Interleukin-6 is an efficacious marker of axonal transport disruption during experimental glaucoma and stimulates neuritogenesis in cultured retinal ganglion cells

It is increasingly recognised that chronically activated glia contribute to the pathology of various neurodegenerative diseases, including glaucoma. One means by which this can occur is through the release of neurotoxic, proinflammatory factors. In the current study, we therefore investigated the spatio-temporal patterns of expression of three such cytokines, IL-1β, TNFα and IL-6, in a validated rat model of experimental glaucoma. First, only weak evidence was found for increased expression of IL-1β and TNFα following induction of ocular hypertension. Second, and much more striking, was that robust evidence was uncovered showing IL-6 to be synthesised by injured retinal ganglion cells following elevation of intraocular pressure and transported in an orthograde fashion along the nerve, accumulating at sites of axonal disruption in the optic nerve head. Verification that IL-6 represents a novel marker of disrupted axonal transport in this model was obtained by performing double labelling immunofluorescence with recognised markers of fast axonal transport. The stimulus for IL-6 synthesis and axonal transport during experimental glaucoma arose from axonal injury rather than ocular hypertension, as the response was identical after optic nerve crush and bilateral occlusion of the carotid arteries, each of which is independent of elevated intraocular pressure. Moreover, the response of IL-6 was not a generalised feature of the gp130 family of cytokines, as it was not mimicked by another family member, ciliary neurotrophic factor. Finally, further study suggested that IL-6 may be an early part of the endogenous regenerative response as the cytokine colocalised with growth-associated membrane phosphoprotein-43 in some putative regenerating axons, and potently stimulated neuritogenesis in retinal ganglion cells in culture, an effect that was additive to that of ciliary neurotrophic factor. These data comprise clear evidence that IL-6 is actively involved in the attempt of injured retinal ganglion cells to regenerate their axons.

Regulatory mechanism of the light-activable allosteric switch LOV-TAP for the control of DNA binding: a computer simulation study

The spatio-temporal control of gene expression is fundamental to elucidate cell proliferation and deregulation phenomena in living systems. Novel approaches based on light-sensitive multiprotein complexes have recently been devised, showing promising perspectives for the noninvasive and reversible modulation of the DNA-transcriptional activity in vivo. This has lately been demonstrated in a striking way through the generation of the artificial protein construct light-oxygen-voltage (LOV)-tryptophan-activated protein (TAP), in which the LOV-2-Jα photoswitch of phototropin1 from Avena sativa (AsLOV2-Jα) has been ligated to the tryptophan-repressor (TrpR) protein from Escherichia coli. Although tremendous progress has been achieved on the generation of such protein constructs, a detailed understanding of their functioning as opto-genetical tools is still in its infancy. Here, we elucidate the early stages of the light-induced regulatory mechanism of LOV-TAP at the molecular level, using the noninvasive molecular dynamics simulation technique. More specifically, we find that Cys450-FMN-adduct formation in the AsLOV2-Jα-binding pocket after photoexcitation induces the cleavage of the peripheral Jα-helix from the LOV core, causing a change of its polarity and electrostatic attraction of the photoswitch onto the DNA surface. This goes along with the flexibilization through unfolding of a hairpin-like helix-loop-helix region interlinking the AsLOV2-Jα- and TrpR-domains, ultimately enabling the condensation of LOV-TAP onto the DNA surface. By contrast, in the dark state the AsLOV2-Jα photoswitch remains inactive and exerts a repulsive electrostatic force on the DNA surface. This leads to a distortion of the hairpin region, which finally relieves its tension by causing the disruption of LOV-TAP from the DNA.

Limitations of Remotely-sensed Aerosol as a Spatial Proxy for Fine Particulate Matter

Recent research highlights the promise of remotely-sensed aerosol optical depth (AOD) as a proxy for ground-level PM2.5. Particular interest lies in the information on spatial heterogeneity potentially provided by AOD, with important application to estimating and monitoring pollution exposure for public health purposes. Given the temporal and spatio-temporal correlations reported between AOD and PM2.5 , it is tempting to interpret the spatial patterns in AOD as reflecting patterns in PM2.5 . Here we find only limited spatial associations of AOD from three satellite retrievals with PM2.5 over the eastern U.S. at the daily and yearly levels in 2004. We then use statistical modeling to show that the patterns in monthly average AOD poorly reflect patterns in PM2.5 because of systematic, spatially-correlated error in AOD as a proxy for PM2.5 . Furthermore, when we include AOD as a predictor of monthly PM2.5 in a statistical prediction model, AOD provides little additional information to improve predictions of PM2.5 when included in a model that already accounts for land use, emission sources, meteorology and regional variability. These results suggest caution in using spatial variation in AOD to stand in for spatial variation in ground-level PM2.5 in epidemiological analyses and indicate that when PM2.5 monitoring is available, careful statistical modeling outperforms the use of AOD.

TRENDS IN PARTICULATE MATTER AND MORTALITY: AN APPROACH TO THE ASSESSMENT OF UNMEASURED CONFOUNDING

We propose a method for diagnosing confounding bias under a model which links a spatially and temporally varying exposure and health outcome. We decompose the association into orthogonal components, corresponding to distinct spatial and temporal scales of variation. If the model fully controls for confounding, the exposure effect estimates should be equal at the different temporal and spatial scales. We show that the overall exposure effect estimate is a weighted average of the scale-specific exposure effect estimates. We use this approach to estimate the association between monthly averages of fine particles (PM2.5) over the preceding 12 months and monthly mortality rates in 113 U.S. counties from 2000-2002. We decompose the association between PM2.5 and mortality into two components: 1) the association between “national trends” in PM2.5 and mortality; and 2) the association between “local trends,” defined as county-specificdeviations from national trends. This second component provides evidence as to whether counties having steeper declines in PM2.5 also have steeper declines in mortality relative to their national trends. We find that the exposure effect estimates are different at these two spatio-temporalscales, which raises concerns about confounding bias. We believe that the association between trends in PM2.5 and mortality at the national scale is more likely to be confounded than is the association between trends in PM2.5 and mortality at the local scale. If the association at the national scale is set aside, there is little evidence of an association between 12-month exposure to PM2.5 and mortality.

Estimation of vertical groundwater fluxes into a streambed through continuous temperature profile monitoring and the relationship of groundwater fluxes to coaster brook trout spawning habitat

We hypothesized that the spatial distribution of groundwater inflows through river bottom sediments is a critical factor associated with the selection of coaster brook trout (a life history variant of Salvelinus fontinalis,) spawning sites. An 80-m reach of the Salmon Trout River, in the Huron Mountains of the upper peninsula of Michigan, was selected to test the hypothesis based on long-term documentation of coaster brook trout spawning at this site. Throughout this site, the river is relatively similar along its length with regard to stream channel and substrate features. A monitoring well system consisting of an array of 27 wells was installed to measure subsurface temperatures underneath the riverbed over a 13-month period. The monitoring well locations were separated into areas where spawning has and has not been observed. Over 200,000 total temperature measurements were collected from 5 depths within each of the 27 monitoring wells. Temperatures within the substrate at the spawning area were generally cooler and less variable than river temperatures. Substrate temperatures in the non-spawning area were generally warmer, more variable, and closely tracked temporal variations in river temperatures. Temperature data were inverted to obtain subsurface groundwater velocities using a numerical approximation of the heat transfer equation. Approximately 45,000 estimates of groundwater velocities were obtained. Estimated velocities in the spawning and non-spawning areas confirmed that groundwater velocities in the spawning area were primarily in the upward direction, and were generally greater in magnitude than velocities in the non-spawning area. In the non-spawning area there was a greater occurrence of velocities in the downward direction, and velocity estimates were generally lesser in magnitude than in the spawning area. Both the temperature and velocity results confirm the hypothesis that spawning sites correspond to areas of significant groundwater influx to the river bed.

EVALUATION OF THE EVOLVING STRESS FIELD OF THE YELLOWSTONE VOLCANIC PLATEAU FROM 1988 TO 2010 FROM EARTHQUAKE FIRST MOTIONS INVERSION

Within the Yellowstone National Park, Wyoming, the silicic Yellowstone volcanic field is one of the most active volcanic systems all over the world. Although the last rhyolite eruption occurred around 70,000 years ago, Yellowstone is still believed to be volcanically active, due to high hydrothermal and seismic activity. The earthquake data used in this study cover the period of time between 1988 and 2010. Earthquake relocations and a set of 369 well-constrained, double-couple, focal mechanism solutions were computed. Events were grouped according to location and time to investigate trends in faulting. The majority of the events has oblique, normal-faulting solutions. The overall direction of extension throughout the 0.64 Ma Yellowstone caldera looks nearly ENE, consistently with the direction of alignments of volcanic vents within the caldera, but detailed study revealed spatial and temporal variations. Stress-field solutions for different areas and time periods were calculated from earthquake focal mechanism inversion. A well-resolved rotation of σ3 was found, from NNE-SSW near the Hebgen Lake fault zone, to ENE-WSW near Norris Junction. In particular, the σ3 direction changed throughout the years in the Norris Junction area, from being ENE-WSW, as calculated in the study by Waite and Smith (2004), to NNE-SSW, while the other σ3 directions are mostly unchanged over time. The Yellowstone caldera was subject to periods of net uplift and subsidence over the past century, explained in previous studies as caused by expanding or contracting sills, at different depths. Based on the models used to explain these deformation periods, we investigated the relationship between variability in aseismic deformation and seismic activity and faulting styles. Focal mechanisms and P and T axes were divided into temporal and depth intervals, in order to identify spatial or temporal trends in deformation. The presence of “chocolate tablet” structures, with composite dilational faults, was identified in many stages of the deformation history both in the Norris Geyser Basin area and inside the caldera. Strike-slip component movement was found in a depth interval below a contracting sill, indicating the movement of magma towards the caldera.

Effects of Chemical Deicers on Amphibian Communites

Roads and highways present a unique challenge to wildlife as they exhibit substantial impacts on the surrounding ecosystem through the interruption of a number of ecological processes. With new roads added to the national highway system every year, an understanding of these impacts is required for effective mitigation of potential environmental impacts. A major contributor to these negative effects is the deposition of chemicals used in winter deicing activities to nearby surface waters. These chemicals often vary in composition and may affect freshwater species differently. The negative impacts of widespread deposition of sodium chloride (NaCl) have prompted a search for an `environmentally friendly' alternative. However, little research has investigated the potential environmental effects of widespread use of these alternatives. Herein, I detail the results of laboratory tests and field surveys designed to determine the impacts of road salt (NaCl) and other chemical deicers on amphibian communities in Michigan's Upper Peninsula. Using larval amphibians I demonstrate the lethal impacts of a suite of chemical deicers on this sensitive, freshwater species. Larval wood frogs (Lithobates sylvatica) were tolerant of short-term (96 hours) exposure to urea (CH4N2O), sodium chloride (NaCl), and magnesium chloride (MgCl2). However, these larvae were very sensitive to acetate products (C8H12CaMgO8, CH3COOK) and calcium chloride (CaCl2). These differences in tolerance suggest that certain deicers may be more harmful to amphibians than others. Secondly, I expanded this analysis to include an experiment designed to determine the sublethal effects of chronic exposure to environmentally realistic concentrations of NaCl on two unique amphibian species, L. sylvatica and green frogs (L. clamitans). L. sylvatica tend to breed in small, ephemeral wetlands and metamorphose within a single season. However, L. clamitans breed primarily in more permanent wetlands and often remain as tadpoles for one year or more. These species employ different life history strategies in this region which may influence their response to chronic NaCl exposure. Both species demonstrated potentially harmful effects on individual fitness. L. sylvatica larvae had a high incidence of edema suggesting the NaCl exposure was a significant physiologic stressor to these larvae. L. clamitans larvae reduced tail length during their exposure which may affect adult fitness of these individuals. In order to determine the risk local amphibians face when using these roadside pools, I conducted a survey of the spatial distribution of chloride in the three northernmost counties of Michigan. This area receives a relatively low amount of NaCl which is confined to state and federal highways. The chloride concentrations in this region were much lower than those in urban systems; however, amphibians breeding in the local area may encounter harmful chloride levels arising from temporal variations in hydroperiods. Spatial variation of chloride levels suggests the road-effect zone for amphibians may be as large as 1000 m from a salt-treated highway. Lastly, I performed an analysis of the use of specific conductance to predict chloride concentrations in natural surface water bodies. A number of studies have used this regression to predict chloride concentrations from measurements of specific conductance. This method is often chosen in the place of ion chromatography due to budget and time constraints. However, using a regression method to characterize this relationship does not result in accurate chloride ion concentration estimates.

IMPACTS OF CLIMATE CHANGE ON SOIL MICROORGANISMS IN NORTHERN HARDWOOD FORESTS

As global climate continues to change, it becomes more important to understand possible feedbacks from soils to the climate system. This dissertation focuses on soil microbial community responses to climate change factors in northern hardwood forests. Two soil warming experiments at Harvard Forest in Massachusetts, and a climate change manipulation experiment with both elevated temperature and increased moisture inputs in Michigan were sampled. The hyphal in-growth bag method was to understand how soil fungal biomass and respiration respond to climate change factors. Our results from phospholipid fatty acid (PLFA) analyses suggest that the hyphal in-growth bag method allows relatively pure samples of fungal hyphae to be partitioned from bacteria in the soil. The contribution of fungal hyphal respiration to soil respiration was examined in climate change manipulation experiments in Massachusetts and Michigan. The Harvard Forest soil warming experiments in Massachusetts are long-term studies with 8 and 18 years of +5 °C warming treatment. Hyphal respiration and biomass production tended to decrease with soil warming at Harvard Forest. This suggests that fungal hyphae adjust to higher temperatures by decreasing the amount of carbon respired and the amount of carbon stored in biomass. The Ford Forestry Center experiment in Michigan has a 2 x 2 fully factorial design with warming (+4-5 °C) and moisture addition (+30% average ambient growing season precipitation). This experiment was used to examine hyphal growth and respiration of arbuscular mycorrhizal fungi (AMF), soil enzymatic capacity, microbial biomass and microbial community structure in the soil over two years of experimental treatment. Results from the hyphal in-growth bag study indicate that AMF hyphal growth and respiration respond negatively to drought. Soil enzyme activities tend to be higher in heated versus unheated soils. There were significant temporal variations in enzyme activity and microbial biomass estimates. When microbial biomass was estimated using chloroform fumigation extractions there were no differences between experimental treatments and the control. When PLFA analyses were used to estimate microbial biomass we found that biomass responds negatively to higher temperatures and positively to moisture addition. This pattern was present for both bacteria and fungi. More information on the quality and composition of the organic matter and nutrients in soils from climate change manipulation experiments will allow us to gain a more thorough understanding of the mechanisms driving the patterns reported here. The information presented here will improve current soil carbon and nitrogen cycling models.

Species mobility and frequency dynamics in a Swiss limestone grassland

Community dynamics in a calcareous grassland (Mesobrometum) in Egerkingen (Jura mountains, Switzerland) were investigated for 53 non-woody species in 25 1-m2 plots over 6 years. 50 0.0 1-m2 subplots per plot were recorded. The derived variables were spatial frequency, temporal frequency, frequency fluctuation, turnover, and cumulative frequency (each species), and cumulative species richness (all species). Spectra for 53 species of all variables were different for the two investigated spatial scales (0.0 1 m2, 1 m2). The comparison with other investigations of similar grass lands showed that the behaviour of some species is specific for this type of vegetation in general (e.g. Achillea millefolium, Arrhenatherum elatius, Bromus erectus ), but most species behaved in a stand-specific way, i.e. they may play another (similar or completely different) role in another grassland stand. Six spatio-temporal patterns were defined across species. To understand community dynamics, not only the dynamics of mobility but also of frequency fluctuations and spatial distribution of the species are fundamental. In addition, the understanding of temporal behaviour of all species present should be included. Averages always hide important information of vegetation dynamics, as was shown by the present investigation.

BOLD correlates of EEG alpha phase-locking and the fMRI default mode network

Phase locking or synchronization of brain areas is a key concept of information processing in the brain. Synchronous oscillations have been observed and investigated extensively in EEG during the past decades. EEG oscillations occur over a wide frequency range. In EEG, a prominent type of oscillations is alpha-band activity, present typically when a subject is awake, but at rest with closed eyes. The spectral power of alpha rhythms has recently been investigated in simultaneous EEG/fMRI recordings, establishing a wide-range cortico-thalamic network. However, spectral power and synchronization are different measures and little is known about the correlations between BOLD effects and EEG synchronization. Interestingly, the fMRI BOLD signal also displays synchronous oscillations across different brain regions. These oscillations delineate so-called resting state networks (RSNs) that resemble the correlation patterns of simultaneous EEG/fMRI recordings. However, the nature of these BOLD oscillations and their relations to EEG activity is still poorly understood. One hypothesis is that the subunits constituting a specific RSN may be coordinated by different EEG rhythms. In this study we report on evidence for this hypothesis. The BOLD correlates of global EEG synchronization (GFS) in the alpha frequency band are located in brain areas involved in specific RSNs, e.g. the 'default mode network'. Furthermore, our results confirm the hypothesis that specific RSNs are organized by long-range synchronization at least in the alpha frequency band. Finally, we could localize specific areas where the GFS BOLD correlates and the associated RSN overlap. Thus, we claim that not only the spectral dynamics of EEG are important, but also their spatio-temporal organization.

Cortical local and long-range synchronization interplay in human absence seizure initiation

Brain activity relies on transient, fluctuating interactions between segregated neuronal populations. Synchronization within a single and between distributed neuronal clusters reflects the dynamics of these cooperative patterns. Thus absence epilepsy can be used as a model for integrated, large-scale investigation of the emergence of pathological collective dynamics in the brain. Indeed, spike-wave discharges (SWD) of an absence seizure are thought to reflect abnormal cortical hypersynchronization. In this paper, we address two questions: how and where do SWD arise in the human brain? Therefore, we explored the spatio-temporal dynamics of interactions within and between widely distributed cortical sites using magneto-encephalographic recordings of spontaneous absence seizures. We then extracted, from their time-frequency analysis, local synchronization of cortical sources and long-range synchronization linking distant sites. Our analyses revealed a reproducible sequence of 1) long-range desynchronization, 2) increased local synchronization and 3) increased long-range synchronization. Although both local and long-range synchronization displayed different spatio-temporal profiles, their cortical projection within an initiation time window overlap and reveal a multifocal fronto-central network. These observations contradict the classical view of sudden generalized synchronous activities in absence epilepsy. Furthermore, they suggest that brain states transition may rely on multi-scale processes involving both local and distant interactions.

Modeling Calcium Concentration and Biochemical Reactions

Almost all regions of the brain receive one or more neuromodulatory inputs, and disrupting these inputs produces deficits in neuronal function. Neuromodulators act through intracellular second messenger pathways to influence the electrical properties of neurons, integration of synaptic inputs, spatio-temporal firing dynamics of neuronal networks, and, ultimately, systems behavior. Second messengers pathways consist of series of bimolecular reactions, enzymatic reactions, and diffusion. Calcium is the second messenger molecule with the most effectors, and thus is highly regulated by buffers, pumps and intracellular stores. Computational modeling provides an innovative, yet practical method to evaluate the spatial extent, time course and interaction among second messenger pathways, and the interaction of second messengers with neuron electrical properties. These processes occur both in compartments where the number of molecules are large enough to describe reactions deterministically (e.g. cell body), and in compartments where the number of molecules is small enough that reactions occur stochastically (e.g. spines). – In this tutorial, I explain how to develop models of second messenger pathways and calcium dynamics. The first part of the tutorial explains the equations used to model bimolecular reactions, enzyme reactions, calcium release channels, calcium pumps and diffusion. The second part explains some of the GENESIS, Kinetikit and Chemesis objects that implement the appropriate equations. In depth explanation of calcium and second messenger models is provided by reviewing code, both in XPP, Chemesis and Kinetikit, that implements simple models of calcium dynamics and second messenger cascades.

Continental-scale temperature variability during the past two millennia

Past global climate changes had strong regional expression. To elucidate their spatio-temporal pattern, we reconstructed past temperatures for seven continental-scale regions during the past one to two millennia. The most coherent feature in nearly all of the regional temperature reconstructions is a long-term cooling trend, which ended late in the nineteenth century. At multi-decadal to centennial scales, temperature variability shows distinctly different regional patterns, with more similarity within each hemisphere than between them. There were no globally synchronous multi-decadal warm or cold intervals that define a worldwide Medieval Warm Period or Little Ice Age, but all reconstructions show generally cold conditions between ad 1580 and 1880, punctuated in some regions by warm decades during the eighteenth century. The transition to these colder conditions occurred earlier in the Arctic, Europe and Asia than in North America or the Southern Hemisphere regions. Recent warming reversed the long-term cooling; during the period ad 1971–2000, the area-weighted average reconstructed temperature was higher than any other time in nearly 1,400 years.

Effects of amygdala-hippocampal stimulation on synchronization.

Changes in EEG synchronization, i.e., spatio-temporal correlation, with amygdala-hippocampal stimulation were studied in patients with temporal lobe epilepsy. Synchronization was evaluated for high frequency, 130Hz, pseudo-monophasic or biphasic charge-balanced pulses. Desynchronization was most frequently induced by stimulation. There was no correlation between the changes in synchronization and the changes in interictal epileptiform discharge rates. Changes in synchronization do not appear yet to be a marker of stimulation efficiency in reducing seizures.

A multiplex PCR for the simultaneous detection and genotyping of the Echinococcus granulosus complex.

Echinococcus granulosus is characterized by high intra-specific variability (genotypes G1-G10) and according to the new molecular phylogeny of the genus Echinococcus, the E. granulosus complex has been divided into E. granulosus sensu stricto (G1-G3), E. equinus (G4), E. ortleppi (G5), and E. canadensis (G6-G10). The molecular characterization of E. granulosus isolates is fundamental to understand the spatio-temporal epidemiology of this complex in many endemic areas with the simultaneous occurrence of different Echinococcus species and genotypes. To simplify the genotyping of the E. granulosus complex we developed a single-tube multiplex PCR (mPCR) allowing three levels of discrimination: (i) Echinococcus genus, (ii) E. granulosus complex in common, and (iii) the specific genotype within the E. granulosus complex. The methodology was established with known DNA samples of the different strains/genotypes, confirmed on 42 already genotyped samples (Spain: 22 and Bulgaria: 20) and then successfully applied on 153 unknown samples (Tunisia: 114, Algeria: 26 and Argentina: 13). The sensitivity threshold of the mPCR was found to be 5 ng Echinoccoccus DNA in a mixture of up to 1 µg of foreign DNA and the specificity was 100% when template DNA from closely related members of the genus Taenia was used. Additionally to DNA samples, the mPCR can be carried out directly on boiled hydatid fluid or on alkaline-lysed frozen or fixed protoscoleces, thus avoiding classical DNA extractions. However, when using Echinococcus eggs obtained from fecal samples of infected dogs, the sensitivity of the mPCR was low (<40%). Thus, except for copro analysis, the mPCR described here has a high potential for a worldwide application in large-scale molecular epidemiological studies on the Echinococcus genus.