Impact of nonrandom selection mechanisms on the causal effect estimation for two-sample Mendelian randomization methods

Nonrandom selection in one-sample Mendelian Randomization (MR) results in biased estimates and inflated type I error rates only when the selection effects are sufficiently large. In two-sample MR, the different selection mechanisms in two samples may more seriously affect the causal effect estimation. Firstly, we propose sufficient conditions for causal effect invariance under different selection mechanisms using two-sample MR methods. In the simulation study, we consider 49 possible selection mechanisms in two-sample MR, which depend on genetic variants (G), exposures (X), outcomes (Y) and their combination. We further compare eight pleiotropy-robust methods under different selection mechanisms. Results of simulation reveal that nonrandom selection in sample II has a larger influence on biases and type I error rates than those in sample I. Furthermore, selections depending on X+Y, G+Y, or G+X+Y in sample II lead to larger biases than other selection mechanisms. Notably, when selection depends on Y, bias of causal estimation for non-zero causal effect is larger than that for null causal effect. Especially, the mode based estimate has the largest standard errors among the eight methods. In the absence of pleiotropy, selections depending on Y or G in sample II show nearly unbiased causal effect estimations when the casual effect is null. In the scenarios of balanced pleiotropy, all eight MR methods, especially MR-Egger, demonstrate large biases because the nonrandom selections result in the violation of the Instrument Strength Independent of Direct Effect (InSIDE) assumption. When directional pleiotropy exists, nonrandom selections have a severe impact on the eight MR methods. Application demonstrates that the nonrandom selection in sample II (coronary heart disease patients) can magnify the causal effect estimation of obesity on HbA1c levels. In conclusion, nonrandom selection in two-sample MR exacerbates the bias of causal effect estimation for pleiotropy-robust MR methods.     

Thyroid cancer following scalp irradiation: a reanalysis accounting for uncertainty in dosimetry

In the 1940s and 1950s, over 20,000 children in Israel were treated for tinea capitis (scalp ringworm) by irradiation to induce epilation. Follow-up studies showed that the radiation exposure was associated with the development of malignant thyroid neoplasms. Despite this clear evidence of an effect, the magnitude of the dose-response relationship is much less clear because of probable errors in individual estimates of dose to the thyroid gland. Such errors have the potential to bias dose-response estimation, a potential that was not widely appreciated at the time of the original analyses. We revisit this issue, describing in detail how errors in dosimetry might occur, and we develop a new dose-response model that takes the uncertainties of the dosimetry into account. Our model for the uncertainty in dosimetry is a complex and new variant of the classical multiplicative Berkson error model, having components of classical multiplicative measurement error as well as missing data. Analysis of the tinea capitis data suggests that measurement error in the dosimetry has only a negligible effect on dose-response estimation and inference as well as on the modifying effect of age at exposure.     

Identification and utilization of genetic determinants of trait measurement errors in image-based,high-throughput phenotyping

The accuracy of trait measurements greatly affects the quality of genetic analyses. During automated phenotyping, trait measurement errors, i.e. differences between automatically extracted trait values and ground truth, are often treated as random effects that can be controlled by increasing population sizes and/or replication number. In contrast, there is some evidence that trait measurement errors may be partially under genetic control. Consistent with this hypothesis, we observed substantial nonrandom, genetic contributions to trait measurement errors for five maize (Zea mays) tassel traits collected using an image-based phenotyping platform. The phenotyping accuracy varied according to whether a tassel exhibited “open” versus. “closed” branching architecture, which is itself under genetic control. Trait-associated SNPs (TASs) identified via genome-wide association studies (GWASs) conducted on five tassel traits that had been phenotyped both manually (i.e. ground truth) and via feature extraction from images exhibit little overlap. Furthermore, identification of TASs from GWASs conducted on the differences between the two values indicated that a fraction of measurement error is under genetic control. Similar results were obtained in a sorghum (Sorghum bicolor) plant height dataset, demonstrating that trait measurement error is genetically determined in multiple species and traits. Trait measurement bias cannot be controlled by increasing population size and/or replication number.

The accuracy of high-throughput phenotyping can be affected by genetically determined measurement biases, which can alter the results of genetic analyses.     

The Genetic Code Is One in a Million

Statistical and biochemical studies of the genetic code have found evidence of nonrandom patterns in the distribution of codon assignments. It has, for example, been shown that the code minimizes the effects of point mutation or mistranslation: erroneous codons are either synonymous or code for an amino acid with chemical properties very similar to those of the one that would have been present had the error not occurred. This work has suggested that the second base of codons is less efficient in this respect, by about three orders of magnitude, than the first and third bases. These results are based on the assumption that all forms of error at all bases are equally likely. We extend this work to investigate (1) the effect of weighting transition errors differently from transversion errors and (2) the effect of weighting each base differently, depending on reported mistranslation biases. We find that if the bias affects all codon positions equally, as might be expected were the code adapted to a mutational environment with transition/transversion bias, then any reasonable transition/transversion bias increases the relative efficiency of the second base by an order of magnitude. In addition, if we employ weightings to allow for biases in translation, then only 1 in every million random alternative codes generated is more efficient than the natural code. We thus conclude not only that the natural genetic code is extremely efficient at minimizing the effects of errors, but also that its structure reflects biases in these errors, as might be expected were the code the product of selection. Received: 25 July 1997 / Accepted: 9 January 1998     

Are brood sex ratios adaptive?—The effect of experimentally altered brood sex ratio on nestling growth,mortality and recruitment

Brood sex ratios (BSRs) have often been found to be nonrandom in respect of parental and environmental quality, and many hypotheses suggest that nonrandom sex ratios can be adaptive. To specifically test the adaptive value of biased BSRs, it is crucial to disentangle the consequences of BSR and maternal effects. In multiparous species, this requires cross-fostering experiments where foster parents rear offspring originating from multiple broods, and where the interactive effect of original and manipulated BSR on fitness components is tested. To our knowledge, our study on collared flycatchers (Ficedula albicollis) is the first that meets these requirements. In this species, where BSRs had previously been shown to be related to parental characteristics, we altered the original BSR of the parents shortly after hatching by cross-fostering nestlings among trios of broods and examined the effects on growth, mortality and recruitment of the nestlings. We found that original and experimental BSR, as well as the interaction of the two, were unrelated to the fitness components considered. Nestling growth was related only to background variables, namely brood size and hatching rank. Nestling mortality was related only to hatching asynchrony. Our results therefore do not support that the observed BSRs are adaptive in our study population. However, we cannot exclude the possibility of direct effects of experimentally altered BSRs on parental fitness, which should be evaluated in the future. In addition, studies similar to ours are required on various species to get a clearer picture of the adaptive value of nonrandom BSRs.     

Interaction of leachable model compounds and their impact on Chinese hamster ovary cell cultivation

The presence of leachables in biopharmaceutical processes using single-use technologies (SUT) is well known. For the detection and quantification of the latter, extractable studies of SUT are very common nowadays. Although a mixture of compounds is regularly found in extractable studies, research has only been carried out regarding the effect of individual compounds on cell culture and the cumulative effect of a mix of leachables has not been investigated yet. In this study, a set of leachable model compounds (LMCs) was chosen and the effect of the LMCs on a Chinese hamster ovary DG44 cell line producing an IgG antibody was investigated concerning cell growth, cell cycle distribution and productivity. It was shown that even if worst-case concentrations were used, the LMCs solely impact cell growth. Additionally, interaction studies revealed that the inhibiting effect of the mix is lower than the expected cumulative effect. A strong antagonism between the antioxidant butylated hydroxytoluene and the plasticizer Tris(2-ethylhexyl)trimellitate was found using an isobologram analysis.     

Design,synthesis of novel furan appended benzothiazepine derivatives and in vitro biological evaluation as potent VRV-PL-8a and H+/K+ ATPase inhibitors

A series of new of furan derivatised [1,4] benzothiazepine analogues were synthesized starting from 1-(furan-2-yl)ethanone. 1-(Furan-2-yl)ethanone was converted into chalcones by its reaction with various aromatic aldehydes, then were reacted with 2-aminobenzenethiol in acidic conditions to obtain the title compounds in good yields. The synthesized new compounds were characterized by ¹H NMR, ¹³C NMR, Mass spectral studies and elemental analyses. All the new compounds were evaluated for their in vitro VRV-PL-8a and H⁺/K⁺ ATPase inhibitor properties. Preliminary studies revealed that, some molecules amongst the designed series showed promising VRV-PL-8a and H⁺/K⁺ ATPase inhibitor properties. Further, rigid body docking studies were performed to understand possible docking sites of the molecules on the target proteins and the mode of binding. This finding presents a promising series of lead molecules that can serve as prototypes for the treatment of inflammatory related disorder that can mitigate the ulcer inducing side effect shown by other NSAIDs.     

Impact of Habitat-Specific GPS Positional Error on Detection of Movement Scales by First-Passage Time Analysis

Advances in animal tracking technologies have reduced but not eliminated positional error. While aware of such inherent error, scientists often proceed with analyses that assume exact locations. The results of such analyses then represent one realization in a distribution of possible outcomes. Evaluating results within the context of that distribution can strengthen or weaken our confidence in conclusions drawn from the analysis in question. We evaluated the habitat-specific positional error of stationary GPS collars placed under a range of vegetation conditions that produced a gradient of canopy cover. We explored how variation of positional error in different vegetation cover types affects a researcher''s ability to discern scales of movement in analyses of first-passage time for white-tailed deer (Odocoileus virginianus). We placed 11 GPS collars in 4 different vegetative canopy cover types classified as the proportion of cover above the collar (0–25%, 26–50%, 51–75%, and 76–100%). We simulated the effect of positional error on individual movement paths using cover-specific error distributions at each location. The different cover classes did not introduce any directional bias in positional observations (1 m≤mean≤6.51 m, 0.24≤p≤0.47), but the standard deviation of positional error of fixes increased significantly with increasing canopy cover class for the 0–25%, 26–50%, 51–75% classes (SD = 2.18 m, 3.07 m, and 4.61 m, respectively) and then leveled off in the 76–100% cover class (SD = 4.43 m). We then added cover-specific positional errors to individual deer movement paths and conducted first-passage time analyses on the noisy and original paths. First-passage time analyses were robust to habitat-specific error in a forest-agriculture landscape. For deer in a fragmented forest-agriculture environment, and species that move across similar geographic extents, we suggest that first-passage time analysis is robust with regard to positional errors.     

Evolution of indirect reciprocity in groups of various sizes and comparison with direct reciprocity

Recently many studies have investigated the evolution of indirect reciprocity through which cooperative action is returned by a third individual, e.g. individual A helped B and then receives help from C. Most studies on indirect reciprocity have presumed that only two individuals take part in a single interaction (group), e.g. A helps B and C helps A. In this paper, we investigate the evolution of indirect reciprocity when more than two individuals take part in a single group, and compare the result with direct reciprocity through which cooperative action is directly returned by the recipient. Our analyses show the following. In the population with discriminating cooperators and unconditional defectors, whether implementation error is included or not, (i) both strategies are evolutionarily stable and the evolution of indirect reciprocity becomes more difficult as group size increases, and (ii) the condition for the evolution of indirect reciprocity under standing reputation criterion where the third individuals distinguish between justified and unjustified defections is more relaxed than that under image scoring reputation criterion in which the third individuals do not distinguish with. Furthermore, in the population that also includes unconditional cooperators, (iii) in the presence of errors in implementation, the discriminating strategy is evolutionarily stable not only under standing but also under image scoring if group size is larger than two. Finally, (iv) in the absence of errors in implementation, the condition for the evolution of direct reciprocity is equivalent to that for the evolution of indirect reciprocity under standing, and, in the presence of errors, the condition for the evolution of direct reciprocity is very close to that for the evolution of indirect reciprocity under image scoring.     

How Does Our Motor System Determine Its Learning Rate?

Motor learning is driven by movement errors. The speed of learning can be quantified by the learning rate, which is the proportion of an error that is corrected for in the planning of the next movement. Previous studies have shown that the learning rate depends on the reliability of the error signal and on the uncertainty of the motor system’s own state. These dependences are in agreement with the predictions of the Kalman filter, which is a state estimator that can be used to determine the optimal learning rate for each movement such that the expected movement error is minimized. Here we test whether not only the average behaviour is optimal, as the previous studies showed, but if the learning rate is chosen optimally in every individual movement. Subjects made repeated movements to visual targets with their unseen hand. They received visual feedback about their endpoint error immediately after each movement. The reliability of these error-signals was varied across three conditions. The results are inconsistent with the predictions of the Kalman filter because correction for large errors in the beginning of a series of movements to a fixed target was not as fast as predicted and the learning rates for the extent and the direction of the movements did not differ in the way predicted by the Kalman filter. Instead, a simpler model that uses the same learning rate for all movements with the same error-signal reliability can explain the data. We conclude that our brain does not apply state estimation to determine the optimal planning correction for every individual movement, but it employs a simpler strategy of using a fixed learning rate for all movements with the same level of error-signal reliability.     

Immunological and chemical studies on porcine submaxillary mucins

Porcine submaxillary mucins (PSM) were classified as A, H, AH, and — types according to their ability to inhibit the hemagglutination of human blood group systems. Of 210 glands examined, the following blood group distribution was observed: 35% A, 35% H, 1% AH, and 29%—. The A-type mucin was an effective inhibitor at microgram concentrations, whereas the H-types showed considerably weaker hemagglutination inhibition.
Rabbit antisera to the A and H mucins could be used for typing of human blood groups A and H; no cross-reactivity was observed with the other types. The A-type PSM antisera were adsorbed by human red blood cells of the A type only. Immunodiffusion and immunoelectrophoretic studies of rabbit antisera to crude mucins revealed many antigenic components, but antisera to purified mucin preparations usually developed only two precipitin bands, one of which showed up only after staining for proteins.
PSM was prepared from individual glands of known blood group types by a slight modification of a previously described method. Chemical analyses showed that the variation in composition of individual glands within a given blood group type was larger than could be attributable to experimental error. In addition, chemical variations were also observed among mucins of A, H, and — blood group types.     

Robustness to failure of assumptions of tests for a common slope amongst several allometric lines--a simulation study

In allometry, researchers are commonly interested in estimating the slope of the major axis or standardized major axis (methods of bivariate line fitting related to principal components analysis). This study considers the robustness of two tests for a common slope amongst several axes. It is of particular interest to measure the robustness of these tests to slight violations of assumptions that may not be readily detected in sample datasets. Type I error is estimated in simulations of data generated with varying levels of nonnormality, heteroscedasticity and nonlinearity. The assumption failures introduced in simulations were difficult to detect in a moderately sized dataset, with an expert panel only able to correct detect assumption violations 34-45% of the time. While the common slope tests were robust to nonnormal and heteroscedastic errors from the line, Type I error was inflated if the two variables were related in a slightly nonlinear fashion. Similar results were also observed for the linear regression case. The common slope tests were more liberal when the simulated data had greater nonlinearity, and this effect was more evident when the underlying distribution had longer tails than the normal. This result raises concerns for common slopes testing, as slight nonlinearities such as those in simulations are often undetectable in moderately sized datasets. Consequently, practitioners should take care in checking for nonlinearity and interpreting the results of a test for common slope. This work has implications for the robustness of inference in linear models in general.     

Effects of sample size and intraspecific variation in phylogenetic comparative studies: a meta‐analytic review

Comparative analyses aim to explain interspecific variation in phenotype among taxa. In this context, phylogenetic approaches are generally applied to control for similarity due to common descent, because such phylogenetic relationships can produce spurious similarity in phenotypes (known as phylogenetic inertia or bias). On the other hand, these analyses largely ignore potential biases due to within‐species variation. Phylogenetic comparative studies inherently assume that species‐specific means from intraspecific samples of modest sample size are biologically meaningful. However, within‐species variation is often significant, because measurement errors, within‐ and between‐individual variation, seasonal fluctuations, and differences among populations can all reduce the repeatability of a trait. Although simulations revealed that low repeatability can increase the type I error in a phylogenetic study, researchers only exercise great care in accounting for similarity in phenotype due to common phylogenetic descent, while problems posed by intraspecific variation are usually neglected. A meta‐analysis of 194 comparative analyses all adjusting for similarity due to common phylogenetic descent revealed that only a few studies reported intraspecific repeatabilities, and hardly any considered or partially dealt with errors arising from intraspecific variation. This is intriguing, because the meta‐analytic data suggest that the effect of heterogeneous sampling can be as important as phylogenetic bias, and thus they should be equally controlled in comparative studies. We provide recommendations about how to handle such effects of heterogeneous sampling.     

Some new indole–coumarin hybrids; Synthesis,anticancer and Bcl-2 docking studies

Hybrid molecules have attracted attention for their improved biological activity, selectivity and lesser side effects profile, distinct from their individual components. In the quest for novel anticancer drug entities, three series of indole–coumarin hybrids – 3-(1-benzyl-1H-indol-2-yl)-2H-chromen-2-ones, 2-(2-oxo-2H-chromen-3-yl)-1H-indole-3-carbaldehydes and 2-(2-oxo-2H-chromen-3-yl)-1H-indole-3-carboxylic acids were synthesized. All the synthesized compounds were characterized by spectral techniques like IR, ¹H NMR, ¹³C NMR, mass spectrometry and elemental analysis. In silico docking studies of synthesized molecules with apoptosis related gene Bcl-2 that is recognized to play an important role in tumerogenesis were carried out. Dose-dependent cytotoxic effect of the compounds in human breast adenocarcinoma (MCF-7) and normal cell lines were assessed using MTT assay and compared with that of the standard marketed drug, Vincristine. Compound 4c had a highly lipophilic bromine substituent capable of forming halogen bond and was identified as a potent molecule both in docking as well as cytotoxicity studies. Flow cytometric cell cycle analysis of 4c exhibited apoptotic mode of cell death due to cell cycle arrest in G2/M phase. Structure activity relationship of these hybrid molecules was also studied to determine the effect of steric and electronic properties of the substituents on cell viability.     

Chemical scent constituents in feces of wild Iberian wolves (Canis lupus signatus)

In spite of the importance of feces in scent marking and intraspecific chemical communication of many mammals, only a few studies have examined the chemical constituents of feces that could have a signaling function. We described here the chemicals found in feces recently deposited in the wild by adult and pup Iberian wolves (Canis lupus signatus). By means of analyses using gas chromatography–mass spectrometry (GC–MS), we identified 77 compounds in feces of adult wolves, mainly heterocyclic aromatic organic compounds, such as indole or phenol, but also steroids, such as cholesterol, carboxylic acids and their esters between n-C₃ and n-C₁₈, aldehydes, alcohols and significant quantities of squalene and α-tocopherol, which would increase the chemical stability of feces on humid substrates. Some of the odoriferous compounds are very likely originated in the anal-sac secretions, and later incorporated onto feces during defecation. However, the abundance of the main odoriferous compounds in feces could be explained by the result of the bacterial action on food components. Feces of pups only had 26 compounds, most of these were also found in adults’ feces, but four compounds were only found in pups’ feces, and there were also age-related differences respect to the proportions of shared chemicals. These differences could be partly due to the absence of anal-sac glands in pups, which would not need to scent mark substrates.     

Interaction between model membranes and a new class of surfactants with antioxidant function.

The effect of two series of amphiphilic quaternary ammonium salts on some properties of phospholipid membranes was studied. The compounds of one series, N-benzyl-N,N-dimethyl-N-alkyl ammonium bromides, exert a destructive effect on membranes and are treated as reference compounds. The compounds of the other series, N-(3,5-di-t-butyl-4-hydroxy)benzyl-N,N-dimethyl-N-alkyl ammonium bromides, are derivatives of the former ones, exhibit antioxidant properties, and do only relatively slight damage to the membranes. The aim of the work was to explain the difference in molecular interaction with membranes between the two kinds of hydrophobic compounds. Thermodynamic methods, a new mixing technique, and monolayer and quantum calculation methods were used. It has been shown that the antioxidant molecules are less hydrophobic than those of the reference compounds and disturb the membrane organization to a lesser extent. On the basis of monolayer data, we suggest that the studied antioxidant behaves like a substitutional impurity, whereas the reference behaves like an interstitial one.     

Chemical composition of essential oils of four Eucalyptus species and their phytotoxicity on silverleaf nightshade (Solanum elaeagnifolium Cav.) in Australia

Phytotoxicity and chemical composition of essential oils from four selected Eucalyptus species in Australia were investigated. Essential oils had stronger inhibitory effects on germination and seedling growth of silverleaf nightshade (Solanum elaeagnifolium Cav.) when compared with a commercial eucalyptus oil and with 1,8-cineole. E. salubris oil had the highest inhibition index for silverleaf nightshade germination, root growth and shoot growth, while E. spathulata had the lowest inhibitory effect except root growth. Gas chromatography-mass spectrometry analysis revealed 56 compounds present in E. salubris oil, with 1,8-cineole (57.6?%), ??-pinene (10.9?%) and p-cymene (8.3?%) predominant. E. dundasii oil contained 55 identified compounds with 1,8-cineole (65.5?%) and ??-pinene (19.9?%) being the richest fractions. There were 56 compounds identified from E. brockwayii oil with ??-pinene (31.1?%), isopentyl isovalerate (20.2?%) and 1,8-cineole (16.9?%) as the most abundant components. E. spathulata oil contained 60 compounds, predominantly 1,8-cineole (52.9?%) and ??-pinene (31.0?%). Further study is required to determine the phytoxicity of the individual identified compounds on silverleaf nightshade and whether the observed phytotoxicity is attributable to a single compound or to the synergistic effects of several compounds.     

Uncertainties of synchrotron microCT-based digital volume correlation bone strain measurements under simulated deformation

Digital Volume Correlation (DVC) is used to measure internal displacements and strains in bone. Recent studies have shown that Synchrotron radiation micro-computed tomography (SR-microCT) can improve the accuracy and precision of DVC. However, only zero-strain or virtually-moved test have been used to quantify the DVC uncertainties, leading to potential underestimation of the measurement errors.In this study, for the first time, the uncertainties of a global DVC approach have been evaluated on repeated SR-microCT scans of bovine cortical bone (voxel size: 1.6 μm), which were virtually deformed for different magnitudes and along different directions.The results showed that systematic and random errors of the normal strain components along the deformation direction were higher than the errors along unstrained directions. The systematic percentage errors were smaller for larger virtual deformations. The random percentage error was in the order of 10% of the virtual deformation. However, higher errors were localized at the boundary of the volumes of interest, perpendicular to the deformation direction. When only the central region of the samples was considered (100 µm layers removed from the borders where the deformation was applied), the errors in the direction of virtual deformation were comparable to the errors in the unstrained directions.In conclusion, the method presented to estimate the uncertainties of DVC is suitable for testing anisotropic specimens as cortical bone. The good agreement between the uncertainties in measurements of strain components obtained with this approach and with the simpler zero-strain-test suggests that the latter is adequate in the tested deformation scenarios.     

Effect of Male Accessory Gland Products on Egg Laying in Gastropod Molluscs

In internally fertilizing animals, seminal fluid is usually added to the spermatozoa, together forming the semen or ejaculate. Besides nourishing and activating sperm, the components in the seminal fluid can also influence female physiology to augment fertilization success of the sperm donor. While many studies have reported such effects in species with separate sexes, few studies have addressed this in simultaneously hermaphroditic animals. This video protocol presents a method to study effects of seminal fluid in gastropods, using a simultaneously hermaphroditic freshwater snail, the great pond snail Lymnaea stagnalis, as model organism. While the procedure is shown using complete prostate gland extracts, individual components (i.e., proteins, peptides, and other compounds) of the seminal fluid can be tested in the same way. Effects of the receipt of ejaculate components on egg laying can be quantified in terms of frequency of egg laying and more subtle estimates of female reproductive performance such as egg numbers within each egg masses. Results show that seminal fluid proteins affect female reproductive output in this simultaneous hermaphrodite, highlighting their importance for sexual selection.