High-throughput sequencing for community analysis: the promise of DNA barcoding to uncover diversity, relatedness, abundances and interactions in spider communities

Large-scale studies on community ecology are highly desirable but often difficult to accomplish due to the considerable investment of time, labor and, money required to characterize richness, abundance, relatedness, and interactions. Nonetheless, such large-scale perspectives are necessary for understanding the composition, dynamics, and resilience of biological communities. Small invertebrates play a central role in ecosystems, occupying critical positions in the food web and performing a broad variety of ecological functions. However, it has been particularly difficult to adequately characterize communities of these animals because of their exceptionally high diversity and abundance. Spiders in particular fulfill key roles as both predator and prey in terrestrial food webs and are hence an important focus of ecological studies. In recent years, large-scale community analyses have benefitted tremendously from advances in DNA barcoding technology. High-throughput sequencing (HTS), particularly DNA metabarcoding, enables community-wide analyses of diversity and interactions at unprecedented scales and at a fraction of the cost that was previously possible. Here, we review the current state of the application of these technologies to the analysis of spider communities. We discuss amplicon-based DNA barcoding and metabarcoding for the analysis of community diversity and molecular gut content analysis for assessing predator-prey relationships. We also highlight applications of the third generation sequencing technology for long read and portable DNA barcoding. We then address the development of theoretical frameworks for community-level studies, and finally highlight critical gaps and future directions for DNA analysis of spider communities

Novel Approach to Mass Tort Class Actions: The Billion Dollar Settlement in the Sulzer Artificial Hip and Knee Litigation: A Symposium

This is a transcript of a two hour symposium which deals with the Sulzer knee and hip replacement class action. A copy of the settlement is included as an appendix. The settlement in the U.S. District Court for the N.D. Ohio was unique and creative approach to resolving a mass tort class action. In a novel move, Sulzer agreed to open its books to an independent review firm to determine how much the firm could pay without going bankrupt. The number was $1 billion. As negotiated by the parties and approved by the court, the final settlement provides compensation for each member of the class based on a variety of factors, such as whether the member has undergone - or is likely to undergo - a revision to replace the defective part. Professor Susan Becker made the introductory remarks. The panel members were all involved in the Sulzer knee and hip replacement class action. R. Eric Kennedy served as lead plaintiffs\u27 counsel. The Honorable Kathleen McDonald O\u27Malley of the U.S. District Court for the Northern District of Ohio presided over the Sulzer class action litigation and settlement. Sidney A. Backstrom and Richard F. Scruggs were the defense counsel. James J. McMonagle served as the Claims Administrator overseeing distribution of the Sulzer class action settlement funds

Novel Approach to Mass Tort Class Actions: The Billion Dollar Settlement in the Sulzer Artificial Hip and Knee Litigation: A Symposium

This is a transcript of a two hour symposium which deals with the Sulzer knee and hip replacement class action. A copy of the settlement is included as an appendix. The settlement in the U.S. District Court for the N.D. Ohio was unique and creative approach to resolving a mass tort class action. In a novel move, Sulzer agreed to open its books to an independent review firm to determine how much the firm could pay without going bankrupt. The number was $1 billion. As negotiated by the parties and approved by the court, the final settlement provides compensation for each member of the class based on a variety of factors, such as whether the member has undergone - or is likely to undergo - a revision to replace the defective part. Professor Susan Becker made the introductory remarks. The panel members were all involved in the Sulzer knee and hip replacement class action. R. Eric Kennedy served as lead plaintiffs\u27 counsel. The Honorable Kathleen McDonald O\u27Malley of the U.S. District Court for the Northern District of Ohio presided over the Sulzer class action litigation and settlement. Sidney A. Backstrom and Richard F. Scruggs were the defense counsel. James J. McMonagle served as the Claims Administrator overseeing distribution of the Sulzer class action settlement funds

Standardization of electroencephalography for multi-site, multi-platform and multi-investigator studies: Insights from the canadian biomarker integration network in depression

Subsequent to global initiatives in mapping the human brain and investigations of neurobiological markers for brain disorders, the number of multi-site studies involving the collection and sharing of large volumes of brain data, including electroencephalography (EEG), has been increasing. Among the complexities of conducting multi-site studies and increasing the shelf life of biological data beyond the original study are timely standardization and documentation of relevant study parameters. We presentthe insights gained and guidelines established within the EEG working group of the Canadian Biomarker Integration Network in Depression (CAN-BIND). CAN-BIND is a multi-site, multi-investigator, and multiproject network supported by the Ontario Brain Institute with access to Brain-CODE, an informatics platform that hosts a multitude of biological data across a growing list of brain pathologies. We describe our approaches and insights on documenting and standardizing parameters across the study design, data collection, monitoring, analysis, integration, knowledge-translation, and data archiving phases of CAN-BIND projects. We introduce a custom-built EEG toolbox to track data preprocessing with open-access for the scientific community. We also evaluate the impact of variation in equipment setup on the accuracy of acquired data. Collectively, this work is intended to inspire establishing comprehensive and standardized guidelines for multi-site studies

Tissue-Specific Education of Decidual NK Cells.

During human pregnancy, fetal trophoblast cells invade the decidua and remodel maternal spiral arteries to establish adequate nutrition during gestation. Tissue NK cells in the decidua (dNK) express inhibitory NK receptors (iNKR) that recognize allogeneic HLA-C molecules on trophoblast. Where this results in excessive dNK inhibition, the risk of pre-eclampsia or growth restriction is increased. However, the role of maternal, self-HLA-C in regulating dNK responsiveness is unknown. We investigated how the expression and function of five iNKR in dNK is influenced by maternal HLA-C. In dNK isolated from women who have HLA-C alleles that carry a C2 epitope, there is decreased expression frequency of the cognate receptor, KIR2DL1. In contrast, women with HLA-C alleles bearing a C1 epitope have increased frequency of the corresponding receptor, KIR2DL3. Maternal HLA-C had no significant effect on KIR2DL1 or KIR2DL3 in peripheral blood NK cells (pbNK). This resulted in a very different KIR repertoire for dNK capable of binding C1 or C2 epitopes compared with pbNK. We also show that, although maternal KIR2DL1 binding to C2 epitope educates dNK cells to acquire functional competence, the effects of other iNKR on dNK responsiveness are quite different from those in pbNK. This provides a basis for understanding how dNK responses to allogeneic trophoblast affect the outcome of pregnancy. Our findings suggest that the mechanisms that determine the repertoire of iNKR and the effect of self-MHC on NK education may differ in tissue NK cells compared with pbNK.This work was supported by Wellcome Trust Grants 090108/Z/09/Z and 085992/Z/08/Z, as well as by British Heart Foundation Grant PG/09/077/27964. P.R.K. was the recipient of a Wellcome Trust Ph.D. studentship.This is the final version of the article. It first appeared from the American Association of Immunologists via http://dx.doi.org/10.4049/​jimmunol.150122

Cationic Peptide Exposure Enhances Pulsed-Electric-Field-Mediated Membrane Disruption

Background: The use of pulsed electric fields (PEFs) to irreversibly electroporate cells is a promising approach for destroying undesirable cells. This approach may gain enhanced applicability if the intensity of the PEF required to electrically disrupt cell membranes can be reduced via exposure to a molecular deliverable. This will be particularly impactful if that reduced PEF minimally influences cells that are not exposed to the deliverable. We hypothesized that the introduction of charged molecules to the cell surfaces would create regions of enhanced transmembrane electric potential in the vicinity of each charged molecule, thereby lowering the PEF intensity required to disrupt the plasma membranes. This study will therefore examine if exposure to cationic peptides can enhance a PEF’s ability to disrupt plasma membranes. Methodology/Principal Findings We exposed leukemia cells to 40 μs PEFs in media containing varying concentrations of a cationic peptide, polyarginine. We observed the internalization of a membrane integrity indicator, propidium iodide (PI), in real time. Based on an individual cell’s PI fluorescence versus time signature, we were able to determine the relative degree of membrane disruption. When using 1–2 kV/cm, exposure to >50 μg/ml of polyarginine resulted in immediate and high levels of PI uptake, indicating severe membrane disruption, whereas in the absence of peptide, cells predominantly exhibited signatures indicative of no membrane disruption. Additionally, PI entered cells through the anode-facing membrane when exposed to cationic peptide, which was theoretically expected. Conclusions/Significance: Exposure to cationic peptides reduced the PEF intensity required to induce rapid and irreversible membrane disruption. Critically, peptide exposure reduced the PEF intensities required to elicit irreversible membrane disruption at normally sub-electroporation intensities. We believe that these cationic peptides, when coupled with current advancements in cell targeting techniques will be useful tools in applications where targeted destruction of unwanted cell populations is desired

Rapid and cost-effective generation of single specimen multilocus barcoding data from whole arthropod communities by multiple levels of multiplexing

In light of the current biodiversity crisis, molecular barcoding has developed into an irreplaceable tool. Barcoding has been considerably simplified by developments in high throughput sequencing technology, but still can be prohibitively expensive and laborious when community samples of thousands of specimens need to be processed. Here, we outline an Illumina amplicon sequencing approach to generate multilocus data from large collections of arthropods. We reduce cost and effort up to 50-fold, by combining multiplex PCRs and DNA extractions from pools of presorted and morphotyped specimens and using two levels of sample indexing. We test our protocol by generating a comprehensive, community wide dataset of barcode sequences for several thousand Hawaiian arthropods from 14 orders, which were collected across the archipelago using various trapping methods. We explore patterns of diversity across the Archipelago and compare the utility of different arthropod trapping methods for biodiversity explorations on Hawaii, highlighting undergrowth beating as highly efficient method. Moreover, we show the effects of barcode marker, taxonomy and relative biomass of the targeted specimens and sequencing coverage on taxon recovery. Our protocol enables rapid and inexpensive explorations of diversity patterns and the generation of multilocus barcode reference libraries across whole ecosystems

A prospective evaluation of the predictive value of faecal calprotectin in quiescent Crohn’s disease

Background: The faecal calprotectin (FC) test is a non-invasive marker for gastrointestinal inflammation. Aim: To determine whether higher FC levels in individuals with quiescent Crohn’s disease are associated with clinical relapse over the ensuing 12 months.<p></p> Methods: A single centre prospective study was undertaken in Crohn's disease patients in clinical remission attending for routine review. The receiver operating characteristic (ROC) curve for the primary endpoint of clinical relapse by 12 months, based on FC at baseline, was calculated. Kaplan-Meier curves of time to relapse were based on the resulting optimal FC cutoff for predicting relapse.<p></p> Results: Of 97 patients recruited, 92 were either followed up for 12 months without relapsing, or reached the primary endpoint within that period. Of these, 10 (11%) had relapsed by 12 months. The median FC was lower for non-relapsers, 96µg/g (IQR 39-237), than for relapsers, 414µg/g (IQR 259-590), (p=0.005). The area under the ROC curve to predict relapse using FC was 77.4%. An optimal cutoff FC value of 240µg/g to predict relapse of quiescent Crohn’s had sensitivity of 80.0% and specificity of 74.4%. Negative predictive value was 96.8% and positive predictive value was 27.6%. FC≥240μg/g was associated with likelihood of relapse 5.7 (95% CI 1.9-17.3) times higher within 2.3 years than lower values (p=0.002).<p></p> Conclusions: In this prospective dataset, FC appears to be a useful, non-invasive tool to help identify quiescent Crohn’s disease patients at a low risk of relapse over the ensuing 12 months. FC of 240µg/g was the optimal cutoff in this cohort.<p></p&gt

Alternative Mechanisms for Tn5 Transposition

Bacterial transposons are known to move to new genomic sites using either a replicative or a conservative mechanism. The behavior of transposon Tn5 is anomalous. In vitro studies indicate that it uses a conservative mechanism while in vivo results point to a replicative mechanism. To explain this anomaly, a model is presented in which the two mechanisms are not independent—as widely believed—but could represent alternate outcomes of a common transpositional pathway