Carcharodon carcharias), by measuring the stable carbon and nitrogen isotope composition of vertebral growth bands to create lifetime records for 15 individuals from California. Isotopic variations in white shark diets can reflect within-region differences among prey (most importantly related to trophic level), as well as differences in baseline values among the regions in which sharks forage, and both prey and habitat preferences may shift with age. The magnitude of isotopic variation among sharks in our study (>5‰ for both elements) is too great to be explained solely by geographic differences, and so must reflect differences in prey choice that may vary with sex, size, age and location. Ontogenetic patterns in δ15N values vary considerably among individuals, and one third of the population fit each of these descriptions: 1) δ15N values increased throughout life, 2) δ15N values increased to a plateau at ∼5 years of age, and 3) δ15N values remained roughly constant values throughout life. Isotopic data for the population span more than one trophic level, and we offer a qualitative evaluation of diet using shark-specific collagen discrimination factors estimated from a 3+ year captive feeding experiment . We assess the degree of individuality with a proportional similarity index that distinguishes specialists and generalists. The isotopic variance is partitioned among differences between-individual (48%), within-individuals (40%), and by calendar year of sub-adulthood (12%). Our data reveal substantial ontogenetic and individual dietary variation within a white shark population.There is growing evidence for individuality in dietary preferences and foraging behaviors within populations of various species. This is especially important for apex predators, since they can potentially have wide dietary niches and a large impact on trophic dynamics within ecosystems. We evaluate the diet of an apex predator, the white shark ( Although diet is often treated as a species-level trait, variation in diet composition and foraging behavior occurs within most species. This variation can be attributed to at least three factors–habitat-specific variation in prey availability; differences in the cost-benefit ratios of potential prey among the sexes, or age- or size-classes of consumers; and phenotypic variation among what often appear to be otherwise similar individuals Carcharodon carcharias) are apex predators that can have cascading effects on marine ecosystems White sharks  13C/12C and 15N/14N gradients throughout the eastern basin of the Pacific Ocean controlled by a variety of factors related to primary production at the base of the food web 13C-depleted deep water to the surface and nutrient-driven phytoplankton blooms off the California coast increase the baseline carbon isotope values ∼2–3‰ relative to less productive offshore areas 2) and processes of biological incorporation or geochemical cycling  vary with productivity regimes 13C-enriched nearshore habitats on the Pacific Rim have carbon isotope values ∼2‰ greater than those foraging offshore The stable isotope composition of a tissue reflects a temporal integration of dietary and environmental inputs , and can thus be used as a natural tracer for foraging variation. The most commonly used stable isotope ratios in dietary studies are Assessments of consumer diet with stable isotope analysis must address these spatial and trophic variations among potential prey. This technique has been used to chronicle patterns of diet and habitat use in a variety of marine predators, including marine mammals , which were preserved in alcohol until sampling . While sWhite shark vertebral centra grow by accretion in concentric rings and have annual growth bands that are used to age individuals 13C and δ15N values were referenced to V-PDB and AIR, respectively. Replicates of a gelatin standard within each analysis session allowed for mass and drift corrections. Comparisons of this standard within and between runs yielded SD of <0.2‰ and <0.3‰ for δ13C and δ15N values (n = 77), respectively.X is the element of interest, h is the high mass number, and R is the high mass-to-low mass ratio. Units are parts per thousand  deviations from a standard. The δ13C values of Earth surface carbon reservoirs that occurred over the past 70 years 13C values did not change constantly through this period, two atmosphere-derived rates (0.05‰ per year for 1937–1960 and 0.022‰ per year for 1961–2000) were used to correct samples to 2000, the year of death for the most recent individuals Because of the temporal span of our data, we corrected for anthropogenic changes in the δ15N values varied significantly with age using a one-way analysis of variance (ANOVA). These age classes were based on length data from observation and tagging studies of white sharks near pinniped rookeries. The smaller white sharks observed at Año Neuvo Island, Southeast Farallon Islands, Tomales Bay, and Point Reyes are approximately 300–350 cm long ∞ is the maximum (or asymptotic) length, L0 is length at birth (t = 0), and k is an empirically derived growth constant. According to VBGF, 300 cm corresponds to white sharks that are 5 years old th year as “young” and to ≥6 years old as “sub-adult to adult.”To test for the existence of an ontogenetic shift at the population level in a generalized linear model, we established 2 age classes and had individuals as a source of variation to determine if δ15N value (assuming random variation but no prevailing trend with age); ii) a continuous increase in δ15N values with age, fit using a first-order polynomial; and iii) a non-linear, asymptotic increase in δ15N values with age, fit using VBGF . We removed statistically significant outlier data  prior to final fitting. We tested the statistical significance of each function at á = 0.05, and used degrees-of-freedom-adjusted R2 values to evaluate goodness of fit for each model. We then assigned individuals to one of three classes based on which of these models  was significant and provided the best fit. For individuals exhibiting an asymptotic increase, we designated the age of transition from young to sub-adult through adult diets as the point at which the VBGF function equaled 90% of the asymptotic value. For individuals exhibiting no significant ontogenetic shift and having high δ15N values in early growth bands, we sampled and analyzed portions of the central vertebra, which form from maternal resources before parturition, to assess the potential of metabolic turnover within the vertebra.We next evaluated individual differences in ontogenetic dietary variation. For each shark, we fit three alternative functions to the time series data: i) a constant δWe compiled isotopic data for potential prey gleaned from studies in northeastern Pacific ecosystems to provide a dietary context for our sub-adult to adult isotopic data from white sharks. We limited our assessment to qualitative patterns, rather than estimating specific proportions of dietary inputs, because the large number of potential prey and their variance would likely produce indeterminate results While our shark data span from 1950–2000, the prey data are relatively recent and thus may not reflect secular shifts in baseline isotope values due to climate change or changes in productivity. This source of variation is difficult to constrain, but there is little evidence for major secular shifts , and variation of this magnitude will not affect our qualitative assessment of diet.To evaluate niche overlap, we used Pianka’s measure i and j), accounting for multivariate covariance and density distributions [g(x)]. We measured the degree of isotopic niche overlap, or “proportional similarity,” between each individual’s sub-adult/adult isotopic range and the population-level isotopic range . Averaged across individuals, this proportional similarity index , while generalists would have extensive overlap (ijw ≈ 1).dex wij,  allowed 13C and δ15N values  were selected based on minimal Akaike Information Criterion (AIC) values We also assessed the effects of individual differences on sub-adult/adult isotopic values within a generalized linear model. This analysis allowed us to assess the relative amount of variance explained by differences among individuals vs. variation within individuals. We included a temporal category, “calendar year of sub-adulthood,” defined as the calendar year a shark reached 6 years of age. The three categories for calendar year of sub-adulthood were based on pinniped populations: before the passage of the Marine Mammal Protection Act (pre-1972), during the period when pinniped populations were increasing (1972–1986), and after pinniped populations doubled from pre-1972 counts (post-1986) 13C and δ15N values, known as trophic discrimination factors, which reflect preferential sorting during metabolism and incorporation into tissues 13C and 15N. The average carbon and nitrogen discrimination factors that are widely used are 0.4‰ (SD = 1.3‰) and 3.4‰ (SD = 1.0‰), respectively There are offsets between prey and consumer δTriakis semifasciata) fed squid over 1250 days hXconsumer value in We conducted a controlled feeding study with captive leopard sharks , confirming that there is an ontogenetic shift in dietary preferences or habitat use in the northeastern Pacific white shark population. Five individuals showed a non-linear, asymptotic increase in δ15N values, with the transition to the sub-adult to adult diet occurring at approximately 4 years of age  before age 6 and linearly increasing or no ontogenetic shift , the average δ15N values in the central vertebra, which are formed prior to parturition, were 0.6–3.4‰ less than the growth bands for ages 1–5  exhibit isotopic ratios consistent with a diet rich in lower trophic level prey, whereas other individuals  appear to consume primarily high trophic level prey. One individual, WS 21, was an outlier from the population pattern, with low δ13C and δ15N values.A bivariate plot of isotopic data from sub-adult/adult growth bands illustrates the dietary diversity within the northeast Pacific white shark population . The popijw<0.5), but a few having high values (ijw<0.8) suggesting more generalized diets . The combined variance in δ13C and δ15N values was explained largely by differences among individuals (48%) and within-individual effects (40%). The calendar year of sub-adulthood accounted for 12% of the variance in a model with both isotope values weighted equally. Post hoc comparisons using the Tukey HSD test indicated the significance of all pairwise differences between calendar year of sub-adulthood categories for both isotopes .Our data reveal that northeast Pacific white sharks occupy a wide isotopic niche, as expected for a generalist population. However, closer inspection reveals a range of strategies among individuals, which may relate to sex, location, size, or individuality, as illustrated by the size and placement of individual bivariate confidence ellipses relative to the population . The shaed diets . The modpulation . The gen13C and δ15N values from the outermost 12 mm did not differ significantly among sharks  near the birthmark, where the corpus calcerum changes angle, was −15.4‰ (0.3) and 18.5‰ (0.5), respectively, which is significantly different from the outermost bands that represent body tissues in steady state with the captive squid diet . Based on the average isotopic value of the sharks’ diet  are 4.2‰ (0.7) and 2.5‰ (1.1), respectively.Because dietary carbon and nitrogen incorporate relatively slowly into shark tissues ks’ diet , the verIsotopic analysis of white shark dietary patterns reveals ontogenetic and among-individual variation. This finding contrasts with previous dietary assessments based on coastal observations and stable isotope data, which suggest that nearshore pinnipeds were the preferred prey for this population 15N values with age reported here is consistent with this scenario. We expected the time series from all sharks to exhibit a trend of early increase in δ15N values, followed by a plateau once individuals had switched to a high-trophic level adult diet. While this pattern was evident for some individuals, it was not the dominant trend in our sample . These shark-specific collagen discrimination factors are greater than other tissues  13C-enriched compared to other amino acids 13C values are greater than muscle. In contrast to our study, previously published shark vertebrae discrimination factors were obtained from relatively short growth periods (<200 days) and collagen was not isolated within the calcified vertebral tissue The trophic enrichment used for white shark prey comparisons was based on the leopard shark discrimination factors  are likely foraging on pinnipeds when near shore. However, intermediate isotopic values suggest that there are some offshore inputs . Other individuals  that also have intermediate isotope values, but with little to no ontogenetic shift and a low degree of specialization, are likely opportunistic, non-specialized foragers. One distinct outlier among the sharks in our population is WS 21, which had low δ13C and δ15N values throughout its lifetime  mostly encompass the white shark data, but overlap of a consumer’s δThe isotopic data in aggregate suggest that northeast Pacific white sharks are generalists at the population level, but further analysis reveals a high degree of individual specialization within the population. Individual differences within the white shark population can be attributed to changes in prey preference and foraging location with ontogeny. A core constraint on our analysis of niche occupancy and breadth is that if isotopic values differ between two specimens, then either prey type or foraging location (or both) must differ between the specimens. The converse is not true, however. Isotopic similarity between two specimens could result from consumption of the same prey in the same location, but also from fortuitous combinations of different prey types in different locations. Because we do not attempt to specify the particular prey, our assessment of individuality is a conservative measure and reflects minimal niche differences.ijw) quantified and compared isotopic variation within each individual to the population’s isotopic distribution. The ijw values for the sampled population indicated 8 specialists (ijw = 0–0.33), 4 generalists (ijw = 0.73–0.90), and 3 animals with intermediate values  and isotopic niche breadth , as these two metrics are not necessarily correlated. For example, isotopic niche breadth was similar for specimens 42094–2 and 42898 PR  and after drilling (right) on specimen WS101. Ages are also noted next to the growth bands.(TIF)Click here for additional data file.File S2The compiled prey isotope values from the literature and unpublished data used in (DOC)Click here for additional data file.File S313C values for the 15 white sharks analyzed for this study. Individuals are in the same A, B, and C groups as The ontogeny of δ(TIF)Click here for additional data file.


Expression of the SodA superoxide dismutase (MnSOD) in Escherichia coli is regulated by superoxide concentration through the SoxRS system and also by Fur (Ferric uptake regulator) through a mixed incoherent feed forward loop (FFL) containing the RyhB small regulatory RNA. In this work I theoretically analyze the function of this feed forward loop as part of the network controlling expression of the two cytoplasmic superoxide dismutases, SodA and SodB. I find that feed forward regulation allows faster response to superoxide stress at low intracellular iron levels compared to iron rich conditions. That is, it can conditionally modulate the response time of a superimposed transcriptional control mechanism. Most of the cells exposed to oxygen possess enzymes that provide protection to superoxide toxicity by catalyzing dismutation of superoxide to hydrogen peroxide and oxygen. Extracellular superoxide is produced for instance by phagocytic leukocytes to inactivate invading microorganisms, or by heterotrophic bacteria, affecting global biogeochemistry Escherichia coli has three superoxide dismutases  which require different metal cofactors and are regulated in different ways The highly reactive superoxide  sodB transcription is independent of Fe-Fur fur strains Transcription of Models section. Using this model I compared the steady state and dynamic behaviors of the natural system, where SodA expression is regulated by RyhB and Fe-Fur through a FFL, with a hypothetical control where SodA production does not depend on FeFur or RyhB concentration.I have developed a mathematical model to study the characteristics of feed-forward regulation of SodA expression. The model is described in detail in the fur strains Similar SodA activities were reported in wild type cells growing in iron rich conditions and in ΔFirst I explored the steady-state levels of SodA at intermediate Fe-Fur levels in the absence of superoxide stress. Simulations showed that SodA mRNA levels were kept in a relatively narrow range as Fe-Fur levels were changed from 2 to 2000 nM . Unlike To explore how SodA mRNA levels respond to changes in iron availability, I simulated sudden changes in Fe-Fur levels . SodA exThe opposite effect was observed when the Fe-Fur concentration was suddenly increased . In thisfur cells, similar fold activation was observed in wild type and Δfur cells in the presence of paraquat sodA promoter. Because SoxS in intrinsically unstable with an in vitro half-life of about 2 minutes sodA transcription rate  E. coli , suggesting that the fast response may help in inactivating superoxide molecules generated by the immune system.In conclusion, the FFL allows faster superoxide stress response and better adaptation in iron restricted environments, such as mammalian hosts. The same regulatory system is present in pathogenic strains (e.g. S), SodA mRNA (Am), and SodB mRNA (Bm). Similar to previous models of sRNA regulation The dynamical variables I keep track of in our model are the concentrations of RyhB . In this term, Sα is the maximal production rate of RyhB per cell generation time DK ( = 0.02 μM) is the binding constant of Fe-Fur to its operator site Sτ ( = 30/ln2 min) Gτ ( = 40/ln2 min) represents the passive degradation and dilution (by cell devision) of RyhB, respectively. The last two terms represent the active degradation of the RyhB-mRNA complexes by RNaseE The first term represents the production of RyhB, which is repressed by Fe-Fur . The next two terms represent dilution and passive degradation (Aτ = 11.8/ln2 min) Aδ, was chosen to be 0.0019/min to obtain similar SodA mRNA levels in the absence and in the presence of Fe-Fur Based on The dynamics of SodB is modeled by the following equation:Bτ = 8.8/ln2 min) Bδ, was chosen to be 0.014/min to reproduce the experimental observation that the SodB level in Δfur cells is 13% of the wild type level The first term represents the production of SodB. The next two terms represent dilution and passive degradation  was chosen based on the reported level of SodB mRNA (about 5 molecules/cell) There are several other RyhB target mRNAs exist in the cell, which, similar to SodB, are not regulated directly by Fur. Therefore Aα, and Sα were chosen in such a way that Aα+Bα = Sα/4 (representing the upper bound for SαAα = 7/cell generation). I compare the above natural system to a hypothetical one where SodA levels depend only on superoxide concentration and not on Fe-Fur or RyhB levels. In this case, AC = αA/3.3 to match the Fur-Fe regulated SodA level.The values of


We determined the Ea for four typical lignocellulose degrading enzymes in the A and B horizons of seven soils covering six different soil orders. We also elucidated which soil properties predicted any measurable differences in Ea. β-glucosidase, cellobiohydrolase, phenol oxidase and peroxidase activities were measured at five temperatures, 4, 21, 30, 40, and 60°C. Ea was calculated using the Arrhenius equation. β-glucosidase and cellobiohydrolase Ea values for both A and B horizons in this study were similar to previously reported values, however we could not make a direct comparison for B horizon soils because of the lack of data. There was no consistent relationship between hydrolase enzyme Ea and the environmental variables we measured. Phenol oxidase was the only enzyme that had a consistent positive relationship between Ea and pH in both horizons. The Ea in the arctic and subarctic zones for peroxidase was lower than the hydrolases and phenol oxidase values, indicating peroxidase may be a rate limited enzyme in environments under warming conditions. By including these six soil types we have increased the number of soil oxidative enzyme Ea values reported in the literature by 50%. This study is a step towards better quantifying enzyme kinetics in different climate zones.Enzyme dynamics are being incorporated into soil carbon cycling models and accurate representation of enzyme kinetics is an important step in predicting belowground nutrient dynamics. A scarce number of studies have measured activation energy (E Several studies have demonstrated that enzymes produced by microorganisms in colder climates have lower temperature optima in situ temperature range, with enzymes in colder climates having more flexible structures than enzymes in warmer climates a of enzymatic reactions in soils from different climates because isoenzymes for the same reaction do have different substrate affinities a. Extracellular enzymes can be sorbed to clays a of soil enzymes aa for four typical lignocellulose degrading enzymes in surface and subsurface horizons of seven soils covering six different soil orders. We also elucidated which soil properties predicted any measurable differences in Ea. We hypothesized that Ea for all enzymes would be greatest in cold regions (arctic and subarctic), followed by temperate and tropical regions. Tropical regions have very stable temperatures so that enzymes may be adapted to the constant warm temperatures whereas temperatures fluctuate daily and seasonally in the temperate and cold regions. In addition, we hypothesized that (2) the ambient temperature 30 days prior to sampling would influence the enzyme pool, so that potential enzyme activity would be lower in soils collected during warmer periods compared to colder periods due to enzymatic efficiency, and (3) Ea for oxidative enzymes would be greater than hydrolytic enzymes, regardless of climate regions, due to the difference in substrate complexity.Surprisingly, a scarce amount of studies have been carried out to determine the ESoil samples were collected from a broad range of climatic zones across the western hemisphere . From eaField moist soil samples were composited, 2 mm sieved, and then stored at −10°C for a maximum of 1 week until enzyme analyses could be performed. Gravimetric water content (GWC) was determined in triplicate by oven drying the pre-weighed subsamples for 24 hr at 105°C, and then reweighing each subsample. Total carbon and nitrogen analyses were performed on air dried, ground soil using a LECO TruSpec CN analyzer . Particle size analysis was performed using the Buoycous hydrometer method in situ soil pH, however Wang et al. (2012b) have demonstrated that most soil enzymes have pH optima around 5. Also, oxidative enzyme activity is very difficult to measure at a higher pH due to abiotic oxidation of substrates, which can lead to incorrect calculation of Ea. Through laboratory trials, it was determined that a pH 5 buffer would be an adequate pH for these soils despite the wide range of in situ soil pH  and cellobiohydrolase , and two oxidases, peroxidase  and phenol oxidase   soil pH . For eveThe hydrolase assays were performed in replicates of eight in black 96-well microplates. The blank wells on each plate received 250 µL of acetate buffer, reference-standard wells received 200 µL of acetate buffer and 50 µL of 10 µM 4-methylumbelliferone (MUB) standard, and negative-control wells received 200 µL acetate buffer and 50 µL 200 µM 4-MUB-linked substrates (4-MUB-β-D-glucoside and 4-MUB-β-D-cellobioside). For each soil, quench-control wells received 200 µL of soil suspension and 50 µL 10 µM 4-MUB standard, sample control wells received 200 µL soil suspension and 50 µL of 50 mM acetate buffer. Activity assay wells received 200 µL of soil suspension and 50 µL of 200 µM 4-MUB-linked substrate. After incubating for 2 h, each black microplate received 10 µL 0.5N NaOH in every well in order to raise the pH and enhance the fluorescence to a detectable level. Fluorescence was then detected at an excitation wavelength 365 nm, emission wavelength 450 nm, and sensitivity of 50 with a BioTec Synergy™MX Multi-Mode Microplate Reader.2O2. After incubation for 24 h, clear microplates were read spectrophotometrically at 460 nm with a BioTec Synergy™MX Multi-Mode Microplate Reader.The oxidase assays were performed in clear 96-well UV microplates. For each plate, 8 blank wells received 250 µL of acetate buffer and 16 reference-standard wells received 200 µL acetate buffer and 50 µL of L-3,4-dihydroxyphenylalanine (L-DOPA). For each soil, 8 homogenate control wells received 200 µL of soil suspension and 50 µL of acetate buffer and 16 assay wells received 200 µL soil suspension and 50 µL L-DOPA. Additionally, each well of the peroxidase assay plates received 10 µL of 0.3% H−1 h−1). An Arrhenius plot was created to estimate activation energy according to the Arrhenius equation:a is the activation energy, R is the gas constant and T is the absolute temperature (°K)For enzymatic assays, the activities for both hydrolases and oxidases were expressed as nmol of substrate converted per g dry soil per h , using the three enzyme subsamples from each location.Relationships between Ea tended to be higher in the B horizon compared to the A horizon horizon, except in the arctic system where they were similar (P<0.05). Phenol oxidase Ea, was consistently greater in the A horizon than B horizon soil in the tropics (P<0.05). Cellobiohydrolase and peroxidase Ea showed no discernible trend with soil depth.In most cases, β-glucosidase E similar ; P<0.05.a for all enzymes was affected by biome type (P<0.1 and 0.05). In the A horizon, Ea for the hydrolases was similar for arctic and subarctic biomes, while in the B horizon the hydrolase's Ea for the subarctic was more similar to temperate and tropical biomes. In the A horizon, within a biome the Ea of the hydrolytic enzymes was similar, whereas the Ea of the two oxidative enzymes were very different from each other. In the B horizon, the Ea of both hydrolases (β-glucosidase and cellobiohydrolase) were always lower in the arctic biome (Alaska) than subarctic, temperate and tropical biomes (P<0.1 and 0.05). Phenol oxidase Ea was similar in arctic and temperate biomes in both horizons, and lowest in the subarctic biome (P<0.1).Ea of β-glucosidase and cellobiohydrolase have positive relationships with average air temperature in the month preceding sampling and mean annual temperature . B horizon enzyme activity increased with assay temperature, however there was no clear trend for activity with average air temperature (data not shown).There was no clear relationship between potential hydrolase activities and average air temperature in the month preceding sampling , but there is a large portion of the terrestrial carbon stored below. It is unclear why enzyme studies tend to ignore enzymes dynamics below the first 15–20 cm, perhaps it is assumed that enzymes in the B horizon would respond similarly to those in the A horizon or that they are less affected by environmental disturbance due to their depth.In several cases the Ea and the environmental variables we measured. We found that in the A horizon there were fewer relationships between Ea and the environmental variables we used  compared to the B horizon. Only the positive linear relationship between phenol oxidase Ea and pH was maintained in both A and B horizons. There are other environmental variables  not measured in this study that could be measured in the future to determine their influence if any over Ea.Across enzymes there was no consistent relationship between Ea for oxidative enzymes is scant from soil environments a estimates coming from pure cultures of isolated enzymes a and 14 values for peroxidase Ea were identified in a in soil samples ranged from 30.5 kJ mol−1−1−1a in this study averaged across all climate regions was lower, 32 kJ mol−1, than previously estimated, but particularly in the arctic and subarctic regions, which had an average Ea of 19 kJ mol−1. Phenol oxidase activity ranged from 29–102 kJ mol−1 at our study sites and averaged 59.5 KJ mol−1 which was similar to previously recorded phenol oxidase Ea values 37–57 kJ mol−1−1a in colder regions was counter to our hypothesis that enzymes in colder regions would be more temperature sensitive than those in warmer region, however this does not hold true for phenol oxidase. The large difference in Ea between peroxidase and phenol oxidase indicate that not all oxidative enzymes respond similarly to temperature, whereas the two hydrolytic enzymes had similar temperature responses. Grouping oxidative enzymes together, as we did in our hypothesis, was incorrect because peroxidases appear to be much less temperature sensitive than hydrolytic enzymes, whereas phenol oxidases are much more temperature sensitive.The information to date on Ea than peroxidase indicating that the phenol oxidase reactions are more sensitive to temperature increases than peroxidase reactions, thus phenol oxidase reaction rates may increase more than peroxidase reaction rates with warming. Peroxidase also had very low Ea compared to the hydrolytic enzymes, indicating that the reaction rate is less sensitive to changes in temperature. In this study, peroxidase was the dominant oxidative enzyme in most environments, thus if temperatures rise, it may be the rate limiting step in decomposition because of its reduced sensitivity to temperature.In the soils we used, phenol oxidase had a greater EP<0.2). There was very little measurable peroxidase and no phenol oxidase activity in the soils from the warmer locations during the 4°C incubations and no measurable phenol oxidase activity in soils warmer locations during the 21°C incubations. This may be due to the structural conformation of the peroxidase and phenol oxidase enzymes. Enzymes in warmer locations tend to have more rigid or stable conformations whereas enzymes in colder environments tend to have more fluid conformations Enzyme activity for both oxidative enzymes showed a distinct trend with decreasing activity as the 30 day average temperature increased in accordance with our hypothesis that there would be lower potential enzyme activity in soils collected during warmer time periods because of increased enzymatic efficiency ; P<0.2. a calculations are made by adding simple substrates to soil slurries. The substrates are similar in structure to the substrates depolymerized in nature by enzymes but different in complexity. As mentioned earlier, plant material is made up of many different substrates but primarily lignin and cellulose which form a lignocelluloses complex. The lignocellulose complex has the lignin and cellulose intertwined so that enzymes need to work in conjunction to break down the material. The substrates we add in assays are single substrates, not in a complex, so the enzymes likely break down the substrates at a faster rate than in nature. Since Ea is calculated using activity rates the use of single substrates may decrease the Ea over what occurs in nature when a consortium of enzymes are required to complete the depolymerization of lignocelluloses complexes.Enzyme activity and thus Ea across the globe, out of the thirty possible enzymes to be assayed we selected only four, but we did select enzymes representative of two major groups, hydrolases and oxidases. Enzyme activity has been measured in arctic and tropical biomes, however the Ea for enzymes in these systems has rarely been measured before a values reported in the literature by fifty percent. This study is a step towards better understanding and comparing enzyme kinetics in different climate zones. Also, it points out that the classification of enzymes by reaction types may not be indicative of their responses to temperature. Enzyme dynamics are being incorporated into models This study is by no means a comprehensive list of enzyme EFigure S1Activation energy for all enzymes in both soil horizons. Ea in the A horizon (a–d), B horizon (e–h), and combined A and B horizons (i–l) across four soil characteristics: clay, pH, average air temperature (°C) for the month preceding sampling, and mean annual temperature (MAT). Significant and marginally significant linear and polynomial regressions are shown for each enzyme and each soil characteristic, P<0.2.(TIF)Click here for additional data file.


Mosquitoes, which evade contact with long-lasting insecticidal nets and indoor residual sprays, by feeding outdoors or upon animals, are primary malaria vectors in many tropical countries. They can also dominate residual transmission where high coverage of these front-line vector control measures is achieved. Complementary strategies, which extend insecticide coverage beyond houses and humans, are required to eliminate malaria transmission in most settings. The overwhelming diversity of the world's malaria transmission systems and optimal strategies for controlling them can be simply conceptualized and mapped across two-dimensional scenario space defined by the proportion of blood meals that vectors obtain from humans and the proportion of human exposure to them which occurs indoors. Indoor residual spraying (IRS) and long-lasting insecticidal nets (LLIN) can dramatically reduce malaria transmission, but will not be sufficient to completely eliminate it from most endemic tropical settings, even if effective drugs and vaccines are available, primarily because of vectors which evade contact with domestic applications of insecticides . At highπ) [πi) and measured in the field by weighting the observed indoor (i) and outdoor (o) biting rates at each period of the night by the surveyed mean proportion of humans that are in these two compartments at that time [πi,0) in the pre-intervention scenario (Ω) before the effective scale up of those interventions (Ω = 0). De facto protective coverage of humans  with LLINs, or any other form of personal protection against indoor exposure, is therefore defined slightly more specifically than before [Ch; estimated as the reported nightly usage rate) and this proportion of personal human exposure which is practically and directly preventable with an LLIN [Exophagic and zoophagic vectors can therefore comprise an increasingly important fraction of residual transmission in settings where high demographic coverage of LLIN or IRS has successfully suppressed predominant species that primarily feed indoors upon humans –11. For π) . In the hat time –16. Wherhat time –11 so thn before ,4,12, asa. However, as illustrated in figure 1b, only 40 per cent de facto protective coverage of humans is achieved in a scenario with 80 per cent demographic coverage, when only 50 per cent of human exposure occurs indoors.Obviously, the lower the proportion of exposure to a given mosquito population that occurs indoors, the lower will be the impact of LLINs or IRS upon the transmission it mediates, and the more persistent and prominent those populations will be in residual vector systems. Current demographic indicators of coverage for LLINs and IRS often grossly over-represent the degree of insecticidal hazard to which vector mosquitoes are exposed. A conventional demographic view of the current global target of 80 per cent LLIN use among all age groups  is presefigure 1b. The baseline human blood index  is defined as the population-wide mean proportion of blood meals that are obtained from humans (h), rather than animals, before the introduction of any intervention (Ω = 0). This parameter can be readily measured in the field and has long been known as an important determinant of malaria epidemiology and intervention impact [Ch,p → 1), because killing them in sufficient numbers to suppress malaria transmission requires high protective coverage of all available blood sources , including animals. This biological indicator of resource coverage is simply the product of the pre-intervention (Ω = 0) human blood index  and the protective coverage of humans  [A, Ah and Ah,p are the total availabilities or kinetic rates of encounter and feeding and attacking all hosts, all humans and all humans while protected, respectively [However, de facto coverage is a biological parameter relating to the coverage of all blood resources that mosquitoes need to thrive and is often even lower than apparent from figure 1n impact . The imps  :1.2wherec illustrates how 80 per cent demographic coverage of human users could result in only 20 per cent coverage of the total blood sources available for mosquitoes when the vector obtains half of its blood meals from animals and is equally likely to feed indoors and outdoors. The impact of LLIN or IRS intervention upon vector populations, and therefore the associated selection pressure for heritable resistance traits, are both directly related to this more biologically meaningful coverage indicator with the following simplified form of previous formulations [γP is the probability of a mosquito surviving all host attack per feeding cycle, while μp and μu represent the mortality probabilities of mosquitoes attacking protected and unprotected hosts, respectively.Figure 1Qh,0 < 0.1) [Plasmodium falciparum and Plasmodium vivax), so the small proportion of a very zoophagic mosquito population that is killed or diverted by these insecticidal products when they encounter humans can be a large proportion of those that actually transmit malaria [Ch,p; equation (1.1)) and the entomologically measured estimate of direct personal protective efficacy against biting exposure (ρ) [ψΩh, is the relative rate of exposure to malaria transmission of the average human (h) community member immediately after rapidly achieving a specific vector control scenario (Ω) defined by the protective coverage and protective efficacy of LLINs or IRS, compared with the average non-user under baseline conditions before scale up [The importance of host preference behaviour is best illustrated by the numerous mosquito species that rarely feed on humans, but which do so often enough to sustain stable malaria transmission (0 < 0 < 0.1) , and are0 < 0.1) . In star0 < 0.1) , LLINs a0 < 0.1) . Neverth0 < 0.1) . This is malaria . As malasure (ρ) :1.4wherescale up .c) [LLINs or IRS are clearly insufficient in themselves to eliminate malaria transmission because de facto protective coverage is attenuated where mosquitoes can readily access blood resources from animals or from humans while they are outdoors c [1–4,12c) ,21. As ic) . The devc) .c represents a simple framework with which the overwhelming diversity of the world's malaria transmission systems, and optimal strategies for controlling them with high coverage (Ch → 1) of adulticides [πi,0 [Qh,0 [πi,0) and upon humans versus animals  [Fortunately, figure 1lticides ,12,20,21es [πi,0 –16 and Q,0 [Qh,0 ,23. For s  ,12, can


Transcranial direct current stimulation (tDCS) has been shown to modulate cortical excitability. A small number of studies suggested that tDCS modulates the response to experimental pain paradigms. No trials have been conducted to evaluate the response of patients already suffering from pain, to an additional experimental pain before and after tDCS. The present study investigated the effect of a single session of anodal, cathodal and sham stimulation (15 mins/1 mA) over the primary motor cortex on the perceived intensity of repeated noxious thermal and electrical stimuli and on elements of quantitative sensory testing  applied to the right hand in 15 patients with chronic low back pain. The study was conducted in a double-blind sham-controlled and cross-over design. No significant alterations of pain ratings were found. Modalities of quantitative sensory testing remained equally unchanged. It is therefore hypothesized that a single 15 mins session of tDCS at 1 mA may not be sufficient to alter the perception of experimental pain and in patients with chronic pain. Further studies applying repetitive tDCS to patients with chronic pain are required to fully answer the question whether experimental pain perception may be influenced by tDCS over the motor cortex. Transcranial direct current stimulation (tDCS) is a non-invasive brain stimulation technique that modulates the neuronal resting membrane potential and thereby affects neuronal activity A pain reducing effect of tDCS on different types of experimental pain has been reported by a number of studies in healthy participants only Stimulation paradigms chosen for studies on the reduction of chronic clinical pain were more consistent: all authors applied anodal tDCS over the motor cortex. A significant pain reduction was reported in patients with fibromyalgia Moreover, the neurophysiological background has still not been fully understood and little is known about the effects of tDCS on experimental pain in patients with chronic pain. As chronic noxious input causes widespread structural changes to the brain In conclusion, it is unknown at present whethertDCS alters the perceived pain intensity of an experimental pain paradigm in patients with chronic pain.anodal or cathodal tDCS is more effective to influence experimental pain in patients with chronic pain.tDCS influences both thermal and electrical pain in the same way.Consequently, this study investigates whether tDCS reduces the perceived intensity of experimental pain  and modalities of quantitative sensory testing  in patients with chronic low back pain.Fifteen patients with chronic low back pain were included into a double-blind sham-controlled crossover trial to study the effects of anodal, cathodal and sham tDCS on experimental thermal and electrical pain.Eligible patients were recruited among participants of a pain management group program for low back pain at a specialized clinic in Germany. Inclusion criteria were: 18 years or older and a minimum of 6 months of low back pain, defined according to the “European Guidelines of Chronic Low Back Pain” Each participant received all three modes of stimulation . The order of stimulation polarity was randomized according to a computer generated randomization list that matched study entry numbers with stimulation codes . Consecutively, ratings to painful electrical stimuli and to an established repetitive heat pain paradigm were recorded  device with a thermode of Peltier elements . The thermode was placed on the participant's right forearm. Initially, warm detection thresholds (WDT) were measured, followed by heat pain thresholds (HPT). The mean threshold temperature of three measurements was calculated.Consecutively, a suprathreshold heat pain paradigm that had been used in a number of previous investigations The stimulation site for subsequent tDCS was determined by identifying the motor cortical representational fields of the right abductor digiti minimi muscle by single pulse TMS to the left motor cortex using a magnetic stimulator  and a figure-of-eight shaped magnetic coil with an outer diameter of 70 mm. The corresponding spot was marked with a felt tip pen.2) consequently resulting in a current density of 0.029 mA/cm2 over the left motor cortex. The stimulation paradigm was chosen according to the most frequently applied tDCS paradigm in previous research on experimental pain The direct current stimulation was delivered by a battery driven device  and applied via saline-soaked sponge electrodes sized 5×7 cm  was added to avoid loss of zero-values  with the factors stimulation mode  and time with respect to the intervention . Additionally, a repeated measures ANOVA was calculated with pain ratings of the 10 successive blocks each with the factors stimulation mode , time with respect to the intervention  and pain rating per block .Changes of mean electrical and thermal pain ratings, WDT and HPT were analyzed separately by a two-way analysis of variance for repeated measures (ANOVA) with the factors stimulation mode  and time with respect to the intervention . Uncorrected paired t-tests were used in all ANOVAs as post-hoc tests in case of significant main effects or interactions.As normal distribution is difficult to ascertain in small samples, the analysis was repeated with non-parametric tests (Friedman's ANOVA and Wilcoxon test). In case of repeated measures ANOVAs, pre-post differences were calculated and inserted into Friedman's ANOVA for the factor STIM . Additionally, all pre and all post results were also inserted into Friedman's ANOVA. Pre-post differences were examined by means of the Wilcoxon test. Elements of the 3 factorial repeated measures ANOVA for all consecutive blocks of repetitive heat pain were not repeated if results were non-significant and results of the non-parametric testing with mean pain ratings for all 10 blocks of repetitive heat pain were also non-significant.Rate of correct blinding was tested as a 2×2 contingency table for statistical significance with the Fisher's exact test. P values<.05 were considered significant. Statistical analysis was performed with SPSS 17 for Windows .Additionally, Pearson's correlation coefficient was calculated for baseline ratings of clinical pain and first electrical and thermal pain ratings and heat pain thresholds recorded immediately thereafter.A total of 15 subjects with chronic low back pain were recruited. One subject (s04) dropped out before completion of the study, because she was unable to tolerate the repetitive heat pain. The remaining 14 subjects  were included into the statistical analysis. An overview of baseline characteristics is given in There was no significant difference of the perceived mean pain intensity before and after tDCS stimulation for the factors time  = 0.85; p = 0.374) and stimulation modality  = 0.93; p = 0.379) and no interaction between STIM*PREPOST  = 1.18; p = 0.323). Non-parametric testing did not differ from these findings. Mean electrical pain ratings are shown in Individual patient responses are shown in Due to technical problems during data acquisition , data of 2 patients could not be collected . Thermal threshold and pain evaluation is therefore based on 11 respectively 12 complete sets of data respectively.post hoc t-tests showed that this effect was driven by the comparison of anodal results before and after stimulation (t(13) = −2.72; p = 0.018). Non-parametric tests confirmed these findings.Mean values for thermal thresholds are given in There were no significant differences of HPT before and after tDCS  = 0.82; p = 0.389) and between treatment modalities  = 1.27; p = 0.306). No interaction between the two factors  = 1.44; p = 0.263) could be found. Non-parametric testing did not differ from parametric testing.a priori difference  = 1.14; p = 0.319) between the three experimental sessions in a one way ANOVA. No significant difference of mean heat pain intensity over all 10 blocks before and after tDCS  = 2.09; p = 0.176) and between stimulation modalities  = 0.47; p = 0.555) could be found  did not show any significant be found . In addiNon-parametric testing did not differ from these findings.Likewise, a three factor repeated measures ANOVA using non-normalized data across all 10 blocks did not yield significant results for the factors PREPOST: F = 1.67; p = 0.222) STIM  = 0.56; p = 0.509) and BLOCK  = 2.87; p = 0.103) and the corresponding interactions  = 0.13; p = 0.767; STIM*BLOCK: F = 0.70; p = 0.554; PREPOST*BLOCK: F = 1.86; p = 0.188; STIM*PREPOST*BLOCK: F = 1.1; p = 0.363). When repeated with normalized data, results were comparable.Individual patient responses are shown in All patients reported that their back pain remained unchanged in intensity and quality after each stimulation session compared to baseline assessment as given in Pearson's correlation coefficient shows no relevant correlation between baseline ratings of clinical pain and first ratings of electrical and thermal pain and heat pain thresholds recorded immediately thereafter .Blinding was successful as confirmed by a non-significant result in Fisher's Exact test (p = 0.45).A single 15 minutes session of anodal or cathodal stimulation at 1 mA over the motor cortex did not significantly alter the perception of noxious thermal and electrical stimuli and thermal pain thresholds applied to the right hand in patients with chronic low back pain. The analysis according to responders and non-responders did not show significant distribution differences across the stimulation modes. Neither cathodal nor anodal or sham stimulation influenced the perceived intensity or quality of the patients' back pain. However, warm detection thresholds were significantly elevated after anodal tDCS.A change in clinical pain was not expected after a single 15 minutes session of tDCS with an intensity of 1 mA and clinical pain was not the main focus of this trial. A pain reducing effect on chronic pain has already been demonstrated by other authors who all applied more than one session of tDCS and commonly used longer durations (20 minutes) and higher intensities (2 mA) All patients stated that they could clearly distinguish between clinical and experimental pain. In addition, there was no relevant correlation between baseline clinical pain and first ratings of experimental thermal and electrical pain. Therefore, it is unlikely that an attentional deficit lead to insufficient discrimination between clinical and experimental pain.post hoc tests supports a careful appraisal of these findings.The only significant finding was an increase in warm detection thresholds after anodal tDCS. Thermal sensation may be more susceptible to cortical excitability changes following tDCS than nociception, especially as tDCS of the primary motor cortex induces widespread central activity changes Our results did not reproduce the effect reported by publications on tDCS for the reduction of experimental pain in healthy participants. Potential explanations for the different response of chronic pain patients to experimental pain compared to healthy participants as reported in previous publications Functional adaptations in chronic pain patients comprise altered cortical activity and changes of the cortical representational fields of the affected body parts The role of the motor cortex in neuromodulation may have been overestimated as effects of motor cortex stimulation have been shown to be conveyed by a network of other interconnected brain areas These results differ from those reported in two other studies on the effects of repetitive transcranial magnetic stimulation (rTMS) on experimental pain in patients suffering from a chronic pain condition We can exclude methodological shortcomings which could explain a missing effect for the following reasons: as previously shown, effects of tDCS on nociception persist for a maximum of 150 minutes after tDCS A larger sample size may have detected an effect that was missed in this study. Chronic low back pain patients are a heterogeneous sample regarding pain presentation and co-morbidities, thereby potentially influencing the effect of an intervention. In addition, the concomitant intake of analgesics antidepressant/antiepileptic drugs in a subgroup of the patients could have influenced our results, although medication was kept constant throughout the study. The participant (s10) who took pregabalin and amitriptylin did not react differently to tDCS than any other participant.Single-session anodal and cathodal direct current stimulation did not modulate the perception of nociceptive experimental stimuli in patients with chronic low back pain. Further research is needed to investigate the effect of higher intensities and repetitive application of tDCS as well as stimulation sites other than the motor cortex.


Drosophila melanogaster provides an ideal system to study the genetics of variation for resistance to oxidative stress.Aerobic organisms are susceptible to damage by reactive oxygen species. Oxidative stress resistance is a quantitative trait with population variation attributable to the interplay between genetic and environmental factors. Drosophila Genetic Reference Panel for a genome-wide association study of acute oxidative stress resistance to two oxidizing agents, paraquat and menadione sodium bisulfite. We found significant genetic variation for both stressors. Single nucleotide polymorphisms (SNPs) associated with variation in oxidative stress resistance were often sex-specific and agent-dependent, with a small subset common for both sexes or treatments. Associated SNPs had moderately large effects, with an inverse relationship between effect size and allele frequency. Linear models with up to 12 SNPs explained 67–79% and 56–66% of the phenotypic variance for resistance to paraquat and menadione sodium bisulfite, respectively. Many genes implicated were novel with no known role in oxidative stress resistance. Bioinformatics analyses revealed a cellular network comprising DNA metabolism and neuronal development, consistent with targets of oxidative stress-inducing agents. We confirmed associations of seven candidate genes associated with natural variation in oxidative stress resistance through mutational analysis.We used 167 wild-derived inbred lines of the We identified novel candidate genes associated with variation in resistance to oxidative stress that have context-dependent effects. These results form the basis for future translational studies to identify oxidative stress susceptibility/resistance genes that are evolutionary conserved and might play a role in human disease. Oxidative stress, or the overabundance of reactive oxygen species (ROS) as an unavoidable consequence of aerobic respiration, has been implicated in aging age-1, mth, and 66shc associated with increased longevity of Caenorhabditis elegans, Drosophila melanogaster and mice Superoxide dismutase (Sod) Catalase (Cat) Sod and Cat transgenes results in enhanced oxidative stress resistance and longevity in specific Drosophila genetic backgrounds Diptericin, Tropomyosin1, and AttacinD. melanogasterGenetic variants associated with susceptibility to oxidative stress in human populations have largely been identified indirectly, as many alleles associated with increased risk for common diseases and aging are in genes in oxidative stress response pathways D. melanogaster Genetic Reference Panel (DGRP), derived from the Raleigh, North Carolina population, has been fully sequenced Drosophila model allows validation of candidate genes and identification of causal SNPs through a variety of genetic approaches, including analysis of mutations.Recently, a population of inbred wild-derived lines comprising the Here, we have used 167 DGRP lines to conduct GWA mapping of acute oxidative stress susceptibility/resistance by measuring survival time on two different oxidative stress inducing agents, paraquat and menadione sodium bisulfite (MSB). We found significant and sex-specific genetic variation in survival time among the lines for both treatments. Our GWA analysis identified several hundred candidate genes, several of which were previously not known to be involved in oxidative stress response and many of which have human homologs. Many of these candidate genes were sex-specific and uniquely susceptible to one of the oxidative stress inducing treatments, but a subset converge on a common network that may represent a central conduit for response to various oxidative stress inducing agents. Finally, we have validated a subset of candidate genes using mutant analysis.Wolbachia infection status Wolbachia infection had a significant effect on survival time on paraquat for both sexes and survival time on MSB in males  and menadione sodium bisulfite (MSB), among 167 DGRP lines in males . Therefo2H) for survival time was 2H = 0.48 on MSB and 2H = 0.36 on paraquat (MFr), which quantify the extent to which the same variants affect a trait in males and females, were significantly different from unity , underscoring sex-specific differences in susceptibility to oxidative stress agents.We found extensive phenotypic and genetic variation for survival time on both paraquat and MSB ; Table 1paraquat , when poparaquat . HoweverPMr = 0.35; males PMr = 0.29; When sexes were assessed separately, pooled across treatments , the effP<10−5. A total of 56 (23) remained significant for paraquat (MSB) when the significance threshold was P<10−6 . For the analyses of survival time on either paraquat and MSB pooled across sexes we found that the SNP effects  were inversely related to minor allele frequencies, such that the less common SNPs had greater effects  of SNPs with SNP by sex interactions for survival on paraquat had sexually antagonistic effects; the remainder had sex-biased or sex-specific effects. The larger number of SNPs affecting both sexes were associated with increased survival of females on paraquat and increased survival of males on MSB; this is consistent with the mean differences in male and female survival on paraquat and MSB.As expected from the quantitative genetic analyses indicating significant genetic variation in sexual dimorphism for survival on paraquat and MSB, we identified 87 (45) SNPs with significant SNP by sex interaction terms for paraquat (MSB) at Our quantitative genetic analysis also indicated significant genetic variation in the magnitude and or direction of the effects of the two treatments. Consistent with this observation, we identified 54 (21) SNPs exhibiting significant SNP by treatment interactions in females . In females, 34 of these SNPs (63%) had opposite effects in the two treatments, while in males 13 (62%) had opposite effects on survival under paraquat and MSB. The remaining SNPs with significant SNP by treatment interactions were treatment-specific or -biased.CG11873, CG32541, enabled (ena), Glutamate receptor binding protein (Grip), and rugose (rg)) were associated with survival time on both paraquat and MSB  have human homologs . Three additional genes, CG13492, nicotine Acetylcholine Receptor α 30D  and tonalli (tna) contained different SNPs that were associated with variation in oxidative stress susceptibility in both sexes ). For MSB, three SNPs were shared between the female and male prediction models . These results show that alleles with large effects on oxidative stress resistance are generally not shared between the sexes. We found no overlap between the genes in the multiple regression models for survival on paraquat and on MSB, which highlights the low correlation in survival in response to the different agents.For paraquat, only two SNPs were included in both the female and male prediction models , ena, fog, homeobrain (hbn), nACRα-30D, and rg) associated with oxidative stress susceptibility/resistance for mutant validation. These genes were chosen based on the significance level of their association with phenotypic variation and on the availability of co-isogenic P-element or Minos-element mutant alleles. All seven genes and corresponding controls were tested for both sexes with both treatments. All seven genes showed a significant difference in survival time on an oxidizing agent between mutant and control for at least one sex (P = 0.0001).We selected seven candidate genes  and/or compounds between our candidate genes, we identified a network significantly enriched (P = 0.005) for 42 genes that includes 17 candidate genes from the genome wide association analysis  that were significantly associated with oxidative stress susceptibility in both sexes; two genes  that were significantly associated with oxidative stress susceptibility in both treatments; and three genes  with low P-values. Mutations in all seven genes were significant in at least one sex or treatment  also have human homologs implicated in Alzheimer's disease lat, has a human homolog that has been associated with schizophrenia Rapgap1 and numb have human homologs with tumor suppressor activity mei-41 and mus201Btk29A has been implicated in gout We identified a cellular network comprising 42 genes, including 17 of our candidate genes, centered on DNA metabolism and neural development . Twelve Drosophila is complex, sexually dimorphic and dependent on the oxidative stress inducing agent. Candidate genes associated with oxidative stress susceptibility/resistance fall largely in gene ontology categories associated with DNA metabolism and nervous system development and function. While we were successful in identifying many SNPs associated with oxidative stress resistance, some of the completely recessive alleles detected in our study presumably do not contribute to variation in outbred populations. Future work using progeny from crosses and outbred populations will be necessary in order to detect dominance effects that contribute to oxidative stress susceptibility, resulting in a more complete understanding of the genetic architecture underlying this trait. This study and future work regarding oxidative stress susceptibility/resistance in D. melanogaster allow the identification of evolutionarily conserved target genes and gene networks that can serve as a blueprint for future translational studies on oxidative stress in people.We have shown that the genetic architecture of oxidative stress susceptibility in Drosophila melanogaster Genetic Reference Panel (DGRP) We used 167 inbred lines of the Drosophila Activity Monitoring System (Trikinetics). Time of death was defined as the last activity count followed by six hours of inactivity. Survival times were expressed as the deviation from a contemporaneous w1118; Canton-S isogenic control line mean. Two oxidative stress inducing agents were administered through diet, paraquat  and menadione sodium bisulfite (MSB). To assay survival time on paraquat, individual flies were exposed to filter paper soaked with 50 µL of a 1% sucrose solution containing 20 mM paraquat. Previous studies showed that a 20 mM paraquat 1% sucrose solution is sufficient to induce oxidative stress related death in Drosophila within 88 hours We measured 24 three-six day-old individual flies per sex per DGRP line per oxidative stress inducing agent for 88 hours using the Wolbachia infection status, previously measured on the DGRP lines Y = μ+S+I+S×I+L(I)+S×L(I)+ε, where I denotes the fixed effect of infection status, S is the fixed effect of sex, L is the random effect of the DGRP line, and ε is the error variance. We also performed reduced analyses for each sex separately. In cases where the effect of Wolbachia was significant, data were corrected, separately by sex.We assessed the effect of Wolbachia-corrected data using ANOVAs of two forms. For Model 1, both treatments were analyzed separately using an ANOVA of the form Y = μ+S+L+S×L+ε, where terms are defined as above. Reduced ANOVAs were also performed for each sex separately. For Model 2, treatments were pooled and sexes were analyzed separately using an ANOVA of the form Y = μ+T+L+T×L+ε, where T is the fixed effect of treatment. For Model 1, we estimated broad-sense heritabilities (2H) as 2H = (2Lσ+2SLσ)/(2Lσ+2SLσ+2Eσ), where 2Lσ, 2SLσ, and 2Eσ are the among-line, sex by line and within-line variance components, respectively. Similarly, broad-sense heritabilities were estimated for Model 2 as 2H = (2Lσ+2TLσ)/(2Lσ+2TLσ+2Eσ), where 2TLσ is the line by treatment interaction. Coefficients of genetic (GCV = 100Gσ/mean) and environmental (ECV = 100Eσ/mean) variance were also computed. For the analyses of each sex separately, H2 = Lσ2/(Lσ2+Wσ2), where Lσ2 is the among line variance component for males or females. We estimated cross-trait (cross-sex) genetic correlations as G = covij/σiσjr, where ijcov is the covariance of line means between traits i and j , and iσ and jσ are the square roots of the among line variance components for the two traits .We partitioned phenotypic variance using the Wolbachia infection status. All segregating sites within lines were treated as missing data. SNPs were filtered based on several criteria: (1) the minor allele had to be represented in at least four DGRP lines; (2) SNPs were excluded if coverage from whole-genome sequencing was less than 2 or greater than 30 We tested survival times on paraquat and MSB for association with 2,481,491 SNPs previously identified by whole-genome sequencing of the DGRP lines Y = μ+M+S+M×S+L(M)+ε, where M is the effect of the SNP, and S and L are as defined above. Reduced analyses of the form Y = μ+M+ε were also performed for males and females separately. Analyses where paraquat and MSB treatments (T) were pooled separately for each sex using the ANOVA model Y = μ+M+T+M×T+L(T)+ε, were also performed. The main effect (a) of each SNP was estimated as one-half the difference in trait mean between marker classes  Each SNP was tested for association with survival time on paraquat and MSB using an ANOVA of the form P<10−5) that were within a gene were incorporated into the model. Only one SNP was included in the model for pairs of SNPs in high LD. The most significant gene-centered SNP was fitted in the model first, and markers were sequentially added until the maximum 2r for variance explained was reached, up to a maximum of 12 markers. Models were fitted for each treatment and performed separately using line mean data from females, males and the average of the two sexes. Once a final model was selected, an ANOVA of the form Y = μ+H+L(H)+ε was performed, where H denotes haplotype and L line. The fraction of the total genetic variance accounted for by the model was estimated as Hσ2/(Hσ2+Lσ2), where Hσ2 is the among-haplotype variance component and Lσ2 is the among-line variance component.Gene-centered forward selection was used to generate multiple regression models in order to identify SNPs that were predictive for survival on paraquat and MSB. Only significant SNPs . Functional annotations of genes are based on Flybase Table S1Wolbachia infection status and mean phenotypic values of paraquat and MSB resistance.(DOC)Click here for additional data file.Table S2Analyses of variance of survival time on paraquat and MSB.(DOC)Click here for additional data file.Table S3Analyses of variance of females and males pooled across treatments.(DOC)Click here for additional data file.Table S4Quantitative genetic analysis of females and males pooled across treatments.(DOC)Click here for additional data file.Table S5GWA analysis results for survival time on paraquat and MSB (sexes pooled).(XLS)Click here for additional data file.Table S6GWA analysis results for females and males (treatments pooled).(XLS)Click here for additional data file.Table S7Candidate genes with human homologs and their role in complex disease.(XLS)Click here for additional data file.Table S8P-values.Validation mutant test (DOC)Click here for additional data file.


However, one common element that is integral to the maintenance of stromal cell environments, either in lymphoid tissue or in extra-lymphoid sites, is the constitutive regulation of stromal cell phenotype and/or function by the lymphotoxin (LT) pathway. Here we discuss how the LT pathway influences stromal cell environments both in homeostasis and in the context of inflammation in lymphoid and non-lymphoid tissues.Stromal cell microenvironments within lymphoid tissues are designed to support immune cell homeostasis and to regulate ongoing immune responses to pathogens. Such stromal cell networks have been best characterized within lymphoid tissues including the spleen and peripheral lymph nodes, and systems for classifying stromal cell phenotypes and functions are emerging. In response to inflammation, stromal cell networks within lymphoid tissues change in order to accommodate and regulate lymphocyte activation. Local inflammation in non-lymphoid tissues can also induce  LTβR is a member of the tumor necrosis factor (TNF) superfamily of receptors and is triggered by two ligands: membrane-bound LTαpathways . During pathways .In the adult animal, stromal cell phenotype and function must be constitutively maintained for the lifetime of the host in order to maintain the integrity of lymphoid tissue, and much of this maintenance is accomplished by continual LTβR signaling . The celThese findings have important implications for how we view stromal cells. First, it suggests that stromal cells are highly dynamic and rely on continual input from LTαβ-expressing cells. Second, since LTαβ is up-regulated on activated lymphocytes , lymphocFigure 1 are examples of LN stroma that are under LT control in both the steady state and during inflammation.Lymph nodes are composed of a variety of stromal cell types whose phenotype and function are being increasingly elucidated . In genein situ . Mice that receive LTβR-Ig treatment have hypo-cellular LN due to the requirement of LTβR signaling in regulating the expression of sulfotransferase enzymes that mediate post-translational modification of PNAd. Without these modifications, PNAd is aberrantly expressed in HEV and naive L-selectiniciently . A similiciently . Recentliciently . WhetherThe LT pathway plays a critical role in regulation of IgA production in the gut , and thi+ cells  that extend their processes into the SCS lumen to pick up Ag complexes  infection, lymphoid tissue architecture becomes disorganized but is eventually restored in a manner that depends on LTαβ expression on LTi-like innate lymphoid cells . In addiIn the LN, inflammation also greatly increases the size of the LN and this LN hypertrophy is accompanied by endothelial cell proliferation that can be promoted by the production of VEGF. FRC is a source of VEGF and this is dependent on LTαβ/LTβR signaling  as well As mentioned, mature primary FDCs are located throughout B cell follicles and rely on constitutive, low-level LTβR signaling . During Inflammation in peripheral tissues can create an environment that is permissive to the formation of follicle-like structures (FLS). These structures have been observed in a wide variety of settings and display differing levels of organization, and in some cases have been shown to support local immune responses . In thisInducible bronchus-associated lymphoid tissues iBALT;  are FLS LTα-/- mice have shown that LTβR signaling is not required for iBALT formation or induction of CXCL13, CCL19, and CCL21 during acute inflammation (+IL-17+ cells are necessary to initiate iBALT formation (+IL-17+ cells are insufficient for optimal organization and maintenance of iBALT which instead is dependent on LTβR signaling.Unlike LN and PP which require LTαβ–LTβR signaling for their formation, studies using ammation . Insteadormation . HoweverFollicle-like structures have been documented at sites of chronic inflammation in several autoimmune diseases including: rheumatoid arthritis, Sjörgen’s syndrome, systemic lupus erythematosus, and Multiple Sclerosis MS; . There iA role for the LT pathway in attenuating clinical disease has previously been described in the rodent model of MS, experimental autoimmune encephalomyelitis EAE; . PharmacIt is clear that LTβR-generated signaling underpins the maintenance and in some cases the function of stromal cell types within lymphoid tissues. Not discussed here are examples of how LTβR signaling is also important in myeloid/DC biology , and DC The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Both components can drive the synthesis of ATP at the chloroplast ATP synthase, but the ΔpH component also plays a key role in regulating photosynthesis, down-regulating the efficiency of light capture by photosynthetic antennae via the qE mechanism, and governing electron transfer at the cytochrome b6f complex. Differential partitioning of pmf into ΔpH and Δψ has been observed under environmental stresses and proposed as a mechanism for fine-tuning photosynthetic regulation, but the mechanism of this tuning is unknown. We show here that putrescine can alter the partitioning of pmf both in vivo (in Arabidopsis mutant lines and in Nicotiana wild type) and in vitro, suggesting that the endogenous titer of weak bases such as putrescine represents an unrecognized mechanism for regulating photosynthetic responses to the environment.The light reactions of photosynthesis store energy in the form of an electrochemical gradient of protons, or proton motive force ( In contrast, under environmental stresses—e.g., high light, low CO2/O2, when photoprotection is advantageous—pmf can be predominantly stored as ΔpH, maximizing lumen acidification for a given pmf.Differential partitioning of the thylakoid pmf is partitioned into Δψ and ΔpH remains unclear, but in vitro experiments and modeling have established that at least three factors are critical pmf partitioning in response to environmental changes, because thylakoid membrane capacitance and lumen buffering capacity are thought to change only slowly, whereas plastids can rapidly alter the ionic balance by regulating ion channels or ion pumps The mechanism by which thylakoid More recently, Ioannidis et al. 2(CH2)4NH2], which, along with spermidine and spermine, constitute the major polyamines in plants. Polyamines are important or even essential for many cellular processes, such as cell growth and stress tolerance pmf in isolated thylakoids pmf. However, weak bases in the presence of high concentrations of counterions, which are permeable through ion channels, can dissipate both the Δψ and ΔpH components of pmfin vivo is likely to be small pmf.Putrescine (Put) is a diamine [NHin vivo, we assessed the fractions of pmf stored as Δψ and ΔpH using in vivo spectroscopic techniques pmf, whereas the slowly recovering phase is attributed specifically to the ΔpH portion. Deconvolution, as indicated, yields an estimate of the fraction of pmf stored as ΔpH and Δψ (ΔpH-related phase (or decrease of Δψ) upon partial depletion of putrescine, which was reversed by putrescine infiltration  deficient in putrescine synthesis. Plants completely lacking putrescine are not viable adc2-2−1). Estimates of pmf partitioning using the decay of the ECS adc2-2 led to a ∼15% smaller fraction of pmf attributable to Δψ [or a larger fraction stored as ΔpH , and its effects have been attributed to its ability to bind anions, nucleic acids, and other negatively charged molecules or domains pmf partitioning by scavenging anions that would otherwise permeate the thylakoid and dissipate Δψ. To test these possibilities, we assayed the effects of putrescine on Δψ/pmf using ECS assays pmf on putrescine concentration is shown in 2) (solution of low ionic strength), the fraction of pmf stored as Δψ was about 0.2 for control, indicating that a significant decline occurred after isolation in comparison to the in vivo conditions (Δψ/pmf∼0.5)  . This de/pmf>0.9 . Our decThe fact that putrescine decreased ΔpH even in a solution with low concentration of scavangeable anions, supports the operation of the BWB mechanism, and is also consistent with the effects of exogenously added weak bases in photosynthetic prokaryotes pmf stored as Δψ and ΔpH  Arabidopsis grown under high salt stress, photosynthesis would likely need to operate under conditions where the ionic strength inside the plastid is high. In this case, pmf storage would be heavily biased toward ΔpH formation 2 were not limiting. Thus, the accumulation of putrescine observed in plants grown under high salt stress Arabidopsis through adc2 induction pmf toward Δψ and optimizing the regulation of energy transduction. In line with this view, blocking this up-regulation of putrescine during salt stress, e.g., in the adc-2-1 mutant of Arabidopsis, leads to increased sensitivity to salt stress, which is restored upon addition of putrescine adc increased tolerance to drought In pmf partitioning in vivo via the BWB mechanism, possibly operating as a part of the regulatory network of photosynthesis. Putrescine levels in the leaf are known to be regulated at several levels, including rates of synthesis, catabolism, conversion to spermidine, conjugation, intracellular or extracellular transport, gene expression, and/or allostery E response to pmf, optimizing the balance between energy transduction and dissipation under a variety of stress conditions.We present evidence that putrescine plays a role in modulating E, ECSt, and Δψ/pmf at room temperature using wild-type (Wt) Arabidopsis thaliana  plants, the low putrescine adc2-2 mutant Nicotiana tabacum cv Xanthi as previously described −2s−1) and Ft is the level of fluorescence immediately before the saturating pulse. For estimates of pmf partitioning, the ECS was measured over longer dark intervals (60 sec) and deconvoluted as previously described 520 = A520 − 0.5×A535 − 0.5×A505. For more precise deconvolution of ECS signals from Arabidopsis, we derived an equation from empirically determined, relative extinction coefficients at each wavelength, based on methods described previously 520 = −(1.61*A520−0.61*A505−1.17A535).We conducted time-resolved spectroscopic measurements for estimation of LEF, qPutrescine titer in leaves was estimated after benzoylation of the amines, separation in HPLC, and quantitation of the derivatives as previously described 2, 0.3 M sucrose, 10% ficoll. Photosynthetic proton uptake was supported by 30 µM PMS. Spectroscopic measurements were performed, as above, with the sample contained in a cuvette (10-mm path length). For thylakoid samples, a simple deconvolution yielded consistent results: ECS520 = A520 − 1.2×A535.Thylakoids were isolated from market spinach as previously described Figure S1Simplified scheme for the regulation of pmf partitioning by Putrescine.(DOC)Click here for additional data file.Figure S2Assay for putrescine uptake into tobacco leaves by HPLC.(DOC)Click here for additional data file.Figure S3Effects on Δψ/pmf and PSII photochemical efficiency of infiltration of leaves with water.(DOC)Click here for additional data file.Figure S4E) on the ΔpH component of light-induced Effect of putrescine titre on the dependence of energy-dependent antenna down-regulation (qpmf.(DOC)Click here for additional data file.Figure S5E) as a function of total light-induced Energy-dependent antenna down-regulation (qpmf.(DOC)Click here for additional data file.Figure S6H+).Effects of putrescine titre on the apparent proton conductivity of the ATP synthase (g(DOC)Click here for additional data file.


The retinoic acid receptor beta2(RARβ2) is a type of nuclear receptor that is activated by both all-trans retinoic acid and 9-cis retinoic acid, which has been shown to function as a tumor suppressor gene in different types of human tumors. Previous reports demonstrated that the frequency of RARβ2 methylation was significantly higher in prostate cancer patients compared with controls, but the relationship between RARβ2 promoter methylation and pathological stage or Gleason score of prostate cancer remained controversial. Therefore, a meta-analysis of published studies investigating the effects of RARβ2 methylation status in prostate cancer occurrence and association with both pathological stage and Gleason score in prostate cancer was performed in the study. A total of 12 eligible studies involving 777 cases and 404 controls were included in the pooled analyses. Under the random-effects model, the pooled OR of RARβ2 methylation in prostate cancer patients, compared to non-cancer controls, was 17.62 with 95%CI = 6.30–49.28. The pooled OR with the fixed-effects model of pathological stage in RASSF1A methylated patients, compared to unmethylated patients, was 0.67 (95%CI = 0.40–1.09) and the pooled OR of low-GS in RARβ2 methylated patients by the random-effect model, compared to high-GS RARβ2 methylated patients, was 0.54 (95%CI = 0.28–1.04). This study showed that RARβ2 might be a potential biomarker in prostate cancer prevention and diagnosis. The detection of RARβ2 methylation in urine or serum is a potential non-invasive diagnostic tool in prostate cancer. The present findings also require confirmation through adequately designed prospective studies. Prostate cancer is the most commonly diagnosed noncutaneous neoplasia in the world. The disease predominantly affects men after the 6th decade of life and is associated with considerable morbidity and mortality Recently, methylation of CpG islands within the promoter and/or 5′ regions of genes is recognized as a common alteration in cancer-related genes often associated with partial or complete transcriptional disruption The RARβ2 is mapped to chromosomal region 3p24 , which is expressed in most tissues and has been shown to function as a tumor suppressor gene in lung, breast, and gynecological neoplasia To date, RARβ2 methylation has been proved in a number of individual studies, which is detected not only in tissue samples but also in urine and serum samples. The prognostic value of RARβ2 methylation status in prostate cancer patient's diagnosis and the relationship between RARβ2 methylation and pathological stage of prostate cancer and Gleason score remains unclear. Therefore, a systematic review was performed of the literature with meta-analysis to obtain a more accurate evaluation of the role of RARβ2 methylation in prostate cancer management.Studies were identified via an electronic search of PubMed and Google Scholar using the following key words: prostate cancer, PCa, retinoic acid receptor β2, RARβ2, RARbeta2, methylation. We also manually searched the references of these publications in order to retrieve additional studies. Only those published as full-text articles and in English were included as candidates. The search updated on 28, December,2012.Studies were selected for analysis if they met the following criteria: 1) they were original epidemiological studies on the correlation between RARβ2 promoter methylation and the prognosis of prostate cancer patients; 2) RARβ2 methylation status was examined using methylation-specific PCR (MSP) or quantitative MSP (QMSP); 3)the subjects in every study comprised patients and non-cancer controls; 3) studies should be with full text not only abstracts for relevant information extraction; 4) when the same patient population reported in several publications, only the most recent report or the most complete one was included in this analysis to avoid overlapping between cohorts; 5) the numbers of patients and controls in each study should be more than 5 respectively.For each eligible study, we collected information regarding authors, year and source of publication, country of origin, inclusion criteria, exclusion criteria, pathological stage, Gleason score, RARβ2 methylation frequencies in non-cancer controls and patients of prostate cancer and the method for methylation detection. All included studies used non-cancer people as a control group, though some of them did not provide the definition of non-cancer. In studies defining non-cancer people, there are two definitions: (1) normal healthy person; (2) people with benign hyperplasia prostate. Since it is impossible to redefine non-cancer people on a unified standard, we combined non-cancer people in our meta-analysis according to their original group in each individual study. Of these studies, pathological stage ≤ T2 was defined as low-stage, and pathological stage ≥ T3 was defined as high-stage which were defined by clinical differentiation. Gleason score≥7 was defined as high-GS and Gleason score≤6 was defined as low-GS. The final eligible articles selected for further meta-analysis were evaluated independently by two reviewers. Minor discrepancies were resolved by the authors' discussion.The foremost analysis examined the differences in the frequency of RARβ2 methylation between prostate cancer patients and non-cancer people by odds ratio (OR) with the corresponding 95% CI. Moreover, the strength of association between RARβ2 methylation and patients' pathological stage and tumor Gleason score were also assessed by OR with the corresponding 95% CI. To assess heterogeneity across the studies, the statistics analysis for heterogeneity was performedAccording to our inclusion criteria, a total of 12 eligible studiesIn general, the frequencies of RARβ2 methylation were tested in 12 reliable studies. The main results were summarized in Sensitivity analysis revealed that 11 independent studies were the main source of heterogeneityAs shown in The results of our systematic review showed that RARβ2 methylation in prostate cancer was associated with tumor risk as either detected in urine, serum or tissue by MSP or QMSP. However, the RARβ2 methylation was not associated with increased risk for developing pathological stage or Gleason score of prostate cancer in comparison between RARβ2 methylated bladder cancer patients and unmethylated patients.Hypermethylations of the RARβ2 gene having been reported in many studies declared that the frequency of RARβ methylation was found to be significantly higher in patients group compared with controls However, Carmen Jerónimo's study suggested that RARβ2 methylation levels correlated with pathological tumor stage but not with Gleason scoreEarly diagnosis of prostate cancer currently relied on trans-rectal ultrasound guided needle biopsy (10 to 12 cores) in men with increased total PSA (greater than 4.0 ng/ml) and/or abnormal DRE findingsCancer is not a single cell disease. Aberrant cancer cells and their interactive microenvironment are needed for cancer to progress to androgen independence and distant metastasisRARb2 might be silenced not only by DNA methylation but also by histone deacetylation. Acetylation and deacetylation on lysine residues of histone amino-terminal tails had profound effects on gene transcriptionIn conclusion, our meta-analysis suggested that detection of RARβ2 methylation might be a potential biomarker diagnostic tool in prostate cancer. The detection of RARβ2 methylation in urine or serum is a potential non-invasive diagnostic tool in prostate cancer. It is necessary to conduct large sample size studies of the association between RARβ2 methylation and prostate cancer risk, eventually leading to our better understanding.