DWG color parameters., Results: CO(2) production and mold colonization indicate that at temperatures near 12 °C, the aerobic stability of DWG was high and that it can be stored for at least a 10-day period. At temperatures close to 22 °C, the onset of increased microbial activity and visible mold colonization occurred between 4 and 7 days and both activity and mold ratings were very high by the ninth day in all three experiments. At 32 °C, 2 days may be a more appropriate limit for storage., Conclusion: Temperature and time interact in a nonlinear fashion that permits the prediction of DWG stability boundaries. The simple visual appearance of mold appears to be a reasonable indicator that correlates well (r = 0.694) with CO(2) production, a measure of the aerobic stability of DWG., (Published 2012 by John Wiley & Sons, Ltd.)

Background: Obligate bacterial symbionts alter the diets of host animals in numerous ways, but the ecological roles of facultative bacterial residents that colonize insect guts remain unclear. Carabid beetles are a common group of beneficial insects appreciated for their ability to consume insect prey and seeds, but the contributions of microbes to diet diversification in this and similar groups of facultative granivores are largely unknown., Methodology and Principal Findings: Using 16S rRNA gene clone libraries and terminal restriction fragment (tRF) length polymorphism analyses of these genes, we examined the bacterial communities within the guts of facultatively granivorous, adult Harpalus pensylvanicus (Carabidae), fed one of five dietary treatments: 1) an untreated Field population, 2) Seeds with antibiotics (seeds were from Chenopodium album), 3) Seeds without antibiotics, 4) Prey with antibiotics (prey were Acheta domesticus eggs), and 5) Prey without antibiotics. The number of seeds and prey consumed by each beetle were recorded following treatment. Harpalus pensylvanicus possessed a fairly simple gut community of approximately 3-4 bacterial operational taxonomic units (OTU) per beetle that were affiliated with the Gammaproteobacteria, Bacilli, Alphaproteobacteria, and Mollicutes. Bacterial communities of the host varied among the diet and antibiotic treatments. The field population and beetles fed seeds without antibiotics had the closest matching bacterial communities, and the communities in the beetles fed antibiotics were more closely related to each other than to those of the beetles that did not receive antibiotics. Antibiotics reduced and altered the bacterial communities found in the beetle guts. Moreover, beetles fed antibiotics ate fewer seeds, and those beetles that harbored the bacterium Enterococcus faecalis consumed more seeds on average than those lacking this symbiont., Conclusions/significance: We conclude that the relationships between the bacterium E. faecalis and this factultative granivore's ability to consume seeds merit further investigation, and that facultative associations with symbiotic bacteria have important implications for the nutritional ecology of their hosts.

Ground beetles such as Poecilus chalcites (Coleoptera: Carabidae) are beneficial insects in agricultural systems where they contribute to the control of insect and weed pests. We assessed the complexity of bacterial communities occurring in the digestive tracts of field-collected P. chalcites using terminal restriction fragment length polymorphism analyses of polymerase chain reaction-amplified 16S rRNA genes. Bacterial identification was performed by the construction of 16S rRNA gene clone libraries and sequence analysis. Intestinal bacteria in field-collected beetles were then compared to those from groups of beetles that were reared in the lab on an artificial diet with and without antibiotics. Direct cell counts estimated 1.5x10(8) bacteria per milliliter of gut. The digestive tract of field-collected P. chalcites produced an average of 4.8 terminal restriction fragments (tRF) for each beetle. The most abundant clones were affiliated with the genus Lactobacillus, followed by the taxa Enterobacteriaceae, Clostridia, and Bacteriodetes. The majority of the sequences recovered were closely related to those reported from other insect gastrointestinal tracts. Lab-reared beetles produced fewer tRF, an average of 3.1 per beetle, and a reduced number of taxa with a higher number of clones from the family Enterobacteriaceae compared to the field-collected beetles. Antibiotic treatment significantly (p<0.05) reduced the number of tRF per beetle and selected for a less diverse set of bacterial taxa. We conclude that the digestive tract of P. chalcites is colonized by a simple community of bacteria that possess autochthonous characteristics. Laboratory-reared beetles harbored the most common bacteria found in field-collected beetles, and these bacterial communities may be manipulated in the laboratory with the addition of antibiotics to the diet to allow study of functional roles.

Distillers grains are coproduced with ethanol and carbon dioxide during the production of fuel ethanol from the dry milling and fermentation of corn grain, yet there is little basic microbiological information on these materials. We undertook a replicated field study of the microbiology of distillers wet grains (DWG) over a 9 day period following their production at an industrial fuel ethanol plant. Freshly produced DWG had a pH of about 4.4, a moisture content of about 53.5% (wet mass basis), and 4 x 10(5) total yeast cells/g dry mass, of which about 0.1% were viable. Total bacterial cells were initially below detection limits (ca. 10(6) cells/g dry mass) and then were estimated to be approximately 5 x 10(7) cells/g dry mass during the first 4 days following production. Culturable aerobic heterotrophic organisms (fungi plus bacteria) ranged between 10(4) and 10(5) CFU/g dry mass during the initial 4 day period, and lactic acid bacteria increased from 36 to 10(3) CFU/g dry mass over this same period. At 9 days, total viable bacteria and yeasts and (or) molds topped 10(8) CFU/g dry mass and lactic acid bacteria approached 10(6) CFU/g dry mass. Community phospholipid fatty acid analysis indicated a stable microbial community over the first 4 days of storage. Thirteen morphologically distinct isolates were recovered, of which 10 were yeasts and molds from 6 different genera, 2 were strains of the lactic-acid-producing Pediococcus pentosaceus and only one was an aerobic heterotrophic bacteria, Micrococcus luteus. The microbiology of DWG is fundamental to the assessment of spoilage, deleterious effects (e.g., toxins), or beneficial effects (e.g., probiotics) in its use as feed or in alternative applications.

Purpose: Chronic cellular inflammation closely associated with epilepsy without an active infection is a hallmark of Rasmussen encephalitis (RE). RE has typical and defining features lacking in other rare epilepsy patients who also have neocortical lymphocytes without an identifiable cause. A patient with malformations of cortical development had an abrupt change in frequency and epileptic focus after 22 years of a stable seizure disorder. Functional neurosurgery yielded a specimen showing a mixed cellular meningoencephalitis in the absence of a demonstrable infection., Methods: Historical, neurologic, electroencephalographic, pathologic, and literature data were correlated., Results: There was a subarachnoid mixed infiltrate including evidence of dendritic cells in our patient and also cytotoxic T lymphocytes adjacent to karyolytic neurons that corresponded to cells previously demonstrated to damage neurons in RE. Literature review disclosed 42 other cases similar to RE but with heterogeneous findings. The course was more protracted and often more benign than in RE. The inflammation that would have markedly decreased or disappeared in RE over that period was generally still well represented., Conclusions: Our patient has heterogeneous features similar to, yet with differences from, RE. Literature review of chronic cellular inflammatory epileptic encephalopathy cases also similar to RE discloses important differences that may reflect idiosyncratic reactions and pace of the disease rather than a different disease. Comorbidity factors, genetic population traits, and secondary effects of the seizure disorder may lead to an expansion of the initial site of damage by an autoimmune reaction. These cases might best be grouped, probably along with RE, as secondary autoimmune diseases.

The rapid advances in the technology of, and accumulation of pertinent data in, electrophysiological testing has increased exponentially in the past decade. This is attributable to continued advances in computer technology, biomedical engineering, and now the coregistration of the electrophysiological data with neuroimaging results. Knowledge of normal function and electrophysiological response at rest or on stimulation of the central and peripheral nervous systems is important to the neurosurgeon. Only by a basic understanding of normal and abnormal recordings may diagnoses and localizations be achieved. Intraspinal and intracranial surgical procedures are predicated on nontrauma to the neuraxis. This can be accomplished by performing electrophysiological testing to monitor the function of the spinal and cranial nerves, spinal cord, brainstem, basal ganglia, and cerebrum. If the surgeon cannot delineate critical cortex or pathways, he or she will be unable to avoid these areas in the patient.

3-hydroxyphenylacetylene (3-HPA) served as a novel, activity-dependent, fluorogenic and chromogenic probe for bacterial enzymes known to degrade toluene via meta ring fission of the intermediate, 3-methylcatechol. By this direct physiological analysis, cells grown with an aromatic substrate to induce the synthesis of toluene-degrading enzymes were fluorescently labeled.

We report the use of a surface analysis approach, static secondary ion mass spectrometry (SIMS) equipped with a molecular (ReO(4)(-)) ion primary beam, to analyze the surface of intact microbial cells. SIMS spectra of 28 microorganisms were compared to fatty acid profiles determined by gas chromatographic analysis of transesterfied fatty acids extracted from the same organisms. The results indicate that surface bombardment using the molecular primary beam cleaved the ester linkage characteristic of bacteria at the glycerophosphate backbone of the phospholipid components of the cell membrane. This cleavage enables direct detection of the fatty acid conjugate base of intact microorganisms by static SIMS. The limit of detection for this approach is approximately 10(7) bacterial cells/cm(2). Multivariate statistical methods were applied in a graded approach to the SIMS microbial data. The results showed that the full data set could initially be statistically grouped based upon major differences in biochemical composition of the cell wall. The gram-positive bacteria were further statistically analyzed, followed by final analysis of a specific bacterial genus that was successfully grouped by species. Additionally, the use of SIMS to detect microbes on mineral surfaces is demonstrated by an analysis of Shewanella oneidensis on crushed hematite. The results of this study provide evidence for the potential of static SIMS to rapidly detect bacterial species based on ion fragments originating from cell membrane lipids directly from sample surfaces.

Groundwater from an oxic, fractured basalt aquifer was examined for the presence of Archaea. DNA was extracted from cells concentrated from groundwater collected from five wells penetrating the eastern Snake River Plain Aquifer (Idaho, USA). Polymerase chain reaction (PCR) amplification of 16S rDNA was performed with Archaea-specific primers using both nested (ca. 200-bp product) and direct (ca. 600-bp product) PCR approaches. Estimates of the archaeal diversity were made by separating PCR products from all five wells by denaturing gradient gel electrophoresis (DGGE) and phylogenetic analysis of partial 16S rDNA sequences from two wells was performed following cloning procedures. Archaea were detected in all wells and the number of DGGE bands per well ranged from two to nine and varied according to PCR approach. There were 30 unique clonal 16S rDNA partial sequences (ca. 600 bp) within a total of 100 clones that were screened from two wells. Twenty-two of the 16S rDNA fragments recovered from the aquifer were related to the Crenarchaeota and Euryarchaeota kingdoms (one large clade of clones in the former and six smaller clades in the latter), with sequences ranging from 23.7 to 95.4% similar to those found in other investigations. The presence of potentially thermophilic or methanogenic Archaea in this fully oxic aquifer may be related to deep thermal sources or elevated dissolved methane concentrations. Many sequences were similar to those that represent non-thermophilic Crenarchaeota of which there are no known cultured members and therefore no putative function.

Free-living and surface-associated microbial communities in sand-packed columns perfused with groundwater were compared by examination of compositional and functional characteristics. The composition of the microbial communities was assessed by bulk DNA extraction, PCR amplification of 16S ribosomal DNA fragments, separation of these fragments by denaturing gradient gel electrophoresis, and sequence analysis. Community function was assessed by measurement of beta-glucosidase and aminopeptidase extracellular enzyme activities. Free-living populations in the aqueous phase exhibited a greater diversity of phylotypes than populations associated with the solid phase. The attached bacterial community displayed significantly greater beta-glucosidase and aminopeptidase enzyme activities per volume of porous medium than those of the free-living community. On a per-cell basis, the attached community had a significantly higher cell-specific aminopeptidase enzyme activity (1.07 x 10(-7) nmol cell(-1) h(-1)) than the free-living community (5.02 x 10(-8) nmol cell(-1) h(-1)). Conversely, the free-living community had a significantly higher cell-specific beta-glucosidase activity (1.92 x 10(-6) nmol cell(-1) h(-1)) than the surface-associated community (6.08 x 10(-7) nmol cell(-1) h(-1)). The compositional and functional differences observed between these two communities may reflect different roles for these distinct but interacting communities in the decomposition of natural organic matter or biodegradation of xenobiotics in aquifers.

Bench scale column studies were used to examine the partitioning of microorganisms between groundwater and a geologic medium and to examine the effect of hydrogeology (i.e., porous- versus fracture-flow) on organism partitioning. Replicated columns were constructed with intact basalt core segments that contained natural fractures and with the same basalt crushed into particles. The columns were perfused with groundwater, and upon reaching a steady state, the columns were sacrificed and the attached and unattached communities were analyzed by multiple approaches. The analyses included the total number of cells, the phylogenetic affiliation of the cells (i.e., the alpha, beta, and gamma subclasses of the class Proteobacteria and gram positives with high G+C DNA content) by fluorescent in situ hybridization (FISH), number and taxonomic affiliation by fatty acid methyl ester profiles of culturable heterotrophs, most-probable-number estimates of methanotrophs and phenol oxidizers, and whole-community sole carbon source utilization patterns from Biolog GN microplates. In the packed columns, about 99% of the total biomass (per cubic centimeter of porous medium) was attached to the geologic medium. Lack of equitable units precluded a comparison of attached and unattached biomasses in the fractured columns where the attached biomass was expressed per unit of surface area. Compositional differences in the attached and unattached communities were evidenced by (i) the recovery of Pseudomonas stutzeri, an Enterococcus sp., and Bacillus psychrophilus from the groundwater and not from the basalt, (ii) differences between community carbon source utilization patterns, and (iii) the relative abundances of different phylogenetic groups estimated by FISH in both column types. In the packed columns, attached communities were depleted of members of the alpha- and beta-Proteobacteria subclasses in comparison to those in the corresponding groundwater. In the fractured columns, attached communities were enriched in gram-positive Bacteria and gamma-Proteobacteria and depleted of beta-Proteobacteria, in comparison to those in the corresponding groundwater. Segregation of populations and their activities, possibly modified by attachment to geologic media, may influence contaminant fate and transport in the subsurface and impact other in situ applications.

The bacteria colonizing geologic core sections (attached) were contrasted with those found suspended in the groundwater (unattached) by examining the microbiology of 16 depth-paired core and groundwater samples using a suite of culture-independent and culture-dependent analyses. One hundred twenty-two meters was continuously cored from a buried chalcopyrite ore hosted in a biotite-quartz-monzonite porphyry at the Mineral Park Mine near Kingman, Ariz. Every fourth 1.5-m core was acquired using microbiologically defensible methods, and these core sections were aseptically processed for characterization of the attached bacteria. Groundwater samples containing unattached bacteria were collected from the uncased corehole at depth intervals corresponding to the individual cores using an inflatable straddle packer sampler. The groundwater was acidic (pH 2.8 to 5.0), with low levels of dissolved oxygen and high concentrations of sulfate and metals, including ferrous iron. Total numbers of attached cells were less than 10(5) cells g of core material(-1) while unattached cells numbered about 10(5) cells ml of groundwater(-1). Attached and unattached acidophilic heterotrophs were observed throughout the depth profile. In contrast, acidophilic chemolithotrophs were not found attached to the rock but were commonly observed in the groundwater. Attached communities were composed of low numbers (<40 CFU g(-1)) of neutrophilic heterotrophs that exhibited a high degree of morphologic diversity, while unattached communities contained higher numbers (ca. 10(3) CFU ml(-1)) of neutrophilic heterotrophs of limited diversity. Sulfate-reducing bacteria were restricted to the deepest samples of both core and groundwater. 16S ribosomal DNA sequence analysis of attached, acidophilic isolates indicated that organisms closely related to heterotrophic, acidophilic mesophiles such as Acidiphilium organovorum and, surprisingly, to the moderately thermophilic Alicyclobacillus acidocaldarius were present. The results indicate that viable (but possibly inactive) microorganisms were present in the buried ore and that there was substantial distinction in biomass and physiological capabilities between attached and unattached populations.

A BASIC program is offered that reduces data resulting from mixed-species inoculations into Biolog microplates. The procedures of the program are supported by a critical review of the literature relating to Biolog data reduction. The availability of standardized, accelerated data reduction protocols will facilitate study comparisons and allow efficient evaluation of new data reduction approaches.

Objectives: A quantitative on-line analysis of electrical activity in the pallidum of Parkinsonian patients has been developed to determine the focal point of lesioning. Additional recordings are made after the lesioning, to assess residual neural activity. A 3-D volume stereoscopic image system is proposed to display the complex anatomy and to superimpose the electrophysiological data into this system. The purpose of this study is to understand the complex pathophysiology in real-time anatomic/image space and determine the location and effect of lesioning residual energy sites., Material and Methods: Thirty patients undergoing 41 pallidotomies are presented. Neuronal activity from the pallidum is recorded using a semi-microelectrode. Based on this activity, lesioning is performed. Post-lesion recordings are made to determine the necessity of additional lesioning. 3-D volume MR images are acquired pre and postoperatively and compared for accuracy of lesion sites. A 3-D stereoscopic image system has been developed to depict basal ganglia geometry in the last 5 patients. Electrophysiological data are superimposed on this image system to show the surgeon the virtual position of the electrode tip., Results: A stereoscopic 3-D volume MR image system has been developed. This system more efficiently and accurately visualizes and records the coordinates of high neural activity in the pallidum and post lesion residual activity. Post-lesion power analysis was carried out in 30 patients. Additional lesions were indicated in 8 and different trajectories in 6., Conclusion: Real-time visualization of neural recording, both pre and post-lesioning during pallidotomy, facilitates the surgeon's understanding of the spatiotemporal relationships of pathophysiological properties within the globus pallidus.

A computerized method of determining the focal point of electrical activity in the pallidum of parkinsonian patients was developed using on-line quantitative physiological data analysis. Thirty patients in a series of 70 were studied in depth. Neuronal activity was recorded from the pallidum using a semi-microelectrode. The signal is inspected visually while its average power, characteristic frequency and complexity are computed. The target locus was indicated by the highest level of global activity in the vicinity of the electrode (signal power maximum), maximal signal complexity and minimal characteristic frequency. Most often, the vertical coordinate required correction. The postoperative clinical and imaging results have indicated the effectiveness of this method., (Copyright 2001 S. Karger AG, Basel)

Recent interest in microbial diversity has led to increased emphasis on the development of appropriate techniques. Structural diversity encompasses the number and distribution of separate or interacting biological entities responsible for a given function within the overall set of functions of a community. This study evaluated an approach for estimating the relative degree of structural diversity in heterotrophic microbial communities by dilution to extinction of community phenotypic traits. Serial dilutions of environmental samples (rhizosphere, stream) were tested for community phenotypic traits (i.e. carbon source respiration). The non-linear relationship between the number of positive responses (i.e. functional richness or R) and inoculum density in each sample dilution (I) fit the simple rectangular hyperbola model, allowing estimation of the maximal richness (R(max)) and the inoculum density at half-maximal richness (K(I)). The later term appears to be useful in assessing relative structural diversity as evidenced by significantly higher values for communities with higher predicted species diversity. The examination of community functional characteristics across a series of dilutions, particularly in conjunction with other techniques, may be a useful approach for the study of microbial diversity and related ecological parameters such as niche width and metabolic redundancy.

A quantitative on-line analysis of electrical activity in the pallidum of Parkinsonian patients has been developed to determine the focal point of lesioning. A 3D volume image system has been developed to display basal ganglia anatomy and coregister the electrophysiological data within the globus pallidus. Thirty patients undergoing 41 pallidotomies are presented. Neuronal activity from the pallidum is recorded using a semi-microelectrode. Based on this activity, lesioning is performed. Postlesion recordings are made to determine the necessity of additional lesioning. A stereoscopic 3D volume MR image system has been developed that along with on-line signal processing allows visualization of high neural activity in the pallidum and postlesion residual activity., (Copyright 2000 S. Karger AG, Basel)

Microbial abundance, activity, and community-level physiological profiles (CLPP) were examined at centimeter and meter scales in the subsurface environment at a site near Oyster, VA. At the centimeter scale, variations in aerobic culturable heterotrophs (ACH) and glucose mineralization rates (GMR) were highest in the water table zone, indicating that water availability has a major effect on variations in microbial abundance and activity. At the meter scale, ACH and microaerophiles decreased significantly with depth, whereas anaerobic GMR often increased with depth; this may indicate low redox potentials at depth caused by microbial consumption of oxygen. Data of CLPP indicated that the microbial community (MC) in the soybean field exhibited greater capability to utilize multiple carbon sources than MC in the corn field. This difference may reflect nutrient availability associated with different crops (soybean vs corn). By using a regression model, significant spatial and temporal variations were observed for ACH, microaerophiles, anaerobic GMR, and CLPP. Results of this study indicated that water and nutrient availability as well as land use could have a dominant effect on spatial and temporal variations in microbial properties in shallow subsurface environments.

The anterior surgical approach to the cervicothoracic spine presents a challenge to the spine surgeon. To adequately explore the anterior spine from C4 to T4 requires a midsternotomy with extended anterior cervical incision. This approach most adequately provides the extensive cranial-caudal exposure required in dealing with tumors at the cervicothoracic junction. Our surgical experience with five patients harboring tumors in this area of the spine has led us to reflect on the anatomy of the exposure and the corridors of approach at various levels of the cervicothoracic junction. Cadaver dissection and demonstration of the anatomic structures encountered in this exposure and the spinal subdivisions created by these various neurovascular structures will be presented. The anatomy, so demonstrated, will be correlated with the clinical cases.

Frameless 3D volume registration of Magnetic Resonance (MR) and computed (CT) data sets has been described by Kummar et al. [11]. Its use in 3D volume registration for stereotactic planning in patients undergoing pallidotomy is presented. Pre-operative examinations with the stereotactic frame and postoperative examinations without the stereotactic frame can be co-registered and reviewed for accuracy of planned and lesional coordinates.

A 17-year-old male with insulin-dependent diabetes mellitus was referred because of difficulties with diabetic control. Since his diagnosis at age 10, he has been hospitalized more than 60 times for diabetes or its complications, mostly ketoacidosis. He also has short stature, pubertal delay, and hepatomegaly, and on exam was uncooperative and hostile. The long-standing practice of binging and purging followed by vomiting was revealed. His condition was consistent with Mauriac syndrome. Addressing an associated eating disorder may improve diabetes control, but this combination significantly increases the risk of diabetic complications.

Focal seizures arising in the central area require precise anatomic and physiologic mapping of ictal onset. The central sulcus is identified by the callosal grid system whose mid-vertical plane identifies the central sulcus inferiority where the central artery passes into the central sulcus. 5 patients with intractable seizures of central origin where localized with this method. Extent of resection was confirmed on postoperative NRI. The subdural grid was accurately placed on the central sulcus, confirmed by electrophysiologic means. Grid planes compartmentalized ictal onset, and post-operative resection correlated precisely. All patients are seizure-free. Seizures arising in the central area, precisely located, can be treated with good to excellent results. Localization of onset is facilitated by use of the callosal grid system, and allows superimposition of pre-, intra- and post-operative anatomic and physiologic data.