Learning from our GWAS mistakes: from experimental design to scientific method

Many public and private genome-wide association studies that we have analyzed include flaws in design, with avoidable confounding appearing as a norm rather than the exception. Rather than recognizing flawed research design and addressing that, a category of quality-control statistical methods has arisen to treat only the symptoms. Reflecting more deeply, we examine elements of current genomic research in light of the traditional scientific method and find that hypotheses are often detached from data collection, experimental design, and causal theories. Association studies independent of causal theories, along with multiple testing errors, too often drive health care and public policy decisions. In an era of large-scale biological research, we ask questions about the role of statistical analyses in advancing coherent theories of diseases and their mechanisms. We advocate for reinterpretation of the scientific method in the context of large-scale data analysis opportunities and for renewed appreciation of falsifiable hypotheses, so that we can learn more from our best mistakes.     

Overyielding in grassland communities: testing the sampling effect hypothesis with replicated biodiversity experiments

We derive and test some assumptions and predictions of the Sampling Effect Hypothesis (SEH) by examining the relationship between the traits of species in monoculture and their relative abundance in mixture, and by comparing polyculture performance with single-species plots. Although we found a positive relationship between production in monoculture and dominance in mixtures as predicted by the SEH, the relationship had low explanatory power. Counter to predictions, the species with the highest monoculture biomass were not able to strongly dominate all mixtures; instead the dominance of these species decreased with increasing species richness. On average, polycultures did not achieve greater biomass than (transgressively overyield) the species in each mixture, or at each site, that was most productive in monoculture. However, mixture yields did transgressively overyield both the monoculture biomass of the dominant species in the mixtures, and the weighted average of all monocultures (non-transgressive overyielding), both of which were positively related to increasing species richness. The varying responses of different overyielding tests resulted because resource partitioning and positive interactions were often counter-balanced by selection for species with lower biomass than the highest-yielding monocultures. Judging whether or not mixtures overyield therefore depends in part upon which species is the basis for comparison. We present a new general framework for overyielding analysis where every monoculture provides a potential comparison and from which the most relevant tests can be selected.     

Multiple hypothesis testing in proteomics: a strategy for experimental work

In quantitative proteomics work, the differences in expression of many separate proteins are routinely examined to test for significant differences between treatments. This leads to the multiple hypothesis testing problem: when many separate tests are performed many will be significant by chance and be false positive results. Statistical methods such as the false discovery rate method that deal with this problem have been disseminated for more than one decade. However a survey of proteomics journals shows that such tests are not widely implemented in one commonly used technique, quantitative proteomics using two-dimensional electrophoresis. We outline a selection of multiple hypothesis testing methods, including some that are well known and some lesser known, and present a simple strategy for their use by the experimental scientist in quantitative proteomics work generally. The strategy focuses on the desirability of simultaneous use of several different methods, the choice and emphasis dependent on research priorities and the results in hand. This approach is demonstrated using case scenarios with experimental and simulated model data.     

Haemoglobinopathies and malaria: new approaches to an old hypothesis

It is almost 40 years since Haldane proposed that high frequencies of thalassaemia and other hoemoglobinopathies might result because these genetic disorders conferred some protection against malaria In the intervening years convincing clinical, epidemiological and in vitro data have been produced to support this 'malaria hypothesis' for haemoglobin S. However, for the numerous other genetic disorders of haemoglobin that are suspected to have been selected in this way it has been much more difficult to provide clearcut evidence. For example, in the case of beta-thalassaemia some but not all epidemiological studies support the hypothesis, while in vitro studies have failed to demonstrate any impairment of either invasion or growth of P. falciparum in affected red cells under physiological conditions.     

Assessing temporal variations in diversification rates from phylogenies: estimation and hypothesis testing

A new method to estimate the diversification rate of a lineage from a phylogeny of recent species is presented. This uses survival models to analyse the ages of the species as derived from the phylogeny. Survival models can analyse missing data where the exact date of death is unknown (censoring). This approach allows us to include missing data (species not included in a detailed phylogenetic study) in the analysis, provided a minimum age is known for these species. Three models are presented, with emphasis on temporal variation in diversification rates. The maximum likelihood method and Akaike information criteria are used to derive estimators and tests of hypotheses. A simulation study demonstrates that the method is able to detect a temporal variation in diversification rate only when it is present, avoiding type I and type II errors. A lineage with ten species may be sufficient to detect a temporal variation in diversification rate even with 50 per cent of missing data. An application is presented with data from a phylogeny of birds of the genus Ramphocelus.     

Microbial proteases in peptide synthesis: approaches and applications

Biodegradation of polycyclic aromatic hydrocarbons (PAHs) in the environment is often limited due to unfavorable nutrient conditions for the bacteria that use these PAHs as sole source of carbon and energy. Mycobacterium and Sphingomonas are 2 PAH-degrading specialists commonly present in PAH-polluted soil, but not much is known about their specific nutrient requirements. By adding different inorganic supplements of nitrogen (N) and phosphorus (P), affecting the overall carbon/nitrogen/phosphorus ratio of soil in soil slurry degradation tests, we investigated the impact of soil inorganic N and P nutrient conditions on PAH degradation by PAH-degrading Sphingomonas and Mycobacterium strains. The general theoretically calculated C/N/P ratio of 100/10/1 (expressed in moles) allowed rapid PAH metabolization by Sphingomonas and Mycobacterium strains without limitation. In addition, PAH-degradation rate and extent was not affected when ca. ten times lower concentrations of N and P were provided, indicating that Sphingomonas and Mycobacterium strains are capable of metabolizing PAHs under low nutrient conditions. Nor does PAH-degradation seem to be affected by excesses of N and P creating an imbalanced C/N/P ratio. However, supplements of N and P salts increased the salinity of soil slurry solutions and seriously limited or even completely blocked biodegradation.     

Sexual dimorphism in the human pelvis: testing a new hypothesis.

Sexual dimorphism in the human pelvis is inferentially related to parturition. Investigators disagree about the identification and obstetric significance of pelvic dimorphism. Benefiting from a large sample of complete skeletons from the Coimbra Identified Skeletal Collection, we show that the dimensions of the true pelvis (birth canal) that are most sexually dimorphic (that is, the dimensions of females are greater than males) are those which are related to biparietal deformation, which often leads to the death of the human neonate. These dimensions are: the anteroposterior diameter of the inlet (index of dimorphism = 108.41), the transverse diameter of the bispinous midplane (index of dimorphism = 117.13) and the transverse diameter of the outlet (index of dimorphism = 112.3). Therefore, sexual dimorphism in the human pelvis is a reflection of differential selection on the two sexes. These results may stimulate further studies with a fresh approach regarding the fossil and comparative evidence for when and how the modern pattern of birth has evolved.     

Fusion pore conductance: experimental approaches and theoretical algorithms.

Time-resolved admittance measurements provide the basis for studies showing that membrane fusion occurs through the formation and widening of an initially small pore, linking two previously separated aqueous compartments. Here we introduce modifications to this method that correct the cell-pipette (source) admittance for attenuation and phase shifts produced by electrophysiological equipment. Two new approaches for setting the right phase angle are discussed. The first uses the displacement of a patch-clamp amplifier C-slow potentiometer for the calculation of phase. This calculation is based on amplitudes of observed and expected (theoretical) changes in the source admittance. The second approach automates the original phase adjustment, the validity of which we prove analytically for certain conditions. The multiple sine wave approach is modified to allow the calculation of target cell membrane parameters and the conductance of the fusion pore. We also show how this technique can be extended for measurements of the resting potential of the first (voltage-clamped) membrane. We introduce an algorithm for calculation of fusion pore conductance despite a concurrent change in the resistance of the clamped membrane. The sensitivity of the capacitance restoration algorithm to phase shift errors is analyzed, and experimental data are used to demonstrate the results of this analysis. Finally, we show how the phase offset can be corrected "off-line" by restoring the shape of the capacitance increment.     

Microbial biofilms: from ecology to molecular genetics.

Biofilms are complex communities of microorganisms attached to surfaces or associated with interfaces. Despite the focus of modern microbiology research on pure culture, planktonic (free-swimming) bacteria, it is now widely recognized that most bacteria found in natural, clinical, and industrial settings persist in association with surfaces. Furthermore, these microbial communities are often composed of multiple species that interact with each other and their environment. The determination of biofilm architecture, particularly the spatial arrangement of microcolonies (clusters of cells) relative to one another, has profound implications for the function of these complex communities. Numerous new experimental approaches and methodologies have been developed in order to explore metabolic interactions, phylogenetic groupings, and competition among members of the biofilm. To complement this broad view of biofilm ecology, individual organisms have been studied using molecular genetics in order to identify the genes required for biofilm development and to dissect the regulatory pathways that control the plankton-to-biofilm transition. These molecular genetic studies have led to the emergence of the concept of biofilm formation as a novel system for the study of bacterial development. The recent explosion in the field of biofilm research has led to exciting progress in the development of new technologies for studying these communities, advanced our understanding of the ecological significance of surface-attached bacteria, and provided new insights into the molecular genetic basis of biofilm development.     

Assessing macroinvertebrate biodiversity in freshwater ecosystems: advances and challenges in DNA-based approaches

Assessing the biodiversity of macroinvertebrate fauna in freshwater ecosystems is an essential component of both basic ecological inquiry and applied ecological assessments. Aspects of taxonomic diversity and composition in freshwater communities are widely used to quantify water quality and measure the efficacy of remediation and restoration efforts. The accuracy and precision of biodiversity assessments based on standard morphological identifications are often limited by taxonomic resolution and sample size. Morphologically based identifications are laborious and costly, significantly constraining the sample sizes that can be processed. We suggest that the development of an assay platform based on DNA signatures will increase the precision and ease of quantifying biodiversity in freshwater ecosystems. Advances in this area will be particularly relevant for benthic and planktonic invertebrates, which are often monitored by regulatory agencies. Adopting a genetic assessment platform will alleviate some of the current limitations to biodiversity assessment strategies. We discuss the benefits and challenges associated with DNA-based assessments and the methods that are currently available. As recent advances in microarray and next-generation sequencing technologies will facilitate a transition to DNA-based assessment approaches, future research efforts should focus on methods for data collection, assay platform development, establishing linkages between DNA signatures and well-resolved taxonomies, and bioinformatics.     

Microbial communities on the monuments of Moscow and St. Petersburg: biodiversity and trophic relations

Stone monuments situated in the Alexander Nevsky Abbey, the Summer Garden, and the Smolenskoe Cemetery, St. Petersburg, and marble and limestone sculptures and tombstones situated in the Novodevichy Convent and the Donskoy Monastery, Moscow, were investigated for their microbial contamination. The architectural objects studied date to the 12th century. The monuments in the Alexander Nevsky Abbey were found to be severely contaminated with micromycetes belonging to 24 genera (primarily of the class Deuteromycetes). The analysis of the samples taken from the monuments of the Donskoy Monastery by the serial dilution technique showed that they were contaminated with bacteria at a density of (1-1.7) x 10(5) cells/g. This value, however, turned out to be 1 to 2 orders greater when the bacterial population was evaluated by the luciferin-luciferase method. We succeeded in identifying 12 bacterial genera; however, this number may be increased in the course of further studies. Some preventive measures to control the biodeterioration of stone heritage are discussed.