bluuebunny/crossref_metadata_2025_split huggingface dataset:
Lectin‐Array Blotting
Authors: Pazos Raquel , Echevarria Juan , Hernandez Alvaro , Reichardt Niels‐Christian 
Published: September 2017

AbstractAberrant protein glycosylation is a hallmark of cancer, infectious diseases, and autoimmune or neurodegenerative disorders. Unlocking the potential of glycans as disease markers will require rapid and unbiased glycoproteomics methods for glycan biomarker discovery. The present method is a facile and rapid protocol for qualitative analysis of protein glycosylation in complex biological mixtures. While traditional lectin arrays only provide an average signal for the glycans in the mixture, which is usually dominated by the most abundant proteins, our method provides individual lectin binding profiles for all proteins separated in the gel electrophoresis step. Proteins do not have to be excised from the gel for subsequent analysis via the lectin array but are transferred by contact diffusion from the gel to a glass slide presenting multiple copies of printed lectin arrays. Fluorescently marked glycoproteins are trapped by the printed lectins via specific carbohydrate‐lectin interactions and after a washing step their binding profile with up to 20 lectin probes is analyzed with a fluorescent scanner. The method produces the equivalent of 20 lectin blots in a single experiment, giving detailed insight into the binding epitopes present in the fractionated proteins. © 2017 by John Wiley & Sons, Inc.
Automated Tracking of Cell Migration with Rapid Data Analysis
Authors: DuChez Brian J. 
Published: September 2017

AbstractCell migration is essential for many biological processes including development, wound healing, and metastasis. However, studying cell migration often requires the time‐consuming and labor‐intensive task of manually tracking cells. To accelerate the task of obtaining coordinate positions of migrating cells, we have developed a graphical user interface (GUI) capable of automating the tracking of fluorescently labeled nuclei. This GUI provides an intuitive user interface that makes automated tracking accessible to researchers with no image‐processing experience or familiarity with particle‐tracking approaches. Using this GUI, users can interactively determine a minimum of four parameters to identify fluorescently labeled cells and automate acquisition of cell trajectories. Additional features allow for batch processing of numerous time‐lapse images, curation of unwanted tracks, and subsequent statistical analysis of tracked cells. Statistical outputs allow users to evaluate migratory phenotypes, including cell speed, distance, displacement, and persistence, as well as measures of directional movement, such as forward migration index (FMI) and angular displacement. © 2017 by John Wiley & Sons, Inc.
In Vitro Reconstitution of the Endoplasmic Reticulum
Authors: Ferencz Csilla‐Maria , Guigas Gernot , Veres Andreas , Neumann Brigitte , Stemmann Olaf , Weiss Matthias 
Published: September 2017

AbstractReconstitution of cellular organelles in vitro offers the possibility to perform quantitative and qualitative experiments in a controlled environment that cannot be done with the same accuracy in living cells. Following a previous report, the subsequent list of protocols describes how to reconstitute and quantify a tubular ER network in vitro based on purified microsomes from culture cells and cytosol from Xenopus laevis egg extracts. Biological material preparation and reconstitution assays require mostly basic laboratory instrumentation and chemicals, and can be executed without any specific training, making them appealing to a wide range of laboratories. Moreover, to promote conditions that are markedly more reflective of in vivo environments, this method describes for the first time in the literature, the purification of microsomes from HeLa cells in some detail. Basic Protocol 1 in this article describes the reconstitution process on different substrates including the associated fluorescence imaging process. Purification of ER microsomes and cytosol, both of which are needed for this approach, are described in detail in Support Protocols 1 and 2, respectively. Coating of surfaces with polyacrylamide gels is described in Support Protocol 3. Basic Protocol 2 outlines how to segment and skeletonize fluorescence images of ER networks, and how to quantify segment lengths between the network's branching points. The described quantitative evaluation provides a meaningful approach to analyze the topology and geometry of organelle structures. © 2017 by John Wiley & Sons, Inc.
Detecting the Interaction of Peptide Ligands with Plant Membrane Receptors
Authors: Hind Sarah Refi , Hoki Jason S. , Baccile Joshua A. , Boyle Patrick C. , Schroeder Frank C. , Martin Gregory B. 
Published: September 2017

AbstractThe field of plant receptor biology has rapidly expanded in recent years, however the demonstration of direct interaction between receptor‐ligand pairs remains a challenge. Click chemistry has revolutionized small molecule research but lacks popularity in plant research. Here we describe a method that tests for the direct physical interaction of a candidate receptor protein and a peptide ligand. This protocol describes the generation of the ligand probe, transient expression of a receptor protein, enrichment of membrane‐bound receptors, photo‐crosslinking and click chemistry‐mediated reporter addition, and detection of the receptor‐ligand complex. Copper‐based click chemistry confers several advantages, including the versatility to use almost any azide‐containing reporter molecule for detection or visualization of the complex and enables addition of the reporter molecule after receptor‐ligand binding which reduces the need for bulky ligand modifications that could interfere with the interaction. © 2017 by John Wiley & Sons, Inc.
Improved Genetic Transformation of Sugarcane (Saccharum spp.) Embryogenic Callus Mediated by Agrobacterium tumefaciens
Authors: Basso Marcos Fernando , da Cunha Bárbara Andrade Dias Brito , Ribeiro Ana Paula , Martins Polyana Kelly , de Souza Wagner Rodrigo , de Oliveira Nelson Geraldo , Nakayama Thiago Jonas , Augusto das Chagas Noqueli Casari Raphael , Santiago Thais Ribeiro , Vinecky Felipe , Cançado Letícia Jungmann , de Sousa Carlos Antônio Ferreira , de Oliveira Patricia Abrão , de Souza Silvana Aparecida Creste Dias , Cançado Geraldo Magela de Almeida , Kobayashi Adilson Kenji , Molinari Hugo Bruno Correa 
Published: September 2017

AbstractSugarcane (Saccharum spp.) is a monocotyledonous semi‐perennial C4 grass of the Poaceae family. Its capacity to accumulate high content of sucrose and biomass makes it one of the most important crops for sugar and biofuel production. Conventional methods of sugarcane breeding have shown several limitations due to its complex polyploid and aneuploid genome. However, improvement by biotechnological engineering is currently the most promising alternative to introduce economically important traits. In this work, we present an improved protocol for Agrobacterium tumefaciens‐mediated transformation of commercial sugarcane hybrids using immature top stalk‐derived embryogenic callus cultures. The callus cultures are transformed with preconditioned A. tumefaciens carrying a binary vector that encodes expression cassettes for a gene of interest and the bialaphos resistance gene (bar confers resistance to glufosinate‐ammonium herbicide). This protocol has been used to successfully transform a commercial sugarcane cultivar, SP80‐3280, highlighting: (i) reduced recalcitrance and oxidation; (ii) high yield of embryogenic callus; (iii) improved selection; and (iv) shoot regeneration and rooting of the transformed plants. Altogether, these improvements generated a transformation efficiency of 2.2%. This protocol provides a reliable tool for a routine procedure for sugarcane improvement by genetic engineering. © 2017 by John Wiley & Sons, Inc.
Isolation of Novel MAMP‐like Activities and Identification of Cognate Pattern Recognition Receptors in Arabidopsis thaliana Using Next‐Generation Sequencing (NGS)–Based Mapping
Authors: Fan Li , Chae Eunyoung , Gust Andrea A. , Nürnberger Thorsten 
Published: September 2017

AbstractActivation of pattern‐triggered plant immunity requires recognition of microbe‐derived molecular patterns (MAMPs) by plant‐encoded pattern recognition receptors (PRRs). Many plant PRRs are found in selected plant genera only. Transfer of single PRRs or of cassettes expressing several PRRs (PRR stacking) across plant genus boundaries offers the potential to boost disease resistance by improving pathogen recognition features in economically important crop plants. The success of such an approach is most dependent on the availability of a large number of plant PRRs. Here, an efficient method for the identification of novel PRRs in the model plant Arabidopsis thaliana (hereafter, Arabidopsis for simplicity) is described. This method takes advantage of natural variation in microbial pattern sensitivity among hundreds of Arabidopsis accessions currently available. Identification of pattern‐sensitive as well as pattern‐insensitive accessions facilitates next‐generation sequencing (NGS)–assisted mapping of PRRs. This approach is potentially applicable to the identification of PRRs that recognize patterns of any chemical nature. © 2017 by John Wiley & Sons, Inc.
PhenoGraph and viSNE facilitate the identification of abnormal T‐cell populations in routine clinical flow cytometric data
Authors: DiGiuseppe Joseph A. , Cardinali Jolene L. , Rezuke William N. , Pe'er Dana 
Published: September 2017

BackgroundFlow cytometric identification of neoplastic T‐cell populations is complicated by the wide range of phenotypic abnormalities in T‐cell neoplasia, and the diverse repertoire of reactive T‐cell phenotypes. We evaluated whether a recently described clustering algorithm, PhenoGraph, and dimensionality‐reduction algorithm, viSNE, might facilitate the identification of abnormal T‐cell populations in routine clinical flow cytometric data.MethodsWe applied PhenoGraph and viSNE to peripheral blood mononuclear cells labeled with a single 8‐color T/NK‐cell antibody combination. Individual peripheral blood samples containing either a T‐cell neoplasm or reactive lymphocytosis were analyzed together with a cohort of 10 normal samples, which established the location and identity of normal mononuclear‐cell subsets in viSNE displays.ResultsPhenoGraph‐derived subpopulations from the normal samples formed regions of phenotypic similarity in the viSNE display describing normal mononuclear‐cell subsets, which correlated with those obtained by manual gating (r2 = 0.99, P < 0.0001). In 24 of 24 cases of T‐cell neoplasia with an aberrant phenotype, compared with 4 of 17 cases of reactive lymphocytosis (P = 1.4 × 10−7, Fisher Exact test), PhenoGraph‐derived subpopulations originating exclusively from the abnormal sample formed one or more distinct phenotypic regions in the viSNE display, which represented the neoplastic T cells, and reactive T‐cell subpopulations not present in the normal cohort, respectively. The numbers of neoplastic T cells identified using PhenoGraph/viSNE correlated with those obtained by manual gating (r2 = 0.99; P < 0.0001).ConclusionsPhenoGraph and viSNE may facilitate the identification of abnormal T‐cell populations in routine clinical flow cytometric data. © 2017 Clinical Cytometry Society
Association between insulin‐induced weight change and CVD mortality: Evidence from a historic cohort study of 18,814 patients in UK primary care
Authors: Anyanwagu Uchenna , Mamza Jil , Donnelly Richard , Idris Iskandar 
Published: September 2017

AbstractBackgroundThis study explores the association of insulin‐induced weight (wt) gain on cardiovascular outcomes and mortality among patients with type 2 diabetes (T2D) following insulin initiation using real‐world data.MethodsA historical cohort study was performed in 18,814 adults with insulin‐treated T2D derived from the UK The Health Improvement Network database. Based on the average weight change of 5 kg, 1 year postinsulin initiation, patients were grouped into 5 categories (>5 kg wt loss; 1.0‐5.0 kg wt loss; no wt change; 1.0‐5.0 kg wt gain; >5.0 kg wt gain) and followed‐up for 5 years. Cox proportional hazard models and Kaplan‐Meier estimators were fitted to estimate the hazards of a 3‐point composite of nonfatal myocardial infarction, stroke, and all‐cause mortality between categories.ResultsThe median age was 62.8 (IQR: 52.3–71.8) years, HbA1c: 8.6% (IQR: 7.4–9.8) and mean BMI: 31.8 (6.5) kg/m2. The 5 year probability of survival differed significantly within the wt‐change categories (log‐rank test P value = .0005). Only 1963 composite events occurred. Compared with the weight‐neutral group, the risk of composite events was 31% greater in the >5 kg wt‐loss group (aHR: 1.31; 95% CI: 1.02, 1.68), the same in the 1.0 to 5.0 kg wt‐gain category, but nonsignificantly increased in the 1.0 to 5.0 kg wt loss (15%) and >5.0 kg wt gain (13%) categories, respectively. In the obese subgroup, this risk was 50% (aHR: 1.50, 95% CI: 1.08‐2.08) more in the >5 kg weight‐loss group compared with the weight‐neutral group.ConclusionInsulin‐induced weight gain did not translate to adverse cardiovascular outcomes and mortality in patients with T2D. These data provide reassurance on the cardiovascular safety of insulin patients with T2D.
A budget of algal production and consumption by herbivorous fish in an herbivore fisheries management area, Maui, Hawaii
Authors: Kelly Emily L. A. , Eynaud Yoan , Williams Ivor D. , Sparks Russell T. , Dailer Meghan L. , Sandin Stuart A. , Smith Jennifer E. 
Published: September 2017

AbstractThe relative rates of carbon fixed by primary producers vs. consumption by primary consumers shape the community of organisms in ecosystems. On coral reefs, it is important to understand the demography of algae, given known competitive dynamics with reef‐building corals. Numerous studies have shown that fleshy algal abundance is enhanced in the absence of top‐down control by herbivores on coral reefs. However, fewer studies have quantified and compared rates of production by the benthic fleshy algal community and consumption by herbivores. Here, we estimate a budget for fleshy algal growth and herbivorous fish consumption on a Hawaiian coral reef based upon integration of field‐measured and taxonomically specific variables. Data were collected at Kahekili Herbivore Fisheries Management Area, Maui, established in 2009, in which taking of herbivorous fish and urchins is prohibited. Daily algal production was determined by quantifying benthic community composition, standing stock of algal biomass, and growth rates of common algal components. Consumption was determined using distributions of biomass and size classes of herbivorous fish species, consumption rates of herbivores on different algal species, and herbivore bite sizes. Our results show that throughout the first five years of herbivore protection, algal production consistently exceeded the grazing capacity of the herbivorous fish assemblage, but by a diminishing margin since 2010 (consumption 20.8% of production) to the end of the study in 2014 (consumption 67.0% of production). Further, larger size classes of herbivorous fishes in the scraper/excavator herbivore guild contributed more to consumption in later years, which could have additional feedbacks that promote reef‐building taxa. Projecting the budget using data from a neighboring decades‐long protected herbivorous fish assemblage indicated that the production and consumption budget for Kahekili could become balanced in future with continued management.
Is initial post‐disturbance regeneration indicative of longer‐term trajectories?
Authors: Gill Nathan S. , Jarvis Daniel , Veblen Thomas T. , Pickett Steward T. A. , Kulakowski Dominik 
Published: September 2017

AbstractThe ability to estimate and model future vegetation dynamics is a central focus of contemporary ecology and is essential for understanding future ecological trajectories. It is therefore critical to understand when the influence of initial post‐disturbance regeneration versus stochastic processes dominates long‐term post‐disturbance ecological processes. Often, conclusions about post‐disturbance dynamics are based upon initial regeneration in the years immediately after disturbances. However, the degree to which initial post‐disturbance regeneration indicates longer‐term trends is likely to be contingent on the types, intensities, and combinations of disturbances, as well as pre‐disturbance ecosystem structure and composition. Our relatively limited understanding of why initial post‐disturbance regeneration is sometimes a poor predictor of future ecosystem trajectories represents a critical gap in post‐disturbance ecological forecasting. We studied the composition and density of regeneration of tree species following wind blowdown in 1997, wildfire in 2002, and compounded disturbances by blowdown and wildfire in subalpine forests of Colorado. We examined regeneration of Picea engelmannii, Abies lasiocarpa, Pinus contorta, and Populus tremuloides in 180 permanent plots across 12 sites (classified by pre‐disturbance age and composition) in 2003, 2010, and 2015. At sites that were blown down but not burned, regeneration was dense and dominated by Picea and Abies. At these sites, regeneration observed from 2003 to 2005 (hereafter initial regeneration) was also highly predictive of regeneration 5–10 yr later. In contrast, at sites that were burned and sites that were blown down and burned, regeneration was less dense and dominated by a mix of species. At these sites, initial regeneration was a poor predictor of longer‐term trends as species dominance and overall density fluctuated over the 13‐yr period. These findings call into question our ability to confidently predict ecosystem trajectories based upon observations made in the years immediately after large, severe disturbances such as wildfires and compounded disturbances. As compounded disturbances become more common under climatically driven changes in disturbance regimes, post‐disturbance ecosystem trajectories may become increasingly stochastic and unpredictable.
Bayesian networks elucidate interactions between fire and other drivers of terrestrial fauna distributions
Authors: Hradsky Bronwyn A. , Penman Trent D. , Ababei Dan , Hanea Anca , Ritchie Euan G. , York Alan , Di Stefano Julian 
Published: September 2017

AbstractFire is a major driver of community composition and habitat structure and is extensively used as an ecological management tool in flammable landscapes. Interactions between fire and other processes that affect animal distributions, however, cause variation in faunal responses to fire and limit our ability to identify appropriate fire management regimes for biodiversity conservation. Bayesian networks (BNs) have not previously been used to examine terrestrial faunal distributions in relation to fire, but offer an alternative statistical framework for modeling complex environmental relationships as they explicitly capture interactions between predictor variables. We developed a conceptual model of the interactions between drivers of faunal distributions in fire‐affected landscapes, and then used a non‐parametric BN modeling approach to describe and quantify these relationships for a suite of terrestrial native mammal species. We also tested whether BNs could be used to predict these species’ distributions using only remote‐sensed or mapped variables. Data were collected at 113 sites across 47,000 ha of continuous eucalypt forest in the Otway Ranges, southeastern Australia; time‐since‐fire (TSF) ranged from six months to 74 yr. Habitat complexity increased with TSF and forest wetness. Critical‐weight‐range (35–5500 g) marsupials and rodents were generally more likely to occur at long unburnt sites with high habitat complexity, and in wetter forest types. In contrast, large grazers and browsers preferred less complex habitats and younger or drier forest. Species occurrences were more strongly affected by habitat complexity than TSF, coarse woody debris cover, or invasive predator (Vulpes vulpesorFelis catus) occurrence. Bayesian network models effectively discriminated between the presence and absence of most native mammal species, even when only provided with data on remote‐sensed or mapped variables (i.e., without field‐assessed data such as habitat complexity). Non‐parametric BNs are an effective technique for explicitly modeling the complex and context‐dependent influence of fire history on faunal distributions, and may reduce the need to collect extensive field data on habitat structure and other proximate drivers.
Evaluation of an acute oral gavage method for assessment of pesticide toxicity in terrestrial amphibians
Authors: Fort Douglas J. , Mathis Michael B. , Kee Faith , Whatling Paul , Clerkin David , Staveley Jane , Habig Clifford 
Published: September 2017

Abstract

Development of an acute oral toxicity test with a terrestrial-phase amphibian was considered necessary to remove the uncertainty within the field of agrochemical risk assessments. The bullfrog (Lithobates catesbeianus) was selected for use as it is a representative of the family Ranidae and historically this species has been used as an amphibian test model species. Prior to definitive study, oral gavage methods were developed with fenthion and tetraethyl pyrophosphate. Dimethoate and malathion were subsequently tested with both male and female juvenile bullfrogs in comprehensive acute oral median lethal dose (LD50) studies. Juvenile bullfrogs were administered a single dose of the test article via oral gavage of a single gelatin capsule of dimethoate technical (dimethoate) or neat liquid Fyfanon® Technical (synonym malathion), returned to their respective aquaria, and monitored for survival for 14 d. The primary endpoint was mortality, whereas behavioral responses, food consumption, body weight, and snout–vent length (SVL) were used to evaluate indications of sublethal toxicity (secondary endpoints). Acute oral LD50 values (95% fiducial interval) for dimethoate were 1459 (1176–1810, males) and 1528 (1275–1831, females), and for malathion they were 1829 (1480–2259, males) and 1672 (1280–2183, females) mg active substance/kg body weight, respectively. Based on the results of these studies, the methodology for the acute oral gavage administration of test items to terrestrial-phase amphibians was demonstrated as being a practical method of providing data for risk assessments. Environ Toxicol Chem 2018;37:436–450. © 2017 SETAC