bluuebunny/crossref_metadata_2025 huggingface dataset:
Fire and grass cover influence occupancy patterns of rare rodents and feral cats in a mountain refuge: implications for management
Authors: McDonald Peter J. , Stewart Alistair , Schubert Andrew T. , Nano Catherine E. M. , Dickman Chris R. , Luck Gary W.
Published: May 2016

Context
Feral cats (Felis catus) are implicated in the ongoing decline of Australian mammals. New research from northern Australia suggests that predation risk from feral cats could be managed by manipulating fire regimes to increase grass cover.

Aims
We investigate the role of fire history and hummock grass cover in the occurrence of feral cats and rare rodents, including the critically endangered central rock-rat (Zyzomys pedunculatus), in a mountain refuge in central Australia.

Methods
We installed 76 camera stations across four sites in the West MacDonnell National Park and used occupancy modelling to evaluate the influence of recent fire (within 5 years), hummock grass cover and ruggedness on feral cat and rodent occupancy.

Key results
Occupancy of the central rock-rat was positively associated with areas burnt within the past 5 years – a relationship probably driven by increased food resources in early succession vegetation. In contrast, the desert mouse (Pseudomys desertor) was detected at locations with dense hummock grass that had remained unburnt over the same period. Feral cats were widespread across the study area, although our data suggest that they forage less frequently in areas with dense hummock grass cover.

Conclusions
Our results suggest that fire management and grass cover manipulation can be used as a tool for rodent conservation in this environment and potentially elsewhere in arid Australia.

Implications
Creating food-rich patches within dense hummock grasslands may allow central rock-rats to increase occupancy while simultaneously affording them protection from predation. Landscape-scale wildfire resulting in a single post-fire vegetation age class is likely to be unfavourable for native rodents in this environment.
Ecology and conservation of the northern hopping-mouse (Notomys aquilo)
Authors: Diete Rebecca L. , Meek Paul D. , Dickman Christopher R. , Leung Luke K.-P.
Published: May 2016

The northern hopping-mouse (Notomys aquilo) is a cryptic and enigmatic rodent endemic to Australia’s monsoonal tropics. Focusing on the insular population on Groote Eylandt, Northern Territory, we present the first study to successfully use live traps, camera traps and radio-tracking to document the ecology of N. aquilo. Searches for signs of the species, camera trapping, pitfall trapping and spotlighting were conducted across the island during 2012–15. These methods detected the species in three of the 32 locations surveyed. Pitfall traps captured 39 individuals over 7917 trap-nights. Females were significantly longer and heavier, and had better body condition, than males. Breeding occurred throughout the year; however, the greatest influx of juveniles into the population occurred early in the dry season in June and July. Nine individuals radio-tracked in woodland habitat utilised discrete home ranges of 0.39–23.95 ha. All individuals used open microhabitat proportionally more than was available, and there was a strong preference for eucalypt woodland on sandy substrate rather than for adjacent sandstone woodland or acacia shrubland. Camera trapping was more effective than live trapping at estimating abundance and, with the lower effort required to employ this technique, it is recommended for future sampling of the species. Groote Eylandt possibly contains the last populations of N. aquilo, but even there its abundance and distribution have decreased dramatically in surveys over the last several decades. Therefore, we recommend that the species’ conservation status under the Environment Protection and Biodiversity Conservation Act 1999 be changed from ‘vulnerable’ to ‘endangered’.
The effects of language immersion on the bilingual lexicon
Authors: Mok Peggy P.K. , Yu Alan C.L.
Published: May 2016

Previous studies have consistently found an asymmetry where priming in the L1-L2 direction is stronger than that in the L2-L1 direction. However, some studies showed that an L2 immersion environment could attenuate bilingual speakers’ access to the L1 and result in a ‘bilingual disadvantage’. This study investigated how language immersion modulates the priming effects of late adult bilingual speakers. We compared late Chinese-English bilingual speakers with high L2 (English) proficiency in an L1 environment and those in an L2 immersion environment. Both semantic and translation priming in same-language and cross-language conditions were investigated. The results showed no ‘bilingual disadvantage’ of the immersed participants. The priming asymmetry was weakened for the immersed participants who were more comparable in their reaction time to different language conditions. Both semantic and translation priming were found in L1-L2 and L2-L1 directions, suggesting that both types of priming are similar in nature in the bilingual lexicon.
Teasing apart factors influencing executive function performance in bilinguals and monolinguals at different ages
Authors: Gathercole Virginia C. Mueller , Thomas Enlli Môn , Viñas Guasch Nestor , Kennedy Ivan , Prys Cynog , Young Nia , Roberts Emily J. , Hughes Emma K. , Jones Leah
Published: May 2016

This study attempts to tease apart a variety of factors that may contribute to performance on executive function tasks. Data from the Simon task is re-examined to determine the contributions of age, SES, language proficiency/vocabulary, general cognitive performance, and bilingualism on performance. The results suggest influence from a variety of factors, with a major contribution from relative age and from language proficiency, as measured by vocabulary. Bilingualism showed some effect in relation to older adults’ accuracy of performance, in both congruent and incongruent conditions, but not to reaction time.
Gender Differences in the Predictive Validity of a Violence Risk Screening Tool: A Prospective Study in an Acute Psychiatric Ward
Authors: Eriksen Bjørn Magne S. , Bjørkly Stål , Færden Ann , Friestad Christine , Hartvig Pål , Roaldset John Olav
Published: May 2016

In this one-year prospective, naturalistic study in an acute psychiatric facility, we explored gender differences between 230 men and 284 women regarding the predictive validity of the Violence risk screening-10 (V-RISK-10). Hospital staff recorded violent incidents during hospital stays (range 1–176 days; median: 5.5 days) resulting in 50 men (22%) and 30 women (11%) being recorded for violence. The predictive validity was significant for both genders, with ROC-AUCs of .79 for males and .80 for females. Scoring at cut-off or beyond indicated a two-fold increased risk of becoming violent for men and a three-fold risk for women. A lower score indicated a five-fold decrease in risk for both genders. Estimates of explained variance for the prediction model were higher for men. Findings indicate gender differences for risk factors of violence and highlight the need of further research on this issue.
Interferon-γ facilitates hepatic antiviral T cell retention for the maintenance of liver-induced systemic tolerance
Authors: Zeng Zhutian , Li Lu , Chen Yongyan , Wei Haiming , Sun Rui , Tian Zhigang
Published: May 2016

Persistent exposure to liver pathogens leads to systemic antigen-specific tolerance, a major cause of chronicity during hepatotropic infection. The mechanism regarding how this systemic tolerance is maintained remains poorly elucidated. In a well established mouse model of hepatitis B virus (HBV) persistence–induced systemic tolerance, we observed that interferon-γ (IFN-γ) deficiency led to complete loss of tolerance, resulting in robust anti-HBV responses upon peripheral vaccination. The recovery of vaccine-induced anti-HBV responses was mainly caused by the retained antigen-specific CD4+ T cells rather than decreased functional inhibitory cells in the periphery. Mechanistically, HBV persistence induced sustained hepatic CD4+ T cell–derived IFN-γ production. IFN-γ was found to promote CXCL9 secretion from liver-resident macrophages. This T cell chemokine facilitated the retention of antiviral CD4+ T cells in the liver in a CXCR3-dependent manner. Hepatic sequestrated antiviral CD4+ T cells subsequently underwent local apoptotic elimination partially via cytotoxic T lymphocyte–associated protein 4 ligation. These findings reveal an unexpected tolerogenic role for IFN-γ during viral persistence in the liver, providing new mechanistic insights regarding the maintenance of systemic antigen-specific tolerance during HBV persistence.
Class-switched anti-insulin antibodies originate from unconventional antigen presentation in multiple lymphoid sites
Authors: Wan Xiaoxiao , Thomas James W. , Unanue Emil R.
Published: May 2016

Autoantibodies to insulin are a harbinger of autoimmunity in type 1 diabetes in humans and in non-obese diabetic mice. To understand the genesis of these autoantibodies, we investigated the interactions of insulin-specific T and B lymphocytes using T cell and B cell receptor transgenic mice. We found spontaneous anti-insulin germinal center (GC) formation throughout lymphoid tissues with GC B cells binding insulin. Moreover, because of the nature of the insulin epitope recognized by the T cells, it was evident that GC B cells presented a broader repertoire of insulin epitopes. Such broader recognition was reproduced by activating naive B cells ex vivo with a combination of CD40 ligand and interleukin 4. Thus, insulin immunoreactivity extends beyond the pancreatic lymph node–islets of Langerhans axis and indicates that circulating insulin, despite its very low levels, can have an influence on diabetogenesis.
Detection of Incipient Germination in Malting Barley with a Starch Viscosity Method and a Proposed Ethanol Emission Method
Authors: Buckley Wayne T. , Izydorczyk Marta S. , Legge William G.
Published: May 2016

The risk of germination loss during storage in two‐row malting barley can be reduced by identifying grain lots that have undergone incipient germination (IG) during harvest. A method based on starch viscosity that utilizes a Rapid Visco Analyzer (RVA) is currently available for IG analysis. A new potential method, based on the measurement of ethanol emission (EE) from whole barley, may be more efficient and less expensive than the RVA procedure. Three storage experiments were performed: experiments 1 and 3 at 25°C and 80% rh, and experiment 2 in unheated, uninsulated buildings. Decline in germination energy (GE) varied from <1% to about 80% during storage. In experiments 1 and 3, R2 for GE loss (expressed as [weeks to 5% loss of GE]0.2) versus EE was 0.76 in both cases, whereas R2 for GE loss versus RVA viscosity was 0.64 and 0.68, respectively. In experiment 2, the greatest loss of GE was associated with high temperatures and relative humidity in July and August. EE performed as well as RVA in the prediction of germination loss in all three trials. Although further evaluation of the EE method is required, it appears to be a promising alternative to the RVA technique.
Antioxidant Potentials and Phenolic Composition of Tef Varieties: An Indigenous Ethiopian Cereal
Authors: Shumoy Habtu , Raes Katleen
Published: May 2016

Tef, Eragrostis tef (Zucc.) Trotter, is a cereal crop originated and diversified in Ethiopia, where it is used to produce a range of food products. This study aimed to profile and quantify the phenolic composition and antioxidant potential of seven tef grain varieties. Soluble and bound phenolics ranged from 37 to 71 and from 226 to 376 mg of gallic acid equivalent/100 g dry basis (db), and soluble and bound flavonoid contents varied between 36 and 64 and between 113 and 258 mg of catechin equivalent/100 g db, respectively. Protocatechuic, vanillic, syringic, p‐coumaric, sinapic, ferulic, and rosmarinic acids, catechin, and naringenin were detected at least in three of the varieties studied. The dominant phenolic compounds were catechin, rosmarinic acid, and ferulic acid in the soluble extracts, whereas ferulic, rosmarinic, and p‐coumaric acids were the dominant ones in the bound extract. Gallic, caffeic, and salicylic acids were not detected in any of the varieties studied. The majority (>84%) of tef grain phenolics were found in bound form, contributing >84% of total 2,2‐diphenyl‐1‐picrylhydrazyl antioxidative capacity and >80% of total ferric reducing antioxidant power. These results clearly demonstrated the differences in phenolic profile among tef grain varieties. These results are relevant for developing healthy and nutritious tef‐based food products.
Charcoal Canker of Pear, Plum, and Quince Trees Caused by Biscogniauxia rosacearum sp. nov. in Southern Italy
Authors: Raimondo Maria Luisa , Lops Francesco , Carlucci Antonia
Published: May 2016

The genus Biscogniauxia is paraphyletic to members of the family Xylariaceae and includes at least 52 species to date that are mainly pathogens of dicotyledonous angiosperm trees. Most of these are forest trees, such as those in the genera Acacia, Acer, Alnus, Eucalyptus, Fraxinus, Populus, and Quercus, and other species of minor importance. Biscogniauxia spp. have been reported as endophytes or secondary invaders that attack only stressed plants. During a survey in rosaceous orchards in southern Italy, several charcoal cankers were observed and stroma samples were collected. A collection of 31 Biscogniauxia isolates was analyzed. Their phylogenetic relationships were determined through study of the internal transcribed spacer, β-tubulin, and actin gene sequences. Combining morphological, cultural, and molecular data, a new species of Biscogniauxia is described here as Biscogniauxia rosacearum. This new species was isolated for the first time from rosaceous hosts in Apulia. Pathogenicity tests showed that it causes symptoms on stems when artificially inoculated and produces stromata on the bark surface.
Characterization of Disease Resistance Loci in the USDA Soybean Germplasm Collection Using Genome-Wide Association Studies
Authors: Chang Hao-Xun , Lipka Alexander E. , Domier Leslie L. , Hartman Glen L.
Published: May 2016

Genetic resistance is a key strategy for disease management in soybean. Over the last 50 years, soybean germplasm has been phenotyped for resistance to many pathogens, resulting in the development of disease-resistant elite breeding lines and commercial cultivars. While biparental linkage mapping has been used to identify disease resistance loci, genome-wide association studies (GWAS) using high-density and high-quality markers such as single nucleotide polymorphisms (SNPs) has become a powerful tool to associate molecular markers and phenotypes. The objective of our study was to provide a comprehensive understanding of disease resistance in the United States Department of Agriculture Agricultural Research Service Soybean Germplasm Collection by using phenotypic data in the public Germplasm Resources Information Network and public SNP data (SoySNP50K). We identified SNPs significantly associated with disease ratings from one bacterial disease, five fungal diseases, two diseases caused by nematodes, and three viral diseases. We show that leucine-rich repeat (LRR) receptor-like kinases and nucleotide-binding site-LRR candidate resistance genes were enriched within the linkage disequilibrium regions of the significant SNPs. We review and present a global view of soybean resistance loci against multiple diseases and discuss the power and the challenges of using GWAS to discover disease resistance in soybean.
Interactions Between Frankliniella fusca and Pantoea ananatis in the Center Rot Epidemic of Onion (Allium cepa)
Authors: Dutta Bhabesh , Gitaitis Ronald , Barman Apurba , Avci Utku , Marasigan Kathleen , Srinivasan Rajagopalbabu
Published: May 2016

An Enterobacteriaceae bacterium, Pantoea ananatis (Serrano) Mergaert, is the causal agent of an economically important disease of onion, center rot. P. ananatis is transmitted by an onion-infesting thrips, Frankliniella fusca (Hinds). However, interactions between F. fusca and P. ananatis as well as transmission mechanisms largely remain uncharacterized. This study investigated P. ananatis acquisition by thrips and transstadial persistence. Furthermore, the effects of bacterial acquisition on thrips fitness were also evaluated. When thrips larvae and adults were provided with acquisition access periods (AAP) on peanut leaflets contaminated with the bacterium, an exponentially positive relationship was observed between AAP and P. ananatis acquisition (R2 ≥ 0.77, P = 0.01). P. ananatis persisted in thrips through several life stages (larvae, pupae, and adult). Despite the bacterial persistence, no significant effects on thrips fitness parameters such as fecundity and development were observed. Immunofluorescence microscopy of adult thrips with P. ananatis-specific antibody after 48 h AAP on contaminated food revealed that the bacterium was localized only in the gut. These results suggested that the pathogen is not circulative and could be transmitted through feces. Mechanical inoculation of onion seedlings with fecal rinsates produced center rot symptoms, whereas inoculation with rinsates potentially containing salivary secretions did not. These results provide evidence for stercorarian transmission (transmission through feces) of P. ananatis by F. fusca.
Discovery of a new repeat family in the Callithrix jacchus genome
Authors: Konkel Miriam K. , Ullmer Brygg , Arceneaux Erika L. , Sanampudi Sreeja , Brantley Sarah A. , Hubley Robert , Smit Arian F.A. , Batzer Mark A.
Published: May 2016

We identified a novel repeat family, termed Platy-1, in the Callithrix jacchus (common marmoset) genome that arose around the time of the divergence of platyrrhines and catarrhines and established itself as a repeat family in New World monkeys (NWMs). A full-length Platy-1 element is ∼100 bp in length, making it the shortest known short interspersed element (SINE) in primates, and harbors features characteristic of non-LTR retrotransposons. We identified 2268 full-length Platy-1 elements across 62 subfamilies in the common marmoset genome. Our subfamily reconstruction and phylogenetic analyses support Platy-1 propagation throughout the evolution of NWMs in the lineage leading to C. jacchus. Platy-1 appears to have reached its amplification peak in the common ancestor of current day marmosets and has since moderately declined. However, identification of more than 200 Platy-1 elements identical to their respective consensus sequence, and the presence of polymorphic elements within common marmoset populations, suggests ongoing retrotransposition activity. Platy-1, a SINE, appears to have originated from an Alu element, and hence is likely derived from 7SL RNA. Our analyses illustrate the birth of a new repeat family and its propagation dynamics in the lineage leading to the common marmoset over the last 40 million years.
Zygotes segregate entire parental genomes in distinct blastomere lineages causing cleavage-stage chimerism and mixoploidy
Authors: Destouni Aspasia , Zamani Esteki Masoud , Catteeuw Maaike , Tšuiko Olga , Dimitriadou Eftychia , Smits Katrien , Kurg Ants , Salumets Andres , Van Soom Ann , Voet Thierry , Vermeesch Joris R.
Published: May 2016

Dramatic genome dynamics, such as chromosome instability, contribute to the remarkable genomic heterogeneity among the blastomeres comprising a single embryo during human preimplantation development. This heterogeneity, when compatible with life, manifests as constitutional mosaicism, chimerism, and mixoploidy in live-born individuals. Chimerism and mixoploidy are defined by the presence of cell lineages with different parental genomes or different ploidy states in a single individual, respectively. Our knowledge of their mechanistic origin results from indirect observations, often when the cell lineages have been subject to rigorous selective pressure during development. Here, we applied haplarithmisis to infer the haplotypes and the copy number of parental genomes in 116 single blastomeres comprising entire preimplantation bovine embryos (n = 23) following in vitro fertilization. We not only demonstrate that chromosome instability is conserved between bovine and human cleavage embryos, but we also discovered that zygotes can spontaneously segregate entire parental genomes into different cell lineages during the first post-zygotic cleavage division. Parental genome segregation was not exclusively triggered by abnormal fertilizations leading to triploid zygotes, but also normally fertilized zygotes can spontaneously segregate entire parental genomes into different cell lineages during cleavage of the zygote. We coin the term “heterogoneic division” to indicate the events leading to noncanonical zygotic cytokinesis, segregating the parental genomes into distinct cell lineages. Persistence of those cell lines during development is a likely cause of chimerism and mixoploidy in mammals.
Coverslip Cleaning and Functionalization for Total Internal Reflection Fluorescence Microscopy
Authors: Kudalkar Emily M. , Deng Yi , Davis Trisha N. , Asbury Charles L.
Published: May 2016

Total internal reflection fluorescence (TIRF) microscopy allows visualization of biological events at the single-molecule level by restricting excitation to a precise focal plane near the coverslip and eliminating out-of-focus fluorescence. The quality of TIRF imaging relies on a high signal-to-noise ratio and therefore it is imperative to prevent adherence of molecules to the glass coverslip. Nonspecific interactions can make it difficult to distinguish true binding events and may also interfere with accurate quantification of background noise. In addition, nonspecific binding of the fluorescently tagged protein will lower the effective working concentration, thereby altering values used to calculate affinity constants. To prevent spurious interactions, we thoroughly clean the surface of the coverslip and then functionalize the glass either by applying a layer of silane or by coating with a lipid bilayer.
Preparation of Reactions for Imaging with Total Internal Reflection Fluorescence Microscopy
Authors: Kudalkar Emily M. , Davis Trisha N. , Asbury Charles L.
Published: May 2016

Here we present our standard protocol for studying the binding of kinetochore proteins to microtubules as a paradigm for designing single-molecule total internal reflection fluorescence (TIRF) microscopy experiments. Several aspects of this protocol require empirical optimization, including the method for anchoring the polymer or substrate to the coverslip, the type and amount of blocking protein to prevent nonspecific protein adsorption to the glass, the appropriate protein concentration, the laser power, and the duration of imaging. Our method uses bovine serum albumin and κ-casein as blocking agents to coat any imperfections in the coverslip silanization and thereby prevent protein adsorption to the coverslip. Protein concentration and duration of imaging must be optimized for each experiment and protein of interest. Ideally, a range is determined that allows for resolution of single complexes binding to microtubules to ensure proper measurement of kinetic off rates and diffusion along microtubules. Excessively high concentrations may lead to overlapping binding of proteins on microtubules, making it impossible to resolve single binding events. The duration of imaging must be long enough to capture very low off rates (long residence time on microtubules) and we typically image at 10 frames/sec for 200 sec. The laser power can be adjusted to prevent photobleaching, but must be high enough to achieve a sufficient signal/noise ratio.
Data Analysis for Total Internal Reflection Fluorescence Microscopy
Authors: Asbury Charles L.
Published: May 2016

In the microscopes we use to analyze total internal reflection fluorescence (TIRF), the emitted fluorescence is split chromatically, using dichroic filters, into either two or three different colors (“channels”). In our two-color instrument, the green emission wavelengths (405–488 nm; for imaging green fluorescent protein [GFP]-tagged proteins) and far-red emission wavelengths (650–800 nm; for imaging Alexa-647-labeled microtubules) are projected onto the upper and lower halves, respectively, of a single camera. A single filter can be swapped to collect near-red wavelengths (561–640 nm; for imaging mCherry, or Alexa-568-labeled microtubules) instead of far-red. Our three-color instrument is very similar except that the green, near-red, and far-red color ranges are projected onto three separate cameras. In either case, the different colors can be imaged simultaneously. Typically, we collect images at 10 frames/sec for ∼200 sec. We have developed a series of semiautomated image analysis programs, written in LabView, to obtain the brightness, residence time, and mobility of individual particles bound to single microtubules. The basic analysis steps are straightforward and could also be implemented using ImageJ or Matlab. For convenience, this protocol describes the analysis of a single microtubule. Data from many microtubules across many experimental trials are needed to obtain robust conclusions that are independent of stochastic and trial-to-trial variability.
The Use of Lysosomotropic Dyes to Exclude Lysosomal Membrane Permeabilization
Authors: Repnik Urška , Česen Maruša Hafner , Turk Boris
Published: May 2016

Progressive lowering of pH is characteristic for the endocytic pathway and enables efficient degradation of molecules by hydrolytic enzymes at its distal end. The existence of the proton gradient over the endosomal/lysosomal membranes depends on the action of the vacuolar ATPase (v-ATPase). During lysosomal membrane permeabilization (LMP), protons leak through the destabilized membrane, resulting in loss of the pH gradient. Here, we present a protocol showing how this effect can be detected by staining cells with lysosomotropic dyes, which accumulate in acidic organelles after protonation. During LMP, cells lose the ability to retain these dyes and therefore appear pale. Among the most commonly used lysosomotropic dyes are LysoTracker reagents and acridine orange. Cells can be analyzed with a fluorescence microscope; however, flow-cytometric analysis enables fast, objective, and reliable evaluation of differences between samples. Advantages of the technique include the fact that sample preparation is relatively simple and can be scaled-up to test several different compounds or conditions. However, as we will discuss, cells treated with v-ATPase inhibitors also lose the pH gradient across lysosomal membranes and cannot be stained with lysosomotropic dyes, although this is not accompanied by LMP. Therefore, merely observing loss of staining is not in itself a proof of LMP.
Characterizing Cas9 Protospacer-Adjacent Motifs with High-Throughput Sequencing of Library Depletion Experiments
Authors: Braff Jonathan L. , Yaung Stephanie J. , Esvelt Kevin M. , Church George M.
Published: May 2016

This protocol outlines a general approach for characterizing the protospacer-adjacent motifs (PAMs) of Cas9 orthologs. It uses a three-plasmid system: One plasmid carries Cas9 and its tracrRNA, a second targeting vector contains the spacer and repeat, and the third plasmid encodes the targeted sequence (as the protospacer) with varying PAM sequences. It leverages the Cas9 nuclease activity to cleave and destroy plasmids that bear a compatible PAM. The level of depletion of a library of targeted plasmids after Cas9-mediated selection can then be assessed by deep sequencing to reveal candidate PAMs for downstream validation.
Extraction of Chromosomal DNA from Schizosaccharomyces pombe
Authors: Murray Johanne M. , Watson Adam T. , Carr Antony M.
Published: May 2016

Extraction of DNA from Schizosaccharomyces pombe cells is required for various uses, including templating polymerase chain reactions (PCRs), Southern blotting, library construction, and high-throughput sequencing. To purify high-quality DNA, the cell wall is removed by digestion with Zymolyase or Lyticase and the resulting spheroplasts lysed using sodium dodecyl sulfate (SDS). Cell debris, SDS, and SDS–protein complexes are subsequently precipitated by the addition of potassium acetate and removed by centrifugation. Finally, DNA is precipitated using isopropanol. At this stage, purity is usually sufficient for PCR. However, for more sensitive procedures, such as restriction enzyme digestion, additional purification steps, including proteinase K digestion and phenol–chloroform extraction, are recommended. All of these steps are described in detail here.
Colony Polymerase Chain Reaction with Schizosaccharomyces pombe
Authors: Murray Johanne M. , Watson Adam T. , Carr Antony M.
Published: May 2016

When screening a large number of individual Schizosaccharomyces pombe strains by polymerase chain reaction (PCR), a rapid “colony PCR” approach may be used. Numerous colony PCR protocols are available, and fundamental to them all is that the colony must be fresh (grown overnight) and that as few cells as possible are used. In this protocol, we present three reliable methods for preparing S. pombe cells for colony PCR.
G-Protein Modulation of Voltage-Gated Ca2+ Channels from Isolated Adult Rat Superior Cervical Ganglion Neurons
Authors: Lu Van B. , Ikeda Stephen R.
Published: May 2016

Sympathetic neurons isolated from adult rat superior cervical ganglia (SCG) are a well-established model to study G-protein modulation of voltage-gated Ca2+ channels (VGCCs). SCG neurons can be easily dissociated and are amendable to heterologous expression of genes, including genetic tools to study G-protein signaling pathways, within a time frame to maintain good spatial voltage-clamp control of membrane potential during electrophysiological recordings (8–36 h postdissociation). This protocol focuses on examining G-protein modulation of VGCCs; however, the procedures and experimental setup for acute application of agonists can be applied to study modulation of other ion channels (e.g., M-current, G-protein-coupled inwardly rectifying K+ channels). We also discuss some common sources of artifacts that can arise during acute drug application onto dissociated neurons, which can mislead interpretation of results.
Classical Genetics with Saccharomyces cerevisiae
Authors: Amberg David C. , Burke Daniel J.
Published: May 2016

The budding yeast Saccharomyces cerevisiae is an outstanding experimental model organism that has been exploited since the early part of the twentieth century for studies in biochemistry and genetics. It has been the premiere experimental system for modern functional genomics and continues to make important contributions to many areas of biology. Here we discuss its many virtues as an organism for classical genetic research.
Single-Molecule Total Internal Reflection Fluorescence Microscopy
Authors: Kudalkar Emily M. , Davis Trisha N. , Asbury Charles L.
Published: May 2016

The advent of total internal reflection fluorescence (TIRF) microscopy has permitted visualization of biological events on an unprecedented scale: the single-molecule level. Using TIRF, it is now possible to view complex biological interactions such as cargo transport by a single molecular motor or DNA replication in real time. TIRF allows for visualization of single molecules by eliminating out-of-focus fluorescence and enhancing the signal-to-noise ratio. TIRF has been instrumental for studying in vitro interactions and has also been successfully implemented in live-cell imaging. Visualization of cytoskeletal structures and dynamics at the plasma membrane, such as endocytosis, exocytosis, and adhesion, has become much clearer using TIRF microscopy. Thanks to recent advances in optics and commercial availability, TIRF microscopy is becoming an increasingly popular and user-friendly technique. In this introduction, we describe the fundamental properties of TIRF microscopy and the advantages of using TIRF for single-molecule investigation.
Characterization of Cas9–Guide RNA Orthologs
Authors: Braff Jonathan L. , Yaung Stephanie J. , Esvelt Kevin M. , Church George M.
Published: May 2016

In light of the multitude of new Cas9-mediated functionalities, the ability to carry out multiple Cas9-enabled processes simultaneously and in a single cell is becoming increasingly valuable. Accomplishing this aim requires a set of Cas9–guide RNA (gRNA) pairings that are functionally independent and insulated from one another. For instance, two such protein–gRNA complexes would allow for concurrent activation and editing at independent target sites in the same cell. The problem of establishing orthogonal CRISPR systems can be decomposed into three stages. First, putatively orthogonal systems must be identified with an emphasis on minimizing sequence similarity of the Cas9 protein and its associated RNAs. Second, the systems must be characterized well enough to effectively express and target the systems using gRNAs. Third, the systems should be established as orthogonal to one another by testing for activity and cross talk. Here, we describe the value of these orthogonal CRISPR systems, outline steps for selecting and characterizing potentially orthogonal Cas9–gRNA pairs, and discuss considerations for the desired specificity in Cas9-coupled functions.
Strategies for Investigating G-Protein Modulation of Voltage-Gated Ca2+ Channels
Authors: Lu Van B. , Ikeda Stephen R.
Published: May 2016

G-protein-coupled receptor modulation of voltage-gated ion channels is a common means of fine-tuning the response of channels to changes in membrane potential. Such modulation impacts physiological processes such as synaptic transmission, and hence therapeutic strategies often directly or indirectly target these pathways. As an exemplar of channel modulation, we examine strategies for investigating G-protein modulation of CaV2.2 or N-type voltage-gated Ca2+ channels. We focus on biochemical and genetic tools for defining the molecular mechanisms underlying the various forms of CaV2.2 channel modulation initiated following ligand binding to G-protein-coupled receptors.
Long-Distance Transport of Thiamine (Vitamin B1) Is Concomitant with That of Polyamines
Authors: Martinis Jacopo , Gas-Pascual Elisabet , Szydlowski Nicolas , Crèvecoeur Michèle , Gisler Alexandra , Bürkle Lukas , Fitzpatrick Teresa B.
Published: May 2016

Thiamine (vitamin B1) is ubiquitous and essential for cell energy supply in all organisms as a vital metabolic cofactor, known for over a century. In plants, it is established that biosynthesis de novo is taking place predominantly in green tissues and is furthermore limited to plastids. Therefore, transport mechanisms are required to mediate the movement of this polar metabolite from source to sink tissue to activate key enzymes in cellular energy generating pathways but are currently unknown. Similar to thiamine, polyamines are an essential set of charged molecules required for diverse aspects of growth and development, the homeostasis of which necessitates long-distance transport processes that have remained elusive. Here, a yeast-based screen allowed us to identify Arabidopsis (Arabidopsis thaliana) PUT3 as a thiamine transporter. A combination of biochemical, physiological, and genetic approaches permitted us to show that PUT3 mediates phloem transport of both thiamine and polyamines. Loss of function of PUT3 demonstrated that the tissue distribution of these metabolites is altered with growth and developmental consequences. The pivotal role of PUT3 mediated thiamine and polyamine homeostasis in plants, and its importance for plant fitness is revealed through these findings.
Predator–prey mass ratio revisited: does preference of relative prey body size depend on individual predator size?
Authors: Tsai Cheng‐Han , Hsieh Chih‐hao , Nakazawa Takefumi
Published: May 2016

Quantifying predator–prey body size relationships is key to understanding food webs. Food web models often assume that all individuals of predator species prefer the same relative body size of prey, using a single constant called preferred predator–prey mass ratio (preferred PPMR). In contrast, empirical studies have shown that relative prey body size in diet varies with individual predator size, challenging the food web models based on size‐invariant preferred PPMR and their predictions.

We point out that this apparent inconsistency arises because empirical PPMR in those previous studies has been measured only through dietary data (i.e. realized PPMR rather than preferred PPMR) without considering the effects of environmental prey availability, suggesting the possibility that preferred PPMR may be in fact independent of individual predator size.

Here, we present a new approach to revisit the assumption of size‐invariant preferred PPMR in food web models. The approach compares two measures of PPMR calculated from prey compositions in predator diet and environmental prey composition, respectively (i.e. realized PPMR vs. environmental PPMR). The deviations between realized and environmental PPMRs are considered as a proxy of individual variations in relative prey size preference (i.e. preferred PPMR). We apply this idea to long‐term dietary data of an omnivorous predatory fish species collected from a lake ecosystem over four decades.

Our results showed that the preferred PPMR is independent of individual predator size when the foraging mode (i.e. the major prey type) of the predator is considered while the realized PPMR is size‐dependent regardless of the foraging mode, especially when analysed analogously to previous empirical studies.

We suggest that the apparent inconsistency between theoretical assumption and empirical observation of PPMR is due to the conceptual and methodological confusion and could be resolved by distinguishing between preferred and realized PPMRs. Further, in contrast to the previous arguments based on realized PPMR, we provide the first empirical support for size‐invariant preferred PPMR. Future studies are encouraged to apply our ideas to other species/systems to test the robustness of size‐invariant preferred PPMR and to better describe food web models.

A lay summary is available for this article.
Using functional responses to quantify interaction effects among predators
Authors: Wasserman Ryan J. , Alexander Mhairi E. , Dalu Tatenda , Ellender Bruce R. , Kaiser Horst , Weyl Olaf L. F.
Published: May 2016

Predator diversity alterations have been observed in most ecosystems as a result of the loss and/ or addition of species. This has implications for predator–prey dynamics as non‐trophic interactions among predators, so‐called multiple predator effects (MPE), are known to influence predation success. In addition, there is often a density‐dependent relationship between prey availability and prey consumption (functional response). WhileMPEinvestigations are common in the literature, functional responses have rarely been incorporated into this field of predation ecology.

Here, we outline an experimental procedure that incorporates functional responses into multiple predator effect studies. Using three fish species with different functional traits as model predators (bluegillLepomis macrochirus,southern mouthbrooderPseudocrenilabrus philanderand banded tilapiaTilapia sparrmanii), we assess intra‐ and interspecific predator interaction outcomes on predator–prey dynamics. This was done by contrasting observed functional responses of heterospecific and conspecific combinations of predators with expected responses based on those of individual predators.

Multipredator combinations produced variable results. Bluegill were the only species in which observed conspecific multipredator functional responses matched those of expected based on individual performance (prey risk neutral effects). In contrast, prey risk reduction was observed for both mouthbrooder and tilapia conspecific multipredator trials. Heterospecific combinations revealed strong prey risk reduction effects for mouthbrooder–tilapia and bluegill–tilapia trials, while mouthbrooder–bluegill multipredator functional responses combined additively. These results are discussed within the context of behavioural traits of the species and the development of a trait‐based predictive framework.

Using a functional response approach allowed for the assessment of multiple predator effects across a range of prey densities. We propose that the incorporation of within‐guild predator combinations into classic functional response investigations will enhance predictive capacity development in competition and predation ecology.

Alay summaryis available for this article.
Resist or Comply: The Power Dynamics of Organizational Routines during Mergers
Authors: Safavi Mehdi , Omidvar Omid
Published: May 2016

The role of power and agency in the development of organizational routines is under‐theorized. In this paper, we draw on an in‐depth qualitative case study of a merger between two academic institutions, a college of art and a university, and examine the diverging responses of two organizational routines (admissions and budgeting) during the course of the merger to understand how power dynamics contribute to resistance/compliance of routines. Our findings suggest that the differences in routines’ responses to a merger initiative can be explained by applying Bourdieu's theory of practice and by employing the concepts of field and symbolic capital to unpack power relations in the context of organizational routines, and to disclose why some routine participants can exercise their agency while others cannot. We find that (a) the field within which a routine operates and (b) the actors’ symbolic capital and position‐taking during change implementation shape routines’ responses to organizational change initiatives.
Social Identification and Corporate Irresponsibility: A Model of Stakeholder Punitive Intentions
Authors: Antonetti Paolo , Maklan Stan
Published: May 2016

Scholars hypothesize that retaliations against corporate social irresponsibility (CSI) are more likely when observers share the social identity of the victims. We present a model that explains in‐group bias against irresponsibility and identify collective narcissism as a moderator of this effect. Experiment 1 demonstrates that the effect of identity on retaliations is mediated by the perceived similarity of the victims which reinforces feelings of sympathy towards the victims and anger towards the corporation. These emotions drive stakeholders’ attitudes and retaliations. Our study shows that appraisals of the victims of CSI are an important antecedent of stakeholders’ emotions and behavioural intentions. Our evidence also demonstrates that sympathy, an emotion neglected by past research in this area, has a unique effect on individuals’ reactions. Experiment 2 demonstrates that social identity biases in individual punitive intentions are moderated by individuals’ level of collective narcissism. Collective narcissists see out‐group victims as very dissimilar from the self, whereas individuals with low levels of collective narcissism do not differentiate between victims of CSI on the basis of their identity. We extend knowledge on stakeholders’ reactions to CSI and offer insights to organizations promoting campaigns against irresponsible behaviour or managing the fallout from cases of corporate irresponsibility.
Network Positions and the Probability of Being Acquired: An Empirical Analysis in the Biopharmaceutical Industry
Authors: Mazzola Erica , Perrone Giovanni , Kamuriwo Dzidziso Samuel
Published: May 2016

This paper examines the relationship between the firm's direct ties, its inter‐firm network prominence and its likelihood of being acquired. The authors argue that firm's direct ties and prominence enhance the firm's visibility and signal its quality – and thus foster the firm's likelihood of being acquired. However, higher levels of direct ties and prominence, by providing access to resources and the firm's status, respectively, increase the firm's ability to remain independent and thus reduce its likelihood of being acquired. Thus, the authors posit the overall relation as an inverted U‐shaped. Furthermore, they show that, for firms that undergo an initial public offering, the aforementioned relation becomes much weaker. The hypotheses are empirically tested in the biopharmaceutical industry and important theoretical and managerial implications are discussed.
A Validation Study of Bullet and Cartridge Case Comparisons Using Samples Representative of Actual Casework
Authors: Smith Tasha P. , Andrew Smith G. , Snipes Jeffrey B.
Published: May 2016

The foundation of firearm and tool mark identification is that no two tools should produce the same microscopic marks on two separate objects that they would be inaccurately or wrongly identified. Studies addressing the validity of identification infrequently employ tests that mirror realistic casework scenarios. This study attempted to do so using a double‐blind process, reducing test‐taking bias. Test kits including bullets and cartridge cases but not the associated firearms were completed by 31 analysts from 22 agencies. Analysis of the results demonstrated an overall error rate of 0.303%, sensitivity of 85.2%, and specificity of 86.8%. Variability in performance across examiners is addressed, and the effect of examiners’ years of experience on identification accuracy is explored. Finally, the article discusses the importance of studies using realistic case work scenarios when validating the field's performance and in providing courts with usable indicators of the accuracy of firearm and tool mark identification.
Does the diversification rate of endemic birds of mainland China follow abrupt, gradual shifting or constant patterns?
Authors: CHEN Youhua
Published: May 2016

In this brief report, time‐varying (including both gradual and abrupt change) and time‐constant diversification models are fitted on a phylogeny of endemic birds of mainland China to test the diversification patterns of endemic birds in the region. The results show that phylogeny of endemic birds is best quantified by a constant‐rate diversification model through model comparison. Limitations of the study are discussed. In particular, ignorance of non‐endemic taxa and the limited sampling of endemic taxa could influence the conclusions of the study.
Effects of low‐oxygen conditions on embryo growth in the painted turtle, Chrysemys picta
Authors: CORDERO Gerardo A. , KARNATZ Matthew L. , SVENDSEN Jon C. , GANGLOFF Eric J.
Published: May 2016

Low‐oxygen conditions (hypoxia; <21% O2) are considered unfavorable for growth; yet, embryos of many vertebrate taxa develop successfully in hypoxic subterranean environments. Although enhanced tolerance to hypoxia has been demonstrated in adult reptiles, such as in the painted turtle (Chrysemys picta), its effects on sensitive embryo life stages warrant attention. We tested the hypothesis that short‐term hypoxia negatively affects growth during day 40 of development in C. picta, when O2 demands are highest in embryos. A brief, but severe, hypoxic event (5% O2 for 0.5 h) moderately affected embryo growth, causing a 13% reduction in mass (relative to a normoxic control). The same condition had no effect during day 27; instead, a nearly anoxic event (1% O2 for 72 h) caused a 5% mass reduction. All embryos survived the egg incubation period. Our study supports the assumption that reptilian embryos are resilient to intermittently low O2 in subterranean nests. Further work is needed to ascertain responses to suboptimal O2 levels while undergoing dynamic changes in developmental physiology.