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.     

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.     

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 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.     

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 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.     

Natural and accelerated recovery from brain damage: experimental and theoretical approaches

The goal of the Caltech group is to gain insight into the processes that occur within the primate nervous system during dexterous reaching and grasping and to see whether natural recovery from local brain damage can be accelerated by artificial means. We will create computational models of the nervous system embodying this insight and explain a variety of clinically observed neurological deficits in human subjects using these models.     

Experimental design in phylogenetics: testing predictions from expected information

Taxon and character sampling are central to phylogenetic experimental design; yet, we lack general rules. Goldman introduced a method to construct efficient sampling designs in phylogenetics, based on the calculation of expected Fisher information given a probabilistic model of sequence evolution. The considerable potential of this approach remains largely unexplored. In an earlier study, we applied Goldman's method to a problem in the phylogenetics of caecilian amphibians and made an a priori evaluation and testable predictions of which taxon additions would increase information about a particular weakly supported branch of the caecilian phylogeny by the greatest amount. We have now gathered mitogenomic and rag1 sequences (some newly determined for this study) from additional caecilian species and studied how information (both expected and observed) and bootstrap support vary as each new taxon is individually added to our previous data set. This provides the first empirical test of specific predictions made using Goldman's method for phylogenetic experimental design. Our results empirically validate the top 3 (more intuitive) taxon addition predictions made in our previous study, but only information results validate unambiguously the 4th (less intuitive) prediction. This highlights a complex relationship between information and support, reflecting that each measures different things: Information is related to the ability to estimate branch length accurately and support to the ability to estimate the tree topology accurately. Thus, an increase in information may be correlated with but does not necessitate an increase in support. Our results also provide the first empirical validation of the widely held intuition that additional taxa that join the tree proximal to poorly supported internal branches are more informative and enhance support more than additional taxa that join the tree more distally. Our work supports the view that adding more data for a single (well chosen) taxon may increase phylogenetic resolution and support in weakly supported parts of the tree without adding more characters/genes. Altogether our results corroborate that, although still underexplored, Goldman's method offers a powerful tool for experimental design in molecular phylogenetic studies. However, there are still several drawbacks to overcome, and further assessment of the method is needed in order to make it better understood, more accessible, and able to assess the addition of multiple taxa.     

Molecular and technological approaches to evaluate strain biodiversity in Hanseniaspora uvarum of wine origin

AIMS: The characterization by molecular and physiological methods of wild apiculate strains, isolated from 'Aglianico del Vulture' grape must. METHODS AND RESULTS: The restriction analysis of 18S rDNA allowed the identification of strains at the species level, which were predominantly Hanseniaspora uvarum. The RAPD analysis and the evaluation of technological traits, such as the metabolic and enzymatic activities, were useful to evaluate the polymorphism of this species. CONCLUSIONS: The RAPD analysis clustered the wild H. uvarum strains in four main genetic groups and a very high phenotypic variability confirmed this genetic polymorphism. The technological variables, which determined the strain biodiversity differed significantly, demonstrating that these technological traits are strain dependent. A certain correlation was found between the strain behaviour and its isolation zone, indicating the influence of the environment on the genetic patrimony of the population. SIGNIFICANCE AND IMPACT OF THE STUDY: The genetic and technological biodiversity recorded among H. uvarum wild strains represents the basis for organizing a collection of apiculate strains exhibiting oenological characteristics at different levels, such as high/low production of secondary compounds, and, therefore, potentially useful for a selection programme.