Ready-to-use DNA extracted with a CTAB method adapted for herbarium specimens and mucilaginous plant tissue

This report summarizes major changes in previously published protocols for DNA extraction to improve the quality of DNA extracted from plants. Here, we highlight the critical modifications in the original protocols. The efficiency of these changes results in high-quality DNA ready to use in a variety of phytogenetically distant plant families, in particular species with mucopolysaccharides. The DNA obtained can be used without further purification in various molecular biology assays, including direct sequencing and AFLP and RAPD (random-amplified polymorphic DNA) analyses. The effectiveness of this method is proven by the amplification and sequencing of PCR products of up to 1 kb with DNA extracted from herbarium tissue ≥60 years old. This versatility is not usually found in DNA extraction protocols. In addition, this method is quick, adaptable to standard laboratories, and most important, safer and more cost-effective.     

From information to understanding: the role of model organism databases in comparative and functional genomics

Data integration is key to functional and comparative genomics because integration allows diverse data types to be evaluated in new contexts. To achieve data integration in a scalable and sensible way, semantic standards are needed, both for naming things (standardized nomenclatures, use of key words) and also for knowledge representation. The Mouse Genome Informatics database and other model organism databases help to close the gap between information and understanding of biological processes because these resources enforce well-defined nomenclature and knowledge representation standards. Model organism databases have a critical role to play in ensuring that diverse kinds of data, especially genome-scale data sets and information, remain useful to the biological community in the long-term. The efforts of model organism database groups ensure not only that organism-specific data are integrated, curated and accessible but also that the information is structured in such a way that comparison of biological knowledge across model organisms is facilitated.     

Achieving Target 2 of the Global Strategy for Plant Conservation: building a preliminary assessment of vascular plant species using data from herbarium specimens

The Global Strategy for Plant Conservation calls for a preliminary assessment of the conservation status of all known plant species by the year 2010. To date insufficient progress has been made on meeting this target. New efforts to develop a preliminary list beyond using the full IUCN criteria in plant assessments are needed. Here we present an algorithm that provides a preliminary assessment of the conservation status of plant species using data from herbarium specimens. We use Hawaiian specimen data from the United States National Herbarium to calibrate the parameters of the algorithm and then use specimen data from the Arecaceae, Commelinaceae, Gesneriaceae and Heliconiaceae as examples of the application of the algorithm. The algorithm was calibrated to insure 95% accuracy in placing the Hawaiian plant species into previously and independently determined threatened categories. Our results indicate that 28% of the Hawaiian taxa, 27% of the species of Arecaceae, 45% of the species of Commelinaceae, 32% of the species of Gesneriaceae, and 35% of the species of Heliconiaceae are Not Threatened and will not need any further evaluation for the preliminary assessment. Species identified here as Potentially Extinct and Potentially Threatened can be further assessed by additional herbarium material and/or conservation specialists for final evaluation using other assessment strategies (e.g., regional and national lists, taxonomic expert assessment, etc.).     

A new approach for a modular valuation of LCAs

Goal Scope and Background

Qualitative valuation methods carefully try to avoid an aggregation across impact categories. However, such an aggregation often helps in obtaining a clear result for the valuation (which product scores better?). This article presents a new valuation method that uses an iterative approach. The application is demonstrated by the help of a case study for electric motors in trains.

Methods / Main Features

The approach combines two existing, unique valuation methods described earlier in literature, which both are of a rather non-aggregating nature, in line with ISO requirements, and were designed to be performed by LCA experts. The method is implemented in a computer software. Besides constants used within the method, the software needs as input solely indicator values from the Impact Assessment.

Results and Discussion

The iterative nature of these methods itself, and especially the combination of these methods, helps in achieving a valuation result for the LCA with not more subjective and aggregating elements than necessary. Subjective elements are clearly separated from others. The algorithm seems highly sensitive to changes in impact categories regarded as important ones. The implementation in software greatly eases the application of the method by transferring routine work from LCA experts to a machine. It ensures a reproducible result and prevents erroneous steps in a rather complicated valuation procedure. It further helps in hiding the complexity of the method from the user.

Conclusion

The approach of combining valuation methods in LCAs seems a fruitful one, and shows benefits when implemented in computer software, in terms of usability, and in terms of a more reproducible application. Care has to be taken to make sure users know what they do when performing an automated valuation procedure.

Outlook

We see three ways for extending the approach, namely: (i) become part of a toolbox of different valuation procedures; (ii) explicitly cope with uncertainty, and (iii) include different values for normalisation, in different regions worldwide. The software will be made available also in a stand alone version.

     

Historical ozone concentrations and flavonoid levels in herbarium specimens of the Antarctic moss Bryum argenteum

Depletion of stratospheric ozone since the mid 1970s has led to significant increases in ultraviolet B (UVB) irradiation over Antarctica. The detrimental effects of UVB on plants are many, but plants produce photoprotective flavonoids that reduce cellular damage. We used herbarium samples of the moss Bryum argenteum collected in Antarctica to compare the levels of flavone aglycones in plants collected before and after the formation of the ozone hole. The interpretation of historical data is difficult, because environmental conditions immediately before sample collection are unknown. Factors such as cloud cover can have a significant influence on UVB dose at ground level, modifying the flavonoid content of the specimen and adding considerable variability to the results. Nevertheless, our results revealed significant relationships between total flavone concentration and pooled year classes ( P = 0.001). Furthermore, regression analysis showed a significant negative relationship of total flavone concentration and the level of ozone immediately before the time of collection ( P = 0.016). In addition, the ratio of luteolin (an ortho -dihydroxylated flavone) to apigenin (a monohydroxylated flavone) increased significantly with several environmental parameters. These included (a) increasing modelled midday UVB radiation ( P = 0.002), (b) increasing modelled midday UVB/PAR ratio ( P < 0.001), and (c) decreasing ozone concentration ( P < 0.001). We emphasise the utility of this ratio in interpreting the historical ozone trends rather than relying on changes in total flavone concentrations alone. These results illustrate that herbarium specimens may reveal historical levels of UVB radiation.     

Changes in plant collection practices from the 16th to 21st centuries: implications for the use of herbarium specimens in global change research

Background and AimsHerbaria were recently advertised as reliable sources of information regarding historical changes in plant traits and biotic interactions. To justify the use of herbaria in global change research, we asked whether the characteristics of herbarium specimens have changed during the past centuries and whether these changes were due to shifts in plant collection practices.MethodsWe measured nine characteristics from 515 herbarium specimens of common European trees and large shrubs collected from 1558 to 2016. We asked botanists to rank these specimens by their scientific quality, and asked artists to rank these specimens by their beauty.Key ResultsEight of 11 assessed characteristics of herbarium specimens changed significantly during the study period. The average number of leaves in plant specimens increased 3-fold, whereas the quality of specimen preparation decreased. Leaf size negatively correlated with leaf number in specimens in both among-species and within-species analyses. The proportion of herbarium sheets containing plant reproductive structures peaked in the 1850s. The scientific value of herbarium specimens increased until the 1700s, but then did not change, whereas their aesthetic value showed no systematic trends.ConclusionsOur findings strongly support the hypothesis that many characteristics of herbarium specimens have changed systematically and substantially from the 16th to 21st centuries due to changes in plant collection and preservation practices. These changes may both create patterns which could be erroneously attributed to environmental changes and obscure historical trends in plant traits. The utmost care ought to be taken to guard against the possibility of misinterpretation of data obtained from herbarium specimens. We recommend that directional changes in characters of herbarium specimens which occurred during the past 150‒200 years, primarily in specimen size and in the presence of reproductive structures, are accounted for when searching for the effects of past environmental changes on plant traits.     

An integrated approach to optimization of Escherichia coli fermentations using historical data

Using a fermentation database for Escherichia coli producing green fluorescent protein (GFP), we have implemented a novel three-step optimization method to identify the process input variables most important in modeling the fermentation, as well as the values of those critical input variables that result in an increase in the desired output. In the first step of this algorithm, we use either decision-tree analysis (DTA) or information theoretic subset selection (ITSS) as a database mining technique to identify which process input variables best classify each of the process outputs (maximum cell concentration, maximum product concentration, and productivity) monitored in the experimental fermentations. The second step of the optimization method is to train an artificial neural network (ANN) model of the process input-output data, using the critical inputs identified in the first step. Finally, a hybrid genetic algorithm (hybrid GA), which includes both gradient and stochastic search methods, is used to identify the maximum output modeled by the ANN and the values of the input conditions that result in that maximum. The results of the database mining techniques are compared, both in terms of the inputs selected and the subsequent ANN performance. For the E. coli process used in this study, we identified 6 inputs from the original 13 that resulted in an ANN that best modeled the GFP fluorescence outputs of an independent test set. Values of the six inputs that resulted in a modeled maximum fluorescence were identified by applying a hybrid GA to the ANN model developed. When these conditions were tested in laboratory fermentors, an actual maximum fluorescence of 2.16E6 AU was obtained. The previous high value of fluorescence that was observed was 1.51E6 AU. Thus, this input condition set that was suggested by implementing the proposed optimization scheme on the available historical database increased the maximum fluorescence by 55%.     

An integrated approach for the analysis of biological pathways using mixed models

Gene class, ontology, or pathway testing analysis has become increasingly popular in microarray data analysis. Such approaches allow the integration of gene annotation databases, such as Gene Ontology and KEGG Pathway, to formally test for subtle but coordinated changes at a system level. Higher power in gene class testing is gained by combining weak signals from a number of individual genes in each pathway. We propose an alternative approach for gene-class testing based on mixed models, a class of statistical models that: a) provides the ability to model and borrow strength across genes that are both up and down in a pathway, b) operates within a well-established statistical framework amenable to direct control of false positive or false discovery rates, c) exhibits improved power over widely used methods under normal location-based alternative hypotheses, and d) handles complex experimental designs for which permutation resampling is difficult. We compare the properties of this mixed models approach with nonparametric method GSEA and parametric method PAGE using a simulation study, and illustrate its application with a diabetes data set and a dose-response data set.     

Harvesting Betulaceae sequences from GenBank to generate a new chronogram for the family

Betulaceae, with 120–150 species in six genera, are a family of Fagales that occurs mainly in the Northern Hemisphere. Previous studies of the evolution of Alnus, Betula, Carpinus, Corylus, Ostrya and Ostryopsis have relied on a relatively small number of sequence data and molecular clocks with fixed‐point calibrations. We exploited GenBank to construct Betulaceae matrices of up to 900 sequence accessions and 9300 nucleotides of nuclear and plastid DNA; we also computed species consensus sequences to build 46‐ and 29‐species matrices that strike a balance between species sampling and nucleotide sampling. Trees were rooted on Ticodendraceae and Casuarinaceae, and divergence times were inferred under relaxed and strict molecular clocks, using alternative fossil constraints. The data support the traditional two subfamilies, Betuloideae (Alnus, Betula) and Coryloideae, and show that Ostryopsis is sister to Ostrya/Carpinus. The fossil record and molecular clocks calibrated with alternating fossils indicate that the stem lineage of Betulaceae dates back to the Upper Cretaceous, the two subfamilies to the Palaeocene and the most recent common ancestors of each of the living genera to the mid‐ to late Miocene. A substitution rate shift in Coryloideae between 25 and 15 Mya preceded the mid‐Miocene climatic optimum and may be linked to temperate niches that became available following the mid‐Miocene. © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 172 , 465–477.     

Design,construction, and optimization of a novel,modular, and scalable incubation chamber for continuous viral inactivation

We designed, built or 3D printed, and screened tubular reactors that minimize axial dispersion to serve as incubation chambers for continuous virus inactivation of biological products. Empirical residence time distribution data were used to derive each tubular design's volume equivalent to a theoretical plate (VETP) values at a various process flow rates. One design, the Jig in a Box (JIB), yielded the lowest VETP, indicating optimal radial mixing and minimal axial dispersion. A minimum residence time (MRT) approach was employed, where the MRT is the minimum time the product spends in the tubular reactor. This incubation time is typically 60 minutes in a batch process. We provide recommendations for combinations of flow rates and device dimensions for operation of the JIB connected in series that will meet a 60‐min MRT. The results show that under a wide range of flow rates and corresponding volumes, it takes 75 ± 3 min for 99% of the product to exit the reactor while meeting the 60‐min MRT criterion and fulfilling the constraint of keeping a differential pressure drop under 5 psi. Under these conditions, the VETP increases slightly from 3 to 5 mL though the number of theoretical plates stays constant at about 1326 ± 88. We also demonstrated that the final design volume was only 6% ± 1% larger than the ideal plug flow volume. Using such a device would enable continuous viral inactivation in a truly continuous process or in the effluent of a batch chromatography column. Viral inactivation studies would be required to validate such a design. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:954–965, 2017     

Streamlining scenario analysis and optimization of key choices in value chains using a modular LCA approach

Purpose

The environmental performance of products or services is often a result of a number of key decisions that shape their life cycles (e.g., techology choices). This paper introduces a modular LCA approach that is capable of reducing the effort involved in performing scenario analyses and optimization when several key choices along a product’s value chain lead to many alternative life cycles.

Methods

The main idea is that the value chain of a product can be divided into interconnected but exchangeable modules, which together represent a full life cycle. A module is comprised of unit processes from the practitioner’s LCI database. The inputs, outputs, and system boundaries of each module can be tailored to the context of the studied system. Alternatives arise whenever multiple modules produce substitutable products. Unlike in conventional LCI databases, no copies are necessary to represent the same process with different inputs. A module-product matrix is used to store this information. It can be used as a basis for an automated scenario analysis of all alternatives or as an input to an optimization model.

Results and discussion

Our approach is illustrated in two case studies: (1) Passenger car fuel choices are modeled by 15 modules representing 33 alternative value chains for diesel, petrol, natural gas and electric cars. The automated comparison of LCA results indicates that electric mobility is often the preferable option from a climate perspective, but impacts depend strongly on the electricity source. (2) A dynamic optimization model including stocks is built from eight modules to analyze the optimal use of wood for material and energy applications. Results indicate that although direct substitution benefits are higher for energy applications, cascading use of wood can maximize environmental performance over the entire life cycle.

Conclusions

The modular LCA approach permits an efficient modeling and comparison of alternative product life cycles, enabling practitioners to focus on key decisions. It can be applied to exploit a potential that is hidden in LCI databases, which is that they contain many specific inventories but not all useful combinations in the context of scenario analyses. The user-defined level of abstraction that is introduced through modules can be helpful in the communication of LCA results. The modular approach also facilitates the integration of LCA and optimization as well as other industrial ecology methods. An open source software is provided to enable others to apply and further develop our implementation of a modular LCA approach.

     

Explaining invasiveness from the extent of native range: new insights from plant atlases and herbarium specimens

Aim

We tested the relationship between the extent of the native range and the success (number of occurrences) in the introduced range of European vascular plant species naturalized in the province of Québec (Canada). We hypothesized that the performance of models linking native range size and species invasiveness can be improved if residence time and climate tolerance are taken into account.

Methods

The extent of the native range (Europe, Asia) was estimated using plant atlases. The number of occurrences in the introduced range (Québec) was estimated using the number of herbarium specimens stored in herbaria. Herbarium specimens were also used to obtain residence time. Plant hardiness was used as an indicator of the suitability of a species to the climate of the introduced range. Multiple linear regression models, corrected to take into account phylogenetic biases, were used to calculate correlations between the extent of the native range and the number of occurrences in the introduced range.

Results

The larger the native distribution area in Eurasia, the greater the number of occurrences (herbarium specimens) in Québec. The shorter the residence time and the less hardy the plant, the fewer the number of occurrences. In all models tested, the phylogenetic structure explained a significant proportion of the variance, but its influence decreased as the number of species or area studied (Europe versus Eurasia) increased.

Main conclusions

The extent of the native range is a good explanatory variable for the invasion success of vascular plants, especially once other factors (residence time, climate tolerance, phylogeny) are taken into account. Thus, a model using these variables could be used by environmental managers to flag species warranting further investigation. With the emergence of online databases, gathering the required information is becoming easier and cheaper. As online databases continue to improve and new analytical tools are developed, this approach will become even more powerful.

     

A scalable and flexible approach for investigating the genomic landscapes of phylogenetic incongruence

Analyses of DNA sequence datasets have repeatedly revealed inconsistencies in phylogenetic trees derived with different data. This is termed phylogenetic incongruence, and may arise from a methodological failure of the inference process or from biological processes, such as horizontal gene transfer, incomplete lineage sorting, and introgression. To better understand patterns of incongruence, we developed a method (PartFinder) that uses likelihood ratios applied to sliding windows for visualizing tree-support changes across genome-sequence alignments, allowing the comparative examination of complex phylogenetic scenarios among many species. As a pilot, we used PartFinder to investigate incongruence in the Homo-Pan-Gorilla group as well as Platyrrhini using high-quality bacterial artificial chromosome (BAC)-derived sequences as well as assembled whole-genome shotgun sequences. Our simulations verified the sensitivity of PartFinder, and our results were comparable to other studies of the Homo-Pan-Gorilla group. Analyses of the whole-genome alignments reveal significant associations between support for the accepted species relationship and specific characteristics of the genomic regions, such as GC-content, alignment score, exon content, and conservation. Finally, we analyzed sequence data generated for five platyrrhine species, and found incongruence that suggests a polytomy within Cebidae, in particular. Together, these studies demonstrate the utility of PartFinder for investigating the patterns of phylogenetic incongruence.     

New approach for evaluating habitat stability using scarce records for both historical and contemporary specimens: a case study using Carabidae specimen records

Natural history collections, such as specimen records, are crucial resources for conservation and habitat management. However, these data are usually scarce compared to physical environmental data (e.g., digital terrain maps) that we often have little species data and a lot of physical environmental data with which to evaluate habitats. In this paper, we propose a method for evaluating habitat stability using scarce natural history records and abundant physical environmental data. We used both historical and contemporary specimen records of carabid beetles (areas in which records of the same species were recorded during both periods) and evaluated the attributes of these areas using terrain characteristics. We found two common terrain characteristics among the occupied areas: large total river length and low variation in elevation. These terrain characteristics suggest that habitats of carabid species have been conserved in disturbed and wet environments for a long time. These results are consistent with the ecological characteristics of carabid beetles. Our study shows that scarce natural history collections, combined with ingenuity, can be useful for evaluating habitats.     

Molecular markers in Viola L. subsect. Viola: Application and taxonomic implications for the identification of dubious herbarium specimens

Abstract

The authors report the use of nuclear [internal transcribed spacer (ITS) sequences 1 and 2] and chloroplast DNA (trnS-trnG intergenic spacer sequences) in Viola subsect. Viola for separate tracking of maternal lineages and detecting dubious herbarium specimens. A phylogenetic investigation carried out on ITS data after removal of all material with possible hybrid origin showed that V. hirta is a monophyletic unit, whereas V. odorata includes at least V. collina and V. jaubertiana, as well as three sequences of V. alba subsp. dehnhardtii from literature. In some, among the sampled individuals, the morphological attribution to one species is contradicted by nuclear DNA, which indicated a wide distance from other non-specific individuals from different locations and closer proximity to a different species. Chloroplast DNA data for the same individuals, on the contrary concurred with morphological evidence. These findings confirm document univocal correlation between our chosen chloroplast sequences and the studied taxa at species or subspecies level; these sequences have the appropriate variability range to be employed for detection of the maternal lineage of unknown Viola samples.     

An approach to integrated data assessment in a proficiency test on the enumeration of Escherichia coli

Aims: To develop appropriate statistical approaches to plan and evaluate proficiency tests for the enumeration of Escherichia coli, addressing, in particular, a possible but frequently unavoidable lack of test sample homogeneity. Methods and Results: Each of 50 laboratories analysed two samples of a stabilized suspension of E. coli in duplicate, using various media, inoculation methods, and incubation times and conditions. In parallel, the E. coli suspension was tested by the organiser for homogeneity and stability. Escherichia coli counts followed a log‐normal distribution. After eliminating, by Youden analysis, two data sets that were considered outliers and eight data sets for underperformance of the laboratories (substantial lack of repeatability), the standard deviation of the mean was about 0·06 log₁₀ units. There was no evidence of bimodality of the data. Lack of homogeneity of distribution of bacteria had a strong effect on measurement uncertainty, in addition to laboratory bias and method repeatability. The homogeneity decreases during storage of the individual test vials; this effect could be modelled by the known kinetics of inactivation of micro‐organisms. The results were confirmed by Monte Carlo simulations. Conclusions: By a tailored analysis of proficiency testing data, it is possible to distinguish the effect of lack of homogeneity, laboratory bias and method repeatability, on the measurement uncertainty. Significance and Impact of the Study: A statistic tool is provided to solve problems related to lack of stability of microbiological test material and to separate the effects of sample inhomogeneity from the performance of the individual laboratory.     

Taverna: a tool for the composition and enactment of bioinformatics workflows

MOTIVATION: In silico experiments in bioinformatics involve the co-ordinated use of computational tools and information repositories. A growing number of these resources are being made available with programmatic access in the form of Web services. Bioinformatics scientists will need to orchestrate these Web services in workflows as part of their analyses. RESULTS: The Taverna project has developed a tool for the composition and enactment of bioinformatics workflows for the life sciences community. The tool includes a workbench application which provides a graphical user interface for the composition of workflows. These workflows are written in a new language called the simple conceptual unified flow language (Scufl), where by each step within a workflow represents one atomic task. Two examples are used to illustrate the ease by which in silico experiments can be represented as Scufl workflows using the workbench application.     

Gene selection for tumor classification using a novel bio-inspired multi-objective approach

Identifying the informative genes has always been a major step in microarray data analysis. The complexity of various cancer datasets makes this issue still challenging. In this paper, a novel Bio-inspired Multi-objective algorithm is proposed for gene selection in microarray data classification specifically in the binary domain of feature selection. The presented method extends the traditional Bat Algorithm with refined formulations, effective multi-objective operators, and novel local search strategies employing social learning concepts in designing random walks. A hybrid model using the Fisher criterion is then applied to three widely-used microarray cancer datasets to explore significant biomarkers which reveal the effectiveness of the proposed method for genomic analysis. Experimental results unveil new combinations of informative biomarkers have association with other studies.     

Sensitive and specific detection of Trypanosoma cruzi DNA in clinical specimens using a multi-target real-time PCR approach

Background

The laboratory diagnosis of Chagas disease is challenging because the usefulness of different diagnostic tests will depend on the stage of the disease. Serology is the preferred method for patients in the chronic phase, whereas PCR can be successfully used to diagnose acute and congenital cases. Here we present data using a combination of three TaqMan PCR assays to detect T. cruzi DNA in clinical specimens.

Methods/Principal Findings

Included in the analysis were DNA extracted from 320 EDTA blood specimens, 18 heart tissue specimens, 6 umbilical cord blood specimens, 2 skin tissue specimens and 3 CSF specimens. For the blood specimens both whole blood and buffy coat fraction were analyzed. The specimens were from patients living in the USA, with suspected exposure to T. cruzi through organ transplantation, contact with triatomine bugs or laboratory accidents, and from immunosuppressed patients with suspected Chagas disease reactivation. Real-time PCR was successfully used to diagnose acute and Chagas disease reactivation in 20 patients, including one case of organ-transmitted infection and one congenital case. Analysis of buffy coat fractions of EDTA blood led to faster diagnosis in six of these patients compared to whole blood analysis. The three real-time PCR assays produced identical results for 94% of the specimens. The major reason for discrepant results was variable sensitivity among the assays, but two of the real-time PCR assays also produced four false positive results.

Conclusions/Significance

These data strongly indicate that at least two PCR assays with different performances should be combined to increase the accuracy. This evaluation also highlights the benefit of extracting DNA from the blood specimen''s buffy coat to increase the sensitivity of PCR analysis.