Application of the principles of enzyme kinetics to clonal growth rate assays: an approach for delineating interactions among growth promoting agents.

The interaction of mitogenic factors on a single cell type and the comparative activity of a given factor in diverse cell types have been studied by applying the principles of Michaelis-Menten kinetics to clonal growth data. Such comparisons are facilitated by derivation of two parameters; Km mitogen, the mitogen concentration that gives half-maximal clonal growth and a theoretical maximal growth rate, RMAX T. Both parameters are analogous to the Km and VMAX as applied to enzymatic reactions. Use of these parameters permits meaningful comparisons between cells with different growth rates. Using kinetic analysis of dose-response data, we found that normal human epithelial cells require 200 times more fetal bovine serum protein (FBSP) than a malignant line to multiply at their respective half-maximal rates. Further, the Km FBSP of normal cells was reduced to that of the malignant line by the inclusion of growth factors (EGF or FGF, and hydrocortisone) in the medium. On the other hand, even though greater levels of serum were required when growth factors and hydrocortisone were not present, their inclusion did not alter RMAX T. Interactions between mitogenic factors were shown to be unidirectional. Although EGF reduced the Km FBSP, FBSP did not change the Km EGF. The same type of analysis revealed that hydrocortisone, which potentiated the mitogenic activity of EGF did not change the Km EGF. Kinetic analysis of cell growth should prove useful in studies on the relation between growth and tumor promotion as well as in the evaluation of growth-inhibiting chemotherapeutic agents.     

A novel alternative to F-tests for ecological studies

Differences or similarities in the variance of fitness traits are crucial in several biological disciplines, e.g. ecological, toxicological, developmental and evolutionary studies. For example the variance of traits can be utilized as a biomarker of differences in environmental conditions. In the absence of environmental variability, the differences of the variance of a trait can be interpreted as differences of the genetic background. Several tests and transformations are utilized when testing differences between variances. There is, however, a biological tendency for the variance to scale proportionally to the square of the mean (scaling effect) which can considerably bias the results of the tests. We propose a novel method which allows for a more precise correction of the scaling effect and proper comparisons among treatment groups and between investigations. This is relevant for all data sets of distributions with different means and suggests the reanalysis of comparisons among treatment groups. This correction will provide a more reliable method when using bioindicators.     

Why should we constrain stress and limitation? Why conceptual terms deserve broad definitions

In his recent essay, Ch. Körner (2003) suggested that we need to clarify and narrow the meaning of limitation and stress. Current definitions and usage of these terms are not in any great need of revision. Limiting the use of ‘stress’ to only extreme situations is inappropriate because sometimes stress is indeed weak. What is needed is to use the terms within a comparative framework, a framework based on comparisons along gradients and among taxa. Recent studies on the factors controlling diversity have used such a comparative framework, and have increased our understanding of the relative roles of various types of stress. Redefining and narrowing general terms that relate to common processes would limit our ability to make comparisons and generalizations, and this should be avoided.     

Diagnosing causes of bird population declines

The value to bird conservation of determining the causes of population declines is considered and the diagnostic methods available are reviewed, with examples. Diagnosis of the cause or causes of a decline in bird numbers is likely to be helpful in deciding the priority of conservation actions, though actions which aim to reverse the changes in external conditions which caused the decline need not be the most effective in initiating recovery.
The methods for diagnosing causes of declines in bird numbers with the widest application make use of comparisons between geographical areas or time periods with different trends. Correlations between trends in numbers and measurements of external factors are examined across areas or periods or both. The danger of spurious correlations is minimized by drawing up a list of plausible causes based on studies of the natural history of the species. The effects of all of these candidates should be examined, subject to availability of data. The consistency of observed changes over time, or differences among areas, in survival rate or breeding success with the postulated demographic mechanism of the decline should be examined.
Conclusions based on correlations across geographical areas between trends in numbers and external factors may be misleading if birds are able to move between the areas selected for comparison and if their pattern of settlement depends upon external factors thought to be implicated in the decline.
Manipulative experiments should be carried out to test conclusions drawn from correlative studies. However, it must be recognized that the capacity of birds to move between areas means that experiments may measure effects of manipulations on settlement patterns or distribution rather than population size. Experiments that appear well designed in terms of controls and replication may be misleading when applied to the conservation of bird populations if their geographical scale is inappropriate.     

Incubation and embryonic development affect the color of eggs of Mountain Bluebirds

Technological and theoretical advances over the past two decades have allowed researchers to quantify eggshell color in ways that were not previously possible. However, differences among studies in the timing of color measurements during laying or incubation and inclusion of data from inviable eggs may affect the results of these studies as well as conclusions based on comparisons among studies. To determine the effect of the timing of color measurements, we compared the color of the eggs of Mountain Bluebirds (Sialia currucoides) during both laying/early incubation and late incubation. We also assessed the influence of egg viability on eggshell color by comparing viable and inviable eggs from the same clutch. We found that all color metrics investigated (blue‐green and UV chroma, brightness, and hue) were significantly different between early and late incubation, and viable and inviable eggs. However, color metrics of eggs measured during early and late incubation and of viable and inviable eggs in the same clutch were correlated. Our results suggest that the timing of color measurements and the viability of eggs have important effects on eggshell color and, therefore, in future studies, investigators should always provide information about the timing of measurements and the viability of eggs measured. Our results also suggest that comparisons among studies where eggshell color was measured at different times and/or the viability of eggs was not determined or reported are possible, given that the color metrics of viable and inviable eggs and of eggs measured at different times in our study were correlated, but those comparisons should be interpreted with caution.     

Complexities of Using Wild versus Captive Activity Budget Comparisons for Assessing Captive Primate Welfare

Activity budget comparisons between groups or individuals in the wild and those in captivity are commonly used to determine the range of wild-type behaviors that nonhuman animals in captivity perform. These comparisons are conducted with the view that individuals displaying a greater range of wild-type behaviors have enhanced welfare. Such comparisons have a greater appeal when it comes to primates because increased levels of anthropomorphism toward primates lead to welfare concerns. However, the validity of such comparisons has not been assessed. From the reviewed literature, some of the main complexities identified when defining “good welfare” using activity budgets were the ideas of behavioral motivation and adaptation, obstacles when using the wild as a benchmark for good welfare, study methods causing possible variances in behavior, and social and environmental factors. It is proposed that such influencing factors would be controlled for adequately in future studies with the use of multiple indices to measure welfare and methods that infer positive emotional states. It is hoped we can then build evidence of an animal’s emotional state and allow welfare to be inferred to a higher degree.     

Global meta‐analysis of native and nonindigenous trophic traits in aquatic ecosystems

Ecologists have recently devoted their attention to the study of species traits and their role in the establishment and spread of nonindigenous species (NIS). However, research efforts have mostly focused on studies of terrestrial taxa, with lesser attention being dedicated to aquatic species. Aquatic habitats comprise of interconnected waterways, as well as exclusive introduction vectors that allow unparalleled artificial transport of species and their propagules. Consequently, species traits that commonly facilitate biological invasions in terrestrial systems may not be as represented in aquatic environments. We provide a global meta‐analysis of studies conducted in both marine and freshwater habitats. We selected studies that conducted experiments with native and NIS under common environmental conditions to allow detailed comparisons among species traits. In addition, we explored whether different factors such as species relatedness, functional feeding groups, latitude, climate, and experimental conditions could be linked to predictive traits. Our results show that species with traits that enhance consumption and growth have a substantially increased probability of establishing and spreading when entering novel ecosystems. Moreover, traits associated with predatory avoidance were more prevalent in NIS and therefore favour invasive species in aquatic habitats. When we analysed NIS interacting with taxonomically distinctive native taxa, we found that consumption and growth were particularly important traits. This suggests that particular attention should be paid to newly introduced species for which there are no close relatives in the local biota. Finally, we found a bias towards studies conducted in temperate regions, and thus, more studies in other climatic regions are needed. We conclude that studies aiming at predicting future range shifts should consider trophic traits of aquatic NIS as these traits are indicative of multiple interacting mechanisms involved in promoting species invasions.     

Variability of the behavioral laterality in Teleostei (Pisces)

Factors causing variability of behavioral laterality in Teleostei are reviewed. The laterality has been revealed in many fish species belonging to various families. The best ever demonstrated example of the laterality is the different use of the right and left eyes when a fish responds to different visual objects. Magnitude and sign of the laterality differ in fishes of different species, gender, and age. Also, an observed laterality depends on how familiar a stimulus is to fishes and what it means to them, as well as their motivational level and various behavioral traits. Therefore, comparisons of the laterality among different fish species should be based on experimental methods that also take into account those behavioral differences among them that are not directly linked to the laterality.     

Improving efficiency of inferences in randomized clinical trials using auxiliary covariates

Summary .   The primary goal of a randomized clinical trial is to make comparisons among two or more treatments. For example, in a two-arm trial with continuous response, the focus may be on the difference in treatment means; with more than two treatments, the comparison may be based on pairwise differences. With binary outcomes, pairwise odds ratios or log odds ratios may be used. In general, comparisons may be based on meaningful parameters in a relevant statistical model. Standard analyses for estimation and testing in this context typically are based on the data collected on response and treatment assignment only. In many trials, auxiliary baseline covariate information may also be available, and it is of interest to exploit these data to improve the efficiency of inferences. Taking a semiparametric theory perspective, we propose a broadly applicable approach to adjustment for auxiliary covariates to achieve more efficient estimators and tests for treatment parameters in the analysis of randomized clinical trials. Simulations and applications demonstrate the performance of the methods.     

Statistical methods suitable for the analysis of plant tissue culture data

Statistical analyses are an essential part of biological research. Statistical methods are available to biological researchers that range from very simple to extremely complex. Therefore, caution should be used when selecting a statistical method. When possible it is best to avoid complicated statistical procedures that are difficult to interpret and may hinder the researcher's ability to make treatment comparisons. Instead a method should be chosen that compliments a logical and practical treatment design. Statistics should be used as a tool to compare treatments of interest and should not dictate the treatments. Experimental designs should take into account the eventual analysis, otherwise one could conceive of a design that could not be analyzed or, when analyzed, would not answer the desired questions. Therefore, time should be spent before conducting an experiment to plan an experimental design and analysis that best compliments the treatment scheme and questions to be answered. The purpose of this paper is to present examples of experimental designs, means separation procedures, data transformations and presentation methods suitable for plant cell and tissue culture data.Abbreviations ANOVA analysis of variance - BA benzyladenine - CV coefficient of variation - DF degrees of freedom - IAA indole-3-acetic acid - IBA indole-3-butyric acid - LOF lack-of-fit - MSE mean square error - P-ITB phenyl indole-3-thiolobutyrate - S standard deviation - SE standard error of the mean - TDZ thidiazuron     

Proposed terminology and reporting units for sea ice algal assemblages

Summary Many terms and units are used to describe the algae associated with sea ice. Most of these terms are open to misinterpretation and have been frequently misused. The use of a number of different units when reporting on experimental studies makes it difficul, if not impossible, to compare studies done by different investigators. In an attempt to avoid these ambiguities and to make comparisons easier, we here suggest some standard terms and repoting units that should be used when discussing ice algal assemblages.     

The jury is out on "guilt by association" trials.

The availability of comprehensive protein-protein interaction maps will significantly enhance medical research and aid the functional characterisation of novel genes. To date, the largest scale studies of protein-protein interactions have used the yeast two hybrid method. In this review we take a closer look at the different approaches used in these studies and discuss some key considerations that should be taken into account when designing high throughput interaction mapping projects.     

DNA fingerprinting data and the problem of non-independence among pairwise comparisons

Multilocus DNA fingerprinting is commonly used to assess genetic similarity within and between geographically disjunct populations. Typically, the proportion of DNA fingerprinting bands shared between two individuals ( S _XY) is calculated for all possible pairwise comparisons and the resulting data analyzed parametrically to test differences in mean band-sharing among groups. The degree to which covariation among interdependent S _XY values ( S _ab - S _bc) biases the analyses is often unknown. Here, we assess the extent of covariation in four DNA fingerprinting studies and evaluate the effectiveness of two corrective procedures, a permutation test and a subsampling routine using only independent pairwise comparisons drawn without replacement from the overall data. Covariation among interdependent S _XY values was significantly greater than zero in every data set examined, including those from a bee, a rodent, and two passerine birds. Permutation tests did not correct for interdependence and yielded significance values nearly identical to those derived from uncorrected parametric procedures. In contrast, the subsampling procedure yielded corrected estimates of the standard error that were two to four times larger than those derived parametrically. As a result, comparisons that were significant using parametric tests were either non-significant or only marginally significant with the subsampling routine. We conclude that interdependence among S _XY values poses a substantial obstacle to hypothesis testing that must be addressed in future studies.     

Paternal levels of DNA damage in spermatozoa and maternal parity influence offspring mortality in an endangered ungulate

Understanding which factors influence offspring mortality rates is a major challenge since it influences population dynamics and may constrain the chances of recovery among endangered species. Most studies have focused on the effects of maternal and environmental factors, but little is known about paternal factors. Among most polygynous mammals, males only contribute the haploid genome to their offspring, but the possibility that sperm DNA integrity may influence offspring survival has not been explored. We examined several maternal, paternal and individual factors that may influence offspring survival in an endangered species (Gazella cuvieri). Levels of sperm DNA damage had the largest impact upon offspring mortality rates, followed by maternal parity. In addition, there was a significant interaction between these two variables, so that offspring born to primiparous mothers were more likely to die if their father had high levels of sperm DNA damage, but this was not the case among multiparous mothers. Thus, multiparous mothers seem to protect their offspring from the deleterious effects of sperm DNA damage. Since levels of sperm DNA damage seem to be higher among endangered species, more attention should be paid to the impact of this largely ignored factor among the viability of endangered species.     

On the indices of plant–plant competition and their pitfalls

The index of relative competition intensity (RCI) has serious built‐in biases, due to its asymptotic behavior when competition intensity is high and its tendency to obtain very low values when plants with neighbors intact perform better than neighbor removal plants. These biases have been partially corrected in the index of relative neighbor effect (RNE), but the existence of fixed upper and lower bounds (?1≤RNE≤+1) still creates problems and biases in communities where the average intensity of competition or facilitation is high and plant performance pronouncedly varies in space. The third commonly used index, the logarithm of response ratio (lnRR), is mathematically and statistically sound, but when computed from pair‐wise comparisons between neighbor removal and control plants, this index reflects the geometric mean of the treatment effect. Moreover, linear patterns in lnRR reflect exponential patterns in the intensity of competition. As the interest of ecologists usually focuses on arithmetic means, we propose a corrected index of relative competition intensity, CRCI=arc sin (RNE). This index is fairly linear within the observed ranges of competition and facilitation, and for the range of competition intensities where RNE behaves reasonably, the two indices obtain almost identical values. We compared the performance of the four indices, using both imagined and real data, the latter from systems where the responses of plants to neighbor removal ranged from weak to moderate, so that RNE and CRCI were expected to behave similarly. The indices were computed both from pooled data for each community and as averages of pair‐wise comparisons. lnRR and CRCI were found to behave in a consistent and bias‐free manner, yielding similar results regardless of method of computation. This was, by and large, the case with RNE, too, but as the values of indices grew, the values from pair‐wise comparisons became increasingly smaller than values computed from pooled data. RCI yielded grossly aberrant results in computations based on pair‐wise comparisons. Therefore, the further use of RCI is unadvisable and studies where RCI has been derived from pair‐wise comparisons should be excluded from meta‐analyses.     

Potential of 13C and 15N labeling for studying protein-protein interactions using Fourier transform infrared spectroscopy.

In this study, we examine the interaction between two bacterial proteins, namely HPr and IIAmtl of the Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system, using FTIR spectroscopy. In an interaction involving a 1:1 molar ratio of these two proteins, when they are unlabeled, the overlap of absorbance of the amide I band arising from the peptide group vibrations of the two proteins is such that it is not possible to determine the contribution which each protein makes to the absorbance. Uniform 15N labeling has little effect on the frequency of the amide I band although there is a significant shift of the amide II band. However, we show that uniform (90%) 13C labeling produces a large shift of bands associated with the carbonyl moiety, especially the amide I band. This opens up windows in different regions of the infrared spectrum. Thus, when the same mixture of the two bacterial proteins is made where one of the proteins is uniformly 13C-labeled (in our case HPr), the amide I maxima of this protein shifts by approximately 45 cm-1 toward lower frequency and reveals the previously overlapped amide I band of the unlabeled IIAmtl. This application of 13C labeling shows the potential of studying protein-protein interactions using FTIR spectroscopy. With thoughtful selection of systems and labeling strategies, numerous studies with proteins should be possible. These could include, among others, enzyme-substrate and protein-ligand interactions.     

Molecular and quantitative genetic divergence among populations of house mice with known evolutionary histories

Evolutionary biologists have long been interested in the processes influencing population differentiation, but separating the effects of neutral and adaptive evolution has been an obstacle for studies of population subdivision. A recently developed method allows tests of whether disruptive (ie, spatially variable) or stabilizing (ie, spatially uniform) selection is influencing phenotypic differentiation among subpopulations. This method, referred to as the F(ST) vs Q(ST) comparison, separates the total additive genetic variance into within- and among-population components and evaluates this level of differentiation against a neutral hypothesis. Thus, levels of neutral molecular (F(ST)) and quantitative genetic (Q(ST)) divergence are compared to evaluate the effects of selection and genetic drift on phenotypic differentiation. Although the utility of such comparisons appears great, its accuracy has not yet been evaluated in populations with known evolutionary histories. In this study, F(ST) vs Q(ST) comparisons were evaluated using laboratory populations of house mice with known evolutionary histories. In this model system, the F(ST) vs Q(ST) comparisons between the selection groups should reveal quantitative trait differentiation consistent with disruptive selection, while the F(ST) vs Q(ST) comparisons among lines within the selection groups should suggest quantitative trait differentiation in agreement with drift. We find that F(ST) vs Q(ST) comparisons generally produce the correct evolutionary inference at each level in the population hierarchy. Additionally, we demonstrate that when strong selection is applied between populations Q(ST) increases relative to Q(ST) among populations diverging by drift. Finally, we show that the statistical properties of Q(ST), a variance component ratio, need further investigation.     

Iminothiol/thiourea tautomeric equilibrium in thiourea lipids impacts DNA compaction by inducing a cationic nucleation for complex assembly

Our research on lipidic vectors for transfection led us to develop thiourea lipids able to interact with DNA. Hence, we developed a series of lipopolythioureas based on the strong hydrogen bond donor ability of thiourea. More recently we have reported a branched hydroxylated bis-thiourea derivative with interesting transfecting properties. The last step of the syntheses involved a strong acidic condition, leading to an unstable product upon storage. Therefore we designed a new synthesis in mild acidic conditions. Though they exhibit the same mass, the lipids obtained in the two different conditions differ by their interaction with DNA. We therefore explored the physicochemical properties of these two lipids by different means that we describe in this article. In order to insure easier and reliable ¹³C-NMR studies of the thiourea group we have designed the synthesis of the corresponding ¹³C-labeled thiourea lipids. We have thus shown that when the lipid was submitted to mildly acidic medium; only the thiourea group was observed; while a thiourea/charged and/or uncharged iminothiol tautomeric equilibrium formed when the last step of the synthesis was submitted to low pH. NMR experiments showed that this tautomeric equilibrium could not form in polar solvents. However, UV experiments on the liposomal form of the lipopolythiourea showed the presence of the tautomers. Lipid/DNA interaction consequently differed according to the acidic treatment applied. Eventually, these results revealed that on this particular thiourea lipid, electrostatic interactions due to cationic thioureas are likely to be responsible for DNA compaction and that this tautomeric form of the thiourea could be stabilised by hydrogen bonds in a supramolecular assembly. Nevertheless, this does not reflect a general thiourea lipid/DNA interaction as other thiourea lipids that are able to compact DNA do not undergo an acidic treatment during the final stage of their synthesis.