Gaps in the canopy: the missing dimension in vegetation dynamics

Canopy gaps are important as entry points for new genotypes and new species into many types of vegetation, yet little is known about them in any type of vegetation but forests. Forest gaps are too large for manipulative experiments to be readily undertaken, and hitherto grassland gaps have been too small to be easily mapped. Preliminary results from mapping small (>1 cm) grassland gaps with a new fibre-optic device suggest that experiments need to be performed at a smaller physical scale than has hitherto been achieved.     

The state of the art in the analysis of two-dimensional gel electrophoresis images

Software-based image analysis is a crucial step in the biological interpretation of two-dimensional gel electrophoresis experiments. Recent significant advances in image processing methods combined with powerful computing hardware have enabled the routine analysis of large experiments. We cover the process starting with the imaging of 2-D gels, quantitation of spots, creation of expression profiles to statistical expression analysis followed by the presentation of results. Challenges for analysis software as well as good practices are highlighted. We emphasize image warping and related methods that are able to overcome the difficulties that are due to varying migration positions of spots between gels. Spot detection, quantitation, normalization, and the creation of expression profiles are described in detail. The recent development of consensus spot patterns and complete expression profiles enables one to take full advantage of statistical methods for expression analysis that are well established for the analysis of DNA microarray experiments. We close with an overview of visualization and presentation methods (proteome maps) and current challenges in the field. An erratum to this article can be found at     

Certain methodical aspects of experiments studying the nutrition of caterpillars of the Gypsy moth

It is shown that there are a number of factors that are important to consider when planning the experiments on studying the trophic parameters of the caterpillars of the Gypsy moth, first of all, the population characteristics of the species including the sex and age structure that change annually. The final results of the study are also influenced by some procedures during the experiments, particularly, the duration (especially in the longest instars of the caterpillars) and volume of the sampling, which is supposed to grow as the caterpillars instar increases. The expediency is examined for using the individual growing of the caterpillars to study the species nutrition.     

Population of on-pathway intermediates in the folding of ubiquitin

The role that intermediate states play in protein folding is the subject of intense investigation and in the case of ubiquitin has been controversial. We present fluorescence-detected kinetic data derived from single and double mixing stopped-flow experiments to show that the F45W mutant of ubiquitin (WT*), a well-studied single-domain protein and most recently regarded as a simple two-state system, folds via on-pathway intermediates. To account for the discrepancy we observe between equilibrium and kinetic stabilities and m-values, we show that the polypeptide chain undergoes rapid collapse to an intermediate whose presence we infer from a fast lag phase in interrupted refolding experiments. Double-jump kinetic experiments identify two direct folding phases that are not associated with slow isomerisation reactions in the unfolded state. These two phases are explained by kinetic partitioning which allows molecules to reach the native state from the collapsed state via two possible competing routes, which we further examine using two destabilised ubiquitin mutants. Interrupted refolding experiments allow us to observe the formation and decay of an intermediate along one of these pathways. A plausible model for the folding pathway of ubiquitin is presented that demonstrates that obligatory intermediates and/or chain collapse are important events in restricting the conformational search for the native state of ubiquitin.     

Statistical approach to combining the results of similar experiments, with application to the hematologic effects of extremely-low-frequency electric field exposures

A large proportion of scientific effort in investigating the possible biological effects of exposure to extremely-low-frequency (ELF) fields consists of laboratory studies on experimental animals. Most experiments in which hematologic properties are measured show no statistically significant effect due to exposure. However, some studies show significant effects which, in general, are not clearly reproducible. A difficult question must then be addressed: Are these relatively few indications of ELF effects statistical artifacts due to the increased risk of a type I error in multiple studies, or is there a real biological effect that is undetected in most studies due to the relatively small sample sizes commonly used? A statistical approach for examining the accumulated results of multiple experiments which results in a single test for treatment effect is presented. The technique requires very mild assumptions, and is valid for experiments that vary widely in specific characteristics such as exposure level, duration, and laboratory. The method is applied to the results of a collection of hematologic and serum chemistry experiments, and the combined results indicate the existence of experimental effects on some end points.     

Evaluation of the key odorants of foods by dilution experiments, aroma models and omission.

The state of the art in aroma analysis is reviewed with emphasis on aroma-recombination studies using synthetic blends of odorants (aroma models) which have been prepared on the basis of analytical data. The model that matches the original aroma is the starting material for omission experiments which are performed to establish the odorants that actually contribute to the aroma. These experiments are discussed in detail for the aromas of two wine varieties, three olive oils of different provenance, French fries, boiled beef and coffee. The results indicate that odorants with higher odour activity values (OAV, the ratio of the concentration to the odour threshold) are frequently essential for the aroma. However, there are exceptions where odorants with high OAVs are suppressed in the aroma and compounds with lower OAVs are important contributors. These findings are discussed in the light of model experiments which have been reported in the literature to obtain an insight into the perceptual interactions of odorants in mixtures.     

Biodiversity–ecosystem function experiments reveal the mechanisms underlying the consequences of biodiversity change in real world ecosystems

In a recent Forum paper, Wardle (Journal of Vegetation Science, 2016) questions the value of biodiversity–ecosystem function (BEF) experiments with respect to their implications for biodiversity changes in real world communities. The main criticism is that the previous focus of BEF experiments on random species assemblages within each level of diversity has ‘limited the understanding of how natural communities respond to biodiversity loss.’ He concludes that a broader spectrum of approaches considering both non‐random gains and losses of diversity is essential to advance this field of research. Wardle's paper is timely because of recent observations of frequent local and regional biodiversity changes across ecosystems. While we appreciate that new and complementary experimental approaches are required for advancing the field, we question criticisms regarding the validity of BEF experiments. Therefore, we respond by briefly reiterating previous arguments emphasizing the reasoning behind random species composition in BEF experiments. We describe how BEF experiments have identified important mechanisms that play a role in real world ecosystems, advancing our understanding of ecosystem responses to species gains and losses. We discuss recent examples where theory derived from BEF experiments enriched our understanding of the consequences of biodiversity changes in real world ecosystems and where comprehensive analyses and integrative modelling approaches confirmed patterns found in BEF experiments. Finally, we provide some promising directions in BEF research.     

Experimental analysis of soft‐tissue fossilization: opening the black box

Taphonomic experiments provide important insights into fossils that preserve the remains of decay‐prone soft tissues, tissues that are usually degraded and lost prior to fossilization. These fossils are among the most scientifically valuable evidence of ancient life on Earth, giving us a view into the past that is much less biased and incomplete than the picture provided by skeletal remains alone. Although the value of taphonomic experiments is beyond doubt, a lack of clarity regarding their purpose and limitations, and ambiguity in the use of terminology, are hampering progress. Here we distinguish between processes that promote information retention and those that promote information loss, in order to clarify the distinction between fossilization and preservation. Recognizing distinct processes of decay, mineralization and maturation, the sequence in which they act, and the potential for interactions, has important consequences for analysis of fossils, and for the design of taphonomic experiments. The purpose of well‐designed taphonomic experiments is generally to understand decay, maturation and preservation individually, thus limiting the number of variables involved. Much work remains to be done, but these methodologically reductionist foundations will allow researchers to build towards more complex taphonomic experiments and a more holistic understanding and analysis of the interactions between decay, maturation and preservation in the fossilization of non‐biomineralized remains. Our focus must remain on the key issue of understanding what exceptionally preserved fossils reveal about the history of biodiversity and evolution, rather than on debating the scope and value of an experimental approach.     

Simple experiments to understand the ionic origins and characteristics of the ventricular cardiac action potential

Electrophysiological experiments are helpful for students to understand the role of electrical activity in heart function. Papillary muscle, which belongs to the ventricle, offers the advantage of being easily studied using glass microelectrodes. In addition, there is commercially available software that simulates ventricular electrical activity and can help overcome some difficulties, such as voltage clamp experiments, which need expensive apparatus when used for studies on living preparations. Here, we present a class practical session that is taken by undergraduate students at our University. In the first part of this class, students record action potentials from papillary muscles with the use of glass microelectrodes, and they change extracellular conditions to study the ionic basis of the action potential. In the second part of the class, students simulate action potentials using the Oxsoft Heart model (v. 4.0) and model their previous experiments on papillary muscle to quantify the effects. In particular, the model is very helpful in promoting understanding of the effect that extracellular potassium has on cardiac action potential by simulating voltage clamp experiments. This twin approach of papillary muscle experiments and computer modeling leads to a good understanding of the functioning of the action potential and can help introduce discussion of some abnormal cardiac functioning.     

Normalization of high dimensional genomics data where the distribution of the altered variables is skewed

Genome-wide analysis of gene expression or protein binding patterns using different array or sequencing based technologies is now routinely performed to compare different populations, such as treatment and reference groups. It is often necessary to normalize the data obtained to remove technical variation introduced in the course of conducting experimental work, but standard normalization techniques are not capable of eliminating technical bias in cases where the distribution of the truly altered variables is skewed, i.e. when a large fraction of the variables are either positively or negatively affected by the treatment. However, several experiments are likely to generate such skewed distributions, including ChIP-chip experiments for the study of chromatin, gene expression experiments for the study of apoptosis, and SNP-studies of copy number variation in normal and tumour tissues. A preliminary study using spike-in array data established that the capacity of an experiment to identify altered variables and generate unbiased estimates of the fold change decreases as the fraction of altered variables and the skewness increases. We propose the following work-flow for analyzing high-dimensional experiments with regions of altered variables: (1) Pre-process raw data using one of the standard normalization techniques. (2) Investigate if the distribution of the altered variables is skewed. (3) If the distribution is not believed to be skewed, no additional normalization is needed. Otherwise, re-normalize the data using a novel HMM-assisted normalization procedure. (4) Perform downstream analysis. Here, ChIP-chip data and simulated data were used to evaluate the performance of the work-flow. It was found that skewed distributions can be detected by using the novel DSE-test (Detection of Skewed Experiments). Furthermore, applying the HMM-assisted normalization to experiments where the distribution of the truly altered variables is skewed results in considerably higher sensitivity and lower bias than can be attained using standard and invariant normalization methods.     

Characterization of the arginine repressor from Salmonella typhimurium and its interactions with the carAB operator.

Primer extension experiments showed that the argR gene, encoding the arginine repressor in Salmonella typhimurium, is transcribed from a single promoter that is negatively regulated by arginine. A repressor overproducing strain was constructed and the repressor was purified to homogeneity. Gel filtration, sedimentation and cross-linking studies established that the native repressor is a hexamer of identical 17,000 Mr subunits. Gel retardation experiments indicate that the apparent dissociation constant for repressor/carAB operator is 6 x 10(-12) M. These experiments showed that arginine is essential for binding of the repressor to the DNA and that pyrimidine nucleotides have no significant effect on this binding. These results indicate that the effect of pyrimidines on expression of the arginine sensitive "downstream" carAB promoter is not directly mediated by the arginine repressor. These experiments also suggest that a single hexamer binds to the carAB operator, which carries two previously defined "ARG box" sequences that characterize operators for arg genes. Gel retardation experiments with DNA fragments carrying the individual ARG boxes showed that both boxes are required for effective binding of the hexameric repressor to the operator, indicating that the ARG boxes comprise a single binding site for the repressor. Analysis of the potential secondary structure of the arginine repressor does not reveal any of the recognizable structural motifs common to a number of DNA-binding proteins. A combination of DNase I, premethylation interference, depurination and hydroxyl radical footprinting techniques were employed to characterize the interactions of the repressor with the carAB operator, with the results suggesting that the repressor predominantly interacts with A.T residues in this region. Comparative DNA sequence analysis of the known arginine operators of enteric bacteria further indicates that the specificity of interaction may be based more on the precise distance between two defined A.T-rich regions rather than on the specific nucleotide sequence.     

Fluorescent protein FRET: the good, the bad and the ugly

Dynamic protein interactions play a significant part in many cellular processes. A technique that shows considerable promise in elucidating such interactions is F?rster resonance energy transfer (FRET). When combined with multiple, colored fluorescent proteins, FRET permits high spatial resolution assays of protein-protein interactions in living cells. Because FRET signals are usually small, however, their measurement requires careful interpretation and several control experiments. Nevertheless, the use of FRET in cell biological experiments has exploded over the past few years. Here we describe the physical basis of FRET and the fluorescent proteins appropriate for these experiments. We also review the approaches that can be used to measure FRET, with particular emphasis on the potential artifacts associated with each approach.     

Yeast genomic databases and the challenge of the post-genomic era

Since the completion of the yeast genome sequence in 1996, three genomic databases, the Saccharomyces Genome Database, the Yeast Proteome Database, and MIPS (produced by the Munich Information Center for Protein Sequences), have organized published knowledge of yeast genes and proteins onto the framework of the genome. Now, post-genomic technologies are producing large-scale datasets of many types, and these pose new challenges for knowledge integration. This review first examines the structure and content of the three genomic databases, and then draws from them and other resources to examine the ways knowledge from the literature, genome, and post-genomic experiments is stored, integrated, and disseminated. To better understand the impact of post-genomic technologies, 20 collections of post-genomic data were analyzed relative to a set of 243 previously uncharacterized genes. The results indicate that post-genomic technologies are providing rich new information for nearly all yeast genes, but data from these experiments is scattered across many Web sites and the results from these experiments are poorly integrated with other forms of yeast knowledge. Goals for the next generation of databases are set forth which could lead to better access to yeast knowledge for yeast researchers and the entire scientific community. Electronic Publication     

Testing the functional significance of microbial composition in natural communities

Ecologists have long studied the relationship between biotic composition and ecosystem functioning in larger organisms; however, only recently has this relationship been investigated widely in microorganisms. Recent studies are reviewed within a framework of three experimental approaches that are often used to study larger organisms: environmental treatment, common garden, and reciprocal transplant experiments. Although the composition of microorganisms cannot be easily manipulated in the field, applying these approaches to intact microbial communities can begin to tease apart the effects of microbial composition from environmental parameters on ecosystem functioning. The challenges in applying these approaches to microorganisms are highlighted and it is discussed how the experimental approach and duration affects a study's interpretation. In general, long-term environmental treatment experiments identify correlative relationships between microbial composition and ecosystem functioning, whereas short-term common garden experiments demonstrate that microbial composition influences ecosystem functioning. Finally, reciprocal transplants simultaneously test for interactive effects of the environment and composition on functioning. The studies reviewed provide evidence that, at least in some cases, microbial composition influences ecosystem functioning. It is concluded that whole-community experiments offer a way to test whether information about microbial composition will help predict ecosystem responses to global change.     

Scale-down and optimization studies of the gluconic acid fermentation by Gluconobacter oxydans

To simulate production-scale conditions of gluconic acid fermentation by Gluconobacter oxydans, different experimental setups are presented in this study. From the determination of the time constants of a production-scale reactor, it can be concluded that mixing and oxygen transfer are the rate-limiting mechanisms. This results in oxygen concentration gradients which were simulated in a one-compartment reactor in which the oxygen concentration was fluctuated by a fluctuated gassing with air and nitrogen. It could be concluded that only very long periods of absence of oxygen (ca. 180 s) results in lower specific oxygen uptake rates by Gluconobacter oxydans. From scale-down studies carried out in a two-compartment system to simulate a production-scale reactor more accurately, it could be concluded that not only the residence time in the aerated part of the system is important, but the liquid flow in between the different parts of the reactor is also an essential parameter. It could also be concluded that the microorganisms are not influenced negatively by the fluctuated oxygen concentrations with respect to their maximal oxidation capacity. The two-compartment system can also be used for optimization experiments in which the "aerated" compartment was gassed with pure oxygen. From these experiments it was concluded that also a short residence of the cells at high oxygen concentrations diminished the growth and product formation rates. These experiments show the necessity of the scale-down experiments if optimization is carried out. The two-compartment system presented in this study is a very attractive tool for reliable scale-down experiments.     

Probing the presently tenuous link between comets and the origin of life

A unique set of millimeter-wave experiments for future cometary space missions is discussed. These experiments could yield answers to many basic questions about the presently undetermined nature of cometary nuclei and inner comae. This same set of experiments, designed to do fundamental cometary research, could simultaneously provide information on whether the accepted biological requirements necessary for the development of life are met in comets.     

Combining Information from Similar Experiments: I. Statistical Issues

A general method for combining information from similar experiments is illustrated in the case where two independent experiments are designed to estimate a dose-response curve. By accounting for variability across experiments, the proposed method avoids inferential pitfalls such as extended forms of Simpson's paradox. The validity of the method is supported by seven fundamental assumptions about data from replicated experiments. In contrast, an example indicates that failing to reject a preliminary test of equal distributions is inadequate justification for pooling data from two experiments. Methods that account for the variability across experiments in terms of known covariates are also discussed.