15N2 measurement of nitrogen fixation by legumes and actinorhizals: theory and practice

A gas-tight chamber has been constructed to calibrate the ¹⁵N isotope dilution method against direct ¹⁵N₂ measurements. The theoretical basis for such estimates is given, and the practical problems associated with the experiments are discussed.     

Assessing differential gene expression with small sample sizes in oligonucleotide arrays using a mean-variance model

The identification of the genes that are differentially expressed in two-sample microarray experiments remains a difficult problem when the number of arrays is very small. We discuss the implications of using ordinary t-statistics and examine other commonly used variants. For oligonucleotide arrays with multiple probes per gene, we introduce a simple model relating the mean and variance of expression, possibly with gene-specific random effects. Parameter estimates from the model have natural shrinkage properties that guard against inappropriately small variance estimates, and the model is used to obtain a differential expression statistic. A limiting value to the positive false discovery rate (pFDR) for ordinary t-tests provides motivation for our use of the data structure to improve variance estimates. Our approach performs well compared to other proposed approaches in terms of the false discovery rate.     

Meta - and combined - QTL analysis of different experiments on immune traits in chickens

Meta and/or combined QTL analysis from multiple studies can improve quantitative trait loci (QTL) position estimates compared to the individual experiments. Hereby we present results of a meta-analysis of QTL on chicken chromosome 9, 14 and 18 using data from three separate experiments and joint QTL analysis for chromosome 14 and 18. Meta QTL analysis uses information from multiple QTLs studies. Joint QTL analysis is based on combining raw data from different QTL experimental populations. QTLs under the study were related to specific antibody response to keyhole lymphet hemocyanin (KLH), and natural antibodies to environmental antigens, lipopolisaccharide (LPS) and lipoteichoic acid (LTA). Meta QTL analysis resulted in narrowing down the confidence interval for two QTLs on GGA14. The first one for natural antibodies against LTA and the second one for specific antibody response toward KLH. Also, a confidence interval of a QTL for natural antibodies against LPS located on GGA18 was narrowed down. Combined QTL analysis was successful for two QTLs: for specific antibody response toward KLH on GGA14, and for natural antibodies against LPS on GGA18. The greatest statistical power for QTL detection in joint analysis was achieved when raw data from segregating half–sib families from different populations under the study was used.     

13C metabolic flux analysis: optimal design of isotopic labeling experiments

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Highlights► Design of isotopic labeling experiments determines the precision of flux estimates. ► New approaches were developed to select optimal isotopic tracers and measurements. ► Tandem mass spectrometry provides more informative data for ¹³C flux analysis. ► Parallel labeling experiments can improve the quality of flux estimates. ► New methods are needed for optimal design of parallel labeling experiments.     

The exact values of the probability of fixation of underdominant chromosomal rearrangements.

A numerical analysis of the probability of fixation of a chromosomal mutation with partial sterility of the heterozygote in a single population is performed. Three different genetic models are considered: the first model entails constant selection against the heterozygote and is the model almost universally used in previous works; in the other two models selection against the heterozygote depends on its frequency. The exact values of the fixation probability are found by iterating transition matrices with genotype specification. Differences in results among models are small. The exact values found in the first model are compared to estimates obtained from approximations. Solutions based on diffusion models give good approximations when selection against the heterozygote is low, especially if the population is very small. For the higher values of the selection coefficient against the heterozygote, the estimates are rather imprecise, especially when the populations are not very small.     

Bacterivory Rate Estimates and Fraction of Active Bacterivores in Natural Protist Assemblages from Aquatic Systems

Unlike the fraction of active bacterioplankton, the fraction of active bacterivores (i.e., those involved in grazing) during a specified time period has not been studied yet. Fractions of protists actively involved in bacterivory were estimated assuming that the distributions of bacteria and fluorescently labeled bacteria (FLB) ingested by protists follow Poisson distributions. Estimates were compared with experimental data obtained from FLB uptake experiments. The percentages of protists with ingested FLB (experimental) and the estimates obtained from Poisson distributions were similar for both flagellates and ciliates. Thus, the fraction of protists actively grazing on natural bacteria during a given time period could be estimated. The fraction of protists with ingested bacteria depends on the incubation time and reaches a saturating value. Aquatic systems with very different characteristics were analyzed; estimates of the fraction of protists actively grazing on bacteria ranged from 7 to 100% in the studied samples. Some nanoflagellates appeared to be grazing on specific bacterial sizes. Evidence indicated that there was no discrimination for or against bacterial surrogates (i.e., FLB); also, bacteria were randomly encountered by bacterivorous protists during these short-term uptake experiments. These analyses made it possible to estimate the ingestion rates from FLB uptake experiments by counting the number of flagellates containing ingested FLB. These results represent the first reported estimates of active bacterivores in natural aquatic systems; also, a proposed protocol for estimating in situ ingestion rates by protists represents a significant improvement and simplification to the current protocol and avoids the tedious work of counting the number of ingested FLB per protist.     

Parameter Identifiability and Sensitivity Analysis Predict Targets for Enhancement of STAT1 Activity in Pancreatic Cancer and Stellate Cells

The present work exemplifies how parameter identifiability analysis can be used to gain insights into differences in experimental systems and how uncertainty in parameter estimates can be handled. The case study, presented here, investigates interferon-gamma (IFNγ) induced STAT1 signalling in two cell types that play a key role in pancreatic cancer development: pancreatic stellate and cancer cells. IFNγ inhibits the growth for both types of cells and may be prototypic of agents that simultaneously hit cancer and stroma cells. We combined time-course experiments with mathematical modelling to focus on the common situation in which variations between profiles of experimental time series, from different cell types, are observed. To understand how biochemical reactions are causing the observed variations, we performed a parameter identifiability analysis. We successfully identified reactions that differ in pancreatic stellate cells and cancer cells, by comparing confidence intervals of parameter value estimates and the variability of model trajectories. Our analysis shows that useful information can also be obtained from nonidentifiable parameters. For the prediction of potential therapeutic targets we studied the consequences of uncertainty in the values of identifiable and nonidentifiable parameters. Interestingly, the sensitivity of model variables is robust against parameter variations and against differences between IFNγ induced STAT1 signalling in pancreatic stellate and cancer cells. This provides the basis for a prediction of therapeutic targets that are valid for both cell types.     

Meta-analysis of results from quantitative trait loci mapping studies on pig chromosome 4

Meta-analysis of results from multiple studies could lead to more precise quantitative trait loci (QTL) position estimates compared to the individual experiments. As the raw data from many different studies are not readily available, the use of results from published articles may be helpful. In this study, we performed a meta-analysis of QTL on chromosome 4 in pig, using data from 25 separate experiments. First, a meta-analysis was performed for individual traits: average daily gain and backfat thickness. Second, a meta-analysis was performed for the QTL of three traits affecting loin yield: loin eye area, carcass length and loin meat weight. Third, 78 QTL were selected from 20 traits that could be assigned to one of three broad categories: carcass, fatness or growth traits. For each analysis, the number of identified meta-QTL was smaller than the number of initial QTL. The reduction in the number of QTL ranged from 71% to 86% compared to the total number before the meta-analysis. In addition, the meta-analysis reduced the QTL confidence intervals by as much as 85% compared to individual QTL estimates. The reduction in the confidence interval was greater when a large number of independent QTL was included in the meta-analysis. Meta-QTL related to growth and fatness were found in the same region as the FAT1 region. Results indicate that the meta-analysis is an efficient strategy to estimate the number and refine the positions of QTL when QTL estimates are available from multiple populations and experiments. This strategy can be used to better target further studies such as the selection of candidate genes related to trait variation.     

Phylogenetic influence on mating call preferences in female túngara frogs, Physalaemus pustulosus

We evaluated how various phylogenetic models for estimating ancestral characters can influence studies of behavioural evolution. Previously we used a single model of evolution to estimate the values of call characters at ancestral nodes for the Physalaemus pustulosus species group and some close relatives (Ryan & Rand 1995, Science, 269, 390-392). We then synthesized these ancestral calls and measured the females' responses to such calls in phonotaxis experiments. We repeated the above procedure to determine the sensitivity of these results and conclusions to various models used to estimate the ancestral call characters. We asked whether: (1) different models gave different call estimates for the same nodes; (2) different call estimates at the same node were perceived as different by females; and (3) differences in female responses influenced previous conclusions. We used seven different models that varied in at least one of the following parameters: tree topology (bifurcating versus pectinate in-group trees), algorithms (local squared-change versus squared-change parsimony), tempo (gradual or punctuated evolution), and outgroups (two or three outgroup taxa used). Although different models often gave different call estimates for the same node, these different estimates often were not perceived as different by the females. These data reinforce our previous conclusions that: (1) the range of female preferences exceeds the known variation of the conspecific call; (2) females do not discriminate between the conspecific call and the call of their most recent ancestor; and (3) female responses may be context dependent, given that females differ in their responses to the same signal variation in discrimination and recognition experiments. Copyright 1999 The Association for the Study of Animal Behaviour.     

Ammonium uptake and mineralization in prairie streams: chamber incubation and short-term nutrient addition experiments

1. We used two separate approaches to estimate ambient ammonium cycling in the north and south branches of Kings Creek, a prairie stream. Chamber experiments were conducted to determine ammonium uptake and mineralization rates associated with epilithic biofilms and filamentous algae collected from the streams. A series of short-term whole-stream ammonium addition experiments were also conducted to estimate the rate of uptake at ambient stream concentrations, based on the relationship between ammonium concentrations and uptake rates.
2. Chamber experiments were scaled up to whole-stream levels, resulting in ambient gross uptake estimates of 0.08  μ g⁻² s⁻¹ for the north branch and 0.16  μ g⁻² s⁻¹ for the south branch. The substrata-specific estimates of mineralization were higher than uptake in both streams.
3. Substrata-specific measurements indicated that ammonium uptake is higher in riffle habitats than in pools habitats. The results of the short-term ammonium addition experiments support these findings.
4. Short-term ammonium addition experiments show that uptake rates saturate with increasing ammonium concentrations. The observed saturation of uptake rates is consistent with a Michaelis–Menten relationship.
5. Scaled estimates of uptake from the chamber experiments were similar to estimates of ambient ammonium uptake based on the whole-stream experiments, and were comparable with previous estimates of ammonium uptake and mineralization made by using stable isotope tracer methods in Kings Creek.     

Measuring tolerance to herbivory: accuracy and precision of estimates made using natural versus imposed damage

Abstract.— Tolerance to herbivory (the ability of a plant to incur herbivore damage without a corresponding reduction in fitness) can be measured using either naturally occurring or imposed herbivore damage. After briefly reviewing some of the advantages and disadvantages of these approaches, we present calculations describing the degree to which estimates of tolerance will be biased by environmental variables that affect both herbivory and fitness. With naturally occurring herbivory the presence of environmental variables that are correlated with herbivory and fitness will result in biased estimates of tolerance. In contrast, estimates obtained from experiments in which herbivory is artificially imposed will be unbiased; however, under a wide range of parameter values these estimates will be less precise than estimates obtained from experiments in which herbivory is not manipulated.     

Radioimmunoassay of luteinizing hormone in the baboon (Papio hamadryas)

A heterologous radioimmunoassay (RIA) for luteinizing hormone (LH) consisting of a cynomolgus LH tracer and an antiserum raised against human chorionic gonadotropin (cynLH:anti-hCG) fulfilled the recognized criteria of reliability when applied to baboon (Papio hamadryas) plasma and pituitary extracts obtained in different endocrine conditions. This RIA is 5.5 times more sensitive than the ovine (oLH:anti-oLH) system, yields estimates of baboon LH (bLH) fairly close to those obtained by in vitro bioassay, and recognizes all bioactive molecular species of bLH present in male and female pituitary extracts. However, the system yields slightly but significantly lower estimates of bLH than the in vitro bioassay.     

Flume tank experiments to estimate the volume filtered by highspeed plankton samplers and to assess the effect of net clogging

Two types of flowmeter were used to measure the velocity ofwater passing into Lowestoft high-speed tow nets and hence toestimate the volume filtered. Miniature (Streamflo) flowmeterswere used to estimate the average velocity through the circularopening plane by measuring the velocity at several points alongthe radius of the opening and taking a weighted average of these.The velocity varies along the radius of the opening and becausethe Braystoke internal flowmeters, which are used routinelyin field deployments, only measure the velocity in the centralpart of the opening, they do not give an accurate estimate ofthe average velocity through the opening plane. This will resultin biassed estimates of volume filtered unless a correctionis applied. Linear models are used to show that the correctiondepends on the type of gear and the degree of clogging of thenet, but is independent of water velocity. Clogging was simulatedin the experiments using impervious patches of material, andcan be estimated in the field from the ratio of internal:externalflowmeter readings. Corrections for bias and estimates of filtrationefficiency are given for three gear configurations under a rangeof clogging states. Further experiments are needed to resolveremaining uncertainty over estimates of filtration efficiency.     

Effects of Nosema bombi and its treatment fumagillin on bumble bee (Bombus occidentalis) colonies

We examined the effects of Nosema bombi (Microsporidia: Nosematidae) on colonies of bumble bees, Bombus occidentalis Greene (Hymenoptera: Apidae), used to pollinate tomatoes in commercial greenhouses. We assessed methods of detecting N. bombi and tested the effectiveness of fumagillin to control this parasite. N. bombi did not affect adult population size or amount of brood in B. occidentalis colonies. Fumagillin was not effective against N. bombi at the doses we tested, and frass samples did not provide accurate estimates of the intensity of N. bombi infections. The number of N. bombi spores per bee was highly variable among bumble bees within colonies, and accurate estimates could only be obtained by sampling a large proportion of bees in each colony. Therefore, whole bee and frass sampling is useful for determining if N. bombi is present or absent, but not for obtaining accurate estimates of the intensity of N. bombi infections.     

Technical note: Tree truthing: How accurate are substrate estimates in primate field studies?

Field studies of primate positional behavior typically rely on ground‐level estimates of substrate size, angle, and canopy location. These estimates potentially influence the identification of positional modes by the observer recording behaviors. In this study we aim to test ground‐level estimates against direct measurements of support angles, diameters, and canopy heights in trees at La Suerte Biological Research Station in Costa Rica. After reviewing methods that have been used by past researchers, we provide data collected within trees that are compared to estimates obtained from the ground. We climbed five trees and measured 20 supports. Four observers collected measurements of each support from different locations on the ground. Diameter estimates varied from the direct tree measures by 0–28 cm (Mean: 5.44 ± 4.55). Substrate angles varied by 1–55° (Mean: 14.76 ± 14.02). Height in the tree was best estimated using a clinometer as estimates with a two‐meter reference placed by the tree varied by 3–11 meters (Mean: 5.31 ± 2.44). We determined that the best support size estimates were those generated relative to the size of the focal animal and divided into broader categories. Support angles were best estimated in 5° increments and then checked using a Haglöf clinometer in combination with a laser pointer. We conclude that three major factors should be addressed when estimating support features: observer error (e.g., experience and distance from the target), support deformity, and how support size and angle influence the positional mode selected by a primate individual. individual. Am J Phys Anthropol, 2012. © 2012 Wiley Periodicals, Inc.     

Identification of insulin receptor systems: assessing the impact of model selection and measurement error on precision of parameter estimates using Monte Carlo study

An extensive Monte Carlo study has been carried out in order to study the effect of measurement error on the precision of parameter estimates of an insulin binding system. Hypothetical radioimmunoassay experiments were generated for insulin binding to erythrocytes. The design of experiments followed strictly the protocol of real experiments. Randomly generated error was added to the synthetic data. The standard technique, a weighted non-linear regression analysis, was employed to re-estimate parameters of a model of two receptor sites and a model of negative co-operativity. As the original parameter values were known, the differences between original and estimated values was studied for (a) measurement error in the range from 0-17%, (b) random initial estimates and (c) error-free non-specific binding. In addition, analytical estimates of parameter precision were compared with the true between-experiment variation of parameter estimates. At the measurement error of 12%, a one site model is recommended to estimate the high affinity population of the two sites model. Plausible results can be expected in 90% of experiments, the between-experiment variation being approximately 30%. The model of two receptor sites gives approximately two thirds of plausible results. The high affinity population can be estimated with the between-experiment variation of 40%, the low affinity population is virtually unidentifiable with the between-experiment variation of approximately 100% and parameter estimates biased to higher values. Only half of the results obtained from the model of negative co-operativity are plausible, the variation in parameter estimates ranges from 90-150% and estimates are biased to higher values. At the level of 12% measurement error, random initial estimates do not significantly affect the estimation process, provided initial estimates are selected from a feasible range. At the same measurement error, the error-free non-specific binding does not improve the results, indicating that the mean of six replicates may be taken as a reliable estimate of non-specific binding. The analytical estimates of the coefficient of variation systematically underestimates the true between-experiments coefficient of variation, the difference has been found to be about 50%.     

On estimating critical population size for an endangered species in the presence of environmental stochasticity

A stationary second order autoregressive process with Gaussian noise, which was linked to survivorship and reproductive success by logistic transformations, was used as a model for an environmental process. Computer experiments in Monte Carlo integration, with the objective of exploring the sensitivity of estimates of mean critical population size to variations in the parameters of the environmental process, were then conducted. These experiments suggest that estimates of mean critical population size are very sensitive to the form of the autocorrelation function of the stationary environmental process. For the most part, those experiments in which the autocorrelation function was strictly positive not only resulted in the largest estimates of mean critical population size but also led to the highest levels of environmental stochasticity as measured by its coefficient of variation. As in previous work, these experiments suggest that concerted efforts should be made to model those environmental factors that are critical to the survivability of an endangered species in assessing its chances for continued existence.     

Assessing the effect of an influenza vaccine in an encouragement design

Many randomized experiments suffer from noncompliance. Some of these experiments, so-called encouragement designs, can be expected to have especially large amounts of noncompliance, because encouragement to take the treatment rather than the treatment itself is randomly assigned to individuals. We present an extended framework for the analysis of data from such experiments with a binary treatment, binary encouragement, and background covariates. There are two key features of this framework: we use an instrumental variables approach to link intention-to-treat effects to treatment effects and we adopt a Bayesian approach for inference and sensitivity analysis. This framework is illustrated in a medical example concerning the effects of inoculation for influenza. In this example, the analyses suggest that positive estimates of the intention-to-treat effect need not be due to the treatment itself, but rather to the encouragement to take the treatment: the intention-to-treat effect for the subpopulation who would be inoculated whether or not encouraged is estimated to be approximately as large as the intention-to-treat effect for the subpopulation whose inoculation status would agree with their (randomized) encouragement status whether or not encouraged. Thus, our methods suggest that global intention-to-treat estimates, although often regarded as conservative, can be too coarse and even misleading when taken as summarizing the evidence in the data for the effects of treatments.     

Vaccine effectiveness against SARS-CoV-2 infection or COVID-19 hospitalization with the Alpha,Delta, or Omicron SARS-CoV-2 variant: A nationwide Danish cohort study

BackgroundThe continued occurrence of more contagious Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) variants and waning immunity over time require ongoing reevaluation of the vaccine effectiveness (VE). This study aimed to estimate the effectiveness in 2 age groups (12 to 59 and 60 years or above) of 2 or 3 vaccine doses (BNT162b2 mRNA or mRNA-1273) by time since vaccination against SARS-CoV-2 infection and Coronavirus Disease 2019 (COVID-19) hospitalization in an Alpha-, Delta-, or Omicron-dominated period.Methods and findingsA Danish nationwide cohort study design was used to estimate VE against SARS-CoV-2 infection and COVID-19 hospitalization with the Alpha, Delta, or Omicron variant. Information was obtained from nationwide registries and linked using a unique personal identification number. The study included all previously uninfected residents in Denmark aged 12 years or above (18 years or above for the analysis of 3 doses) in the Alpha (February 20 to June 15, 2021), Delta (July 4 to November 20, 2021), and Omicron (December 21, 2021 to January 31, 2022) dominated periods. VE estimates including 95% confidence intervals (CIs) were calculated (1-hazard ratio∙100) using Cox proportional hazard regression models with underlying calendar time and adjustments for age, sex, comorbidity, and geographical region. Vaccination status was included as a time-varying exposure. In the oldest age group, VE against infection after 2 doses was 90.7% (95% CI: 88.2; 92.7) for the Alpha variant, 82.3% (95% CI: 75.5; 87.2) for the Delta variant, and 39.9% (95% CI: 26.3; 50.9) for the Omicron variant 14 to 30 days since vaccination. The VE waned over time and was 73.2% (Alpha, 95% CI: 57.1; 83.3), 50.0% (Delta, 95% CI: 46.7; 53.0), and 4.4% (Omicron, 95% CI: −0.1; 8.7) >120 days since vaccination. Higher estimates were observed after the third dose with VE estimates against infection of 86.1% (Delta, 95% CI: 83.3; 88.4) and 57.7% (Omicron, 95% CI: 55.9; 59.5) 14 to 30 days since vaccination. Among both age groups, VE against COVID-19 hospitalization 14 to 30 days since vaccination with 2 or 3 doses was 98.1% or above for the Alpha and Delta variants. Among both age groups, VE against COVID-19 hospitalization 14 to 30 days since vaccination with 2 or 3 doses was 95.5% or above for the Omicron variant. The main limitation of this study is the nonrandomized study design including potential differences between the unvaccinated (reference group) and vaccinated individuals.ConclusionsTwo vaccine doses provided high protection against SARS-CoV-2 infection and COVID-19 hospitalization with the Alpha and Delta variants with protection, notably against infection, waning over time. Two vaccine doses provided only limited and short-lived protection against SARS-CoV-2 infection with Omicron. However, the protection against COVID-19 hospitalization following Omicron SARS-CoV-2 infection was higher. The third vaccine dose substantially increased the level and duration of protection against infection with the Omicron variant and provided a high level of sustained protection against COVID-19 hospitalization among the +60-year-olds.

Mie Agermose Gram and colleagues estimate vaccine effectiveness against infection and COVID-19 hospitalization with the Alpha, Delta or Omicron variant in Denmark.     

The value of constant surveillance in a risky environment

In risky environments, where threats are unpredictable and the quality of information about threats is variable, all individuals face two fundamental challenges: balancing vigilance against other activities, and determining when to respond to warning signals. The solution to both is to obtain continuous estimates of background risk, enabling vigilance to be concentrated during the riskiest periods and informing about the likely cost of ignoring warnings. Human surveillance organizations routinely produce such estimates, frequently derived from indirect cues. Here we show that vigilant individuals in an animal society (the pied babbler, Turdoides bicolor) perform a similar role. We ask (i) whether, in the absence of direct predator threats, pied babbler sentinels react to indirect information associated with increased risk and whether they communicate this information to group mates; (ii) whether group mates use this information to adjust their own vigilance, and whether this influences foraging success; and (iii) whether information provided by sentinels reduces the likelihood of inappropriate responses to alarm calls. Using playback experiments, we show that: (i) sentinels reacted to indirect predator cues (in the form of heterospecific alarm calls) by giving graded surveillance calls; (ii) foragers adjusted their vigilance in reaction to changes in surveillance calls, with substantial effects on foraging success; and (iii) foragers reduced their probability of responding to alarm calls when surveillance calls indicated lowered risk. These results demonstrate that identifying attacks as they occur is only part of vigilance: equally important is continuous surveillance providing information necessary for individuals to make decisions about their own vigilance and evasive action. Moreover, they suggest that a major benefit of group living is not only the increased likelihood of detecting threats, but a marked improvement in the quality of information available to each individual.