bluuebunny/crossref_metadata_2025 huggingface dataset:
A Chromosome-level Sequence Assembly Reveals the Structure of theArabidopsis thalianaNd-1 Genome and its Gene Set
Authors: Pucker Boas , Holtgräwe Daniela , Stadermann Kai Bernd , Frey Katharina , Huettel Bruno , Reinhardt Richard , Weisshaar Bernd
Published: September 2018

Background

In addition to the BAC-based reference sequence of the accession Columbia-0 from the year 2000, several short read assemblies of THE plant model organismArabidopsis thalianawere published during the last years. Also, a SMRT-based assembly of Landsbergerectahas been generated that identified translocation and inversion polymorphisms between two genotypes of the species.

Results

Here we provide a chromosome-arm level assembly of theA. thalianaaccession Niederzenz-1 (AthNd-1_v2c) based on SMRT sequencing data. The best assembly comprises 69 nucleome sequences and displays a contig length of up to 16 Mbp. Compared to an earlier Illumina short read-based NGS assembly (AthNd-1_v1), a 75 fold increase in contiguity was observed for AthNd-1_v2c. To assign contig locations independent from the Col-0 gold standard reference sequence, we used genetic anchoring to generate ade novoassembly. In addition, we assembled the chondrome and plastome sequences.

Conclusions

Detailed analyses of AthNd-1_v2c allowed reliable identification of large genomic rearrangements betweenA. thalianaaccessions contributing to differences in the gene sets that distinguish the genotypes. One of the differences detected identified a gene that is lacking from the Col-0 gold standard sequence. Thisde novoassembly extends the known proportion of theA. thalianapan-genome.
Budding yeast complete DNA replication after chromosome segregation begins
Authors: Ivanova Tsvetomira , Maier Michael , Missarova Alsu , Ziegler-Birling Céline , Carey Lucas B. , Mendoza Manuel
Published: September 2018

To faithfully transmit genetic information, cells must replicate their entire genome before division. This is thought to be ensured by the temporal separation of replication and chromosome segregation. Here we show that in a substantial fraction of unperturbed yeast cells, DNA replication finishes during anaphase, late in mitosis. High cyclin-Cdk activity inhibits replication in metaphase, and the decrease in cyclin-Cdk activity during mitotic exit allows DNA replication to finish at difficult-to-replicate regions. Replication during late mitosis correlates with elevated mutation rates, including copy number variation. Thus, yeast cells temporally overlap replication and chromosome segregation during normal growth, possibly allowing cells to maximize population-level growth rate while simultaneously exploring greater genetic space.

One Sentence Summary

Completion of DNA replication is coupled to downregulation of Cyclin-Dependent Kinase during mitotic exit.
Differences in Therapeutic Efficacy in Pancreatic Cancer Between Interstitial and Superficial Light Delivery Strategies in Targeted Photo Therapy
Authors: De Magalhães Nzola ,   HTL(ASCP)CM
Published: September 2018

The purpose of this study was to determine if therapeutic efficacy of a Cetuximab based near-infrared (NIR) targeted photo therapy (TPT) was dependent on light delivery strategies. We examined the cytotoxic effects of TPT in a pancreatic cancer mouse model, when administered to tumors interstitially and superficially.

A subcutaneous mouse model of pancreatic cancer using BXPC-3 -GFP cells was established in male athymic (nu/nu) mice. The mice received intravenous (IV) injection of Cetuximab-IR700DX, 24 hours prior to near-infrared light irradiation. Interstitial illumination was administered at a 400mW/cm fixed power output, at a light dose of 100 J/cm to half the mice and at 300 J/cm to the remaining mice. Superficial illumination was administered at a 150mw/cm2fixed power density at a dose of 50 J/cm2to half the mice, and at 250 J/cm2to the other half. Cellular damage and decrease in cell viability was determined by the decrease in GFP fluorescence intensity levels in whole animal images and in relative intensity measurements.

Interstitially administered TPT resulted in greater long-term permanent damage (72 hours post treatment) to tumor cells (0% recovery at low dose, and 11% recovery at high dose) compared to superficially administered TPT (1% recovery at low dose, and 44% recovery at high dose). While these results demonstrated that near-infrared targeted photo therapy efficacy was dependent on the type of light delivery strategy, overall, both superficial and interstitial Cet-IR700DX based near-infrared targeted photo therapy can effect significant long-term damage (less signal recovery) to pancreatic cancer cellsin vivoat lower doses regimens, compared to higher dose regimens (higher signal recovery).
Distinct pathogenic genes causing intellectual disability and autism exhibit overlapping effects on neuronal network development
Authors: Frega Monica , Selten Martijn , Mossink Britt , Keller Jason M. , Linda Katrin , Moerschen Rebecca , Qu Jieqiong , Koerner Pierre , Jansen Sophie , Bijvank Elske , Oudakker Astrid , Kleefstra Tjitske , Bokhoven Hans van , Zhou Huiqing , Schubert Dirk , Kasri Nael Nadif
Published: September 2018

An intriguing question in medical biology is how mutations in functionally distinct genes can lead to similar clinical phenotypes. For example, patients with mutations in distinct epigenetic regulators EHMT1, MBD5, MLL3 or SMARCB1 share the core clinical features of intellectual disability (ID), autism spectrum disorder (ASD) and facial dysmorphisms. To elucidate how these phenotypic similarities are reflected by convergence at the molecular, cellular and neuronal network level, we directly compared the effects of their loss of function in neurons. Interestingly, knockdown of each gene resulted in hyperactive neuronal networks with altered patterns of synchronized activity. At the single-cell level, we found genotype-specific changes in intrinsic excitability and excitatory-inhibitory balance, but in all cases leading to increased excitability. Congruent with our physiological findings, we identified dysregulated genes that converge on biological and cellular pathways related to neuronal excitability and synaptic function, including genes previously implicated in ID/ASD. Yet, our data suggests that the common cellular phenotypes depend on the ensemble of dysregulated genes engaged in neuronal excitability rather than the direction of transcriptional changes of individual genes. The demonstration of increasing convergence from molecular pathways to neuronal networks may be a paradigm for other types of ID/ASD.
A Type I Restriction-Modification System Associated withEnterococcus faeciumSubspecies Separation
Authors: Huo Wenwen , Adams Hannah M. , Trejo Cristian , Badia Rohit , Palmer Kelli L.
Published: September 2018

The gastrointestinal colonizerEnterococcus faeciumis a leading cause of hospital-acquired infections. Multidrug-resistant (MDR)E. faeciumare particularly concerning for infection treatment. Previous comparative genomic studies revealed that subspecies referred to as Clade A and Clade B exist withinE. faecium. MDRE. faeciumbelong to Clade A, while Clade B consists of drug-susceptible fecal commensalE. faecium. Isolates from Clade A are further grouped into two sub-clades, A1 and A2. In general, Clade A1 isolates are hospital epidemic isolates whereas Clade A2 isolates are isolates from animals and sporadic human infections. Such phylogenetic separation indicates that reduced gene exchange occurs between the clades. We hypothesize that endogenous barriers to gene exchange exist betweenE. faeciumclades. Restriction-modification (R-M) systems are such barriers in other microbes. We utilized bioinformatics analysis coupled with second generation and third generation deep sequencing platforms to characterize the methylome of two representativeE. faeciumstrains, one from Clade A1 and one from Clade B. We identified a Type I R-M system that is Clade A1-specific, is active for DNA methylation, and significantly reduces transformability of Clade A1E. faecium. Based on our results, we conclude that R-M systems act as barriers to horizontal gene exchange inE. faeciumand propose that R-M systems contribute toE. faeciumsubspecies separation.

IMPORTANCE

Enterococcus faeciumis a leading cause of hospital-acquired infections around the world. Rising antibiotic resistance in certainE. faeciumlineages leaves fewer treatment options. The overarching aim of the attached work was to determine whether restriction-modification (R-M) systems contribute to the structure of theE. faeciumspecies, wherein hospital-epidemic and non-hospital-epidemic isolates have distinct evolutionary histories and highly resolved clade structures. R-M provides bacteria with a type of innate immunity to horizontal gene transfer (HGT). We identified a Type I R-M system that is enriched in the hospital-epidemic clade and determined that it is active for DNA modification activity and significantly impacts HGT. Overall, this work is important because it provides a mechanism for the observed clade structure ofE. faeciumas well as a mechanism for facilitated gene exchange among hospital-epidemicE. faecium.
Identification of novel protein lysine acetyltransferases inEscherichia coli
Authors: Christensen David G. , Meyer Jesse G. , Baumgartner Jackson T. , D’Souza Alexandria K. , Nelson William C. , Payne Samuel H. , Kuhn Misty L. , Schilling Birgit , Wolfe Alan J.
Published: September 2018

Post-translational modifications, such as Nε-lysine acetylation, regulate protein function. Nε-lysine acetylation can occur either non-enzymatically or enzymatically. The non-enzymatic mechanism uses acetyl phosphate (AcP) or acetyl coenzyme A (AcCoA) as acetyl donors to modify an Nε-lysine residue of a protein. The enzymatic mechanism uses Nε-lysine acetyltransferases (KATs) to specifically transfer an acetyl group from AcCoA to Nε-lysine residues on proteins. To date, only one KAT (YfiQ, also known as Pka and PatZ) has been identified inE. coli. Here, we demonstrate the existence of 4 additionalE. coliKATs: RimI, YiaC, YjaB, and PhnO. In a genetic background devoid of all known acetylation mechanisms (most notably AcP and YfiQ) and one deacetylase (CobB), overexpression of these putative KATs elicited unique patterns of protein acetylation. We mutated key active site residues and found that most of them eliminated enzymatic acetylation activity. We used mass spectrometry to identify and quantify the specificity of YfiQ and the four novel KATs. Surprisingly, our analysis revealed a high degree of substrate specificity. The overlap between KAT-dependent and AcP-dependent acetylation was extremely limited, supporting the hypothesis that these two acetylation mechanisms play distinct roles in the post-translational modification of bacterial proteins. We further showed that these novel KATs are conserved across broad swaths of bacterial phylogeny. Finally, we determined that one of the novel KATs (YiaC) and the known KAT (YfiQ) can negatively regulate bacterial migration. Together, these results emphasize distinct and specific non-enzymatic and enzymatic protein acetylation mechanisms present in bacteria.

Importance

Nε-lysine acetylation is one of the most abundant and important post-translational modifications across all domains of life. One of the best-studied effects of acetylation occurs in eukaryotes, where acetylation of histone tails activates gene transcription. Although bacteria do not have true histones, Nε-lysine acetylation is prevalent; however, the role of these modifications is mostly unknown. We constructed anE. colistrain that lacked both known acetylation mechanisms to identify four new Nε-lysine acetyltransferases (RimI, YiaC, YjaB, and PhnO). We used mass spectrometry to determine the substrate specificity of these acetyltransferases. Structural analysis of selected substrate proteins revealed site-specific preferences for enzymatic acetylation that had little overlap with the preferences of the previously reported acetyl-phosphate non-enzymatic acetylation mechanism. Finally, YiaC and YfiQ appear to regulate flagellar-based motility, a phenotype critical for pathogenesis of many organisms. These acetyltransferases are highly conserved and reveal deeper and more complex roles for bacterial post-translational modification.
Innolysins: A novel approach to engineer endolysins to kill Gram-negative bacteria
Authors: Zampara Athina , Holst Sørensen Martine C. , Grimon Dennis , Antenucci Fabio , Briers Yves , Brøndsted Lone
Published: September 2018

Bacteriophage-encoded endolysins degrading the essential peptidoglycan of bacteria are promising alternative antimicrobials to handle the global threat of antibiotic resistant bacteria. However, endolysins have limited use against Gram-negative bacteria, since their outer membrane prevents access to the peptidoglycan. Here we present Innolysins, a novel concept for engineering endolysins that allows the enzymes to pass through the outer membrane, hydrolyse the peptidoglycan and kill the target bacterium. Innolysins combine the enzymatic activity of endolysins with the binding capacity of phage receptor binding proteins (RBPs). As our proof of concept, we used phage T5 endolysin and receptor binding protein Pb5, which binds irreversibly to the phage receptor FhuA involved in ferrichrome transport inEscherichia coli. In total, we constructed twelve Innolysins fusing endolysin with Pb5 or the binding domain of Pb5 with or without flexible linkers in between. While the majority of the Innolysins maintained their muralytic activity, Innolysin#6 also showed bactericidal activity againstE. colireducing the number of bacteria by 1 log, thus overcoming the outer membrane barrier. Using anE. coli fhuAdeletion mutant, we demonstrated that FhuA is required for bactericidal activity, supporting that the specific binding of Pb5 to its receptor onE. coliis needed for the endolysin to access the peptidoglycan. Accordingly, Innolysin#6 was able to kill other bacterial species that carry conserved FhuA homologs such asShigella sonneiandPseudomonas aeruginosa. In summary, the Innolysin approach expands recent protein engineering strategies allowing customization of endolysins by exploiting phage RBPs to specifically target Gram-negative bacteria.

IMPORTANCE

The extensive use of antibiotics has led to the emergence of antimicrobial resistant bacteria responsible for infections causing more than 50,000 deaths per year across Europe and the US. In response, the World Health Organization has stressed an urgent need to discover new antimicrobials to control in particular Gram-negative bacterial pathogens, due to their extensive multi-drug resistance. However, the outer membrane of Gram-negative bacteria limits the access of many antibacterial agents to their targets. Here, we developed a new approach, Innolysins that enable endolysins to overcome the outer membrane by exploiting the binding specificity of phage receptor binding proteins. As proof of concept, we constructed Innolysins againstE. coliusing the endolysin and the receptor binding protein of phage T5. Given the rich diversity of phage receptor binding proteins and their different binding specificities, our proof of concept paves the route for creating an arsenal of pathogen specific alternative antimicrobials.
Human CtIP forms a tetrameric dumbbell-shaped particle which binds and bridges complex DNA end structures for double-strand break repair
Authors: Wilkinson O.J. , Martín-González A. , Kang H.J. , Northall S.J. , Wigley D.B. , Moreno-Hererro F. , Dillingham M.S.
Published: September 2018

CtIP is involved in the resection of double-stranded DNA breaks during the S and G2 phases of the cell cycle for repair by homologous recombination. Acting in concert with the MRN complex, it plays a particularly important role in handling complex DNA end structures by localised nucleolytic processing of DNA termini in preparation for longer range resection. Here we show that human CtIP is a tetrameric protein adopting a dumbbell architecture in which DNA binding domains are connected by long coiled-coils. The protein complex binds two short DNA duplexes with high affinity and bridges DNA molecules in trans. DNA binding is potentiated by dephosphorylation and is not specific for DNA end structures per se. However, the affinity for linear DNA molecules is increased if the DNA terminates with more complex structures including forked ssDNA overhangs and model nucleoprotein conjugates. This work provides a biochemical and structural basis for the function of CtIP at complex DNA breaks.
PhenoMeNal: Processing and analysis of Metabolomics data in the Cloud
Authors: Peters Kristian , Bradbury James , Bergmann Sven , Capuccini Marco , Cascante Marta , de Atauri Pedro , Ebbels Timothy M D , Foguet Carles , Glen Robert , Gonzalez-Beltran Alejandra , Guenther Ulrich , Handakas Evangelos , Hankemeier Thomas , Haug Kenneth , Herman Stephanie , Holub Petr , Izzo Massimiliano , Jacob Daniel , Johnson David , Jourdan Fabien , Kale Namrata , Karaman Ibrahim , Khalili Bita , Khonsari Payam Emami , Kultima Kim , Lampa Samuel , Larsson Anders , Ludwig Christian , Moreno Pablo , Neumann Steffen , Novella Jon Ander , O’Donovan Claire , Pearce Jake TM , Peluso Alina , Pireddu Luca , Piras Marco Enrico , Reed Michelle AC , Rocca-Serra Philippe , Roger Pierrick , Rosato Antonio , Rueedi Rico , Ruttkies Christoph , Sadawi Noureddin , Salek Reza M , Sansone Susanna-Assunta , Selivanov Vitaly , Spjuth Ola , Schober Daniel , Thévenot Etienne A. , Tomasoni Mattia , van Rijswijk Merlijn , van Vliet Michael , Viant Mark R , Weber Ralf J. M. , Zanetti Gianluigi , Steinbeck Christoph
Published: September 2018

Background

Metabolomics is the comprehensive study of a multitude of small molecules to gain insight into an organism’s metabolism. The research field is dynamic and expanding with applications across biomedical, biotechnological and many other applied biological domains. Its computationally-intensive nature has driven requirements for open data formats, data repositories and data analysis tools. However, the rapid progress has resulted in a mosaic of independent – and sometimes incompatible – analysis methods that are difficult to connect into a useful and complete data analysis solution.

Findings

The PhenoMeNal (Phenome and Metabolome aNalysis) e-infrastructure provides a complete, workflow-oriented, interoperable metabolomics data analysis solution for a modern infrastructure-as-a-service (IaaS) cloud platform. PhenoMeNal seamlessly integrates a wide array of existing open source tools which are tested and packaged as Docker containers through the project’s continuous integration process and deployed based on a kubernetes orchestration framework. It also provides a number of standardized, automated and published analysis workflows in the user interfaces Galaxy, Jupyter, Luigi and Pachyderm.

Conclusions

PhenoMeNal constitutes a keystone solution in cloud infrastructures available for metabolomics. It provides scientists with a ready-to-use, workflow-driven, reproducible and shareable data analysis platform harmonizing the software installation and configuration through user-friendly web interfaces. The deployed cloud environments can be dynamically scaled to enable large-scale analyses which are interfaced through standard data formats, versioned, and have been tested for reproducibility and interoperability. The flexible implementation of PhenoMeNal allows easy adaptation of the infrastructure to other application areas and ‘omics research domains.
Estimation of realized rates of genetic gain and indicators for breeding program assessment
Authors: Rutkoski J.E.
Published: September 2018

Routine estimation of the rate of genetic gain (ΔGt) realized by a breeding program has been proposed as a means to monitor its effectiveness. Several methods of realizedΔGtestimation have been utilized in other studies, but none have been objectively evaluated in a plant breeding context. Stochastic simulations of 80 rice (Oryza sativa) breeding programs over 28 years were done to generate data used to evaluate five methods of realizedΔGtestimation in terms of error, precision, efficiency and correlation between true and predicted annual mean breeding values. Two indicators ofΔGt, the expectedΔGtand the average number of equivalent complete generations (EqCg), were described and evaluated. At best, estimates of realizedΔGtwere over or underestimated by 15% and 27% when considering all 28 years and the past 15 years of breeding respectively. The best methods were the control population, estimated breeding value, and ERA trial methods. Among these, correlations between true and estimatedΔGtwere at best 0.59, indicating that these methods cannot very accurately rank breeding programs in terms of realizedΔGt. The expectedΔGtand the average EqCg were shown to be useful indicators for determining if a non-zero genetic gain is expected. Determining which of the three best realizedΔGtestimation methods evaluated, if any, would be appropriate for any given breeding program should be done with careful consideration of the objectives, resources, seed stocks, and structure of the data available.
A SKI subcomplex specifically required for the degradation of ribosome-free RNA regions
Authors: Zhang Elodie , Khanna Varun , Namane Abdelkader , Doyen Antonia , Dacheux Estelle , Turcotte Bernard , Jacquier Alain , Fromont-Racine Micheline
Published: September 2018

The Ski2-Ski3-Ski8 (SKI) complex assists the RNA exosome during the 3’-5’ degradation of cytoplasmic transcripts. Previous reports showed that the SKI complex is involved in the 3’-5’ degradation of mRNA, including 3’ untranslated regions (UTRs), devoid of ribosomes. Paradoxically, we recently showed that the SKI complex directly interacts with ribosomes during the co-translational mRNA decay and that this interaction is necessary for its RNA degradation promoting activity. Here, we characterized a new SKI-associated factor, Ska1, which antagonizes the SKI-ribosome interaction. We showed that the SKI-Ska1-subcomplex is specifically involved in the degradation of ribosome-free RNA regions such as long mRNA 3’UTRs and cytoplasmic lncRNAs. We propose a model in which the SKI-exosome complex first targets ribosome-free RNA 3’ends in its Ska1-associated form. When the complex reaches the mRNA coding sequence, the Ska1-SKI-exosome complex is exchanged for the SKI-exosome, which interacts directly with ribosomes in order to resume the degradation process.
Behavioral Analysis of Substrate Texture Preference in a Leech,Helobdella austinensis
Authors: Kim Rachel C. , Le Dylan , Ma Kenny , Heath-Heckman Elizabeth A. C. , Whitehorn Nathan , Kristan William B. , Weisblat David A.
Published: September 2018

Leeches in the wild are often found on smooth surfaces, such as vegetation, smooth rocks or human artifacts such as bottles and cans, thus exhibiting what appears to be a “substrate texture preference behavior”. Here, we have reproduced this behavior under controlled circumstances, by allowing leeches to step about freely on a range of silicon carbide sandpaper substrates. To begin to understand the neural mechanisms underlying this texture preference behavior, we have determined relevant parameters of leech behavior both on uniform substrates of varying textures, and in a behavior choice paradigm in which the leech is confronted with a choice between rougher and smoother substrate textures at each step. We tested two non-exclusive mechanisms which could produce substrate texture preference: 1) a Diffusion Trap mechanism, in which a leech is more likely to stop moving on a smooth surface than on a rough one, and; 2) an Anterior Choice mechanism, in which a leech is more likely to attach its front sucker (prerequisite for taking a step) to a smooth surface than to a rough one. We propose that both mechanisms contribute to the texture preference exhibited by leeches.
Monitoring the eradication of the highly invasive topmouth gudgeon (Pseudorasbora parva) using a novel eDNA assay
Authors: Robinson Chloe Victoria , de Leaniz Carlos Garcia , Rolla Matteo , Consuegra Sofia
Published: September 2018

Aquatic Invasive Species (AIS) represent an important threat for Biodiversity and are one of the factors determining the ecological integrity of water bodies under the Water Framework Directive. Eradication is one of the most effective tools for the management of invasive species but has important economic and ecological trade-offs and its success needs to be carefully monitored. We assessed the eradication success of the topmouth gudgeon (Pseudorasbora parva), an invasive fish that poses significant risks to endemic aquatic fauna, in four ponds previously treated with the piscicide Rotenone using a novel environmental DNA (eDNA)-qPCR assay. Topmouth gudgeon was detected in all four treated ponds using 750 mL water samples and in three of the ponds using 15 mL samples, despite the eradication treatment. The highly sensitive qPCR assay detected topmouth gudgeon in a significantly greater proportion of sites (77.5%) than eDNA detection methods based on conventional PCR (35%). Our results highlight the difficulties of eradicating invasive fish and the need to incorporate reliable monitoring methods as part of a risk management strategy under the Water Framework Directive.
Improved inference of chromosome conformation from images of labeled loci
Authors: Ross Brian C. , Costello James
Published: September 2018

We previously published a method that infers chromosome conformation from images of fluorescently-tagged genomic loci, for the case when there are many loci labeled with each distinguishable color. Here we build on our previous work and improve the reconstruction algorithm to address previous limitations. We show that these improvements 1) increase the reconstruction accuracy and 2) allow the method to be used on large-scale problems involving several hundred labeled loci. Simulations indicate that full-chromosome reconstructions at 1/2 Mb resolution are possible using existing labeling and imaging technologies. The updated reconstruction code and the script files used for this paper are available at: https://github.com/heltilda/align3d.
Maternal Circulating MiRNAs That Predict Infant FASD Outcomes Influence Placental Maturation
Authors: Tseng Alexander M. , Mahnke Amanda H. , Wells Alan B. , Salem Nihal A. , Allan Andrea M. , Roberts Victoria H.J. , Newman Natali , Walter Nicole A.R. , Kroenke Christopher D. , Grant Kathleen A. , Akison Lisa K. , Moritz Karen M. , Chambers Christina D. , Miranda Rajesh C. ,   CIFASD
Published: September 2018

Prenatal Alcohol exposure (PAE), like other pregnancy complications, can result in placental insufficiency and fetal growth restriction, though the linking causal mechanisms are unclear. We previously identified 11 gestationally-elevated maternal circulating miRNAs that predicted infant growth deficits following PAE. Here, we investigated whether theseHEamiRNAs contribute to the pathology of PAE, by inhibiting trophoblast epithelial-mesenchymal transition (EMT), a pathway critical for placental development. We now report for the first time, that PAE inhibits expression of placental pro-EMT pathway members in both rodents and primates, and thatHEamiRNAs collectively, but not individually, mediate placental EMT inhibition.HEamiRNAs collectively, but not individually, also inhibited cell proliferation and the EMT pathway in cultured trophoblasts, while inducing cell stress, and following trophoblast syncytialization, aberrant endocrine maturation. Moreover, a single intra-vascular administration of the pooled murine-expressedHEamiRNAs, to pregnant mice, decreased placental and fetal growth and inhibited expression of pro-EMT transcripts in placenta. Our data suggests thatHEamiRNAs collectively interfere with placental development, contributing to the pathology of PAE, and perhaps also, to other causes of fetal growth restriction.

Maternal gestational circulating microRNAs, predictive of adverse infant outcomes including growth deficits, following prenatal alcohol exposure, contribute to placental pathology by impairing the EMT pathway in trophoblasts.
Bacteria-mediated stabilization of a panel of Picornaviruses
Authors: Aguilera Elizabeth R. , Nguyen Y , Sasaki Jun , Pfeiffer Julie K.
Published: September 2018

Several viruses encounter various bacterial species within the host and in the environment. Despite these close encounters, the effects of bacteria on picornaviruses specifically is not completely understood. Previous work determined that poliovirus (PV), an enteric virus, has enhanced virion stability when exposed to bacteria or bacterial surface polysaccharides such as lipopolysaccharide. Virion stabilization by bacteria may be important for inter-host transmission since a mutant PV with reduced bacterial binding had a fecal-oral transmission defect in mice. Therefore, we investigated whether bacteria broadly enhance stability of picornaviruses from three different genera: Enterovirus (PV and coxsackievirus B3 (CVB3)), Kobuvirus (Aichi virus) and Cardiovirus (Mengo virus). Furthermore, to delineate strain-specific effects, we examined two strains of CVB3 and a PV mutant with enhanced thermal stability. We determined that specific bacterial strains enhance thermal stability of PV and CVB3, while Mengo virus and Aichi virus are stable at high temperatures in the absence of bacteria. Additionally, we determined that bacteria or lipopolysaccharide can stabilize PV, CVB3, Aichi virus, and Mengo virus during exposure to bleach. These effects are likely mediated through direct interactions with bacteria since viruses bound to bacteria in a pull-down assay. Overall, this work reveals shared and distinct effects of bacteria on a panel of picornaviruses.

IMPORTANCE

Recent studies have shown that bacteria promote infection and stabilization of poliovirus particles, but the breadth of these effects on other members of the Picornaviridae family is unknown. Here, we compared the effect of bacteria on four distinct members of the Picornaviridae family. We found that bacteria reduced inactivation of all of the viruses during bleach treatment, but not all viral strains were stabilized by bacteria during heat treatment. Overall, our data provide insight into how bacteria play differential roles on picornavirus stability.
Full-length transcript characterization of SF3B1 mutation in chronic lymphocytic leukemia reveals downregulation of retained introns
Authors: Tang Alison D. , Soulette Cameron M. , Baren Marijke J van , Hart Kevyn , Hrabeta-Robinson Eva , Wu Catherine J. , Brooks Angela N.
Published: September 2018

SF3B1 is one of the most frequently mutated genes in chronic lymphocytic leukemia (CLL) and is associated with poor patient prognosis. While alternative splicing patterns caused by mutations in SF3B1 have been identified with short-read RNA sequencing, a critical barrier in understanding the functional consequences of these splicing changes is that we lack the full transcript context in which these changes are occurring. Using nanopore sequencing technology, we have resequenced full-length cDNA from CLL samples with and without the hotspot SF3B1 K700E mutation, and a normal B cell. We have developed a workflow called FLAIR (Full-Length Alternative Isoform analysis of RNA), leveraging the full-length transcript sequencing data that nanopore affords. We report results from nanopore sequencing that are concordant with known SF3B1 biology from short read sequencing as well as altered intron retention events more confidently observed using long reads. Splicing analysis of nanopore reads between the SF3B1WT and SF3B1K700E samples identifies alternative upstream 3’ splice sites associated with SF3B1K700E. We also find downregulation of intron retention events in SF3B1K700E relative to SF3B1WT and no difference between CLL SF3B1MT and B cell, suggesting an aberrant intron retention landscape in CLL samples lacking SF3B1 mutation. With full-length isoforms, we are able to better estimate the abundance of RNA transcripts that are productive and will likely be translated versus those that are unproductive. Validation from short-read data also reveals a strong branch point sequence in these downregulated intron retention events, consistent with previously reported branch points associated with mutated SF3B1. As nanopore sequencing has yet to become a routine tool for characterization of the transcriptome, our work demonstrates the potential utility of nanopore sequencing for cancer and splicing research.
2.8 Å resolution cryo-EM structure of human parechovirus 3 in complex with Fab from a neutralizing antibody
Authors: Domanska Aušra , Flatt Justin W. , Jukonen Joonas J.J. , Geraets James A. , Butcher Sarah J.
Published: September 2018

Human parechovirus 3 (HPeV3) infection is associated with sepsis in neonates characterized by significant immune activation and subsequent tissue damage. Strategies to limit infection have been unsuccessful due to inadequate molecular diagnostic tools for early detection and lack of a vaccine or specific antiviral therapy. Towards the latter, we present a 2.8 Å-resolution structure of HPeV3 in complex with fragments from a neutralizing human monoclonal antibody AT12-015 using cryo-EM and image reconstruction. Modeling revealed that the epitope extends across neighboring asymmetric units with contributions from capsid proteins VP0, VP1, and VP3. Antibody decoration was found to block binding of HPeV3 to cultured cells. Additionally at high-resolution, it was possible to model a stretch of RNA inside the virion and from this identify the key features that drive and stabilize protein-RNA association during assembly.

Importance

HPeV3 is receiving increasing attention as a prevalent cause of sepsis-like symptoms in neonates, which despite the severity of disease, there are no effective treatments available. Structural and molecular insights into virus neutralization are urgently needed, especially as clinical cases are on the rise. Towards this goal, we present the first structure of HPeV3 in complex with fragments from a neutralizing monoclonal antibody. At high-resolution it was possible to precisely define the epitope that when targeted, prevents virions from binding to cells. Such an atomic-level description is useful for understanding host-pathogen interaction, viral pathogenesis mechanisms, and for finding potential cures for infection and disease.
A Novel Method for the Capture-based Purification of Whole Viral Native RNA Genomes
Authors: Tan Cedric Chih Shen , Maurer-Stroh Sebastian , Wan Yue , Sessions October Michael , de Sessions Paola Florez
Published: September 2018

Current technologies for targeted characterization and manipulation of viral RNA primarily involve amplification or ultracentrifugation with isopycnic gradients of viral particles to decrease host RNA background. The former strategy is non-compatible for characterizing properties innate to RNA strands such as secondary structure, RNA-RNA interactions, and also for nanopore direct RNA sequencing involving the sequencing of native RNA strands. The latter strategy, ultracentrifugation, causes loss in genomic information due to its inability to retrieve unassembled viral RNA. To address this, we developed a novel application of current nucleic acid hybridization technologies for direct characterization of RNA. In particular, we modified a current enrichment protocol to capture whole viral native RNA genomes for downstream RNA assays to circumvent the abovementioned problems. This technique involves hybridization of biotinylated baits at 500 nucleotides (nt) intervals, stringent washes and release of free native RNA strands using DNase I treatment, with a turnaround time of about 6 h 15 min. RT-qPCR was used as the primary proof of concept that capture-based purification indeed removes host background. Subsequently, capture-based purification was applied to direct RNA sequencing as proof of concept that capture-based purification can be coupled with downstream RNA assays. We report that this protocol was able to successfully purify viral RNA by 561-791 fold. We also report that application of this protocol to direct RNA sequencing yielded a reduction in human host RNA background by 1580 fold, a 99.91% recovery of viral genome with at least 15x coverage, and a mean coverage across the genome of 120x. This report is, to the best of our knowledge, the first description of a capture-based purification method for assays that involve direct manipulation or characterisation of native RNA. This report also describes a successful application of capture-based purification as a direct RNA sequencing strategy that addresses certain limitations of current strategies in sequencing RNA viral genomes.
Mi-2/NuRD complex protects stem cell progeny from mitogenic Notch signaling
Authors: Zacharioudaki Eva , Sanjuan Julia Falo , Bray Sarah
Published: September 2018

To progress towards differentiation, the progeny of stem cells need to extinguish expression of stem cell maintenance genes. Failures in these mechanisms that suppress stem cell programmes can lead to supernumerary stem cells and drive tumorigenesis. In Drosophila neural stem cell lineages, excessive Notch signalling results in supernumerary stem cells causing hyperplasia. But the onset of hyperplasia is considerably delayed implying there are mechanisms that resist the mitogenic signal. Monitoring live the expression of an early NSC marker, the Notch target gene E(spl)mγ, revealed that the normal process of NSC fate attenuation is still initiated even in the presence of excess Notch activity so that the re-emergence of stem cell properties occurs only in older progeny. Screening for factors responsible, we found that depletion of Mi-2 and other members of the NuRD ATP remodeling complex dramatically enhanced the Notch-induced hyperplasia. Under these conditions, E(spl)mγ was no longer extinguished in the stem cell progeny, but instead remained at high levels. We propose that Mi-2 is required for decommissioning stem cell enhancers in their progeny, enabling the switch towards a more differentiated fate and rendering them insensitive to mitogenic factors such as Notch.
EMT network-based feature selection improves prognosis prediction in lung adenocarcinoma
Authors: Shao Borong , Bjaanæs Maria M , Åslaug Helland , Schütte Christof , Conrad Tim
Published: September 2018

Various feature selection algorithms have been proposed to identify cancer prognostic biomarkers. In recent years, however, their reproducibility is criticized. The performance of feature selection algorithms is shown to be affected by the datasets, underlying networks and evaluation metrics. One of the causes is the curse of dimensionality, which makes it hard to select the features that generalize well on independent data. Even the integration of biological networks does not mitigate this issue because the networks are large and many of their components are not relevant for the phenotype of interest. With the availability of multi-omics data, integrative approaches are being developed to build more robust predictive models. In this scenario, the higher data dimensions create greater challenges.

We proposed a phenotype relevant network-based feature selection (PRNFS) framework and demonstrated its advantages in lung cancer prognosis prediction. We constructed cancer prognosis relevant networks based on epithelial mesenchymal transition (EMT) and integrated them with different types of omics data for feature selection. With less than 2.5% of the total dimensionality, we obtained EMT prognostic signatures that achieved remarkable prediction performance (average AUC values >0.8), very significant sample stratifications, and meaningful biological interpretations. In addition to finding EMT signatures from different omics data levels, we combined these single-omics signatures into multi-omics signatures, which improved sample stratifications significantly. Both single- and multi-omics EMT signatures were tested on independent multi-omics lung cancer datasets and significant sample stratifications were obtained.
Improved Characterization of the Solution Kinetics and Thermodynamics of Biotin, Biocytin and HABA Binding to Avidin and Streptavidin
Authors: Delgadillo Roberto F. , Mueser Timothy C. , Zaleta-Rivera Kathia , Carnes Katie A. , González-Valdez José , Parkhurst Lawrence J.
Published: September 2018

The high affinity (KD∼ 10−15M) of biotin to avidin and streptavidin is the essential component in a multitude of bioassays with many experiments using biotin modifications to invoke coupling. Equilibration times suggested for these assays assume that the association rate constant (kon) is approximately diffusion limited (109M−1s−1) but recent single molecule and surface binding studies indicate they are slower than expected (105to 107M−1s−1). In this study, we asked whether these reactions in solution are diffusion controlled, what reaction model and thermodynamic cycle described the complex formation, and the functional differences between avidin and streptavidin. We have studied the biotin association by two stopped-flow methodologies using labeled and unlabeled probes: I) fluorescent probes attached to biotin and biocytin; and II) unlabeled biotin and HABA, 2-(4’-hydroxyazobenzene)-benzoic acid. Native avidin and streptavidin are homo-tetrameric and the association data show no cooperativity between the binding sites. The konvalues of streptavidin are faster than avidin but slower than expected for a diffusion limited reaction in both complexes. Moreover, the Arrhenius plots of the konvalues revealed strong temperature dependence with large activation energies (6-15 kcal/mol) that do not correspond to a diffusion limited process (3-4 kcal/mol). The data suggest that the avidin binding sites are deeper and less accessible than those of streptavidin. Accordingly, we propose a simple reaction model with a single transition state for non-immobilized reactants whose forward thermodynamic parameters complete the thermodynamic cycle in agreement with previously reported studies. Our new understanding and description of the kinetics, thermodynamics and spectroscopic parameters for these complexes will help to improve purification efficiencies, molecule detection, and drug screening assays or find new applications.
Control of eNOS and nNOS through regulation of obligatory conformational changes in the reductase catalytic cycle
Authors: Salerno John C. , Hopper Benjamin L. , Ghosh Dipak. K. , Scott Israel M. , McMurry Jonathan L.
Published: September 2018

Endothelial and neuronal nitric oxide synthases (eNOS, nNOS) are important signal generators in a number of processes including angiogenesis and neurotransmission. The homologous inducible isoform (iNOS) occupies a multitude of conformational states in a catalytic cycle, including subnanosecond input and output states and a distribution of ‘open’ conformations with average lifetimes of ~4.3 ns. In this study, fluorescence lifetime spectroscopy was used to probe conformational states of purified eNOS and nNOS in the presence of chaotropes, calmodulin, NADP+ and NADPH. Two-domain FMN/oxygenase constructs of nNOS were also examined with respect to calmodulin effects. Optical biosensing was used to analyze calmodulin binding in the presence of NADP+ and NADPH. Calmodulin binding induced a shift of the population away from the input and to the open and output states of NOS. NADP+ shifted the population towards the input state. The oxygenase domain, lacking the input state, provided a measure of calmodulin-induced open-output transitions. A mechanism for regulation by calmodulin and an elucidation of the catalytic mechanism are suggested by a ‘conformational lockdown’ model. Calmodulin speeds transitions between input and open and between open and output states, effectively reducing the conformational manifold, speeding catalysis. Conformational control of catalysis involves reorientation of the FMN binding domain, of which fluorescence lifetime is an indicator. The approach described herein is a new tool for biophysical and structural analysis of NOS enzymes, regulatory events and other homologous reductase-containing enzymes.

A note to the reader

This manuscript has over the past several years been submitted to and rejected by several journals, usually on the basis of reviewer opinion that it was not an important enough result to merit inclusion in the journal. Owing to the passing of the first author and the loss of his expertise in fluorescence lifetime spectroscopy, it has become too onerous a task to continually revise the manuscript to suit the whims of reviewers who nevertheless still reject the work. We are thus simply releasing the final form of the manuscript to BioRxiv in the hopes that it finds a readership who will find, as we do, that the results are of value to the field.
Fall Risk Prediction in Multiple Sclerosis Using Postural Sway Measures: A Machine Learning Approach
Authors: Sun Ruopeng , Hsieh Katherine L. , Sosnoff Jacob J.
Published: September 2018

Background

Balance impairment affects over 75% of individuals with multiple sclerosis (MS), and leads to an increased risk of falling. Numerous postural sway metrics have been shown to be sensitive to balance impairment and fall risk in individuals with MS. Yet, there are no guidelines concerning the most appropriate postural sway metrics to monitor impairment. This investigation implemented a machine learning approach to assess the accuracy and feature importance of various postural sway metrics to differentiate individuals with MS from healthy controls as a function of physiological fall risk.

Methods

This secondary data analysis included 153 participants (50 controls and 103 individuals with MS) who underwent posturography based balance assessment (30s eyes open standing on a force platform) and physiological fall risk assessment (Physiological Profile Assessment - PPA). Participants were further classified into four subgroups based on fall risk: controls (n=50, 64.9 ± 4.9 years old, PPA < 1); low-risk MS (n=34, 54.0 ± 13.1 years old, PPA < 1); moderate-risk MS (n=27, 58.3 ± 8.3 years old, 1 ≤ PPA < 2); high-risk MS (n=42, 56.8 ± 9.7 years old, PPA ≥ 2). Twenty common sway metrics were derived following standard procedures, and subsequently used to train a machine learning algorithm (random forest – RF, with 10-fold cross validation) to predict individuals’ fall risk grouping. The feature importance from the RF algorithms was used to select the strongest sway metric for fall risk prediction.

Results and Discussion

The sway-metric based RF classifier had high classification accuracy in discriminating controls from MS individuals (> 86%). Sway sample entropy, a sway regularity metric, was identified as the strongest feature for classification of low-risk MS individuals from healthy controls. Whereas for all other comparisons, mediolateral sway amplitude was identified as the strongest predictor for fall risk groupings. These findings may set the foundation for the development of guidelines for reporting balance impairment in individuals with MS.