Visualizing the microtubule-associated protein tau in the nucleus

Although tau is mainly known as an axonal microtubule-associated protein,many studies indicate that it is not restricted to this subcellular compartment.Assessing tau’s subcellular distribution,however,is not trivial as is evident from transgenic mouse studies.When human tau is over-expressed,it can be immunohistochemically localized to axons and the somatodendritic domain,modeling what is found in neurodegenerative diseases such as Alzheimer’s disease.Yet,in wild-type mice,despite its abundance,tau is difficult to visualize even in the axon.It is even more challenging to detect this protein in the nucleus,where tau has been proposed to protect DNA from damage.To establish a framework for future studies into tau’s nuclear functions,we compared several methods to visualize endogenous nuclear tau in cell lines and mouse brain.While depending on the fixation and permeabilization protocol,we were able to detect nuclear tau in SH-SY5Y human neuroblastoma cells,we failed to do so in N2a murine neuroblastoma cells.As a second method we used subcellular fractionation of mouse tissue and found that in the nucleus tau is mainly present in a hypophosphorylated form.When either full-length or truncated human tau was expressed,both accumulated in the cytoplasm,but were also found in the nuclear fraction.Because subcellular fractionation methods have their limitations,we finally isolated nuclei to probe for nuclear tau and found that the nuclei were free of cytoplasmic contamination.Together our analysis identifies several protocols for detecting tau in the nucleus where it is found in a less phosphorylated form.     

The serpent and the egg: unidirectional evolution of reproductive mode in vipers?

Dollo’s law, that complex characters are not regained in evolution, is a pattern applied to many systems. Recent work has evaluated unidirectional evolution in a number of contexts, and several violations of this law have been documented. These methods have also been criticized for potentially overestimating reversals. We test the hypothesis that the ancestral reproductive mode of oviparity can be regained in vipers, in opposition to Dollo’s law. We use model comparison and ancestral character state reconstruction methods that address recent criticisms, and find evidence both supporting and refuting Dollo’s predictions from different analyses. We discuss our results in the context of unidirectional evolution and review factors required for strong inference of violations of Dollo’s law.     

Do fast food restaurants surrounding schools affect childhood obesity?

In this study, we estimate the effect of fast food environment surrounding schools on childhood body mass index (BMI). We use two methods that arrive at a similar conclusion, but with different implications. Using school distance from the nearest federal highway to instrument for restaurant location, we find the surrounding restaurants to only marginally affect a student’s BMI measure. The effect size also decreases with increasing radial distances from school, 0.016 standard deviations at one-third of a mile and 0.0032 standard deviations at a mile radial distance. This indicates the decreasing influence of restaurants on a child’s BMI as its distance from school increases. On a subset of students who were exogenously assigned to different school food environment, we find no effect of the fast food restaurants. An important contextual aspect is that nearly all schools in this sample observed closed campus policy, which does not allow students to leave campus during lunch hours.     

Applying complementary species vulnerability assessments to improve conservation strategies in the Galapagos Marine Reserve

Marine biodiversity can be protected by identifying vulnerable species and creating marine protected areas (MPAs) to ensure their survival. A wide variety of methods are employed by environmental managers to determine areas of conservation priority, however which methods should be applied is often a subject of debate for practitioners and scientists. We applied two species vulnerability assessments, the International Union for the Conservation of Nature (IUCN) red list of threatened species and FishBase’s intrinsic vulnerability assessment, to fish communities in three coastal habitats (mangrove, rocky and coral) on the island of San Cristobal, Galapagos. When using the IUCN red list of threatened species, rocky reefs hosted the greatest number of vulnerable species, however when applying the FishBase assessment of intrinsic vulnerability mangroves hosted the greatest abundance of ‘very-highly’ vulnerable species and coral ecosystems hosted the greatest abundance of ‘highly’ vulnerable species. The two methods showed little overlap in determining habitat types that host vulnerable species because they rely on different biological and ecological parameters. Since extensive data is required for IUCN red list assessments, we show that the intrinsic vulnerability assessment from FishBase can be used to complement the IUCN red list especially in data-poor areas. Intrinsic vulnerability assessments are based on less data-intensive methods than the IUCN red list, but nonetheless may bridge information gaps that can arise when using the IUCN red list alone. Vulnerability assessments based on intrinsic factors are not widely applied in marine spatial planning, but their inclusion as a tool for forming conservation strategies can be useful in preventing species loss.     

The Science of Animal Suffering

Can suffering in non‐human animals be studied scientifically? Apart from verbal reports of subjective feelings, which are uniquely human, I argue that it is possible to study the negative emotions we refer to as suffering by the same methods we use in ourselves. In particular, by asking animals what they find positively and negatively reinforcing (what they want and do not want), we can define positive and negative emotional states. Such emotional states may or may not be accompanied by subjective feelings but fortunately it is not necessary to solve the problem of consciousness to construct a scientific study of suffering and welfare. Improvements in animal welfare can be based on the answers to two questions: Q1: Will it improve animal health? and Q2: Will it give the animals something they want? This apparently simple formulation has the advantage of capturing what most people mean by ‘improving welfare’ and so halting a potentially dangerous split between scientific and non‐scientific definitions of welfare. It can also be used to validate other controversial approaches to welfare such as naturalness, stereotypies, physiological and biochemical measures. Health and what animals want are thus not just two of many measures of welfare. They provide the definition of welfare against which others can be validated. They also tell us what research we have to do and how we can judge whether welfare of animals has been genuinely improved. What is important, however, is for this research to be done in situ so that it is directly applicable to the real world of farming, the sea or an animal’s wild habitat. It is here that ethology can make major contributions.     

Comparison of methods for the demarcation between earlywood and latewood in tree rings of Norway spruce

The precise demarcation between earlywood and latewood is important for the detailed analysis of intra-annual tree ring features. Different techniques based on visual assessment, wood anatomy analysis and X-ray densitometry have been developed and are currently used for this purpose. Depending on the chosen method, tree species and environmental conditions, the results can significantly vary. Thus, it is important to determine the technique optimal for a particular research. Here, we investigated Norway spruce (Picea abies) tree rings to examine the agreement among the following demarcation methods: (1) direct visual assessment, (2) Mork’s index (anatomical definition of the transition from earlywood to latewood based on cell wall-lumen ratio) and (3) fixed and floating density thresholds applied to intra-ring density profiles. The aim was to modify both the Mork’s criterion and density thresholds on the basis of reference values given by visual identification of earlywood/latewood transition. A total of 231 tree rings were analysed by all methods. Our results showed that the usage of floating threshold (defined for each ring separately based on density profiles) is more reliable in comparison with fixed threshold (the same threshold value used for all tree rings and samples). Statistical analysis revealed the best correspondence between visual identification of earlywood/latewood transition and demarcation based on the standard Mork’s index and the floating density threshold derived as 80 % of maximum latewood density. In terms of Mork’s index calibration, the results showed that to determine latewood cells in Norway spruce trees growing in temperate conditions, it is sufficient to use an index value equal to 0.83. The results are applicable for the studied spruce population growing in a temperate climate. The methodology itself, however, is universal and can help to calibrate criteria for earlywood-latewood demarcation under specific conditions.     

Lipid Extraction Methods for Lipomyces starkeyi

Various lipid extraction methods were applied to Lipomyces starkeyi, freeze-dried, heat-dried and intact cells. It was found that the freeze-dried cells were usually more extractable than other types of cells. A high lipid recovery was obtained by a lytic enzyme (Corticium centrifugum) treatment, conc. HCI treatment and Pedersen’s method using chloroform-methanol (1: 1). The first two methods, however, hydrolyzed phospholipids and released free fatty acids during the extraction of lipids. From the results we have obtained, the best method for lipid extraction from L. starkeyi is that in which the freeze-dried cells are extracted by the Pedersen’s method. The results obtained from the application of these methods to a hydrocarbon-grown yeast are also described.     

Pitfalls in the statistical analysis of microbiome amplicon sequencing data

Microbiome data are characterized by several aspects that make them challenging to analyse statistically: they are compositional, high dimensional and rich in zeros. A large array of statistical methods exist to analyse these data. Some are borrowed from other fields, such as ecology or RNA-sequencing, while others are custom-made for microbiome data. The large range of available methods, and which is continuously expanding, means that researchers have to invest considerable effort in choosing what method(s) to apply. In this paper we list 14 statistical methods or approaches that we think should be generally avoided. In several cases this is because we believe the assumptions behind the method are unlikely to be met for microbiome data. In other cases we see methods that are used in ways they are not intended to be used. We believe researchers would be helped by more critical evaluations of existing methods, as not all methods in use are suitable or have been sufficiently reviewed. We hope this paper contributes to a critical discussion on what methods are appropriate to use in the analysis of microbiome data.     

Mixed integer linear programming for maximum-parsimony phylogeny inference

Reconstruction of phylogenetic trees is a fundamental problem in computational biology. While excellent heuristic methods are available for many variants of this problem, new advances in phylogeny inference will be required if we are to be able to continue to make effective use of the rapidly growing stores of variation data now being gathered. In this paper, we present two integer linear programming (ILP) formulations to find the most parsimonious phylogenetic tree from a set of binary variation data. One method uses a flow-based formulation that can produce exponential numbers of variables and constraints in the worst case. The method has, however, proven extremely efficient in practice on datasets that are well beyond the reach of the available provably efficient methods, solving several large mtDNA and Y-chromosome instances within a few seconds and giving provably optimal results in times competitive with fast heuristics than cannot guarantee optimality. An alternative formulation establishes that the problem can be solved with a polynomial-sized ILP. We further present a web server developed based on the exponential-sized ILP that performs fast maximum parsimony inferences and serves as a front end to a database of precomputed phylogenies spanning the human genome.     

Sample size calculation for multiple testing in microarray data analysis

Microarray technology is rapidly emerging for genome-wide screening of differentially expressed genes between clinical subtypes or different conditions of human diseases. Traditional statistical testing approaches, such as the two-sample t-test or Wilcoxon test, are frequently used for evaluating statistical significance of informative expressions but require adjustment for large-scale multiplicity. Due to its simplicity, Bonferroni adjustment has been widely used to circumvent this problem. It is well known, however, that the standard Bonferroni test is often very conservative. In the present paper, we compare three multiple testing procedures in the microarray context: the original Bonferroni method, a Bonferroni-type improved single-step method and a step-down method. The latter two methods are based on nonparametric resampling, by which the null distribution can be derived with the dependency structure among gene expressions preserved and the family-wise error rate accurately controlled at the desired level. We also present a sample size calculation method for designing microarray studies. Through simulations and data analyses, we find that the proposed methods for testing and sample size calculation are computationally fast and control error and power precisely.     

Large multiple organism gene finding by collapsed Gibbs sampling.

The Gibbs sampling method has been widely used for sequence analysis after it was successfully applied to the problem of identifying regulatory motif sequences upstream of genes. Since then, numerous variants of the original idea have emerged: however, in all cases the application has been to finding short motifs in collections of short sequences (typically less than 100 nucleotides long). In this paper, we introduce a Gibbs sampling approach for identifying genes in multiple large genomic sequences up to hundreds of kilobases long. This approach leverages the evolutionary relationships between the sequences to improve the gene predictions, without explicitly aligning the sequences. We have applied our method to the analysis of genomic sequence from 14 genomic regions, totaling roughly 1.8 Mb of sequence in each organism. We show that our approach compares favorably with existing ab initio approaches to gene finding, including pairwise comparison based gene prediction methods which make explicit use of alignments. Furthermore, excellent performance can be obtained with as little as four organisms, and the method overcomes a number of difficulties of previous comparison based gene finding approaches: it is robust with respect to genomic rearrangements, can work with draft sequence, and is fast (linear in the number and length of the sequences). It can also be seamlessly integrated with Gibbs sampling motif detection methods.     

Possibility of X-ray spectral diagnostics of a plasma in the tracks of fast multicharged ions

A plasma model of the relaxation of a medium within the tracks of heavy ions in condensed matter is proposed that is based on solving time-dependent radiative collisional kinetic equations with the initial condition corresponding to a medium’s state described by the classical model of multiple ionization of the target atoms by the field of fast multicharged ions. It is shown that the plasma model allows one to describe X-ray spectra recorded in the interaction of ion beams with condensed targets. An X-ray spectral method for plasma diagnostics is proposed that is based on the plasma model. The results obtained can also be used to study the initial stage of the formation of defects in solid bodies under the action of individual fast heavy ions.     

Comparative morphology of shark pectoral fins

Sharks vary greatly in morphology, physiology, and ecology. Differences in whole body shape, swimming style, and physiological parameters have previously been linked to varied habitat uses. Pectoral fin morphology has been used to taxonomically classify species and hypotheses on the functional differences in shape are noted throughout the literature; however, there are limited comparative datasets that quantify external and skeletal morphology. Further, fins were previously categorized into two discrete groups based on the amount of skeletal support present: (a) aplesodic, where less than half of the fin is supported and (b) plesodic where greater than half of the fin is supported. These discrete classifications have been used to phylogenetically place species, though the methodology of classification is infrequently described. In this study, we sampled fins from 18 species, 6 families, and 3 orders, which were also grouped into five ecomorphotype classifications. We examined the external morphology, extent of skeletal support, and cross-sectional shape of individual cartilaginous elements. Using phylogenetic comparative methods, we show that fin shape does not differ significantly between ecomorphotypes, suggesting there may be some mechanical constraint. However, we find that the internal anatomy of the fin does vary significantly between ecomorphotypes, especially the extent and distribution of calcification of skeletal support, suggesting that the superficial similarity of fin shapes across ecomorphotypes may belie differences in function. Finally, we find that a number of morphological variables such as number of radials, radial calcification and shape, and fin taper all correlate with the extent of skeletal support. Within these morphospaces, we also describe that some orders/families tend to occupy certain areas with limited overlap. While we demonstrate that there is some mechanical constraint limiting external variations in shark pectoral fin morphology, there are compounding differences in skeletal anatomy that occur within ecomorphotypes which we propose may affect function.     

Identification of related proteins on family, superfamily and fold level

Proteins might have considerable structural similarities even when no evolutionary relationship of their sequences can be detected. This property is often referred to as the proteins sharing only a "fold". Of course, there are also sequences of common origin in each fold, called a "superfamily", and in them groups of sequences with clear similarities, designated "family". Developing algorithms to reliably identify proteins related at any level is one of the most important challenges in the fast growing field of bioinformatics today. However, it is not at all certain that a method proficient at finding sequence similarities performs well at the other levels, or vice versa.Here, we have compared the performance of various search methods on these different levels of similarity. As expected, we show that it becomes much harder to detect proteins as their sequences diverge. For family related sequences the best method gets 75% of the top hits correct. When the sequences differ but the proteins belong to the same superfamily this drops to 29%, and in the case of proteins with only fold similarity it is as low as 15%. We have made a more complete analysis of the performance of different algorithms than earlier studies, also including threading methods in the comparison. Using this method a more detailed picture emerges, showing multiple sequence information to improve detection on the two closer levels of relationship. We have also compared the different methods of including this information in prediction algorithms.For lower specificities, the best scheme to use is a linking method connecting proteins through an intermediate hit. For higher specificities, better performance is obtained by PSI-BLAST and some procedures using hidden Markov models. We also show that a threading method, THREADER, performs significantly better than any other method at fold recognition.     

Precisely incorrect? Monetising the value of ecosystem services

Environmental scientists employ political and economic arguments to argue for the conservation of biodiversity and the maintenance of ecosystem services. However, the economic terminology has a number of connotations which makes its usefulness for the intended effect questionable.On the one hand, the basic assumptions underlying economic valuation are far from realistic and represent rather a caricature of human behaviour. On the other hand, the methods based on these assumptions are manifold and lead to wildly diverging results. Thus the calculated value of ecosystems and their services is not a robust figure, but varies with the valuation method applied (plus a plethora of subjective assumptions). As a result, it is not possible to ‘objectively’ calculate the value of ecosystem services. Fortunately, it is also not necessary to do so. Given the inherent flaws of the valuation process, it seems more promising for biodiversity and its conservation to restrict the economic calculus to the role of a contribution in the implementation process for a set of politically defined targets, rather than using it as the target setting mechanism itself.The paper lists some of the core assumptions, presents a systematic overview of the most relevant valuation methods, illustrates them by providing examples and discusses their limitations. As an alternative, political target setting is suggested, based on a multi-stakeholder, multi-criteria analysis. Market prices play a role in this analysis, as one factor amongst others. For the implementation, cost-effectiveness analysis gives important hints, and economic instruments – inter alia – can play an important role as enforcement mechanisms. However, incentives should be based on criteria of (potential) effectiveness, not on value calculations.     

Cryopreservation of articular cartilage. Part 3: The liquidus-tracking method

Although it is relatively straightforward to cryopreserve living isolated chondrocytes, at the present time there is no satisfactory method to preserve surgical grafts between the time of procurement or manufacture and actual use. In earlier papers we have established that the cryoprotectants dimethyl sulphoxide or propylene glycol do penetrate into this tissue very rapidly. Chondrocytes are not unusually susceptible to osmotic stress; in fact they appear to be particularly resistant. It appears that damage is associated with the formation of ice per se, even at cooling rates that are optimal for the cryopreservation of isolated chondrocytes. We then showed that current methods of cartilage cryopreservation involve the nucleation and growth of ice crystals within the chondrons rather than ice being restricted to the surrounding acellular matrix. This finding established the need to avoid the crystallization of ice—in other words, vitrification. Song and his colleagues have published a vitrification method that is based on the use of one of Fahy’s vitrification formulations. We confirmed the effectiveness of this method but found it to be very dependent on ultra rapid warming. However, we were able to develop a ‘liquidus-tracking’ method that completely avoids the crystallization of ice and does not require rapid warming. The ability of cartilage preserved in this way to incorporate sulphate into newly synthesized glycosaminoglycans (GAGs) approached 70% of that of fresh control cartilage. In this method the rates of cooling and warming can be very low, which is essential for any method that is to be used in Tissue Banks to process the bulky grafts that are required by orthopaedic surgeons. Work is continuing to refine this method for Tissue Bank use.     

SIMULATION‐BASED LIKELIHOOD APPROACH FOR EVOLUTIONARY MODELS OF PHENOTYPIC TRAITS ON PHYLOGENY

Phylogenetic comparative methods (PCMs) have been used to test evolutionary hypotheses at phenotypic levels. The evolutionary modes commonly included in PCMs are Brownian motion (genetic drift) and the Ornstein–Uhlenbeck process (stabilizing selection), whose likelihood functions are mathematically tractable. More complicated models of evolutionary modes, such as branch‐specific directional selection, have not been used because calculations of likelihood and parameter estimates in the maximum‐likelihood framework are not straightforward. To solve this problem, we introduced a population genetics framework into a PCM, and here, we present a flexible and comprehensive framework for estimating evolutionary parameters through simulation‐based likelihood computations. The method does not require analytic likelihood computations, and evolutionary models can be used as long as simulation is possible. Our approach has many advantages: it incorporates different evolutionary modes for phenotypes into phylogeny, it takes intraspecific variation into account, it evaluates full likelihood instead of using summary statistics, and it can be used to estimate ancestral traits. We present a successful application of the method to the evolution of brain size in primates. Our method can be easily implemented in more computationally effective frameworks such as approximate Bayesian computation (ABC), which will enhance the use of computationally intensive methods in the study of phenotypic evolution.