A high‐throughput method to quantify feeding rates in aquatic organisms: A case study with Daphnia

1. Food ingestion is one of the most basic features of all organisms. However, obtaining precise—and high‐throughput—estimates of feeding rates remains challenging, particularly for small, aquatic herbivores such as zooplankton, snails, and tadpoles. These animals typically consume low volumes of food that are time‐consuming to accurately measure.
2. We extend a standard high‐throughput fluorometry technique, which uses a microplate reader and 96‐well plates, as a practical tool for studies in ecology, evolution, and disease biology. We outline technical and methodological details to optimize quantification of individual feeding rates, improve accuracy, and minimize sampling error.
3. This high‐throughput assay offers several advantages over previous methods, including i) substantially reduced time allotments per sample to facilitate larger, more efficient experiments; ii) technical replicates; and iii) conversion of in vivo measurements to units (mL^‐1 hr^‐1 ind^‐1) which enables broad‐scale comparisons across an array of taxa and studies.
4. To evaluate the accuracy and feasibility of our approach, we use the zooplankton, Daphnia dentifera, as a case study. Our results indicate that this procedure accurately quantifies feeding rates and highlights differences among seven genotypes.
5. The method detailed here has broad applicability to a diverse array of aquatic taxa, their resources, environmental contaminants (e.g., plastics), and infectious agents. We discuss simple extensions to quantify epidemiologically relevant traits, such as pathogen exposure and transmission rates, for infectious agents with oral or trophic transmission.

     

Evaluation of urinary trans-3′-hydroxycotinine as a biomarker of children's environmental tobacco smoke exposure

Abstract

The utility of urinary trans-3′-hydroxy cotinine (3HC) as a biomarker of environmental tobacco smoke (ETS) exposure was investigated in comparison with urinary cotinine (COT), the sum (3HC?+?COT), and ratio of the two nicotine metabolites (3HC/COT). Participants were 150 ETS exposed children (aged 1–44 months) and their parents. Child urine samples were collected during 3weekly baseline assessments and at interviews administered 3, 6, 12, and 18 months after baseline. Findings indicate that 3HC and COT can be measured reliably (rho?=?0.96, 0.88) and show equivalent levels of repeated measures stability (rho?=?0.71, 0.75). COT, 3HC, and 3HC?+?COT showed equally strong associations with air nicotine levels, reported ETS contamination, and reported ETS exposure (r=0.60–0.70). The intraclass correlations of 3HC/COT were lower than those for COT or 3HC. Older children had a higher 3HC/COT ratio than younger children (3.5 versus 2.2), and non-Hispanic White children had a higher ratio than African-American children (3.2 versus 1.9). These findings suggest that COT, 3HC, and 3HC?+?COT are approximately equivalent and equally strong biomarkers of ETS exposure in children. Moreover, 3HC/COT may provide a useful indicator to investigate age- and race-related differences in the metabolism of COT and 3HC.     

Comparative analysis of tandem repeats from hundreds of species reveals unique insights into centromere evolution

Background

Centromeres are essential for chromosome segregation, yet their DNA sequences evolve rapidly. In most animals and plants that have been studied, centromeres contain megabase-scale arrays of tandem repeats. Despite their importance, very little is known about the degree to which centromere tandem repeats share common properties between different species across different phyla. We used bioinformatic methods to identify high-copy tandem repeats from 282 species using publicly available genomic sequence and our own data.

Results

Our methods are compatible with all current sequencing technologies. Long Pacific Biosciences sequence reads allowed us to find tandem repeat monomers up to 1,419 bp. We assumed that the most abundant tandem repeat is the centromere DNA, which was true for most species whose centromeres have been previously characterized, suggesting this is a general property of genomes. High-copy centromere tandem repeats were found in almost all animal and plant genomes, but repeat monomers were highly variable in sequence composition and length. Furthermore, phylogenetic analysis of sequence homology showed little evidence of sequence conservation beyond approximately 50 million years of divergence. We find that despite an overall lack of sequence conservation, centromere tandem repeats from diverse species showed similar modes of evolution.

Conclusions

While centromere position in most eukaryotes is epigenetically determined, our results indicate that tandem repeats are highly prevalent at centromeres of both animal and plant genomes. This suggests a functional role for such repeats, perhaps in promoting concerted evolution of centromere DNA across chromosomes.     

The use of adjunctive GPIIb/IIIa inhibitors in patients with unstable angina/non-Q-wave MI undergoing percutaneous coronary intervention

Glycoprotein IIb/IIIa receptor inhibitors represent a relatively new therapeutic approach in the field of antiplatelet therapy. Following the development of abciximab a number of small molecule GPIIb/IIIa inhibitors have been introduced such as tirofiban and eptifibatide. In this fast-moving field the interventional cardiologist needs a framework to guide decision-making for the individual patient. This review covers the efficacy and safety data from the clinical trials of GPIIb/IIIa inhibitors in the context of patients undergoing percutaneous coronary intervention for unstable angina/non-Q-wave myocardial infarction. There is an increasing body of evidence to support the efficacy of GPIIb/IIIa inhibitors in reducing the risk of adverse ischemic events in high and low risk patients undergoing percutaneous coronary intervention. A number of unresolved efficacy and safety issues remain, including the duration of treatment before and after intervention; whether a reduction in the heparin dose would further decrease the risk of hemorrhage without affecting the periprocedural thrombotic rate in patients undergoing PTCA with adjunctive GPIIb/IIIa inhibitors; and the cost-effectiveness of this therapy. When a thorough analysis of cost-effectiveness has been made, it will be easier to advocate the widespread use of these agents in all patients undergoing coronary intervention.     

Analysis of rat testicular protein expression following 91-day exposure to JP-8 jet fuel vapor

We analyzed protein expression in preparations from whole testis in adult male Sprague-Dawley rats exposed for 6 h/d for 91 consecutive days to jet propulsion fuel-8 (JP-8) in the vapor phase (0, 250, 500, or 1000 mg/m(3) +/- 10%), simulating a range of possible human occupational exposures. Whole body inhalation exposures were carefully controlled to eliminate aerosol phase, and subjects were sacrificed within 48 h postexposure. Organ fractions were solubilized and separated via large-scale, high resolution two-dimensional electrophoresis, and gel patterns scanned, digitized and processed for statistical analysis. Seventy-six different testis proteins were significantly increased or decreased in abundance in vapor-exposed groups, compared to controls, and dose-response profiles were often nonlinear. A number of the proteins were identified by peptide mass fingerprinting and related to histopathological or physiological deficits shown in previously published studies to occur with repeated exposure to hydrocarbon fuels or solvents. These results demonstrate a significant effect of JP-8 exposure on protein expression, particularly in protein expression in the rodent testis, and suggest that a 91 d exposure to jet fuel vapor induces changes of equal or greater magnitude to those reported previously for shorter duration JP-8 aerosol exposures.     

Anticancer immunotherapy by CTLA-4 blockade: obligatory contribution of IL-2 receptors and negative prognostic impact of soluble CD25

The cytotoxic T lymphocyte antigen-4 (CTLA-4)-blocking antibody ipilimumab induces immune-mediated long-term control of metastatic melanoma in a fraction of patients. Although ipilimumab undoubtedly exerts its therapeutic effects via immunostimulation, thus far clinically useful, immunologically relevant biomarkers that predict treatment efficiency have been elusive. Here, we show that neutralization of IL-2 or blocking the α and β subunits of the IL-2 receptor (CD25 and CD122, respectively) abolished the antitumor effects and the accompanying improvement of the ratio of intratumoral T effector versus regulatory cells (Tregs), which were otherwise induced by CTLA-4 blockade in preclinical mouse models. CTLA-4 blockade led to the reduction of a suppressive CD4⁺ T cell subset expressing Lag3, ICOS, IL-10 and Egr2 with a concomitant rise in IL-2-producing effector cells that lost FoxP3 expression and accumulated in regressing tumors. While recombinant IL-2 improved the therapeutic efficacy of CTLA-4 blockade, the decoy IL-2 receptor α (IL-2Rα, sCD25) inhibited the anticancer effects of CTLA-4 blockade. In 262 metastatic melanoma patients receiving ipilimumab, baseline serum concentrations of sCD25 represented an independent indicator of overall survival, with high levels predicting resistance to therapy. Altogether, these results unravel a role for IL-2 and IL-2 receptors in the anticancer activity of CTLA-4 blockade. Importantly, our study provides the first immunologically relevant biomarker, namely elevated serum sCD25, that predicts resistance to CTLA-4 blockade in patients with melanoma.     

A Late Devonian isoetalean lycopsid, Otzinachsonia Beerboweri, gen. et sp. nov., from north-central Pennsylvania, USA

Compressions and impressions of an isoetalean lycopsid, comprising lower portions of stems, lobed bases, attached rootlets, and rounded rootlet scars, discovered in Late Devonian (Famennian) rocks of Clinton County, north-central Pennsylvania, Appalachian Basin, USA, are here described as Otzinachsonia beerboweri, gen. et sp. nov. These specimens demonstrate unequivocally the existence of the isoetalean lobe-and-furrow rhizomorphic growth pattern as early as the Late Devonian. They were found in an Archaeopteris- and Rhacophyton-dominated flora at Red Hill, an outcrop of the Duncannon Member of the Catskill Formation. The fossils were found in a dark-gray to greenish-gray lenticular siltstone layer that has an average thickness of 1.0 m. This deposit is interpreted as a floodplain pond. The low-energy nature of the deposit and the fine preservation of the intact rootlets of the specimens imply little or no transport. The plants were probably growing along the edge of the floodplain pond with their lower portions submerged for at least part of the year.     

Toward new therapeutics for skin and soft tissue infections: propargyl-linked antifolates are potent inhibitors of MRSA and Streptococcus pyogenes

Hospital- and community-acquired, complicated skin and soft tissue infections, often attributed to Staphylococcus aureus and Streptococcus pyogenes, present a significant health burden that is associated with increased health care costs and mortality. As these two species are difficult to discern on diagnosis and are associated with differential profiles of drug resistance, the development of an efficacious antibacterial agent that targets both organisms is a high priority. Herein we describe a structure-based drug development effort that has produced highly potent inhibitors of dihydrofolate reductase from both species. Optimized propargyl-linked antifolates containing a key pyridyl substituent display antibacterial activity against both methicillin-resistant S. aureus and S. pyogenes at MIC values below 0.1 μg/mL and minimal cytotoxicity against mammalian cells. Further evaluation against a panel of clinical isolates shows good efficacy against a range of important phenotypes such as hospital- and community-acquired strains as well as strains resistant to vancomycin.     

A robotic molecular method for in situ detection of marine invertebrate larvae

Knowledge of the temporal and spatial abundance of invertebrate larvae is critical to understanding the dispersal capabilities and recruitment potential of marine and aquatic organisms. Traditional microscopic analyses are time-consuming and difficult given the diversity of larval species and a frequent lack of discriminating morphological characteristics. Here, we describe a sensitive rRNA targeted sandwich hybridization assay (SHA) that uses oligonucleotide probes to detect and enumerate the larvae of invasive green crabs (Carcinus maenas), native blue mussels (Mytilus), native barnacles (Balanus) and polychaetes (Osedax and Ophelia) that occur in the Monterey Bay National Marine Sanctuary, California. Laboratory-based assays demonstrate specificity, high sensitivity, and a quantitative response to cultured samples from three of the target organisms. Oligonucleotide probes were then printed in arrays on nitrocellulose membranes and deployed in our robotic Environmental Sample Processor (ESP) to detect larvae in situ and autonomously. We demonstrate that the SHA-detection method and ESP robot can be used for near real-time, in situ detection of larval species in the marine environment.