NMR‐based conformation and dynamics of a tetrasaccharide‐repeating sulfated fucan substituted by different counterions

The sulfated fucan from the sea urchin Lytechinus variegatus is composed of the repetitive sequence [‐3)‐α‐l ‐Fucp‐4( )‐(1‐3)‐α‐l ‐Fucp‐2,4‐di( )‐(1‐3)‐α‐l ‐Fucp‐2( )‐(1‐3)‐α‐l ‐Fucp‐2( )‐(1‐]_n. Conformation (of rings and chains) and dynamics of this tetrasaccharide‐repeating sulfated fucan substituted by Na⁺, Ca²⁺, and Li⁺ as counterions have been examined through experiments of liquid‐state nuclear magnetic resonance spectroscopy. Scalar coupling and nuclear Overhauser effect (NOE)‐based data have confirmed that all composing units occur as ¹C₄ chair conformer regardless of the cation type, unit position within the repeating sequence, and sulfation type. Chain conformation determined by NOE signal pattern assisted by molecular modeling for a theoretical octasaccharide has shown a similar linear 3D structure for the three differently substituted forms. Data derived from spin‐relaxation measurements have indicated a contribution of counterion type to dynamics. The calcium‐based preparation has shown the highest mobility while the sodiated one showed the lowest mobility. The set of results from this work suggests that counterion type can affect the physicochemical properties of the structurally well‐defined sulfated fucan. The counterion effect seems to impact more on the structural mobility than on average conformation of the studied sulfated glycan in solution.     

New insights into the interaction of proteins and disaccharides—The effect of pH and concentration

To gain new insights into the interaction of proteins and disaccharides, we investigated the hydrodynamic radii, , of lysozyme molecules in solution and in a ternary protein‐sugar‐water system by PFG‐NMR. Our approach is based on the assumption that the anhydrobiotic properties of disaccharides like trehalose are based on aggregation of sugar molecules to the proteins, i.e., accumulation of sugar molecules close to the protein, and that this process can be investigated by the experimentally detectable value of the protein. The R_h values are calculated from the experimentally determined diffusion coefficients and the application of a viscosity correction using the inert molecule dioxane as an internal viscosity reference. The experiments were performed as a function of sugar concentration, the overall particle concentration and the pH value. We investigated the disaccharides trehalose and sucrose, mainly for the reason that trehalose has well know cryptobiotic properties while sucrose, which is similar in size and structure, lacks these properties. The results show the formation of a protective sugar shell around the proteins over a wider range of concentrations and pH values in the case of trehalose.     

Contemporary effective population and metapopulation size (Ne and meta‐Ne): comparison among three salmonids inhabiting a fragmented system and differing in gene flow and its asymmetries

We estimated local and metapopulation effective sizes ( and meta‐) for three coexisting salmonid species (Salmo salar, Salvelinus fontinalis, Salvelinus alpinus) inhabiting a freshwater system comprising seven interconnected lakes. First, we hypothesized that might be inversely related to within‐species population divergence as reported in an earlier study (i.e., F_ST: S. salar> S. fontinalis> S. alpinus). Using the approximate Bayesian computation method implemented in ONeSAMP, we found significant differences in () between species, consistent with a hierarchy of adult population sizes (). Using another method based on a measure of linkage disequilibrium (LDNE: ), we found more finite values for S. salar than for the other two salmonids, in line with the results above that indicate that S. salar exhibits the lowest among the three species. Considering subpopulations as open to migration (i.e., removing putative immigrants) led to only marginal and non‐significant changes in , suggesting that migration may be at equilibrium between genetically similar sources. Second, we hypothesized that meta‐ might be significantly smaller than the sum of local s (null model) if gene flow is asymmetric, varies among subpopulations, and is driven by common landscape features such as waterfalls. One ‘bottom‐up’ or numerical approach that explicitly incorporates variable and asymmetric migration rates showed this very pattern, while a number of analytical models provided meta‐ estimates that were not significantly different from the null model or from each other. Our study of three species inhabiting a shared environment highlights the importance and utility of differentiating species‐specific and landscape effects, not only on dispersal but also in the demography of wild populations as assessed through local s and meta‐s and their relevance in ecology, evolution and conservation.     

Characterization of the superoxide anion radical scavenging activity by tetracycline antibiotics in aprotic media

The tetracycline family antibiotics are widely used as human and veterinary treatments. The drugs are effective as antibiotics and also show antimicrobial and non‐microbial action. However, the antioxidant properties of tetracyclines have not been characterized in aprotic media. To better understand their biological functions, the in vitro superoxide anion radical () scavenging activities of tetracycline, chlortetracycline, oxytetracycline, doxycycline and methacycline were characterized, along with a very efficient scavenger, tiron, in dimethyl sulphoxide (DMSO), using ultra‐weak chemiluminescence (CL). We found that tetracycline, chlortetracycline and doxycycline efficiently inhibited CL from the ‐generating system at concentration levels of 0.02–1.0 mmol/L. Methacycline and oxytetracycline were the scavengers at concentration levels of 0.01–0.1 mmol/L, whereas when their concentration was lowered the drugs were capable of generating , leading to CL enhancement. For all the data obtained in this study, the scavenging activity for the compounds tested decreased in the following order: tetracycline > doxycycline > chlortetracycline > tiron methacycline > oxytetracycline. These results indicate that the tetracycline drugs directly alter redox chemistry in aprotic media. Copyright © 2011 John Wiley & Sons, Ltd.     

Characterization of hyaluronic acid and synovial fluid in stagnation point elongational flow

Hyaluronic acid (HA) is an important biomacromolecule, which fulfils a number of vital physiological functions (especially in the joint synovial fluid) and also has consumer and pharmaceutical applications. HA solution properties have already been quite thoroughly characterized in response to steady shear flows but are less well understood in highly deforming extensional flows. In this study, flow‐induced birefringence measurements are made as a function of the strain rate in planar elongational flow at the stagnation point of a cross‐slot device using HA solutions of a range of molecular weights and at dilute concentrations. The results provide macromolecular relaxation times, molecular weight distributions and the extensional viscosities and Trouton ratios of the fluids. The HA relaxation time is found to vary as ^1.8, which is consistent with a partially solvated, expanded coil. An intrinsic Trouton ratio is defined, which varies as ². The measurement of birefringence with strain rate is shown to be highly sensitive to the molecular weight distribution and can resolve subtle changes due to macromolecular degradation and the presence of fracture products. Mechanical degradation experiments in the cross‐slots indicate midchain scission of HA macromolecules, strongly suggesting near full extension of the high‐molecular weight fraction in the stagnation point extensional flow field. Taken together the results suggest a possible method for analysis of the HA in synovial fluid, and this concept is tested using synovial fluid obtained from porcine tarsal joint. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 287–305, 2014.     

Purging putative siblings from population genetic data sets: a cautionary view

Interest has surged recently in removing siblings from population genetic data sets before conducting downstream analyses. However, even if the pedigree is inferred correctly, this has the potential to do more harm than good. We used computer simulations and empirical samples of coho salmon to evaluate strategies for adjusting samples to account for family structure. We compared performance in full samples and sibling‐reduced samples of estimators of allele frequency (), population differentiation () and effective population size (). Results: (i) unless simulated samples included large family groups together with a component of unrelated individuals, removing siblings generally reduced precision of and ; (ii) based on the linkage disequilibrium method was largely unbiased using full random samples but became increasingly upwardly biased under aggressive purging of siblings. Under nonrandom sampling (some families over‐represented), using full samples was downwardly biased; removing just the right ‘Goldilocks’ fraction of siblings could produce an unbiased estimate, but this sweet spot varied widely among scenarios; (iii) weighting individuals based on the inferred pedigree (to produce a best linear unbiased estimator, BLUE) maximized precision of when the inferred pedigree was correct but performed poorly when the pedigree was wrong; (iv) a variant of sibling removal that leaves intact small sibling groups appears to be more robust to errors in inferences about family structure. Our results illustrate the complex challenges posed by presence of family structure, suggest that no single optimal solution exists and argue for caution in adjusting population genetic data sets for the presence of putative siblings without fully understanding the consequences.     

Estimators for QST and coalescence times

Comparisons of to can provide insights into the evolutionary processes that lead to differentiation, or lack thereof, among the phenotypes of different groups (e.g., populations, species), and these comparisons have been performed on a variety of taxa, including humans. Here, I show that for neutrally evolving (i.e., by genetic drift, mutation, and gene flow alone) quantitative characters, the two commonly used estimators have somewhat different interpretations in terms of coalescence times, particularly when the number of groups that have been sampled is small. A similar situation occurs for estimators. Consequently, when observations come from only a small number of groups, which is not an unusual situation, it is important to match estimators appropriately when comparing to .     

Superoxide anion radical scavenging property of catecholamines

The direct effect of the four catecholamines (adrenaline, noradrenaline, dopamine and isoproterenol) on superoxide anion radicals () was investigated. The reaction between 18‐crown‐6‐ether and potassium superoxide in dimethylsulfoxide was used as a source of . The reactivity of catecholamines with was examined using chemiluminescence, reduction of nitroblue tetrazolium and electron paramagnetic resonance spin‐trapping techniques. 5,5‐Dimethyl‐1‐pyrroline‐N‐oxide was included as the spin trap. The results showed that the four catecholamines were effective and efficient in inhibiting chemiluminescence accompanying the potassium superoxide/18‐crown‐6‐ether system in a dose‐dependent manner over the range 0.05–2 mm in the following order: adrenaline > noradrenaline > dopamine > isoproterenol, with, IC₅₀ = 0.15 ± 0.02 mm 0.21 ± 0.03 mm , 0.27 ± 0.03 mm and 0.50 ± 0.04 mm , respectively. The catecholamines examined also exhibited a strong scavenging effect towards when evaluated this property by the inhibition of nitroblue tetrazolium reduction (56–73% at 1 m concentration). A very similar capacity of scavenging was monitored in the 5,5‐dimethyl‐1‐pyrroline‐N‐oxide spin‐trapping assay. The results suggest that catecholamines tested may involve a direct effect on scavenging radicals. Copyright © 2013 John Wiley & Sons, Ltd.     

High‐throughput identification of protein mutant stability computed from a double mutant fitness landscape

The effect of a mutation on protein stability is traditionally measured by genetic construction, expression, purification, and physical analysis using low‐throughput methods. This process is tedious and limits the number of mutants able to be examined in a single study. In contrast, functional fitness effects can be measured in a high‐throughput manner by various deep mutational scanning tools. Using protein GB 1, we have recently demonstrated the feasibility of estimating the mutational stability effect ( G) of single‐substitution based on the functional fitness profile of all double‐substitutions. The principle is to identify genetic backgrounds that have an exhausted stability margin. The functional effect of an additional substitution on these genetic backgrounds can then be used to compute the mutational G based on the biophysical relationship between functional fitness and thermodynamic stability. However, to identify such genetic backgrounds, the approach described in our previous study required a benchmark dataset, which is a set of known mutational G. In this study, a benchmark‐independent approach is developed. The genetic backgrounds of interest are identified using k‐means clustering with the integration of structural information. We further demonstrated that a reasonable approximation of G can also be obtained without taking structural information into account. In summary, this study describes a novel method for computing G from double‐substitution functional fitness profiles alone, without relying on any known mutational G as a benchmark.     

Summary goodness‐of‐fit statistics for binary generalized linear models with noncanonical link functions

Generalized linear models (GLM) with a canonical logit link function are the primary modeling technique used to relate a binary outcome to predictor variables. However, noncanonical links can offer more flexibility, producing convenient analytical quantities (e.g., probit GLMs in toxicology) and desired measures of effect (e.g., relative risk from log GLMs). Many summary goodness‐of‐fit (GOF) statistics exist for logistic GLM. Their properties make the development of GOF statistics relatively straightforward, but it can be more difficult under noncanonical links. Although GOF tests for logistic GLM with continuous covariates (GLMCC) have been applied to GLMCCs with log links, we know of no GOF tests in the literature specifically developed for GLMCCs that can be applied regardless of link function chosen. We generalize the Tsiatis GOF statistic originally developed for logistic GLMCCs, (), so that it can be applied under any link function. Further, we show that the algebraically related Hosmer–Lemeshow () and Pigeon–Heyse (J²) statistics can be applied directly. In a simulation study, , , and J² were used to evaluate the fit of probit, log–log, complementary log–log, and log models, all calculated with a common grouping method. The statistic consistently maintained Type I error rates, while those of and J² were often lower than expected if terms with little influence were included. Generally, the statistics had similar power to detect an incorrect model. An exception occurred when a log GLMCC was incorrectly fit to data generated from a logistic GLMCC. In this case, had more power than or J².     

Age‐related increases in human lymphocyte DNA damage: is there a role of aerobic fitness?

Oxidative stress has been advanced as one of the major causes of damage to DNA and other macromolecules. Although physical exercise may also increase oxidative stress, an important role has been recognized for regular exercise in improving the overall functionality of the body, as indicated by an increase in maximal aerobic uptake (O_2max), and in resistance to cell damage. The aims of this study were 1) to evaluate the association between DNA damage in human lymphocytes and age and 2) to evaluate the association between DNA damage in human lymphocytes and O_2max. The sample was composed of 36 healthy and nonsmoking males, aged from 20 to 84 years. O_2max was evaluated through the Bruce protocol with direct measurement of oxygen consumption. The comet assay was used to evaluate the DNA damage, strand breaks and formamidopyrimidine DNA glycosylase (FPG)‐sensitive sites. We found a positive correlation of age with DNA strand breaks but not with FPG‐sensitive sites. O_2max was significantly inversely related with DNA strand breaks, but this relation disappeared when adjusted for age. A significantly positive relation between O_2max and FPG‐sensitive sites was verified. In conclusion, our results showed that younger subjects have lower DNA strand breaks and higher O_2max compared with older subjects and FPG‐sensitive sites are positively related with O_2max, probably as transient damage due to the acute effects of daily physical activity. Copyright © 2013 John Wiley & Sons, Ltd.     

pKa predictions for proteins,RNAs, and DNAs with the Gaussian dielectric function using DelPhi pKa

We developed a Poisson‐Boltzmann based approach to calculate the values of protein ionizable residues (Glu, Asp, His, Lys and Arg), nucleotides of RNA and single stranded DNA. Two novel features were utilized: the dielectric properties of the macromolecules and water phase were modeled via the smooth Gaussian‐based dielectric function in DelPhi and the corresponding electrostatic energies were calculated without defining the molecular surface. We tested the algorithm by calculating values for more than 300 residues from 32 proteins from the PPD dataset and achieved an overall RMSD of 0.77. Particularly, the RMSD of 0.55 was achieved for surface residues, while the RMSD of 1.1 for buried residues. The approach was also found capable of capturing the large shifts of various single point mutations in staphylococcal nuclease (SNase) from ‐cooperative dataset, resulting in an overall RMSD of 1.6 for this set of pKa's. Investigations showed that predictions for most of buried mutant residues of SNase could be improved by using higher dielectric constant values. Furthermore, an option to generate different hydrogen positions also improves predictions for buried carboxyl residues. Finally, the calculations on two RNAs demonstrated the capability of this approach for other types of biomolecules. Proteins 2015; 83:2186–2197. © 2015 Wiley Periodicals, Inc.     

Effective size of density‐dependent populations in fluctuating environments

Reliable estimates of effective population size are of central importance in population genetics and evolutionary biology. For populations that fluctuate in size, harmonic mean population size is commonly used as a proxy for (multi‐) generational effective size. This assumes no effects of density dependence on the ratio between effective and actual population size, which limits its potential application. Here, we introduce density dependence on vital rates in a demographic model of variance effective size. We derive an expression for the ratio in a density‐regulated population in a fluctuating environment. We show by simulations that yearly genetic drift is accurately predicted by our model, and not proportional to as assumed by the harmonic mean model, where N is the total population size of mature individuals. We find a negative relationship between and N. For a given N, the ratio depends on variance in reproductive success and the degree of resource limitation acting on the population growth rate. Finally, our model indicate that environmental stochasticity may affect not only through fluctuations in N, but also for a given N at a given time. Our results show that estimates of effective population size must include effects of density dependence and environmental stochasticity.     

Sensitivity analysis of effective population size to demographic parameters in house sparrow populations

The ratio between the effective and the census population size, , is an important measure of the long‐term viability and sustainability of a population. Understanding which demographic processes that affect most will improve our understanding of how genetic drift and the probability of fixation of alleles is affected by demography. This knowledge may also be of vital importance in management of endangered populations and species. Here, we use data from 13 natural populations of house sparrow (Passer domesticus) in Norway to calculate the demographic parameters that determine . Using the global variance‐based Sobol’ method for the sensitivity analyses, we found that was most sensitive to demographic variance, especially among older individuals. Furthermore, the individual reproductive values (that determine the demographic variance) were most sensitive to variation in fecundity. Our results draw attention to the applicability of sensitivity analyses in population management and conservation. For population management aiming to reduce the loss of genetic variation, a sensitivity analysis may indicate the demographic parameters towards which resources should be focused. The result of such an analysis may depend on the life history and mating system of the population or species under consideration, because the vital rates and sex–age classes that is most sensitive to may change accordingly.     

Deep soil carbon dynamics are driven more by soil type than by climate: a worldwide meta‐analysis of radiocarbon profiles

The response of soil carbon dynamics to climate and land‐use change will affect both the future climate and the quality of ecosystems. Deep soil carbon (>20 cm) is the primary component of the soil carbon pool, but the dynamics of deep soil carbon remain poorly understood. Therefore, radiocarbon activity (C), which is a function of the age of carbon, may help to understand the rates of soil carbon biodegradation and stabilization. We analyzed the published C contents in 122 profiles of mineral soil that were well distributed in most of the large world biomes, except for the boreal zone. With a multivariate extension of a linear mixed‐effects model whose inference was based on the parallel combination of two algorithms, the expectation–maximization (EM) and the Metropolis–Hasting algorithms, we expressed soil C profiles as a four‐parameter function of depth. The four‐parameter model produced insightful predictions of soil C as dependent on depth, soil type, climate, vegetation, land‐use and date of sampling (). Further analysis with the model showed that the age of topsoil carbon was primarily affected by climate and cultivation. By contrast, the age of deep soil carbon was affected more by soil taxa than by climate and thus illustrated the strong dependence of soil carbon dynamics on other pedologic traits such as clay content and mineralogy.     

speed‐ne: Software to simulate and estimate genetic effective population size (Ne) from linkage disequilibrium observed in single samples

The genetic effective population size, N_e, can be estimated from the average gametic disequilibrium () between pairs of loci, but such estimates require evaluation of assumptions and currently have few methods to estimate confidence intervals. speed‐ne is a suite of matlab computer code functions to estimate from with a graphical user interface and a rich set of outputs that aid in understanding data patterns and comparing multiple estimators. speed‐ne includes functions to either generate or input simulated genotype data to facilitate comparative studies of estimators under various population genetic scenarios. speed‐ne was validated with data simulated under both time‐forward and time‐backward coalescent models of genetic drift. Three classes of estimators were compared with simulated data to examine several general questions: what are the impacts of microsatellite null alleles on , how should missing data be treated, and does disequilibrium contributed by reduced recombination among some loci in a sample impact . Estimators differed greatly in precision in the scenarios examined, and a widely employed estimator exhibited the largest variances among replicate data sets. speed‐ne implements several jackknife approaches to estimate confidence intervals, and simulated data showed that jackknifing over loci and jackknifing over individuals provided ~95% confidence interval coverage for some estimators and should be useful for empirical studies. speed‐ne provides an open‐source extensible tool for estimation of from empirical genotype data and to conduct simulations of both microsatellite and single nucleotide polymorphism (SNP) data types to develop expectations and to compare estimators.     

Effects of chronic elevated nitrate concentrations on the structure and function of river biofilms

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Stable carbon isotopes of methane for real‐time process monitoring in anaerobic digesters

Efficient operation and stability of biogas plants requires continuous monitoring of the digester content. Traditional laboratory analysis of digester sludge is often complex and time‐consuming and shows a delayed response to disruptions within the fermentation process. As a new approach, we applied an online measurement technique (laser absorption spectroscopy) for real‐time monitoring of stable carbon isotopes of methane () in a pilot‐scale biogas digester (3500 L) regularly fed with maize silage. Generally, isotopic composition of methane gives information about specific substrate degradation, that is, methanogenic pathways that reflect the actual digester state. First results of a 2‐wk monitoring experiment show that stable carbon isotopes of methane respond promptly and highly dynamic to changes in the process state of the digester. In combination with other monitoring parameters (methane production rate, concentration of volatile fatty acids, and pH) the fluctuations in can be interpreted as a change in methanogenic pathways due to a high organic loading rate. In this context, might be used as a new parameter tool for monitoring and characterization of the process state of the digester.     

Quantifying the impact of daily and seasonal variation in sap pH on xylem dissolved inorganic carbon estimates in plum trees

In studies on internal CO₂ transport, average xylem sap pH (pH_x) is one of the factors used for calculation of the concentration of dissolved inorganic carbon in the xylem sap ([]). Lack of detailed pH_x measurements at high temporal resolution could be a potential source of error when evaluating [] dynamics. In this experiment, we performed continuous measurements of CO₂ concentration ([CO₂]) and stem temperature (T_stem), complemented with pH_x measurements at 30‐min intervals during the day at various stages of the growing season (Day of the Year (DOY): 86 (late winter), 128 (mid‐spring) and 155 (early summer)) on a plum tree (Prunus domestica L. cv. Reine Claude d'Oullins). We used the recorded pH_x to calculate [] based on T_stem and the corresponding measured [CO₂]. No statistically significant difference was found between mean [] calculated with instantaneous pH_x and daily average pH_x. However, using an average pH_x value from a different part of the growing season than the measurements of [CO₂] and T_stem to estimate [] led to a statistically significant error. The error varied between 3.25 ± 0.01% under‐estimation and 3.97 ± 0.01% over‐estimation, relative to the true [] data. Measured pH_x did not show a significant daily variation, unlike [CO₂], which increased during the day and declined at night. As the growing season progressed, daily average [CO₂] (3.4%, 5.3%, 7.4%) increased and average pH_x (5.43, 5.29, 5.20) decreased. Increase in [CO₂] will increase its solubility in xylem sap according to Henry's law, and the dissociation of [] will negatively affect pH_x. Our results are the first quantifying the error in [] due to the interaction between [CO₂] and pH_x on a seasonal time scale. We found significant changes in pH_x across the growing season, but overall the effect on the calculation of [] remained within an error range of 4%. However, it is possible that the error could be more substantial for other tree species, particularly if pH_x is in the more sensitive range (pH_x > 6.5).     

Soil moisture variations affect short‐term plant‐microbial competition for ammonium,glycine, and glutamate

We tested whether the presence of plant roots would impair the uptake of ammonium (), glycine, and glutamate by microorganisms in a deciduous forest soil exposed to constant or variable moisture in a short‐term (24‐h) experiment. The uptake of ¹⁵NH₄ and dual labeled amino acids by the grass Festuca gigantea L. and soil microorganisms was determined in planted and unplanted soils maintained at 60% WHC (water holding capacity) or subject to drying and rewetting. The experiment used a design by which competition was tested in soils that were primed by plant roots to the same extent in the planted and unplanted treatments. Festuca gigantea had no effect on microbial N uptake in the constant moist soil, but its presence doubled the microbial uptake in the dried and rewetted soil compared with the constant moist. The drying and rewetting reduced by half or more the uptake by F. gigantea, despite more than 60% increase in the soil concentration of . At the same time, the amino acid and ‐ N became equally valued in the plant uptake, suggesting that plants used amino acids to compensate for the lower acquisition. Our results demonstrate the flexibility in plant‐microbial use of different N sources in response to soil moisture fluctuations and emphasize the importance of including transient soil conditions in experiments on resource competition between plants and soil microorganisms. Competition between plants and microorganisms for N is demonstrated by a combination of removal of one of the potential competitors, the plant, and subsequent observations of the uptake of N in the organisms in soils that differ only in the physical presence and absence of the plant during a short assay. Those conditions are necessary to unequivocally test for competition.