bluuebunny/crossref_metadata_2025 huggingface dataset:
Elucidation of Codon Usage Signatures across the Domains of Life
Authors: Novoa Eva Maria , Jaillon Olivier , Jungreis Irwin , Kellis Manolis
Published: September 2018

Due to the degeneracy of the genetic code, multiple codons are translated into the same amino acid. Despite being ‘synonymous’, these codons are not equally used. Selective pressures are thought to drive the choice among synonymous codons within a genome, while GC content, which is generally attributed to mutational drift, is the major determinant of interspecies codon usage bias. Here we find that in addition to the bias caused by GC content, inter-species codon usage signatures can also be detected. More specifically, we show that a single amino acid, arginine, is the major contributor to codon usage bias differences across domains of life. We then exploit this finding, and show that the identified domain-specific codon bias signatures can be used to classify a given sequence into its corresponding domain with high accuracy. Considering that species belonging to the same domain share similar tRNA decoding strategies, we then wondered whether the inclusion of codon autocorrelation patterns might improve the classification performance of our algorithm. However, we find that autocorrelation patterns are not domain-specific, and surprisingly, are unrelated to tRNA reusage, in contrast to the common belief. Instead, our results reveal that codon autocorrelation patterns are a consequence of codon optimality throughout a sequence, where highly expressed genes display autocorrelated ‘optimal’ codons, whereas lowly expressed genes display autocorrelated ‘non-optimal’ codons.
A generalized observation confirmation model to account for false positive error in species detection-nondetection data
Authors: Clare John D. J. , Zuckerberg Benjamin , Townsend Philip A.
Published: September 2018

Spatially-indexed repeated detection-nondetection data is widely collected by ecologists interested in estimating parameters associated with species distribution, relative abundance, phenology, and more while accounting for imperfect detection. Recent model development has focused on accounting for false positive error as well, given growing recognition that misclassification is common across many sampling protocols. To date, however, the development of model-based solutions to false positive error has been largely restricted to occupancy models. We describe a general form of the observation confirmation protocol originally described for occupancy estimation that permits investigators to flexibly and intuitively extend several models for detection-nondetection data to account for false positive error. Simulation results demonstrate that estimators for relative abundance and arrival time exhibit relative bias greater than 20% under realistic levels of false positive prevalence (e.g., 5% of detections are false positive). Bias increases as true and false positives occur in more distinct places or times, but can also be sensitive to the values of the state variables of interest, sampling design, and sampling efficiency. Results from an empirical study focusing on patterns of gray fox relative abundance across Wisconsin, USA suggest that false positive error can also distort estimated spatial patterns often used to guide decision-making. The extended estimators described within typically improve performance at any level of confirmation, and when false positive error occurs at random and constitutes less than 10% of all detections, the estimators are essentially unbiased when more than 50 observations can be confirmed as true or false positives. The generalized form of the observation-confirmation protocol is a flexible model-based solution to false positive error useful for researchers collecting data with sampling devices like trail or smartphone cameras, acoustic recorders, or other techniques where classifications can be reviewed post-hoc.
Deeply conserved susceptibility in a multi-host, multi-parasite system
Authors: Barrow Lisa N , McNew Sabrina M , Mitchell Nora , Galen Spencer C , Lutz Holly L , Skeen Heather , Valqui Thomas , Weckstein Jason D , Witt Christopher C
Published: September 2018

Variation in susceptibility is ubiquitous in multi-host, multi-parasite assemblages, and can have profound implications for ecology and evolution. The extent to which susceptibility is phylogenetically conserved among hosts is poorly understood and has rarely been appropriately tested. We screened for haemosporidian parasites in 3983 birds representing 40 families and 523 species, spanning ~4500 meters elevation in the tropical Andes. To quantify the influence of host phylogeny on infection status, we applied Bayesian phylogenetic multilevel models that included a suite of environmental, spatial, temporal, life history, and ecological predictors. We found evidence of deeply-conserved susceptibility across the avian tree; host phylogeny explained substantial variation in infection rate, and results were robust to phylogenetic uncertainty. Our study suggests that susceptibility is governed, in part, by conserved, latent aspects of anti-parasite defense. This demonstrates the importance of deep phylogeny for understanding the outcomes of present-day ecological interactions.
Detection of residual disease after neoadjuvant therapy in breast cancer using personalized circulating tumor DNA analysis
Authors: McDonald Bradon R. , Contente-Cuomo Tania , Sammut Stephen-John , Odenheimer-Bergman Ahuva , Ernst Brenda , Perdigones Nieves , Chin Suet-Feung , Farooq Maria , Cronin Patricia A. , Anderson Karen S. , Kosiorek Heidi E. , Northfelt Donald W. , McCullough Ann E. , Patel Bhavika K. , Caldas Carlos , Pockaj Barbara A. , Murtaza Muhammed
Published: September 2018

Accurate detection of minimal residual disease (MRD) can guide individualized management of early stage cancer patients, but current diagnostic approaches lack adequate sensitivity. Circulating tumor DNA (ctDNA) analysis has shown promise for recurrence monitoring but MRD detection immediately after neoadjuvant therapy or surgical resection has remained challenging. We have developed TARgeted DIgital Sequencing (TARDIS) to simultaneously analyze multiple patient-specific cancer mutations in plasma and improve sensitivity for minute quantities of residual tumor DNA. In 77 reference samples at 0.03%-1% mutant allele fraction (AF), we observed 93.5% sensitivity. Using TARDIS, we analyzed ctDNA in 34 samples from 13 patients with stage II/III breast cancer treated with neoadjuvant therapy. Prior to treatment, we detected ctDNA in 12/12 patients at 0.002%-1.04% AF (0.040% median). After completion of neoadjuvant therapy, we detected ctDNA in 7/8 patients with residual disease observed at surgery and in 1/5 patients with pathological complete response (odds ratio, 18.5, Fisher’s exact p=0.032). These results demonstrate high accuracy for a personalized blood test to detect residual disease after neoadjuvant therapy. With additional clinical validation, TARDIS could identify patients with molecular complete response after neoadjuvant therapy who may be candidates for nonoperative management.

One Sentence Summary

A personalized ctDNA test achieves high accuracy for residual disease.
Continuous and discrete quantity discrimination in tortoises
Authors: Gazzola Andrea , Vallortigara Giorgio , Pellitteri-Rosa Daniele
Published: September 2018

The ability to estimate quantity, which is crucially important in several aspects of animal behaviour (e.g., foraging), has been extensively investigated in most taxa, with the exception of reptiles. The few studies available, in lizards, report lack of spontaneous discrimination of quantity, which may suggest that reptiles could represent an exception in numerical abilities among vertebrates. We investigated the spontaneous ability of Hermann’s tortoises (Testudo hermanni) to select the larger quantity of food items. Tortoises showed able to choose the larger food item when exposed with two options differing in size (0.25, 0.50, 0.67 and 0.75 ratio) and when presented with two groups differing in numerousness (1 versus 4, 2 versus 4, 2 versus 3 and 3 versus 4 items). The tortoises succeeded in both size and numerousness discrimination, and their performance appeared to depend on the ratio of items to be discriminated (thus following Weber’s Law). These findings in chelonians provide evidence of an ancient system for the extrapolation of numerical magnitudes from given sets of elements, shared among vertebrates.
Preventions of oral self-mutilation in patients with Lesch-Nyhan syndrome
Authors: de Carvalho Lorevanda V. , de Oliveira Luan F. , Chaves Tiago F.
Published: September 2018

Lesch-Nyhan syndrome (LNS) is a rare genetic disorder linked to the X chromosome that implies an inborn error of metabolism, due to deficiency in the purine metabolic pathway, affected individuals present neurological symptoms, such as cognitive and motor impairment, besides behavioral changes such as self and hetero-aggression. This characteristic aggressive behavior leads to insults and self-mutilation, thus requiring methods of self-protection, some invasive and permanent, such as tooth extraction. Noninvasive methods, such as the use of oral protectors, pharmacological therapies and botulinum toxin A applications, are also employed to decrease discomfort and the implications of such behavior. We describe briefly the methods of prevention against oral self-mutilation in patients with Lesch-Nyhan syndrome found in the literature.
Visualizing metabolic network dynamics through time-series metabolomics data
Authors: Buchweitz Lea F. , Yurkovich James T. , Blessing Christoph M. , Kohler Veronika , Schwarzkopf Fabian , King Zachary A. , Yang Laurence , Jóhannsson Freyr , Sigurjónsson Ólafur E. , Rolfsson Óttar , Heinrich Julian , Dräger Andreas
Published: September 2018

New technologies have given rise to an abundance of -omics data, particularly metabolomics data. The scale of these data introduces new challenges for the interpretation and extraction of knowledge, requiring the development of new computational visualization methodologies. Here, we present a new method for the visualization of time-course metabolomics data within the context of metabolic network maps. We demonstrate the utility of this method by examining previously published data for two cellular systems—the human platelet and erythrocyte under cold storage for use in transfusion medicine.

The results comprise two animated videos that allow for new insights into the metabolic state of both cell types. In the case study of the platelet metabolome during storage, the new visualization technique elucidates a nicotinamide accumulation which mirrors that of hypoxanthine and might, therefore, reflect similar pathway usage. This visual analysis provides a possible explanation for why the salvage reactions in purine metabolism exhibit lower activity during the first few days of the storage period. The second case study displays drastic changes in specific erythrocyte metabolite pools at different times during storage at different temperatures.

In conclusion, this new visualization technique introduced in this article constitutes a well-suitable approach for large-scale network exploration and advances hypothesis generation. This method can be applied to any system with data and a metabolic map to promote visualization and understand physiology at the network level. More broadly, we hope that our approach will provide the blueprints for new visualizations of other longitudinal -omics data types.

AUTHOR SUMMARY

Profiling the dynamic state of a metabolic network through the use of time-course metabolomics technologies allows insights into cellular biochemistry. Interpreting these data together at the systems level provides challenges that can be addressed through the development of new visualization approaches. Here, we present a new method for the visualization of time-course metabolomics data that integrates data into an existing metabolic network map. In brief, the metabolomics data are visualized directly on a network map with dynamic elements (nodes that either change size, fill level, or color corresponding with the concentration) while the user controls the time series (i.e., which time point is being displayed) through a graphical interface. We provide short videos that illustrate the utility of this method through its application to existing data sets for the human platelet and erythrocyte. The results presented here give blueprints for the development of visualization methods for other time-course -omics data types that attempt to understand systems-level physiology.
Rapid and stable microbial community assembly in the headwaters of third-order stream
Authors: Teachey Morgan E. , McDonald Jacob M. , Ottesen Elizabeth A.
Published: September 2018

Small streams and their headwaters are a key source of microbial diversity in fluvial systems and serve as an entry point for bacteria from the surrounding landscape. Community assembly processes occurring in these streams shape downstream population structure and nutrient cycles. To elucidate the development and stability of microbial communities along the length of a first through third order stream, fine-scale temporal and spatial sampling regimes were employed along McNutt Creek in Athens, Georgia, USA. 16S rRNA gene libraries were constructed from samples collected on a single day from 19 sites spanning the first 16.76 km of the stream. Selected sites at the upper, mid, and lower reaches of the stream were sampled daily for 11 days to evaluate community variability over time. In a second study, sites at and near the creek’s headwaters were sampled daily for 11 days to understand the initial stages of bacterioplankton community assembly. In all studies, we observed decreasing alpha and beta diversity with increasing downstream distance. These trends were accompanied by the enrichment of a small fraction of taxa found at low abundance in the furthest-upstream environments. Similar sets of taxa consistently increased significantly in relative abundance in downstream samples over time scales ranging from 1 day to 1 year, many of which belong to microbial clades known to be abundant in freshwater environments. These results underpin the importance of headwaters as the site of rapid in-stream selection that results in the reproducible establishment of a highly stable community of freshwater riverine bacteria.

Importance

Headwater streams are critical introduction points of microbial diversity for larger connecting rivers and play key roles in the establishment of taxa that partake in in-stream nutrient cycling. We examined microbial community composition of a first- through third-order stream using fine-scale temporal and spatial regimes. Our results show that the bacterioplankton community develops rapidly and predictably from the headwater population with increasing total stream length. Along the length of the stream, the microbial community exhibits substantial diversity loss and enriches repeatedly for select taxa across days and years, although the relative abundances of individual taxa vary over time and space. This repeated enrichment of a stable stream community likely contributes to the stability and flexibility of downstream communities.
A multi-omics data simulator for complex disease studies and its application to evaluate multi-omics data analysis methods for disease classification
Authors: Chung Ren-Hua , Kang Chen-Yu
Published: September 2018

An integrative multi-omics analysis approach that combines multiple types of omics data including genomics, epigenomics, transcriptomics, proteomics, metabolomics, and microbiomics, has become increasing popular for understanding the pathophysiology of complex diseases. Although many multi-omics analysis methods have been developed for complex disease studies, there is no simulation tool that simulates multiple types of omics data and models their relationships with disease status. Without such a tool, it is difficult to evaluate the multi-omics analysis methods on the same scale and to estimate the sample size or power when planning a new multi-omics disease study. We developed a multi-omics data simulator OmicsSIMLA, which simulates genomics (i.e., SNPs and copy number variations), epigenomics (i.e., whole-genome bisulphite sequencing), transcriptomics (i.e., RNA-seq), and proteomics (i.e., normalized reverse phase protein array) data at the whole-genome level. Furthermore, the relationships between different types of omics data, such as meQTLs (SNPs influencing methylation), eQTLs (SNPs influencing gene expression), and eQTM (methylation influencing gene expression), were modeled. More importantly, the relationships between these multi-omics data and the disease status were modeled as well. We used OmicsSIMLA to simulate a multi-omics dataset for breast cancer under a hypothetical disease model, and used the data to compare the performance among existing multi-omics analysis methods in terms of disease classification accuracy and run time. Our results demonstrated that complex disease mechanisms can be simulated by OmicsSIMLA, and a random forest-based method showed the highest prediction accuracy when the multi-omics data were properly normalized.
The requirement for the amino acid co-germinant duringC. difficilespore germination is influenced by mutations inyabGandcspA
Authors: Shrestha Ritu , Sorg Joseph A.
Published: September 2018

Clostridium difficilespore germination is critical for the transmission of disease.C. difficilespores germinate in response to cholic acid derivatives, such as taurocholate (TA), and amino acids, such as glycine or alanine. Although the bile acid germinant receptor is known, the amino acid germinant receptor has remained elusive. Here, we used EMS mutagenesis to generate mutants with altered requirements for the amino acid co-germinant, similar to the strategy used previously to identify the bile acid receptor, CspC. Surprisingly, we identified strains that do not require amino acids as co-germinants, and the mutant spores germinated in response to TA alone. Upon sequencing these mutants, we identified different mutations inyabG.InC. difficile, yabGexpression is required for the processing of CspBA to CspB and CspA and preproSleC to proSleC during spore formation. A definedyabGmutant exacerbated the EMS mutant phenotype. Moreover, we found that various mutations incspAcaused spores to germinate in the presence of TA alone without the requirement of an amino acid. Thus, our study provides evidence that apart from regulating the CspC levels in the spore, CspA is important for recognition of amino acids as co-germinants duringC. difficilespore germination and that two pseudoproteases (CspC and CspA) function as theC. difficilegerminant receptors.
Unexpected mutual regulation underlies paralogue functional diversification and promotes maturation of a protective epithelial tissue
Authors: Gurska Daniela , Vargas Jentzsch Iris M. , Panfilio Kristen A.
Published: September 2018

InsectHox3/zengenes represent an evolutionary hotspot for changes in function and copy number. Single orthologues are required either for early specification or late morphogenesis of the extraembryonic tissues, which protect the embryo. The tandemly duplicatedzenparalogues of the beetleTribolium castaneumpresent a unique opportunity to investigate both functions in a single species. We dissect the paralogues’ expression dynamics (transcript and protein) and transcriptional targets (RNA-seq after RNAi) throughout embryogenesis. We identify an unexpected role of Tc-Zen2 in repression ofTc-zen1, generating a negative feedback loop that promotes developmental progression. Tc-Zen2 regulation is dynamic, including within co-expressed multigene loci. We also show that extraembryonic development is the major event within the transcriptional landscape of late embryogenesis and provide a global molecular characterization of the extraembryonic serosal tissue. Altogether, we propose that paralogue mutual regulation arose progressively and drove multiple instances ofzensubfunctionalization, leading to complementary extant roles.
Universal Light-Sheet Generation with Field Synthesis
Authors: Chang Bo-Jui , Kittisopikul Mark , Dean Kevin M. , Roudot Philippe , Welf Erik , Fiolka Reto
Published: September 2018

We introduce Field Synthesis, a theorem that can be used to synthesize any scanned or dithered light-sheet, including those used in lattice light-sheet microscopy (LLSM), from an incoherent superposition of one-dimensional intensity distributions. This user-friendly and modular approach offers a drastically simplified optical design, higher light-throughput, simultaneous multicolor illumination, and a 100% spatial duty cycle, thereby providing uncompromised biological imaging with decreased rates of photobleaching.
Multiplex transcriptional characterizations across diverse and hybrid bacterial cell-free expression systems
Authors: Yim Sung Sun , Johns Nathan I. , Park Jimin , Gomes Antonio L.C. , McBee Ross M. , Richardson Miles , Ronda Carlotta , Chen Sway P. , Garenne David , Noireaux Vincent , Wang Harris H.
Published: September 2018

Cell-free expression systems enable rapid prototyping of genetic programs in vitro. However, current throughput of cell-free measurements is often limited by the use of single-channel reporter assays. Here, we describe DNA Regulatory element Analysis by cell-Free Transcription and Sequencing (DRAFTS), a rapid and robust in vitro approach for multiplexed measurement of transcriptional activities from thousands of regulatory sequences in a single reaction. We employed this method in active cell lysates developed from ten diverse bacterial species. Interspecies analysis of transcriptional profiles from >1,000 diverse regulatory sequences revealed functional differences in gene expression that could be predictively modeled. Finally, we constructed and examined the transcriptional capacities of dual-species “hybrid” cell lysates that can simultaneously harness gene expression properties of multiple organisms. We expect that this cell-free multiplex transcriptional measurement approach will improve genetic circuit prototyping in new bacterial chassis for synthetic biology.
Greater strength of selection and higher proportion of beneficial amino acid changing mutations in humans compared to mice andDrosophila melanogaster
Authors: Zhen Ying , Huber Christian D. , Davies Robert W. , Lohmueller Kirk E.
Published: September 2018

Quantifying and comparing the amount of adaptive evolution among different species is key to understanding evolutionary processes. Previous studies have shown differences in adaptive evolution across species; however, their specific causes remain elusive. Here, we use improved modeling of weakly deleterious mutations and the demographic history of the outgroup species and ancestral population and estimate that at least 20% of nonsynonymous substitutions between humans and an outgroup species were fixed by positive selection. This estimate is much higher than previous estimates, which did not correct for the sizes of the outgroup species and ancestral population. Next, we directly estimate the proportion and selection coefficients (p+ands+, respectively) of newly arising beneficial nonsynonymous mutations in humans, mice, andDrosophila melanogasterby examining patterns of polymorphism and divergence. We develop a novel composite likelihood framework to test whether these parameters differ across species. Overall, we reject a model with the samep+ands+of beneficial mutations across species, and estimate that humans have a higherp+s+compared toD. melanogasterand mice. We demonstrate that this result cannot be caused by biased gene conversion or hypermutable CpG sites. In summary, we find the proportion of beneficial mutations to be higher in humans than inD. melanogasteror mice, suggesting that organismal complexity, which increases the number of steps required in adaptive walks, may be a key predictor of the amount of adaptive evolution within a species.
Effectiveness of physical inactivation methods of dengue virus: heat-versus UV-inactivation
Authors: Idris Fakhriedzwan , Muharram Siti Hanna , Zaini Zainun , Diah Suwarni
Published: September 2018

Introduction

Complete inactivation of virus is crucial before samples are manipulated outside of biological containment areas or general cleaning. There are several control methods that could decrease the risk from viral contamination on surfaces, which include chemical disinfectants, heating sterilisation, and ultraviolet germicidal irradiation depending on the nature and properties of the materials to be sterilised. To date, there are limited studies reporting the effectiveness of physical inactivation methods of dengue virus. Therefore, this study was designed to evaluate the effectiveness of two physical inactivation methods, which are heat- and ultraviolet-inactivation, against dengue virus.

Material and methods

All dengue virus serotypes were subjected to heat treatment at various temperatures and exposed to UV light (wavelength of 250-270 nm) at a distance of approximately 75 cm in a Class II Biosafety cabinet (ESCO) at room temperature. The effectiveness of inactivation methods was tested using viability testing on Vero cells and immunofluorescence assay.

Results

Dengue virus can be effectively inactivated by heat treatment at 56°C for at least 30 minutes or at higher temperature. On the other hand, the virus required 45 minutes or longer of ultraviolet light exposure at 75 cm distance from the source to be completely inactivated.

Conclusion

The results indicated that DENV can be effectively inactivated using high temperature, i.e. 56°C or above, and UV light irradiation. This result would serve as guidelines in proper decontamination and control of dengue virus in laboratory settings, provided proper conditions are met.
The High-throughput WAFFL System for Treating and Monitoring Individual Drosophila melanogaster Adults
Authors: Jaime Maria D.L.A. , Karott Sean , Salem Ghadi H. , Krynitsky Jonathan , Garmendia-Cedillos Marcial , Anderson Sarah , Harbison Susan , Pohida Thomas J. , Oliver Brian
Published: September 2018

Non-mammalian model organisms have been essential for our understanding of the mechanisms and control of development, disease, and physiology, but are underutilized in pharmacological phenotypic screening assays due to low throughput compared to cell-based systems. To increase the utility of using Drosophila melanogaster in screening, we have designed the whole animal feeding flat (WAFFL), a novel, flexible, and complete system for feeding, monitoring, and assaying flies in a high throughput format. Our system was conceived keeping in mind the use of off-the-shelf, commercial, 96-well consumables and equipment in order to be amenable to experimental needs. Here we provide an overview of the design and 3-D printing manufacture specifications.
Extracts for Analysis of DNA Replication in a Nucleus-Free System
Authors: Sparks Justin , Walter Johannes C.
Published: September 2018

Frog egg extracts represent a powerful approach with which to dissect molecular mechanisms of vertebrate DNA replication and repair. In the classical approach, sperm chromatin is added to a crude egg lysate to form replication-competent nuclei. We subsequently described a procedure that bypasses the requirement for nuclear assembly in DNA replication. In this method, DNA is first added to a high-speed supernatant (HSS) of egg lysate, which mimics the G1 phase of the cell cycle in that it supports replication licensing. Subsequent addition of a concentrated nucleoplasmic extract (NPE) leads to replication initiation followed by a single complete round of DNA replication. The advantage of the nucleus-free system is that it supports efficient replication of model DNA templates such as plasmids and lambda DNA that can be modified with specific features such as LacI arrays, DNA protein cross-links, or DNA interstrand cross-links. Here, we describe our current protocol for preparation of HSS and NPE. Methods for their use in DNA replication and repair are described elsewhere.
Microinjection of mRNAs and Oligonucleotides
Authors: Moody Sally A.
Published: September 2018

Microinjecting lineage tracers into a single blastomere in the normal, intact embryo identifies the repertoire of cell types derived from it. In order to reveal the full developmental potential of that blastomere or identify the mechanisms by which its fate is determined, one needs to modify its gene expression under controlled experimental conditions. One method by which this is easily accomplished in Xenopus is by microinjecting synthetic mRNAs or antisense oligonucleotides into an identified blastomere to target altered gene expression specifically to its lineage. Xenopus blastomeres are robust and tolerate pressure-driven microinjection up to a few hundred cells, and they efficiently translate exogenously supplied mRNAs. Targeted microinjections, described here, significantly reduce off-target effects of the mRNAs or oligonucleotides. Many types of constructs can be synthesized to provide specific information about gene function. For example, microinjecting mRNA encoding the wild-type gene in its normal expression domain or in an ectopic site tests whether it promotes or represses target genes or alters the formation of tissues of interest. Mutant forms of a gene transcript can illuminate the function of different domains of the encoded protein or show the developmental consequences of a mutation found in a human disease. mRNAs encoding dominant-negative forms of a protein can elicit a functional knockdown and thereby establish the necessity for that gene in a developmental process. Microinjecting antisense morpholino oligonucleotides (MOs) that are designed to block either endogenous mRNA translation or splicing is an effective method to reduce the levels of endogenous protein.
Einsteck Transplants
Authors: Cousin Hélène
Published: September 2018

Einsteck procedure refers to a method whereby the experimenter inserts material into the blastocoel cavity of an early amphibian embryo. This procedure is simpler to perform than other types of grafts, such as Spemann–Mangold, and with practice yields a sizable amount of data suitable for statistical analysis. This protocol for Einsteck transplantation in Xenopus describes the insertion of the gastrula-stage blastopore lip into the blastocoel cavity of a host embryo.
Bulk Electroporation-Mediated Gene Transfer intoXenopusTadpole Brain
Authors: Sáenz de Miera Cristina , Parr Ethan , Denver Robert J.
Published: September 2018

In vivo gene transfer is a powerful tool for investigating protein function and gene regulation in living organisms. Delivery of plasmid DNA to the brain ofXenopustadpoles by bulk electroporation-mediated (EM) gene transfer can be used to study the effects of ectopic gene expression on development, physiology, and behavior. It can also be used to mark cells for lineage tracing, investigate the in vivo function of gene regulatory elements when linked to a reporter gene, and introduce mutations into the genome of transfected cells, among other applications. Bilateral EM gene transfer allows for transfection of both sides of the brain, whereas unilateral EM gene transfer enables analysis of the effects of forced gene expression on one side of the brain, with the other side serving as the control.
DNase-seq to Study Chromatin Accessibility in Early Xenopus tropicalis Embryos
Authors: Cho Jin Sun , Blitz Ira L. , Cho Ken W.Y.
Published: September 2018

Transcriptional regulatory elements are typically found in relatively nucleosome-free genomic regions, often referred to as “open chromatin.” Deoxyribonuclease I (DNase I) can digest nucleosome-depleted DNA (presumably bound by transcription factors), but DNA in nucleosomes or higher-order chromatin fibers is less accessible to the nuclease. The DNase-seq method uses high-throughput sequencing to permit the interrogation of DNase hypersensitive sites (DHSs) across the entire genome and does not require prior knowledge of histone modifications, transcription factor binding sites, or high quality antibodies to identify potentially active regions of chromatin. Here, discontinuous iodixanol gradients are used as a gentle preparation of the nuclei from Xenopus embryos. Short DNase I digestion times are followed by size selection of digested genomic DNA, yielding DHS fragments. These DNA fragments are subjected to real-time quantitative polymerase chain reaction (qPCR) and sequencing library construction. A library generation method and pipeline for analyzing DNase-seq data are also described.
INTACT Proteomics in Xenopus
Authors: Wasson Lauren , Amin Nirav M. , Conlon Frank L.
Published: September 2018

Analysis of the molecular mechanisms driving cell specification, differentiation, and other cellular processes can be difficult due to the heterogeneity of tissues and organs. Therefore, it is critical to isolate pure cell populations in order to properly assess the function of certain cell types in the context of a tissue. This protocol describes use of the INTACT (isolation of nuclei tagged in specific cell types) method in Xenopus, followed by proteomics analysis of nuclear protein complexes. The INTACT protocol utilizes two transgenes: (1) a three-part nuclear targeting fusion (NTF) consisting of a nuclear envelope protein (Nup35) that targets the NTF to the nuclear membrane, an enhanced green fluorescent protein (EGFP) cassette for NTF visualization in live animals, and a biotin ligase receptor protein (BLRP) that provides a substrate for the biotinylation of the NTF, and (2) the E. coli ligase BirA (which biotinylates the NTF) tagged to mCherry (for visualization). Either or both transgenes are driven by a tissue-specific promoter, making this protocol easily adaptable to proteomics analyses of immunoprecipitated complexes from INTACT-isolated nuclei of multiple tissue types to determine the composition of protein complexes in pure cell populations.
Filopodia-Like Structure Formation from Xenopus Egg Extracts
Authors: Fox Helen M. , Gallop Jennifer L.
Published: September 2018

The actin cytoskeleton comprises many different architectures of filaments, including branched networks, parallel bundles and antiparallel fibers. A current challenge is to elucidate how the diverse array of actin regulators, which controls the growth, assembly and turnover of actin filaments, is used to orchestrate cytoskeletal organization and in turn cell shape and movement. Long observed to assemble at cell membranes, actin in Xenopus egg extracts recapitulates membrane-triggered assembly at specific lipid and membrane environments. The use of Xenopus egg extracts has contributed greatly to identifying how constitutively autoinhibited regulatory pathways are activated, which converge on activation of the Arp2/3 complex. Here we describe a protocol for making parallel actin bundles using Xenopus egg extracts from supernatants prepared by high-speed centrifugation. These filopodia-like actin bundles emanate from clusters of actin regulators that self-assemble at phosphatidylinositol (4,5)-bisphosphate-containing supported lipid bilayers. Forming a plasma membrane-mimicking bilayer on glass allows easy, optimizable, high signal-to-noise microscopy at high spatial and temporal resolution. The use of Xenopus egg extracts yields large quantities of active material that can be flexibly tailored to address specific questions, for example, by dilution, addition of fluorescent proteins, antibodies or protein fragments, immunodepletion, addition of small molecule inhibitors, or biochemical fractionation.
Ex Vivo Eye Tissue Culture Methods for Xenopus
Authors: Henry Jonathan J. , Perry Kimberly J. , Hamilton Paul W.
Published: September 2018

Lens regeneration can be studied in whole animals following removal of the original lens (lentectomy). However, culturing a whole animal can be impractical for assays involving small molecule inhibitors or proteins. Ex vivo eye tissue culture is an alternative approach for examining lens regeneration. The ex vivo culture system offers certain advantages when compared to the in vivo regeneration assay, as the percentage of cases showing lens differentiation can exceed that seen in whole animals. This culture system also allows for the treatment of eye tissues in small volumes, which helps ensure reproducibility and reduces the amount (and cost) of small-molecule inhibitors or exogenous proteins, etc., necessary to conduct an experiment. Additionally, different eye tissues can be combined, such as nontransgenic and transgenic tissues (e.g., eyecup and cornea) that carry reporters or inducible transgenes. This approach represents a very useful tool in the analysis of lens regeneration or for simply culturing specific eye tissues, and can be used to culture either Xenopus laevis or Xenopus tropicalis eye tissues.
Assessing Ubiquitylation of Individual Proteins Using Xenopus Extract Systems
Authors: McDowell Gary S. , Philpott Anna
Published: September 2018

Xenopus extract systems have been used to study ubiquitylation of proteins, and to uncover some of the fundamental processes of the ubiquitylation pathway itself. They provide a simple, quick, and robust method for studying ubiquitylation. In this protocol, methods are provided for studying protein ubiquitylation using Xenopus egg or embryo extracts and in vitro radiolabeled proteins. These methods also enable examination of whether proteins undergo noncanonical ubiquitylation, through modification of the protein by covalent linkage to ubiquitin through residues other than lysine, such as cysteine, serine, and threonine.
The Use of Cell-Free Xenopus Extracts to Investigate Cytoplasmic Events
Authors: Gibeaux Romain , Heald Rebecca
Published: September 2018

Experiments using cytoplasmic extracts prepared from Xenopus eggs have made important contributions to our understanding of the cell cycle, the cytoskeleton, and cytoplasmic membrane systems. Here we introduce the extract system and describe methods for visualizing and manipulating diverse cytoplasmic processes, and for assaying the functions, dynamics, and stability of individual factors. These in vitro approaches uniquely enable investigation of events at specific cell cycle states, including the assembly of actin- and microtubule-based structures, and the formation of the endoplasmic reticulum. Maternal stockpiles in extracts recapitulate diverse processes in the near absence of gene expression, and this biochemical system combined with microscopy empowers a wide range of mechanistic investigations.
Time-resolved pair distribution function analysis of disordered materials on beamlines BL04B2 and BL08W at SPring-8
Authors: Ohara Koji , Tominaka Satoshi , Yamada Hiroki , Takahashi Masakuni , Yamaguchi Hiroshi , Utsuno Futoshi , Umeki Takashi , Yao Atsushi , Nakada Kengo , Takemoto Michitaka , Hiroi Satoshi , Tsuji Naruki , Wakihara Toru
Published: September 2018

A dedicated apparatus has been developed for studying structural changes in amorphous and disordered crystalline materials substantially in real time. The apparatus, which can be set up on beamlines BL04B2 and BL08W at SPring-8, mainly consists of a large two-dimensional flat-panel detector and high-energy X-rays, enabling total scattering measurements to be carried out for time-resolved pair distribution function (PDF) analysis in the temperature range from room temperature to 873 K at pressures of up to 20 bar. For successful time-resolved analysis, a newly developed program was used that can monitor and process two-dimensional image data simultaneously with the data collection. The use of time-resolved hardware and software is of great importance for obtaining a detailed understanding of the structural changes in disordered materials, as exemplified by the results of commissioned measurements carried out on both beamlines. Benchmark results obtained using amorphous silica and demonstration results for the observation of sulfide glass crystallization upon annealing are introduced.
Simultaneous X-ray radioscopy/tomography and energy-dispersive diffraction applied to liquid aluminium alloy foams
Authors: Jiménez Catalina , Paeplow Marlen , Kamm Paul H. , Neu Tillmann R. , Klaus Manuela , Wagener Guido , Banhart John , Genzel Christoph , García-Moreno Francisco
Published: September 2018

High-speed X-ray imaging in two dimensions (radioscopy) and three dimensions (tomography) is combined with fast X-ray diffraction in a new experimental setup at the synchrotron radiation source BESSY II. It allows forin situstudies of time-dependent phenomena in complex systems. As a first application, the foaming process of an aluminium alloy was studied in three different experiments. Radioscopy, optical expansion measurements and diffraction were used to correlate the change of foam morphology to the various phases formed during heating of an AlMg15Cu10 alloy to 620°C in the first experiment. Radioscopy was then replaced by tomography. Acquiring tomograms and diffraction data at 2 Hz allows even more details of foam evolution to be captured, for example, bubble size distribution. In a third experiment, 4 Hz tomography yields dynamic insights into fast phenomena in evolving metal foam.
Crystal structure and sequential dehydration transitions of disodium guanosine 5′-monophosphate tetrahydrate
Authors: Tsubonoya Masaaki , Murofushi Akitaka , Yamamura Shigefumi , Sugawara Yoko
Published: September 2018

Disodium guanosine 5′-monophosphate was reported previously to crystallize as both the tetrahydrate and the heptahydrate. We herein report a determination of the molecular and crystal structures of the title tetrahydrated salt, 2Na+·C10H12N5O8P2−·4H2O. It was found that the structure differs markedly from that of the heptahydrate, but greatly resembles that of disodium deoxyguanosine 5′-monophosphate tetrahydrate. The C2′—O2′H moiety of ribose is surrounded by hydrophilic moieties and is disordered over two sites. The sugar puckering mode is O4′-endo-C1′-exo at both sites and the conformation around the C4′—C5′ bond is gauche–trans. Powder X-ray diffraction and thermal analyses revealed that the temperature-controlled transition from the tetrahydrate to the anhydride proceeded through three intermediate phases between 40 and 60 °C at 0% relative humidity. Large induction periods were observed.
The structure and changes of China’s land system
Authors: Shouying Liu
Published: September 2018

Purpose

The purpose of this paper is to analyze the structure and changes of China’s land system. To achieve this aim, the paper is divided into four parts. The first part gives a brief introduction to the structural characteristics of the Chinese land institutional arrangements; the second part analyzes the reform process of the land system in the past 40 years and its path of change; the third part engages the discussion about the historic contribution made by the land institutional change to rapid economic growth and structural changes; and the final part is conclusion and some policy implications.

Design/methodology/approach

After 40 years of reforms and opening up, China has not only created a growth miracle unparalleled for any major country in human history, but also transformed itself from a rural to an urban society. Behind this great transformation is a systemic reform in land institutions. Rural land institutions went from collectively owned to household responsibility system, thereby protecting farmers’ land rights. This process resulted in long-term sustainable growth in China’s agriculture, a massive rural-urban migration and a historical agricultural transformation. The conversion of agricultural land to non-agricultural uses and the introduction of market mechanisms made land a policy tool in driving high economic growth, industrialization and urbanization.

Findings

Research shows that the role of land and its relationship with the economy will inevitably change as China’s economy enters a new stage of medium-to-high speed growth. With economic restructuring, low-cost industrial land will be less effective. Urbanization is also shifting from rapid expansion to endogenous growth so that returns on land capitalization will decrease and risks will increase. Therefore, China must abandon land-dependent growth model through deepening land reforms and adapt a new pattern of economic development.

Originality/value

This paper gives a brief introduction to the structural characteristics of the Chinese land institutional arrangements, analyzes the reform process of the land system in the past 40 years and its path of change, and evaluates the historic contribution made by the land institutional change to rapid economic growth and structural changes.
Optimisation of corona ring design for composite insulator strings
Authors: Aramugam Kalaiselvi , Illias Hazlee Azil , Ching Yern Chee
Published: September 2018

Purpose

The purpose of this paper is to propose an optimum design of a corona ring for insulator strings using optimisation techniques, which are gravitational search algorithm (GSA) and imperialist competitive algorithm (ICA).

Design/methodology/approach

An insulator string model geometry with a corona ring was modelled in a finite element analysis software, and it was used to obtain the electric field distribution in the model. The design was optimised using GSA and ICA. The variables were the corona ring diameter, ring tube diameter and vertical position of the ring along the insulator string.

Findings

Using optimisation method, the minimum electric field magnitude on the insulator string with a corona ring design is lower than without using optimisation method. GSA yields better results than ICA in terms of the optimised corona ring design.

Practical implications

The proposed methods can help in improvement of corona ring design in reducing the electric field magnitude on the energised end of insulator strings.

Originality/value

A new method to design an optimum corona ring for insulator strings, which is using optimisation methods, has been developed in this work.
Student success in teams: intervention, cohesion and performance
Authors: Croy Glen , Eva Nathan
Published: September 2018

Purpose

The purpose of this paper is to design and test an online team intervention for university students, focusing on communication, leadership and team processes, to influence team cohesion and subsequently team assignment performance. It was administered twice as a formative feedback measure and once as a summative evaluation measure across a semester.

Design/methodology/approach

Survey data were collected from 154 university students across four management modules in a large Australian university. Multiple regression analysis was used to test the hypotheses and open-ended questions were used to understand why the team intervention was effective.

Findings

The results showed that the implementation of an effective team intervention leads to higher levels of team cohesion and subsequently team performance. Open-ended responses revealed that the team intervention caused students to develop team-based sills and increase regular contributions.

Practical implications

In order to develop positive team behaviours amongst students in group assignments and increase the effectiveness of team-based learning activities, educators should implement a regular and process focused team contribution intervention, like the one proposed in this study.

Originality/value

This research contributes to the team intervention literature by drawing on the social information processing perspective, to demonstrate how an intervention that is based on the students’ social processing, task focused, regular implementation and formative feedback has a salient effect over team cohesion.