bluuebunny/crossref_metadata_2025 huggingface dataset:
Improved Annotation with de novo Transcriptome Assembly in Four Social Amoeba Species
Authors: Singh Reema , Lawal Hajara M. , Schilde Christina , Glöeckner Gernot , Barton Geoff J. , Schaap Pauline , Cole Christian
Published: May 2016

Background:

Annotation of gene models and transcripts is a fundamental step in genome sequencing projects. Often this is performed with automated prediction pipelines, which can miss complex and atypical genes or transcripts. RNA-seq data can aid the annotation with empirical data. Here we present de novo transcriptome assemblies generated from RNA-seq data in four Dictyostelid species: D. discoideum, P. pallidum, D. fasciculatum and D. lacteum. The assemblies were incorporated with existing gene models to determine corrections and improvement on a whole-genome scale. This is the first time this has been performed in these eukaryotic species.

Results:

An initial de novo transcriptome assembly was generated by Trinity for each species and then refined with Program to Assemble Spliced Alignments (PASA). The completeness and quality were assessed with the Core Eukaryotic Genes Mapping Approach (CEGMA) and Transrate tools at each stage of the assemblies. The final datasets of 11,315-12,849 transcripts contained 5,610-7,712 updates and corrections to >50% of existing gene models including changes to hundreds or thousands of protein products. Putative novel genes are also identified and alternative splice isoforms were observed for the first time in P. pallidum, D. lacteum and D. fasciculatum.

Conclusions:

In taking a whole transcriptome approach to genome annotation with empirical data we have been able to enrich the annotations of four existing genome sequencing projects. In doing so we have identified updates to the majority of the gene annotations across all four species under study and found putative novel genes and transcripts which could be worthy for follow-up. The new transcriptome data we present here will be a valuable resource for genome curators in the Dictyostelia and we propose this effective methodology for use in other genome annotation projects.
Tissue-Driven Hypothesis of Transcriptome Evolution: An Update
Authors: Hallmark Howard T. , Haltom Jeffrey A , Gu Xun
Published: May 2016

In past decade, many reports have demonstrated that tissues in multi-cellular organisms may play important roles to shape the pattern of genome evolution. The tissue-driven hypothesis was then coined, claiming that tissue-specific factor as the common resource of functional constrain may underlie the positive correlations between tissue expression divergence, sequence divergence, or the expression tolerance of duplication divergence. However, the original version of tissue-driven hypothesis cannot rule out the tissue-specific effect of mutational variance. In this perspective, we solve this problem by modifying the evolutionary model that underlies the tissue expression evolution. Reanalysis of the microarray data reanalysis has revealed the relative importancebetween tissue-specific functional constraints and mutational variances in the tissue evolution. Finally, we outline how to utilize RNA-seq technology to further investigate the tissue expression evolution in the case of multiple tissues and species.
The evolution of CHROMOMETHYLASES and gene body DNA methylation in plants
Authors: Bewick Adam J. , Niederhuth Chad E. , Lexiang Ji , Rohr Nicholas A. , Griffin Patrick T. , Leebens-Mack Jim , Schmitz Robert J.
Published: May 2016

Background

The evolution of gene body methylation (gbM), its origins and its functional consequences are poorly understood. By pairing the largest collection of transcriptomes (>1000) and methylomes (77) across Viridiplantae we provide novel insights into the evolution of gbM and its relationship to CHROMOMETHYLASE (CMT) proteins.

Results

CMTs are evolutionary conserved DNA methyltransferases in Viridiplantae. Duplication events gave rise to what are now referred to as CMT1, 2 and 3. Independent losses of CMT1, 2 and 3 in eudicots, CMT2 and ZMET in monocots and monocots/commelinids, variation in copy number and non-neutral evolution suggests overlapping or fluid functional evolution of this gene family. DNA methylation within genes is widespread and is found in all major taxonomic groups of Viridiplantae investigated. Genes enriched with methylated CGs (mCG) were also identified in species sister to angiosperms. The proportion of genes and DNA methylation patterns associated with gbM are restricted to angiosperms with a functional CMT3 or ortholog. However, mCG-enriched genes in the gymnospermPinus taedashared some similarities with gbM genes inAmborella trichopoda. Additionally, gymnosperms and ferns share a CMT homolog closely related to CMT2 and 3. Hence, the dependency of gbM on a CMT most likely extends to all angiosperms and possibly gymnosperms and ferns.

Conclusions

The resulting gene family phylogeny of CMT transcripts from the most diverse sampling of plants to date redefines our understanding of CMT evolution and its evolutionary consequences on DNA methylation. Future, functional tests of homologous and paralogous CMTs will uncover novel roles and consequences to the epigenome.
Centrifuge: rapid and sensitive classification of metagenomic sequences
Authors: Kim Daehwan , Song Li , Breitwieser Florian P. , Salzberg Steven L.
Published: May 2016

Centrifuge is a novel microbial classification engine that enables rapid, accurate and sensitive labeling of reads and quantification of species on desktop computers. The system uses an indexing scheme based on the Burrows-Wheeler transform (BWT) and the Ferragina-Manzini (FM) index, optimized specifically for the metagenomic classification problem. Centrifuge requires a relatively small index (4.2 GB for 4,078 bacterial and 200 archaeal genomes) and classifies sequences at very high speed, allowing it to process the millions of reads from a typical high-throughput DNA sequencing run within a few minutes. Together these advances enable timely and accurate analysis of large metagenomics data sets on conventional desktop computers. Because of its space-optimized indexing schemes, Centrifuge also makes it possible to index the entire NCBI non-redundant nucleotide sequence database (a total of 109 billion bases) with an index size of 69 GB, in contrast to k-mer based indexing schemes, which require far more extensive space. Centrifuge is available as free, open-source software fromwww.ccb.jhu.edu/software/centrifuge
Highly expressed proteins prefer less informative amino acids
Authors: Wang Guang-Zhong
Published: May 2016

The transcriptional and translational systems are essentially information processing systems. However, how to quantify the amount of information decoded during expression remains a mystery. Here, we have proposed a simple method to evaluate the amount of information transcribed and translated during gene expression. We found that although proteins with a high copy number have more information translated, the average number of bits per amino acid is not high. The negative correlation between protein copy number and bits per amino acid indicates the selective pressure to reduce translational errors. Moreover, interacting proteins have similar bits per residue translated. All of these findings highlight the importance of understanding transcription and translation from an information processing perspective.
Potential pitfalls of FISH microscopy as assessment method for anaerobic digesters
Authors: Abendroth Christian , Hahnke Sarah , Klocke Michael , Luschnig Olaf
Published: May 2016

In the present work we investigated how the state of a biogas reactor impacts the enumeration of prokaryotic cells by fluorescencein situhybridisation (FISH). Therefore, the correlation between gas production and FISH hybridisation rates was analysed in different anaerobic digester sludges. High gasification activity coincided with high hybridisation rates. Low hybridisation rates were especially achieved with reactor samples subjected to long starvation periods showing low biogas production.

Based on our findings we conclude that samples for FISH analysis should be fixed as soon as possible to prevent a loss of microbial activity resulting in lower FISH signals. Furthermore, the location of sampling is of importance, since samples from different fermenters within the same biogas plant also varied strongly in their FISH hybridisation rate. Our results indicate that FISH could be a useful method for assessing the metabolic state of microorganisms in anaerobic digester plants.
Reference-free comparison of microbial communities via de Bruijn graphs
Authors: Mangul Serghei , Koslicki David
Published: May 2016

Microbial communities inhabiting the human body exhibit significant variability across different individuals and tissues, and are suggested to play an important role in health and disease. High-throughput sequencing offers unprecedented possibilities to profile microbial community composition, but limitations of existing taxonomic classification methods (including incompleteness of existing microbial reference databases) limits the ability to accurately compare microbial communities across different samples. In this paper, we present a method able to overcome these limitations by circumventing the classification step and directly using the sequencing data to compare microbial communities. The proposed method provides a powerful reference-free way to assess differences in microbial abundances across samples. This method, called EMDeBruijn, condenses the sequencing data into a de Bruijn graph. The Earth Mover's Distance (EMD) is then used to measure similarities and differences of the microbial communities associated with the individual graphs. We apply this method to RNA-Seq data sets from a coronary artery calcification (CAC) study and shown that EMDeBruijn is able to differentiate between case and control CAC samples while utilizing all the candidate microbial reads. We compare these results to current reference-based methods, which are shown to have a limited capacity to discriminate between case and control samples. We conclude that this reference-free approach is a viable choice in comparative metatranscriptomic studies.
Prospects of genomic prediction in the USDA Soybean Germplasm Collection: Historical data creates robust models for enhancing selection of accessions
Authors: Jarquin Diego , Specht James , Lorenz Aaron
Published: May 2016

The identification and mobilization of useful genetic variation from germplasm banks for use in breeding programs is critical for future genetic gain and protection against crop pests. Plummeting costs of next-generation sequencing and genotyping is revolutionizing the way in which researchers and breeders interface with plant germplasm collections. An example of this is the high density genotyping of the entire USDA Soybean Germplasm Collection. We assessed the usefulness of 50K SNP data collected on 18,480 domesticated soybean (G. max) accessions and vast historical phenotypic data for developing genomic prediction models for protein, oil, and yield. Resulting genomic prediction models explained an appreciable amount of the variation in accession performance in independent validation trials, with correlations between predicted and observed reaching up to 0.92 for oil and protein and 0.79 for yield. The optimization of training set design was explored using a series of cross-validation schemes. It was found that the target population and environment need to be well represented in the training set. Secondly, genomic prediction training sets appear to be robust to the presence of data from diverse geographical locations and genetic clusters. This finding, however, depends on the influence of shattering and lodging, and may be specific to soybean with its presence of maturity groups. The distribution of 7,608 non-phenotyped accessions was examined through the application of genomic prediction models. The distribution of predictions of phenotyped accessions was representative of the distribution of predictions for non-phenotyped accessions, with no non-phenotyped accessions being predicted to fall far outside the range of predictions of phenotyped accessions.
Not just methods: User expertise explains the variability of outcomes of genome-wide studies
Authors: Lotterhos Katie E. , François Olivier , Blum Michael G.B.
Published: May 2016

Genome scan approaches promise to map genomic regions involved in adaptation of individuals to their environment. Outcomes of genome scans have been shown to depend on several factors including the underlying demography, the adaptive scenario, and the software or method used. We took advantage of a pedagogical experiment carried out during a summer school to explore the effect of an unexplored source of variability, which is the degree of user expertise.Participants were asked to analyze three simulated data challenges with methods presented during the summer school. In addition to submitting lists, participants evaluated a priori their level of expertise. We measured the quality of each genome scan analysis by computing a score that depends on false discovery rate and statistical power. In an easy and a difficult challenge, less advanced participants obtained similar scores compared to advanced ones, demonstrating that participants with little background in genome scan methods were able to learn how to use complex software after short introductory tutorials. However, in a challenge ofintermediate difficulty, advanced participants obtained better scores. To explain the difference, we introduce a probabilistic model that shows that a larger variation in scores is expected for SNPs of intermediate difficulty of detection. We conclude that practitioners should develop their statistical and computational expertise to follow the development of complex methods. To encourage training, we release the website of the summer school where users can submit lists of candidate loci, which will be scored and compared to the scores obtained by previous users.
TheC. elegansNF2/Merlin Molecule NFM-1 Non-Autonomously Regulates Neuroblast Migration and Interacts Genetically with the Guidance Cue SLT-1/Slit
Authors: Josephson Matthew P. , Aliani Rana , Lundquist Erik
Published: May 2016

During nervous system development, neurons and their progenitors often migrate to their final destinations. InCaenorhabditis elegans, the bilateral Q neuroblasts and their descendants migrate long distances in opposite directions, despite being born in the same posterior region. QR on the right migrates anteriorly and generates the AQR neuron positioned near the head, and QL on the left migrates posteriorly, giving rise to the PQR neuron positioned near the tail. In a screen for genes required for AQR and PQR migration, we identified an allele ofnfm-1, which encodes a molecule similar to vertebrate NF2/Merlin, an important tumor suppressor in humans. Mutations inNF2lead to Neurofibromatosis Type II, characterized by benign tumors of glial tissues. These molecules containFour-point-oneEzrinRadixinMoesin (FERM) domains characteristic of cytoskeletal-membrane linkers, and vertebrate NF2 is required for epidermal integrity. Vertebrate NF2 can also regulate several transcriptional pathways including the Hippo pathway. Here we demonstrate that inC. elegans,nfm-1is required for complete migration of AQR and PQR, and that it likely acts outside of the Q cells themselves in a non-autonomous fashion. We also show a genetic interaction betweennfm-1and theC. elegans Slithomologslt-1, which encodes a conserved secreted guidance cue. In vertebrates,NF2can controlSlit2mRNA levels through the hippo pathway in axon pathfinding, suggesting a conserved interaction of NF2 and Slit2 in regulating migration.
A Key Cytoskeletal Regulator of Ubiquitination Amplifies TGFβ Signaling During Mouse Developmental Vascular Patterning
Authors: Shetty Ronak , Joshi Divyesh , Jain Mamta , Vasudevan Madavan , Paul Jasper Chrysolite , Bhat Ganesh , Banerjee Poulomi , Abe Takaya , Kiyonari Hiroshi , Vijayraghavan K. , Inamdar Maneesha S.
Published: May 2016

Vascular development involvesde novoformation of a capillary plexus, which is then pruned and remodeled by angiogenic events. Cytoskeletal remodeling and directional endothelial migration are essential for developmental and pathological angiogenesis. Smad-dependent TGFβ signaling controls vascular patterning and is negatively regulated by microtubules. Here we show that a positive regulator of TGFβ signaling is essential for developmental vascular patterning and microtubule stability. Rudhira/BCAS3 is known to bind microtubules and to play a nodal role in cytoskeletal remodeling and directional endothelial cell (EC) migration invitro. We demonstrate that the molecular and cellular function of Rudhira is deployed at critical steps in vascular patterning. We generated the first floxed mice forrudhiraand find that global or endothelial knockout ofrudhiraresults in mid-gestation lethality due to aberrant embryonic and extra-embryonic vessel patterning and defective cardiac morphogenesis.Rudhiranull yolk sac ECs show random and retarded migration. Yolk sac transcriptome analysis revealed key mediators of angiogenic processes and TGFβ receptor signaling were perturbed inrudhiranull mutants. Molecular and biochemical analyses showed thatrudhiradepletion reduced microtubule stability but increased expression of pathway inhibitors leading to high levels of SMAD2/3 ubiquitination and reduced activation. These effects were not rescued by exogenous TGFβ. However, TGFβ treatment of wild type ECs increased Rudhira expression. Further, exogenous Rudhira, which promotes directional cell migration, caused increased SMAD2/3 nuclear translocation and reduced inhibitor levels. Therefore, we propose that Rudhira and TGFβ signaling are mutually dependent. Rudhira has a dual function in promoting TGFβ signaling, possibly by sequestering microtubules and simultaneously preventing SMAD2/3 ubiquitination to permit EC migration and vascular patterning. TGFβ signaling and aberrant human Rudhira (Breast Cancer Amplified Sequence 3, BCAS3) expression are both associated with tumour metastasis. Our study identifies a cytoskeletal, cell type-specific modulator of TGFβ signaling important in development and cancer.
Genetic surfing in human populations: from genes to genomes
Authors: Peischl Stephan , Dupanloup Isabelle , Bosshard Lars , Excoffier Laurent
Published: May 2016

Genetic surfing describes the spatial spread and increase in frequency of variants that are not lost by genetic drift and serial migrant sampling during a range expansion. Genetic surfing does not modify the total number of derived alleles in a population or in an individual genome, but it leads to a loss of heterozygosity along the expansion axis, implying that derived alleles are more often in homozygous state. Genetic surfing also affects selected variants on the wave front, making them behave almost like neutral variants during the expansion. In agreement with theoretical predictions, human genomic data reveals an increase in recessive mutation load with distance from Africa, an expansion load likely to have developed during the expansion of human populations out of Africa.
APRICOT: an integrated computational pipeline for the sequence-based identification and characterization of RNA-binding proteins
Authors: Sharan Malvika , Förstner Konrad U. , Eulalio Ana , Vogel Jörg
Published: May 2016

RNA-binding proteins (RBPs) have been established as core components of several post-transcriptional gene regulation mechanisms. Experimental techniques such as cross-linking and co-immunoprecipitation have enabled the identification of RBPs, RNA-binding domains (RBDs), and their regulatory roles in the eukaryotic species such as human and yeast in large-scale. In contrast, our knowledge of the number and potential diversity of RBPs in bacteria is poorer due to the technical challenges associated with the existing global screening approaches.We introduce APRICOT, a computational pipeline for the sequence-based identification and characterization of proteins using RBDs known from experimental studies. The pipeline identifies functional motifs in protein sequences using Position Specific Scoring Matrices and Hidden Markov Models of the functional domains and statistically scores them based on a series of sequence-based features. Subsequently, APRICOT identifies putative RBPs and characterizes them by several biological properties. Here we demonstrate the application and adaptability of the pipeline on large-scale protein sets, including the bacterial proteome ofEscherichia coli.APRICOT showed better performance on various datasets compared to other existing tools for the sequence-based prediction of RBPs by achieving an average sensitivity and specificity of 0.90 and 0.91 respectively. The command-line tool and its documentation are available athttps://pypi.python.org/pypi/bio-apricot
Talin-KANK1 interaction controls the recruitment of cortical microtubule stabilizing complexes to focal adhesions
Authors: Bouchet Benjamin P. , Gough Rosemarie E. , van de Willige Dieudonnée , Ammon York-Christoph , Post Harm , Jacquemet Guillaume , Altelaar A.F. Maarten , Heck Albert J.R. , Goult Benjamin T. , Akhmanova Anna
Published: May 2016

The cross-talk between dynamic microtubules and integrin-based adhesions to the extracellular matrix plays a crucial role in cell polarity and migration. Microtubules regulate the turnover of adhesion sites, and, in turn, focal adhesions promote cortical microtubule capture and stabilization in their vicinity, but the underlying mechanism is unknown. Here, we show that cortical microtubule stabilization sites containing CLASPs, KIF21A, LL5P and liprins are recruited to focal adhesions by the adaptor protein KANK1, which directly interacts with the major adhesion component, talin. Structural studies showed that the conserved KN domain in KANK1 binds to the talin rod domain R7. Perturbation of this interaction, including a single point mutation in talin, which disrupts KANK1 binding but not the talin function in adhesion, abrogates the association of microtubule-stabilizing complexes with focal adhesions. We propose that the talin-KANK1 interaction links the two macromolecular assemblies that control cortical attachment of actin fibers and microtubules.
A scalable strategy for high-throughput GFP tagging of endogenous human proteins
Authors: Leonetti Manuel D. , Sekine Sayaka , Kamiyama Daichi , Weissman Jonathan S. , Huang Bo
Published: May 2016

A central challenge of the post-genomic era is to comprehensively characterize the cellular role of the ∼20,000 proteins encoded in the human genome. To systematically study protein function in a native cellular background, libraries of human cell lines expressing proteins tagged with a functional sequence at their endogenous loci would be very valuable. Here, using electroporation of Cas9/sgRNA ribonucleoproteins and taking advantage of a split-GFP system, we describe a scalable method for the robust, scarless and specific tagging of endogenous human genes with GFP. Our approach requires no molecular cloning and allows a large number of cell lines to be processed in parallel. We demonstrate the scalability of our method by targeting 48 human genes and show that the resulting GFP fluorescence correlates with protein expression levels. We next present how our protocols can be easily adapted for the tagging of a given target with GFP repeats, critically enabling the study of low-abundance proteins. Finally, we show that our GFP tagging approach allows the biochemical isolation of native protein complexes for proteomic studies. Together, our results pave the way for the large-scale generation of endogenously tagged human cell lines for the proteome-wide analysis of protein localization and interaction networks in a native cellular context.

SIGNIFICANCE STATEMENT

The function of a large fraction of the human proteome still remains poorly characterized. Tagging proteins with a functional sequence is a powerful way to access function, and inserting tags at endogenous genomic loci allows the preservation of a near-native cellular background. To characterize the cellular role of human proteins in a systematic manner and in a native context, we developed a method for tagging endogenous human proteins with GFP that is both rapid and readily applicable at a genome-wide scale. Our approach allows studying both localization and interaction partners of the protein target. Our results pave the way for the large-scale generation of endogenously tagged human cell lines for a systematic functional interrogation of the human proteome.
Experimental Granulomatous Pulmonary Nocardiosis in Balb/c Mice
Authors: Mifuji Lira Roque M. , Limón Flores Alberto Yairh , Salinas Carmona Mario César , Ortiz Stern Alejandro
Published: May 2016

Pulmonary nocardiosis is a granulomatous disease with high mortality that affects both immunosuppressed and immunocompetent patients. The mechanisms leading to the establishment and progression of the infection are currently unknown. An animal model to study these mechanisms is sorely needed. We report the first in vivo model of granulomatous pulmonary nocardiosis that closely resembles human pathology. BALB/c mice infected intranasally with two different doses of GFP-expressingNocardia brasiliensisATCC700358 (NbGFP), develop weight loss and pulmonary granulomas. Mice infected with 109CFUs progressed towards death within a week while mice infected with 108CFUs died after five to six months. Histological examination of the lungs revealed that both the higher and lower doses of NbGFP induced granulomas with NbGFP clearly identifiable at the center of the lesions. Mice exposed to 108CFUs and subsequently to 109CFUs were not protected against disease severity but had less granulomas suggesting some degree of protection. Attempts to identify a cellular target for the infection were unsuccessful but we found that bacterial microcolonies in the suspension used to infect mice were responsible for the establishment of the disease. Small microcolonies of NbGFP, incompatible with nocardial doubling times starting from unicellular organisms, were identified in the lung as early as six hours after infection. Mice infected with highly purified unicellular preparations of NbGFP did not develop granulomas despite showing weight loss. Finally, intranasal delivery of nocardial microcolonies was enough for mice to develop granulomas with minimal weight loss. Taken together these results show that Nocardia brasiliensis microcolonies are both necessary and sufficient for the development of granulomatous pulmonary nocardiosis in mice.
Achieving large dynamic range control of gene expression with a compact RNA transcription-translation regulator
Authors: Westbrook Alexandra M. , Lucks Julius B.
Published: May 2016

RNA transcriptional regulators are emerging as versatile components for genetic circuit construction. However, RNA transcriptional regulators suffer from incomplete repression, making their dynamic range less than that of their protein counterparts. This incomplete repression can cause expression leak, which impedes the construction of larger RNA synthetic regulatory networks. Here we demonstrate how naturally derived antisense RNA-mediated transcriptional regulators can be configured to regulate both transcription and translation in a single compact RNA mechanism that functions in Escherichia coli. Using in vivo gene expression assays, we show that a combination of transcriptional termination and RBS sequestration increases repression from 85% to 98% and activation from 10 fold to over 900 fold in response to cognate antisense RNAs. We also show that orthogonal versions of this mechanism can be created through engineering minimal antisense RNAs. Finally, to demonstrate the utility of this dual control mechanism, we use it to reduce circuit leak in an RNA-only transcriptional cascade that activates gene expression as a function of a small molecule input. We anticipate these regulators will find broad use as synthetic biology moves beyond parts engineering to the design and construction of larger and more sophisticated circuits.
Crystal structure of a dimerization domain of human Caprin-1: insights into the assembly of an evolutionarily conserved ribonucleoprotein complex consisting of Caprin-1, FMRP and G3BP1
Authors: Wu Yuhong , Zhu Jiang , Huang Xiaolan , Du Zhihua
Published: May 2016

Caprin-1 plays roles in many important biological processes, including cellular proliferation, innate immune response, stress response and synaptic plasticity. Caprin-1 has been implicated in several human diseases, including osteosarcoma, breast cancer, viral infection, hearing loss and neurodegenerative disorders. The functions of Caprin-1 depend on its molecular-interaction network. Direct interactions have been established between Caprin-1 and the fragile X mental retardation protein (FMRP), Ras GAP-activating protein-binding protein 1 (G3BP1) and theJapanese encephalitis virus(JEV) core protein. Here, crystal structures of a fragment (residues 132–251) of Caprin-1, which adopts a novel all-α-helical fold and mediates homodimerization through a substantial interface, are reported. Homodimerization creates a large and highly negatively charged concave surface suggestive of a protein-binding groove. The FMRP-interacting sequence motif forms an integral α-helix in the dimeric Caprin-1 structure in such a way that the binding of FMRP would not disrupt the homodimerization of Caprin-1. Based on insights from the structures and existing biochemical data, the existence of an evolutionarily conserved ribonucleoprotein (RNP) complex consisting of Caprin-1, FMRP and G3BP1 is proposed. The JEV core protein may bind Caprin-1 at the negatively charged putative protein-binding groove and an adjacent E-rich sequence to hijack the RNP complex.
On the influence of crystal size and wavelength on native SAD phasing
Authors: Liebschner Dorothee , Yamada Yusuke , Matsugaki Naohiro , Senda Miki , Senda Toshiya
Published: May 2016

Native SAD is an emerging phasing technique that uses the anomalous signal of native heavy atoms to obtain crystallographic phases. The method does not require specific sample preparation to add anomalous scatterers, as the light atoms contained in the native sample are used as marker atoms. The most abundant anomalous scatterer used for native SAD, which is present in almost all proteins, is sulfur. However, the absorption edge of sulfur is at low energy (2.472 keV = 5.016 Å), which makes it challenging to carry out native SAD phasing experiments as most synchrotron beamlines are optimized for shorter wavelength ranges where the anomalous signal of sulfur is weak; for longer wavelengths, which produce larger anomalous differences, the absorption of X-rays by the sample, solvent, loop and surrounding medium (e.g.air) increases tremendously. Therefore, a compromise has to be found between measuring strong anomalous signal and minimizing absorption. It was thus hypothesized that shorter wavelengths should be used for large crystals and longer wavelengths for small crystals, but no thorough experimental analyses have been reported to date. To study the influence of crystal size and wavelength, native SAD experiments were carried out at different wavelengths (1.9 and 2.7 Å with a helium cone; 3.0 and 3.3 Å with a helium chamber) using lysozyme and ferredoxin reductase crystals of various sizes. For the tested crystals, the results suggest that larger sample sizes do not have a detrimental effect on native SAD data and that long wavelengths give a clear advantage with small samples compared with short wavelengths. The resolution dependency of substructure determination was analyzed and showed that high-symmetry crystals with small unit cells require higher resolution for the successful placement of heavy atoms.
DNA polymorphism in crystals: three stable conformations for the decadeoxynucleotide d(GCATGCATGC)
Authors: Thirugnanasambandam Arunachalam , Karthik Selvam , Artheswari Gunanithi , Gautham Namasivayam
Published: May 2016

High-resolution structures of DNA fragments determined using X-ray crystallography or NMR have provided descriptions of a veritable alphabet of conformations. They have also shown that DNA is a flexible molecule, with some sequences capable of adopting two different structures. Here, the first example is presented of a DNA fragment that can assume three different and distinct conformations in crystals. The decanucleotide d(GCATGCATGC) was previously reported to assume a single-stranded double-fold structure. In one of the two crystal structures described here the decamer assumes both the double-fold conformation and, simultaneously, the more conventional B-type double-helical structure. In the other crystal the sequence assumes the A-type double-helical conformation. These results, taken together with CD spectra, which were recorded as the decamer was titrated against four metal ions and spermine, indicate that the molecule may exist as a mixed population of structures in solution. Small differences in the environmental conditions, such as the concentration of metal ion, may decide which of these crystallizes out. The results also support the idea that it may be possible for DNA to change its structure to suit the binding requirements of proteins or drugs.
Transferable aspherical atom model refinement of protein and DNA structures against ultrahigh-resolution X-ray data
Authors: Malinska Maura , Dauter Zbigniew
Published: May 2016

In contrast to the independent-atom model (IAM), in which all atoms are assumed to be spherical and neutral, the transferable aspherical atom model (TAAM) takes into account the deformed valence charge density resulting from chemical bond formation and the presence of lone electron pairs. Both models can be used to refine small and large molecules,e.g.proteins and nucleic acids, against ultrahigh-resolution X-ray diffraction data. The University at Buffalo theoretical databank of aspherical pseudo-atoms has been used in the refinement of an oligopeptide, of Z-DNA hexamer and dodecamer duplexes, and of bovine trypsin. The application of the TAAM to these data improves the quality of the electron-density maps and the visibility of H atoms. It also lowers the conventionalRfactors and improves the atomic displacement parameters and the results of the Hirshfeld rigid-bond test. An additional advantage is that the transferred charge density allows the estimation of Coulombic interaction energy and electrostatic potential.
Biochemical and structural characterization ofKlebsiella pneumoniaeoxamate amidohydrolase in the uric acid degradation pathway
Authors: Hicks Katherine A. , Ealick Steven E.
Published: May 2016

HpxW from the ubiquitous pathogenKlebsiella pneumoniaeis involved in a novel uric acid degradation pathway downstream from the formation of oxalurate. Specifically, HpxW is an oxamate amidohydrolase which catalyzes the conversion of oxamate to oxalate and is a member of the Ntn-hydrolase superfamily. HpxW is autoprocessed from an inactive precursor to form a heterodimer, resulting in a 35.5 kDa α subunit and a 20 kDa β subunit. Here, the structure of HpxW is presented and the substrate complex is modeled. In addition, the steady-state kinetics of this enzyme and two active-site variants were characterized. These structural and biochemical studies provide further insight into this class of enzymes and allow a mechanism for catalysis consistent with other members of the Ntn-hydrolase superfamily to be proposed.
Relationships among attachment theory, social capital perspective, personality characteristics, and Facebook self-disclosure
Authors: Aharony Noa
Published: May 2016

Purpose

– Due to the growth of Facebook use, the question arises regarding which factors may influence individuals’ self-disclosure while using the site. The purpose of this paper is to examine: first, to what extent does attachment theory explain users’ self-disclosure on Facebook; second, to what extent does the social capital paradigm explain users’ self-disclosure on Facebook; third, to what extent do personality characteristics explain users’ self-disclosure on Facebook; and fourth, to what extent do demographic details explain users’ self-disclosure on Facebook.

Design/methodology/approach

– Research was conducted in Israel and included 183 library and information science students. Participants were asked to complete the following questionnaires: personal details, Facebook use, importance of Facebook use, demographic disclosure, picture disclosure, disclosure, social capital, attachment, and personality.

Findings

– The current study extends research about Facebook self-disclosure and confirms that attachment theory, as well as social capital, personality traits, and age significantly predict Facebook self-disclosure.

Originality/value

– We should bear in mind that there are certain people who may benefit from disclosing information on Facebook, while there are others who may be at risk, because they reveal more personal information than they intended and the line between the two is fuzzy. Individuals should bear in mind that if they disclose too much information, they might become easy targets for abuse, internet predators, sexual solicitation, sexting, cyberbullying, and online harassment.
Determinants of effective SoLoMo advertising from the perspective of social capital
Authors: Lee Ya-Ching
Published: May 2016

Purpose

– The purpose of this paper is to investigate factors affecting attitudes toward social-local-mobile (SoLoMo) advertising from the perspective of social capital.

Design/methodology/approach

– There were 422 respondents filling out the survey instrument. The research model in this study is tested using SPSS 20 software.

Findings

– The results show that structural, relational, and cognitive dimensions of social capital have impacts on consumer attitudes toward SoLoMo advertising.

Originality/value

– It contributes to the literature by advancing our knowledge about determinants of effective SoLoMo advertising from the perspective of social capital. It also provides constructs that constitute the three dimensions in advertising. The author expands the understanding of the social relations under the context of business to consumer by adding substantial nuances to the understanding of the role of social capital in advertising. Finally, this study provides practical suggestions.
Product advertising and viral stealth marketing in online videos
Authors: Wendt Lars Michael , Griesbaum Joachim , Kölle Ralph
Published: May 2016

Purpose

– In the context of social media marketing, so called viral stealth videos (VSVs) often attract as much or even more attention than videos that directly advertise products (product advertising video (PAV)). However, beyond this, the product or brand-related impact of such videos is not so clear. In this context, the purpose of this paper is to investigate brand perception of PAVs and VSVs in YouTube.

Design/methodology/approach

– The research design is based on an examination of comments of six VSVs and six PAVs on YouTube. Therefore, the content of 1,080 posts was analyzed to capture the topic, the attitude toward the video and the pragmatic intent of posts.

Findings

– Results indicate that there are strong differences with regard to users ' perception of the two analyzed video type segments. The content of VSVs is clearly recognized as positive more often than the content of PAVs. In contrast, only PAVs evoke substantial brand awareness but receive rather mixed results with regards to brand assessment.

Research limitations/implications

– As a whole, the study is widely descriptive and of explorative value. Nevertheless, the research design can be estimated as a first step to measure the brand-related impact of online videos. Ideally, the data generated in the investigation should be combined with traffic and conversion data of the brands’ websites to get an encompassing picture of the marketing related impact of the investigated online videos.

Practical implications

– Seen from a marketers’ perspective, one can recommend PAVs over VSVs as there are hardly any brand-related impacts of VSVs visible in online communication. PAVs are perceived less positively but they are able to evoke brand awareness at least.

Originality/value

– According to the authors’ knowledge this investigation is one of only a few studies that analyzes real online communication in the context of video-based online marketing.
A toolkit for optimizing fish passage barrier mitigation actions
Authors: King Steven , O'Hanley Jesse R. , Newbold Lynda R. , Kemp Paul S. , Diebel Matthew W.
Published: May 2016

The presence of dams, stream–road crossings and other infrastructure often compromises the connectivity of rivers, leading to reduced fish abundance and diversity. The assessment and mitigation of river barriers is critical to the success of restoration efforts aimed at restoring river integrity.

In this study, we present a combined modelling approach involving statistical regression methods and mixed integer linear programming to maximize resident fish species richness within a catchment through targeted barrier mitigation. Compared to existing approaches, our proposed method provides enhanced biological realism while avoiding the use of complex and computationally intensive population/ecosystem models.

To estimate barrier passability quickly and at low cost, we further outline a rapid barrier assessment methodology. The methodology is used to characterize potential passage barriers for various fish species common to the UK but can be readily adapted to different planning areas and other species of interest.

We demonstrate the applicability of our barrier assessment and prioritization approach based on a case study of the River Wey, located in south‐east England. We find that significant increases in species richness can be achieved for modest investment in barrier mitigation. In particular, dams and weirs with low passability located on mid‐ to high‐order streams are identified as top priorities for mitigation.

Synthesis and applications. Our study shows the benefits of combining a coarse resolution barrier assessment methodology with state‐of‐the‐art optimization modelling to cost‐effectively plan fish passage barrier mitigation actions. The modelling approach can help inform on‐the‐ground river restoration decision‐making by providing a recommended course of action that best allocates limited resources in order to restore longitudinal connectivity and maximize ecological gains.
Exon capture optimization in amphibians with large genomes
Authors: McCartney‐Melstad Evan , Mount Genevieve G. , Shaffer H. Bradley
Published: May 2016

Gathering genomic‐scale data efficiently is challenging for nonmodel species with large, complex genomes. Transcriptome sequencing is accessible for organisms with large genomes, and sequence capture probes can be designed from such mRNA sequences to enrich and sequence exonic regions. Maximizing enrichment efficiency is important to reduce sequencing costs, but relatively few data exist for exon capture experiments in nonmodel organisms with large genomes. Here, we conducted a replicated factorial experiment to explore the effects of several modifications to standard protocols that might increase sequence capture efficiency for amphibians and other taxa with large, complex genomes. Increasing the amounts of c0t‐1 repetitive sequence blocker and individual input DNA used in target enrichment reactions reduced the rates of PCR duplication. This reduction led to an increase in the percentage of unique reads mapping to target sequences, essentially doubling overall efficiency of the target capture from 10.4% to nearly 19.9% and rendering target capture experiments more efficient and affordable. Our results indicate that target capture protocols can be modified to efficiently screen vertebrates with large genomes, including amphibians.
Deficits in axon‐associated proteins in prefrontal white matter in bipolar disorder but not schizophrenia
Authors: Shao Li , Golbaz Khashayar , Honer William G , Beasley Clare L
Published: May 2016

Objectives

Brain imaging studies have implicated white matter dysfunction in the pathophysiology of both bipolar disorder (BD) and schizophrenia (SCZ). However, the contribution of axons to white matter pathology in these disorders is not yet understood. Maintenance of neuronal function is dependent on the active transport of biological material, including synaptic proteins, along the axon. In this study, the expression of six proteins associated with axonal transport of synaptic cargoes was quantified in postmortem samples of prefrontal white matter in subjects with BD, those with SCZ, and matched controls, as a measure of axonal dysfunction in these disorders.

Methods

Levels of the microtubule‐associated proteins β‐tubulin and microtubule‐associated protein 6 (MAP6), the motor and accessory proteins kinesin‐1 and disrupted‐in‐schizophrenia 1 (DISC1), and the synaptic cargoes synaptotagmin and synaptosomal‐associated protein‐25 (SNAP‐25) were quantified in white matter adjacent to the dorsolateral prefrontal cortex in subjects with BD (n = 34), subjects with SCZ (n = 35), and non‐psychiatric controls (n = 35) using immunoblotting and an enzyme‐linked immunosorbent assay (ELISA).

Results

Protein expression of β‐tubulin, kinesin‐1, DISC1, synaptotagmin, and SNAP‐25 was significantly lower in subjects with BD compared to controls. Levels of axon‐associated proteins were also lower in subjects with SCZ, but failed to reach statistical significance.

Conclusions

These data provide evidence for deficits in axon‐associated proteins in prefrontal white matter in BD. Findings are suggestive of decreased axonal density or dysregulation of axonal function in this disorder.
New mouse model of acute adult T‐cell leukemia generated by transplantation of AKT, BCLxL, and HBZ‐transduced T cells
Authors: Kasugai Yumiko , Yoshida Noriaki , Ohshima Koichi , Matsuo Keitaro , Seto Masao , Tsuzuki Shinobu
Published: May 2016

Adult T‐cell leukemia/lymphoma (ATL) develops in human T‐cell leukemia virus type 1 (HTLV‐1) carriers. Although the HTLV‐1‐encoded HBZ gene is critically involved, HBZ alone is insufficient and additional, cooperative “hits” are required for the development of ATL. Candidate cooperative hits are being defined, but methods to rapidly explore their roles in ATL development in collaboration with HBZ are lacking. Here, we present a new mouse model of acute type ATL that can be generated rapidly by transplanting in vitro‐induced T cells that have been retrovirally transduced with HBZ and two cooperative genes, BCLxL and AKT, into mice. Co‐transduction of HBZ and BCLxL/AKT allowed these T cells to grow in vitro in the absence of cytokines (Flt3‐ligand and interleukin‐7), which did not occur with any two‐gene combination. Although transplanted T cells were a mixture of cells transduced with different combinations of the genes, tumors that developed in mice were composed of HBZ/BCLxL/AKT triply transduced T cells, showing the synergistic effect of the three genes. The genetic/epigenetic landscape of ATL has only recently been elucidated, and the roles of additional “hits” in ATL pathogenesis remain to be explored. Our model provides a versatile tool to examine the roles of these hits, in collaboration with HBZ, in the development of acute ATL.