C. elegans Positive Butanone Learning, Short-term, and Long-term Associative Memory Assays

The memory of experiences and learned information is critical for organisms to make choices that aid their survival. C. elegans navigates its environment through neuron-specific detection of food and chemical odors1, 2, and can associate nutritive states with chemical odors3, temperature4, and the pathogenicity of a food source5

Genome-wide Purification of Extrachromosomal Circular DNA from Eukaryotic Cells

Extrachromosomal circular DNAs (eccDNAs) are common genetic elements in Saccharomyces cerevisiae and are reported in other eukaryotes as well. EccDNAs contribute to genetic variation among somatic cells in multicellular organisms and to evolution of unicellular eukaryotes. Sensitive methods for detecting eccDNA are needed to clarify how these elements affect genome stability and how environmental and biological factors induce their formation in eukaryotic cells. This video presents a sensitive eccDNA-purification method called Circle-Seq. The method encompasses column purification of circular DNA, removal of remaining linear chromosomal DNA, rolling-circle amplification of eccDNA, deep sequencing, and mapping. Extensive exonuclease treatment was required for sufficient linear chromosomal DNA degradation. The rolling-circle amplification step by φ29 polymerase enriched for circular DNA over linear DNA. Validation of the Circle-Seq method on three S. cerevisiae CEN.PK populations of 1010 cells detected hundreds of eccDNA profiles in sizes larger than 1 kilobase. Repeated findings of ASP3-1, COS111, CUP1, RSC30, HXT6, HXT7 genes on circular DNA in both S288c and CEN.PK suggests that DNA circularization is conserved between strains at these loci. In sum, the Circle-Seq method has broad applicability for genome-scale screening for eccDNA in eukaryotes as well as for detecting specific eccDNA types

Formation of extrachromosomal circular DNA from long terminal repeats of retrotransposons in <i>Saccharomyces cerevisiae</i>

Extrachromosomal circular DNA (eccDNA) derived from chromosomal Ty retrotransposons in yeast can be generated in multiple ways. Ty eccDNA can arise from the circularization of extrachromosomal linear DNA during the transpositional life cycle of retrotransposons, or from circularization of genomic Ty DNA. Circularization may happen through nonhomologous end-joining (NHEJ) of long terminal repeats (LTRs) flanking Ty elements, by Ty autointegration, or by LTR–LTR recombination. By performing an in-depth investigation of sequence reads stemming from Ty eccDNAs obtained from populations of Saccharomyces cerevisiae S288c, we find that eccDNAs predominantly correspond to full-length Ty1 elements. Analyses of sequence junctions reveal no signs of NHEJ or autointegration events. We detect recombination junctions that are consistent with yeast Ty eccDNAs being generated through recombination events within the genome. This opens the possibility that retrotransposable elements could move around in the genome without an RNA intermediate directly through DNA circularization

Stress Induced Protein Changes in Tall Fescue

Tall fescue (Festuca arundinacea Schreb.), the most important pasture grass in Arkansas, exhibits different agricultural properties when it is infected by its mutualistic endophyte Acremonium coenophialum Morgan-Jones and Gams. We postulate that the presence of endophyte exerts a stress on the host that enhances or detracts from the host\u27s ability to express specific genes. We tested this hypothesis by heat stressing infected and non-infected, juvenile and mature tall fescue, and examining their protein profiles by SDS-PAGE analysis. The results indicate that mature, infected, stressed grass produced greater amounts of Rubisco (ribulose bisphosphate carboxylase-oxygenase) than all other treatments. Additionally, the mature, infected, stressed grass exhibited a 20 k Dalton protein band which was not apparent in other treatments. These observations support the possibility that the endophyte prestresses the grass, and they suggest a molecular mechanism for this response

Selective expansion of myeloid and NK cells in humanized mice yields human-like vaccine responses

Mice engrafted with components of a human immune system have become widely-used models for studying aspects of human immunity and disease. However, a defined methodology to objectively measure and compare the quality of the human immune response in different models is lacking. Here, by taking advantage of the highly immunogenic live-attenuated yellow fever virus vaccine YFV-17D, we provide an in-depth comparison of immune responses in human vaccinees, conventional humanized mice, and second generation humanized mice. We demonstrate that selective expansion of human myeloid and natural killer cells promotes transcriptomic responses akin to those of human vaccinees. These enhanced transcriptomic profiles correlate with the development of an antigen-specific cellular and humoral response to YFV-17D. Altogether, our approach provides a robust scoring of the quality of the human immune response in humanized mice and highlights a rational path towards developing better pre-clinical models for studying the human immune response and disease.National Institute of General Medical Sciences (U.S.) (Award T32GM007753)Searle Scholars ProgramArnold and Mabel Beckman Foundation (Young Investigator Program)National Institutes of Health (U.S.) (1DP2OD020839)National Institutes of Health (U.S.) (5U24AI118672)National Institutes of Health (U.S.) (1U54CA217377)National Institutes of Health (U.S.) (1R33CA202820)National Institutes of Health (U.S.) (2U19AI089992)National Institutes of Health (U.S.) (21R01HL134539)National Institutes of Health (U.S.) (2RM1HG006193)National Institutes of Health (U.S.) (2R01HL095791)National Institutes of Health (U.S.) (P01AI039671)Bill & Melinda Gates Foundation (OPP1139972

On the alleged simplicity of impure proof

Roughly, a proof of a theorem, is “pure” if it draws only on what is “close” or “intrinsic” to that theorem. Mathematicians employ a variety of terms to identify pure proofs, saying that a pure proof is one that avoids what is “extrinsic,” “extraneous,” “distant,” “remote,” “alien,” or “foreign” to the problem or theorem under investigation. In the background of these attributions is the view that there is a distance measure (or a variety of such measures) between mathematical statements and proofs. Mathematicians have paid little attention to specifying such distance measures precisely because in practice certain methods of proof have seemed self- evidently impure by design: think for instance of analytic geometry and analytic number theory. By contrast, mathematicians have paid considerable attention to whether such impurities are a good thing or to be avoided, and some have claimed that they are valuable because generally impure proofs are simpler than pure proofs. This article is an investigation of this claim, formulated more precisely by proof- theoretic means. After assembling evidence from proof theory that may be thought to support this claim, we will argue that on the contrary this evidence does not support the claim

Why did Better Place fail?: Range anxiety, interpretive flexibility, and electric vehicle promotion in Denmark and Israel

With almost $1 billion in funding, Better Place was poised to become one of the most innovative companies in the electric mobility market. The system Better Place proposed had two novel prongs; first, to reduce the cost of batteries, and second, to reduce range anxiety, public infrastructure concerns, and long charging times. Yet, despite this seemingly strong combination, Better Place failed to make any progress in Denmark and Israel, the first two markets it operated in, and subsequently declared bankruptcy, selling off its collective assets for less than$500,000. Drawing from science and technology studies and the notion of “interpretive flexibility,” this paper posits several reasons to explain the failure of Better Place, including that Denmark is not as “green” as it seems nor is the Israeli market as attractive as believed, and that Better Place's solution to charging time and range anxiety resolved a psychological, not a functional, barrier of the general public to adopt electric vehicles. Before investigating these two reasons, the paper presents a short history of Better Place and explores the contours of its operations in Denmark and Israel. It then discusses why Better Place “failed” across both countries before concluding with implications for energy planning, policy, and analysis

Mixed experiences and perceptions of psychological treatment for chronic pain in Singapore: Skepticism, ambivalence, satisfaction and potential

There is little research in Southeast Asia focusing on patients’ experiences of seeking psychological treatment for chronic pain. Objective: This study aims to understand the experiences of patients seeking psychological treatment for chronic pain in this region. Setting: Outpatient pain clinic at a tertiary hospital in Singapore. Subjects: People with experiences of attending psychological treatment for chronic pain, including some who were not receiving this type of treatment. Study design and methods: Fifteen inductive semi-structured interviews were conducted to explore patients’ experiences regarding psychological treatment for their chronic pain. Thematic analysis was then applied. Results: Three main themes were identified: ‘Expectations and Realities of Health Professionals’, ‘Patients’ Attitudes and Beliefs’ and ‘Practical and Social Factors.’ From the patients’ perspectives, an empathetic health professional who was willing to listen contributed to a positive treatment seeking experience. Patients felt that health professionals’ lack of knowledge about appropriate treatment contributed to their frustration. Patients could not understand how psychological treatment was related to pain treatment and queried why they were “paying just to talk”. On the other hand, their experiences were quite positive, and they found psychological treatments helpful when they participated in them. Conclusion: Education for both patients and health professionals unfamiliar with psychological treatments for pain may improve access to these treatments

Large-scale Molecular Diet Analysis in a Generalist Marine Mammal Reveals Male Preference for Prey of Conservation Concern

Sex-specific diet information is important in the determination of predator impacts on prey populations. Unfortunately, the diet of males and females can be difficult to describe, particularly when they are marine predators. We combined two molecular techniques to describe haul-out use and prey preferences of male and female harbor seals (Phoca vitulina) from Comox and Cowichan Bay (Canada) during 2012-2013. DNA metabarcoding quantified the diet proportions comprised of prey species in harbor seal scat, and qPCR determined the sex of the individual that deposited each scat. Using 287 female and 260 male samples, we compared the monthly sex ratio with GLMs and analyzed prey consumption relative to sex, season, site, and year with PERMANOVA. The sex ratio between monthly samples differed widely in both years (range = 12%-79% males) and showed different patterns at each haul-out site. Male and female diet differed across both years and sites: Females consumed a high proportion of demersal fish species while males consumed more salmonid species. Diet composition was related to both sex and season (PERMANOVA: R 2 = 27%, p \u3c 0.001; R 2 = 24%, p \u3c 0.001, respectively) and their interaction (PERMANOVA: R 2 = 11%, p \u3c 0.001). Diet differences between males and females were consistent across site and year, suggesting fundamental foraging differences, including that males may have a larger impact on salmonids than females. Our novel combination of techniques allowed for both prey taxonomic and spatiotemporal resolution unprecedented in marine predators

Use of a Generalized Additive Model to Investigate Key Abiotic Factors Affecting Microcystin Cellular Quotas in Heavy Bloom Areas of Lake Taihu

Lake Taihu is the third largest freshwater lake in China and is suffering from serious cyanobacterial blooms with the associated drinking water contamination by microcystin (MC) for millions of citizens. So far, most studies on MCs have been limited to two small bays, while systematic research on the whole lake is lacking. To explain the variations in MC concentrations during cyanobacterial bloom, a large-scale survey at 30 sites across the lake was conducted monthly in 2008. The health risks of MC exposure were high, especially in the northern area. Both Microcystis abundance and MC cellular quotas presented positive correlations with MC concentration in the bloom seasons, suggesting that the toxic risks during Microcystis proliferations were affected by variations in both Microcystis density and MC production per Microcystis cell. Use of a powerful predictive modeling tool named generalized additive model (GAM) helped visualize significant effects of abiotic factors related to carbon fixation and proliferation of Microcystis (conductivity, dissolved inorganic carbon (DIC), water temperature and pH) on MC cellular quotas from recruitment period of Microcystis to the bloom seasons, suggesting the possible use of these factors, in addition to Microcystis abundance, as warning signs to predict toxic events in the future. The interesting relationship between macrophytes and MC cellular quotas of Microcystis (i.e., high MC cellular quotas in the presence of macrophytes) needs further investigation