Automated detection of ripple oscillations in long-term scalp EEG from patients with infantile spasms.

Objective.Scalp high-frequency oscillations (HFOs) are a promising biomarker of epileptogenicity in infantile spasms (IS) and many other epilepsy syndromes, but prior studies have relied on visual analysis of short segments of data due to the prevalence of artifacts in EEG. Here we set out to robustly characterize the rate and spatial distribution of HFOs in large datasets from IS subjects using fully automated HFO detection techniques.Approach.We prospectively collected long-term scalp EEG data from 12 subjects with IS and 18 healthy controls. For patients with IS, recording began prior to diagnosis and continued through initiation of treatment with adrenocorticotropic hormone (ACTH). The median analyzable EEG duration was 18.2 h for controls and 84.5 h for IS subjects (∼1300 h total). Ripples (80-250 Hz) were detected in all EEG data using an automated algorithm.Main results.HFO rates were substantially higher in patients with IS compared to controls. In IS patients, HFO rates were higher during sleep compared to wakefulness (median 5.5 min-1and 2.9 min-1, respectively;p = 0.002); controls did not exhibit a difference in HFO rate between sleep and wakefulness (median 0.98 min-1and 0.82 min-1, respectively). Spatially, IS patients exhibited significantly higher rates of HFOs in the posterior parasaggital region and significantly lower HFO rates in frontal channels, and this difference was more pronounced during sleep. In IS subjects, ACTH therapy significantly decreased the rate of HFOs.Significance.Here we provide a detailed characterization of the spatial distribution and rates of HFOs associated with IS, which may have relevance for diagnosis and assessment of treatment response. We also demonstrate that our fully automated algorithm can be used to detect HFOs in long-term scalp EEG with sufficient accuracy to clearly discriminate healthy subjects from those with IS.

Analyzing mixing systems using a new generation of Bayesian tracer mixing models.

The ongoing evolution of tracer mixing models has resulted in a confusing array of software tools that differ in terms of data inputs, model assumptions, and associated analytic products. Here we introduce MixSIAR, an inclusive, rich, and flexible Bayesian tracer (e.g., stable isotope) mixing model framework implemented as an open-source R package. Using MixSIAR as a foundation, we provide guidance for the implementation of mixing model analyses. We begin by outlining the practical differences between mixture data error structure formulations and relate these error structures to common mixing model study designs in ecology. Because Bayesian mixing models afford the option to specify informative priors on source proportion contributions, we outline methods for establishing prior distributions and discuss the influence of prior specification on model outputs. We also discuss the options available for source data inputs (raw data versus summary statistics) and provide guidance for combining sources. We then describe a key advantage of MixSIAR over previous mixing model software-the ability to include fixed and random effects as covariates explaining variability in mixture proportions and calculate relative support for multiple models via information criteria. We present a case study of Alligator mississippiensis diet partitioning to demonstrate the power of this approach. Finally, we conclude with a discussion of limitations to mixing model applications. Through MixSIAR, we have consolidated the disparate array of mixing model tools into a single platform, diversified the set of available parameterizations, and provided developers a platform upon which to continue improving mixing model analyses in the future.

Quantitative assessment of Pb sources in isotopic mixtures using a Bayesian mixing model.

Lead (Pb) isotopes provide valuable insights into the origin of Pb within a sample, typically allowing for reliable fingerprinting of their source. This is useful for a variety of applications, from tracing sources of pollution-related Pb, to the origins of Pb in archaeological artefacts. However, current approaches investigate source proportions via graphical means, or simple mixing models. As such, an approach, which quantitatively assesses source proportions and fingerprints the signature of analysed Pb, especially for larger numbers of sources, would be valuable. Here we use an advanced Bayesian isotope mixing model for three such applications: tracing dust sources in pre-anthropogenic environmental samples, tracking changing ore exploitation during the Roman period, and identifying the source of Pb in a Roman-age mining artefact. These examples indicate this approach can understand changing Pb sources deposited during both pre-anthropogenic times, when natural cycling of Pb dominated, and the Roman period, one marked by significant anthropogenic pollution. Our archaeometric investigation indicates clear input of Pb from Romanian ores previously speculated, but not proven, to have been the Pb source. Our approach can be applied to a range of disciplines, providing a new method for robustly tracing sources of Pb observed within a variety of environments.

Seasonal and long-term effects of CO2 and O3 on water loss in ponderosa pine and their interaction with climate and soil moisture.

Evapotranspiration (ET) is driven by evaporative demand, available solar energy and soil moisture (SM) as well as by plant physiological activity which may be substantially affected by elevated CO2 and O3. A multi-year study was conducted in outdoor sunlit-controlled environment mesocosm containing ponderosa pine seedlings growing in a reconstructed soil-litter system. The study used a 2 x 2 factorial design with two concentrations of CO2 (ambient and elevated), two levels of O3 (low and high) and three replicates of each treatment. The objective of this study was to assess the effects of chronic exposure to elevated CO2 and O3, alone and in combination, on daily ET. This study evaluated three hypotheses: (i) because elevated CO2 stimulates stomatal closure, O3 effects on ET will be less under elevated CO2 than under ambient CO2; (ii) elevated CO2 will ameliorate the long-term effects of O3 on ET; and (iii) because conductance (g) decreases with decreasing SM, the impacts of elevated CO2 and O3, alone and in combination, on water loss via g will be greater in early summer when SM is not limiting than to other times of the year. A mixed-model covariance analysis was used to adjust the daily ET for seasonality and the effects of SM and photosynthetically active radiation when testing for the effects of CO2 and O3 on ET via the vapor pressure deficit gradient. The empirical results indicated that the interactive stresses of elevated CO2 and O3 resulted in a lesser reduction in ET via reduced canopy conductance than the sum of the individual effects of each gas. CO2-induced reductions in ET were more pronounced when trees were physiologically most active. O3-induced reductions in ET under ambient CO2 were likely transpirational changes via reduced conductance because needle area and root biomass were not affected by exposures to elevated O3 in this study.

Elevated CO2 and O3 effects on fine-root survivorship in ponderosa pine mesocosms.

Atmospheric carbon dioxide (CO(2)) and ozone (O(3)) concentrations are rising, which may have opposing effects on tree C balance and allocation to fine roots. More information is needed on interactive CO(2) and O(3) effects on roots, particularly fine-root life span, a critical demographic parameter and determinant of soil C and N pools and cycling rates. We conducted a study in which ponderosa pine (Pinus ponderosa) seedlings were exposed to two levels of CO(2) and O(3) in sun-lit controlled-environment mesocosms for 3 years. Minirhizotrons were used to monitor individual fine roots in three soil horizons every 28 days. Proportional hazards regression was used to analyze effects of CO(2), O(3), diameter, depth, and season of root initiation on fine-root survivorship. More fine roots were produced in the elevated CO(2) treatment than in ambient CO(2). Elevated CO(2), increasing root diameter, and increasing root depth all significantly increased fine-root survivorship and median life span. Life span was slightly, but not significantly, lower in elevated O(3), and increased O(3) did not reduce the effect of elevated CO(2). Median life spans varied from 140 to 448 days depending on the season of root initiation. These results indicate the potential for elevated CO(2) to increase the number of fine roots and their residence time in the soil, which is also affected by root diameter, root depth, and phenology.

The vitamin riboflavin and its derivative lumichrome activate the LasR bacterial quorum-sensing receptor.

Many bacteria use quorum sensing (QS) as an intercellular signaling mechanism to regulate gene expression in local populations. Plant and algal hosts, in turn, secrete compounds that mimic bacterial QS signals, allowing these hosts to manipulate QS-regulated gene expression in bacteria. Lumichrome, a derivative of the vitamin riboflavin, was purified and chemically identified from culture filtrates of the alga Chlamydomonas as a QS signal-mimic compound capable of stimulating the Pseudomonas aeruginosa LasR QS receptor. LasR normally recognizes the N-acyl homoserine lactone (AHL) signal, N-3-oxo-dodecanoyl homoserine lactone. Authentic lumichrome and riboflavin stimulated the LasR receptor in bioassays and lumichrome activated LasR in gel shift experiments. Amino acid substitutions in LasR residues required for AHL binding altered responses to both AHLs and lumichrome or riboflavin. These results and docking studies indicate that the AHL binding pocket of LasR recognizes both AHLs and the structurally dissimilar lumichrome or riboflavin. Bacteria, plants, and algae commonly secrete riboflavin or lumichrome, raising the possibility that these compounds could serve as either QS signals or as interkingdom signal mimics capable of manipulating QS in bacteria with a LasR-like receptor.

A functional epitope of the pneumococcal surface adhesin A activates nasopharyngeal cells and increases bacterial internalization.

Pneumococcal surface adhesin A (PsaA) is a putative pneumococcal (Pnc) adhesin known to bind to nasopharyngeal (NP) epithelial cells. This study evaluated the effect of peptides within a functional domain of PsaA on NP cells. Detroit 562 NP cells were treated with synthetic peptides derived from PsaA (P4, P6, and P7; 28, 12, and 16 amino acids, respectively). The P4 peptide also binds to NP cells. Analysis of P4-treated NP cells by transmission electron microscopy revealed major cytological changes. Of 9 cytokines analyzed, a 6-fold increase in FGFb secretion at 3 and 6h (11-fold at 12h) was found post-P4 treatment of NP cells. There was a simultaneous reduction in the secreted levels of IL-6, IL-8, and VEGF. We observed enhancement in the adherence of Pnc strains to P4-treated NP cells (2-38-fold increase). Enhancement in adherence (2-fold increase) to P4-treated NP cells was also recorded with other streptococcal species (Streptococcus mitis and Streptococcus pyogenes). Internalization experiments demonstrated that 45% of the adherent bacteria were actually internalized after pretreatment with P4 peptide as compared to controls. Peptide fragments of P4, P6 and P7 did not activate NP cells to the extent of P4 peptide. The P4-mediated enhancement of Pnc adherence was blocked (100%) by anti-P4 antibodies, confirming the specificity of the P4 sequence for NP cell activation. Our data suggests that this functional domain of PsaA contained within the P4 sequence binds and activates NP cells to facilitate Pnc invasion.

Elevated CO(2) and temperature alter net ecosystem C exchange in a young Douglas fir mesocosm experiment.

We investigated the effects of elevated CO(2) (EC) [ambient CO(2) (AC) + 190 ppm] and elevated temperature (ET) [ambient temperature (AT) + 3.6 degrees C] on net ecosystem exchange (NEE) of seedling Douglas fir (Pseudotsuga menziesii) mesocosms. As the study utilized seedlings in reconstructed soil-litter-plant systems, we anticipated greater C losses through ecosystem respiration (R(e)) than gains through gross photosynthesis (GPP), i.e. negative NEE. We hypothesized that: (1) EC would increase GPP more than R(e), resulting in NEE being less negative; and (2) ET would increase R(e) more than GPP, resulting in NEE being more negative. We also evaluated effects of CO(2) and temperature on light inhibition of dark respiration. Consistent with our hypothesis, NEE was a smaller C source in EC, not because EC increased photosynthesis but rather because of decreased respiration resulting in less C loss. Consistent with our hypothesis, NEE was more negative in ET because R(e) increased more than GPP. The light level that inhibited respiration varied seasonally with little difference among CO(2) and temperature treatments. In contrast, the degree of light inhibition of respiration was greater in AC than EC. In our system, respiration was the primary control on NEE, as EC and ET caused greater changes in respiration than photosynthesis.

The shifting baseline of northern fur seal ecology in the northeast Pacific Ocean.

Historical data provide a baseline against which to judge the significance of recent ecological shifts and guide conservation strategies, especially for species decimated by pre-20th century harvesting. Northern fur seals (NFS; Callorhinus ursinus) are a common pinniped species in archaeological sites from southern California to the Aleutian Islands, yet today they breed almost exclusively on offshore islands at high latitudes. Harvest profiles from archaeological sites contain many unweaned pups, confirming the presence of temperate-latitude breeding colonies in California, the Pacific Northwest, and the eastern Aleutian Islands. Isotopic results suggest that prehistoric NFS fed offshore across their entire range, that California populations were distinct from populations to the north, and that populations breeding at temperate latitudes in the past used a different reproductive strategy than modern populations. The extinction of temperate-latitude breeding populations was asynchronous geographically. In southern California, the Pacific Northwest, and the eastern Aleutians, NFS remained abundant in the archaeological record up to the historical period approximately 200 years B.P.; thus their regional collapse is plausibly attributed to historical hunting or some other anthropogenic ecosystem disturbance. In contrast, NFS populations in central and northern California collapsed at approximately 800 years B.P., long before European contact. The relative roles of human hunting versus climatic factors in explaining this ecological shift are unclear, as more paleoclimate information is needed from the coastal zone.

Bole water content shows little seasonal variation in century-old Douglas-fir trees.

Purportedly, large Douglas-fir trees in the American Pacific Northwest use water stored in bole tissues to ameliorate the effects of seasonal summer drought, the water content of bole tissues being drawn down over the summer months and replenished during the winter. Continuous monitoring of bole relative water content (RWC) in two 110-120-year-old Douglas-fir trees with ThetaProbe impedance devices provided an integrated measure of phloem-sapwood water content over 4 years. Seasonal changes in RWC closely tracked cambial activity and wood formation, but lagged changes in soil water content by 2-3 months. The RWC in the combined phloem and sapwood markedly increased during earlywood production in the late spring and early summer to maximum values of 64-77% as plant available soil water (ASW) decreased by approximately 60%. With transition and latewood formation, RWC decreased to minimum values of 59-72%, even as ASW increased in the fall. The difference between minimum RWC in the fall and maximum RWC in midsummer was only approximately 5%. Seasonal changes in bole RWC corresponded to cambial phenology, although decreasing AWS appeared to trigger the shift from earlywood to latewood formation.

Mycoplasma pneumoniae subtype-independent induction of proinflammatory cytokines in THP-1 cells.

Mycoplasma pneumoniae can be divided into two main subtypes depending on the amino acid sequences of the P1 adhesin and the P65 protein, both located in the attachment organelle. Differences between these subtypes in infectivity, virulence and interaction with host cells have not been extensively studied. Using ELISA to measure released protein and real-time PCR to quantify mRNA, we have demonstrated that both M. pneumoniae subtypes significantly increased tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6) and interleukin-8 (IL-8) at comparable levels in THP-1 cells over a 72 h period of time. However, subtype 2 induced a statistically significant increase (P<0.001) in the release of interleukin-1beta at 24 h post-infection compared to subtype 1. These data provide evidence that the induction of proinflammatory cytokine gene and protein expression by M. pneumoniae is not dependent on the infecting subtype.

CO2 and N-fertilization effects on fine-root length, production, and mortality: a 4-year ponderosa pine study.

We conducted a 4-year study of juvenile Pinus ponderosa fine root (< or =2 mm) responses to atmospheric CO2 and N-fertilization. Seedlings were grown in open-top chambers at three CO2 levels (ambient, ambient+175 mumol/mol, ambient+350 mumol/mol) and three N-fertilization levels (0, 10, 20 g m(-2) year(-1)). Length and width of individual roots were measured from minirhizotron video images bimonthly over 4 years starting when the seedlings were 1.5 years old. Neither CO2 nor N-fertilization treatments affected the seasonal patterns of root production or mortality. Yearly values of fine-root length standing crop (m m(-2)), production (m m(-2) year(-1)), and mortality (m m(-2) year(-1)) were consistently higher in elevated CO2 treatments throughout the study, except for mortality in the first year; however, the only statistically significant CO2 effects were in the fine-root length standing crop (m m(-2)) in the second and third years, and production and mortality (m m(-2) year(-1)) in the third year. Higher mortality (m m(-2) year(-1)) in elevated CO2 was due to greater standing crop rather than shorter life span, as fine roots lived longer in elevated CO2. No significant N effects were noted for annual cumulative production, cumulative mortality, or mean standing crop. N availability did not significantly affect responses of fine-root standing crop, production, or mortality to elevated CO2. Multi-year studies at all life stages of trees are important to characterize belowground responses to factors such as atmospheric CO2 and N-fertilization. This study showed the potential for juvenile ponderosa pine to increase fine-root C pools and C fluxes through root mortality in response to elevated CO2.

Estimating the timing of diet shifts using stable isotopes.

Stable isotope analysis has become an important tool in studies of trophic food webs and animal feeding patterns. When animals undergo rapid dietary shifts due to migration, metamorphosis, or other reasons, the isotopic composition of their tissues begins changing to reflect that of their diet. This can occur both as a result of growth and metabolic turnover of existing tissue. Tissues vary in their rate of isotopic change, with high turnover tissues such as liver changing rapidly, while relatively low turnover tissues such as bone change more slowly. A model is outlined that uses the varying isotopic changes in multiple tissues as a chemical clock to estimate the time elapsed since a diet shift, and the magnitude of the isotopic shift in the tissues at the new equilibrium. This model was tested using published results from controlled feeding experiments on a bird and a mammal. For the model to be effective, the tissues utilized must be sufficiently different in their turnover rates. The model did a reasonable job of estimating elapsed time and equilibrial isotopic changes, except when the time since the diet shift was less than a small fraction of the half-life of the slowest turnover tissue or greater than 5-10 half-lives of the slowest turnover tissue. Sensitivity analyses independently corroborated that model estimates became unstable at extremely short and long sample times due to the effect of random measurement error. Subject to some limitations, the model may be useful for studying the movement and behavior of animals changing isotopic environments, such as anadromous fish, migratory birds, animals undergoing metamorphosis, or animals changing diets because of shifts in food abundance or competitive interactions.

Interference in microsphere flow cytometric multiplexed immunoassays for human cytokine estimation.

The present study describes positive and negative interference of human cytokine measurement in multiplexed bead-based immunoassays. Significant differences in measured IL-6 and TNF-alpha values in 30 normal human plasma samples were apparent depending on whether measurements were with a 2-plex assay or embedded in a multiplex of 8-or more cytokine antibody pairs, as well as among the kits of 3-different vendors. Sample diluents containing proprietary blocking ingredients were shown to greatly affect the outcome of measured cytokine values. Additionally, recovery of IL-6 and TNF-alpha from spiked samples suggests significant negative interference from either endogenous antibodies, soluble receptors or anti-cytokine antibodies in 10% and 26% of samples, respectively. While it is evident that multiplexed immunoassays hold great promise for cytokine profiling, there are still important issues needing further study. Especially needed are universally optimized sample diluents, uniformly calibrated standards with mass values, and internal assay controls, which should greatly facilitate intralaboratory accuracy and precision and interlaboratory comparisons of cytokine measurements. Possible causes of interference and remedies are discussed.

Combining sources in stable isotope mixing models: alternative methods.

Stable isotope mixing models are often used to quantify source contributions to a mixture. Examples include pollution source identification; trophic web studies; analysis of water sources for soils, plants; or water bodies, and many others. A common problem is having too many sources to allow a unique solution. We discuss two alternative procedures for addressing this problem. One option is a priori to combine sources with similar signatures so the number of sources is small enough to provide a unique solution. Aggregation should be considered only when isotopic signatures of clustered sources are not significantly different, and sources are related so the combined source group has some functional significance. For example, in a food web analysis, lumping several species within a trophic guild allows more interpretable results than lumping disparate food sources, even if they have similar isotopic signatures. One result of combining mixing model sources is increased uncertainty of the combined end-member isotopic signatures and consequently the source contribution estimates; this effect can be quantified using the IsoError model (http://www.epa.gov/wed/pages/models/isotopes/isoerror1_04.htm). As an alternative to lumping sources before a mixing analysis, the IsoSource mixing model (http://www.epa.gov/wed/pages/models/isosource/isosource.htm) can be used to find all feasible solutions of source contributions consistent with isotopic mass balance. While ranges of feasible contributions for each individual source can often be quite broad, contributions from functionally related groups of sources can be summed a posteriori, producing a range of solutions for the aggregate source that may be considerably narrower. A paleo-human dietary analysis example illustrates this method, which involves a terrestrial meat food source, a combination of three terrestrial plant foods, and a combination of three marine foods. In this case, a posteriori aggregation of sources allowed strong conclusions about temporal shifts in marine versus terrestrial diets that would not have otherwise been discerned.

Microbial products trigger amino acid exudation from plant roots.

Plants naturally cycle amino acids across root cell plasma membranes, and any net efflux is termed exudation. The dominant ecological view is that microorganisms and roots passively compete for amino acids in the soil solution, yet the innate capacity of roots to recover amino acids present in ecologically relevant concentrations is unknown. We find that, in the absence of culturable microorganisms, the influx rates of 16 amino acids (each supplied at 2.5 microm) exceed efflux rates by 5% to 545% in roots of alfalfa (Medicago sativa), Medicago truncatula, maize (Zea mays), and wheat (Triticum aestivum). Several microbial products, which are produced by common soil microorganisms such as Pseudomonas bacteria and Fusarium fungi, significantly enhanced the net efflux (i.e. exudation) of amino acids from roots of these four plant species. In alfalfa, treating roots with 200 microm phenazine, 2,4-diacetylphloroglucinol, or zearalenone increased total net efflux of 16 amino acids 200% to 2,600% in 3 h. Data from (15)N tests suggest that 2,4-diacetylphloroglucinol blocks amino acid uptake, whereas zearalenone enhances efflux. Thus, amino acid exudation under normal conditions is a phenomenon that probably reflects both active manipulation and passive uptake by microorganisms, as well as diffusion and adsorption to soil, all of which help overcome the innate capacity of plant roots to reabsorb amino acids. The importance of identifying potential enhancers of root exudation lies in understanding that such compounds may represent regulatory linkages between the larger soil food web and the internal carbon metabolism of the plant.