bluuebunny/crossref_metadata_2025_split huggingface dataset:
The microbial basis of impaired wound healing: differential roles for pathogens, “bystanders”, and strain-level diversification in clinical outcomes
Authors: Kalan Lindsay , Meisel Jacquelyn S. , Loesche Michael A. , Horwinski Joseph , Soaita Ioana , Chen Xiaoxuan , Gardner Sue E. , Grice Elizabeth A. 
Published: September 2018

ABSTRACTChronic, non-healing wounds are a major complication of diabetes associated with high morbidity and health care expenditures estimated at $9-13 billion annually in the US. Though microbial infection and critical colonization is hypothesized to impair healing and contribute to severe outcomes such as amputation, antimicrobial therapy is inefficacious and the role of microbes in tissue repair, regeneration, and healing remains unclear. Here, in a longitudinal prospective cohort study of 100 subjects with non-infected neuropathic diabetic foot ulcer (DFU), we performed metagenomic shotgun sequencing to elucidate microbial temporal dynamics at strain-level resolution, to investigate pathogenicity and virulence of the DFU microbiome with respect to outcomes, and to determine the influence of therapeutic intervention on the DFU microbiota. Slow healing DFUs were associated with signatures of biofilm formation, host invasion, and virulence. Though antibiotic resistance was widespread at the genetic level, debridement, rather than antibiotic treatment, significantly shifted the DFU microbiome in patients with more favorable outcomes. Primary clinical isolates ofS. aureus, C. striatum, andA. faecalisinduced differential biological responses in keratinocytes and in a murine model of diabetic wound healing, with theS. aureusstrain associated with non-healing wounds eliciting the most severe phenotype. Together these findings implicate strain-level diversification of the wound pathogenS. aureusin chronic wound outcomes, while revealing potential contributions from skin commensals and other previously underappreciated constituents of the wound microbiota.
The Spectrum of Asynchronous Dynamics in Spiking Networks: A Theory for the Diversity of Non-Rhythmic Waking states in Neocortex
Authors: Zerlaut Yann , Zucca Stefano , Panzeri Stefano , Fellin Tommaso 
Published: September 2018

SummaryThe cerebral cortex of awake animals exhibits frequent transitions between diverse non-rhythmic network states. However, it is still unclear how these different activity states emerge within the same network and how each state impacts network function. Here, we demonstrate that model networks of spiking neurons with moderate recurrent interactions dynamically change their asynchronous dynamics depending upon the level of afferent excitation. We found that the model network displayed a spectrum of asynchronous states, ranging from afferent input-dominated (AD) regimes, characterized by unbalanced synaptic currents and sparse firing, to recurrent input-dominated (RD) regimes, characterized by balanced synaptic currents and dense firing. The model predicted regime-specific relationships between several different neural biophysical properties which were all experimentally confirmed by intracellular recordings in the somatosensory cortex of awake mice. Moreover, theoretical analysis showed that AD regimes more precisely encode spatiotemporal patterns of presynaptic activity, while RD regimes better encoded the strength of afferent inputs. These results provide a theoretical foundation for how recurrent neocortical circuits generate non-rhythmic waking states and how these different states modulate the processing of incoming information.
Promoter activity of ORF-less gene cassettes isolated from the oral metagenome
Authors: Tansirichaiya Supathep , Mullany Peter , Roberts Adam P. 
Published: September 2018

AbstractIntegrons are genetic elements consisting of a functional platform for recombination and expression of gene cassettes (GCs). GCs usually carry promoter-less open reading frames (ORFs), encoding proteins with various functions including antibiotic resistance. The transcription of GCs relies mainly on a cassette promoter (PC), located upstream of an array of GCs. Some integron GCs, called ORF-less GCs, contain no identifiable ORF with a small number shown to be involved in antisense mRNA mediated gene regulation.In this study, promoter sequences were identified, usingin silicoanalysis, within GCs PCR amplified from the oral metagenome. The promoter activity of ORF-less GCs was verified by cloning them upstream of agusAreporter, proving they can function as a promoter, presumably allowing bacteria to adapt to multiple stresses within the complex physico-chemical environment of the human oral cavity. A bi-directional promoter detection system was also developed allowing direct identification of clones with promoter-containing GCs on agar plates. Novel promoter-containing GCs were identified from the human oral metagenomic DNA using this construct, called pBiDiPD.This is the first demonstration and detection of promoter activity of ORF-less GCs and the development of an agar plate-based detection system will enable similar studies in other environments.
Screening for insulin-independent pathways that modulate glucose homeostasis identifies androgen receptor antagonists
Authors: Mullapudi Sri Teja , Helker Christian S. M. , Boezio Giulia L.M. , Maischein Hans-Martin , Sokol Anna M. , Graumann Johannes , Guenther Stefan , Matsuda Hiroki , Kubicek Stefan , Carol Yang Yu Hsuan , Stainier Didier Y.R. 
Published: September 2018

AbstractPathways modulating glucose homeostasis independently of insulin would open new avenues to combat insulin resistance and diabetes. Here, we report the establishment, characterization and employment of a vertebrate ‘insulin-free’ model to identify insulin-independent modulators of glucose metabolism. insulin knockout zebrafish recapitulate core characteristics of diabetes and survive only up to larval stages. Utilizing a highly efficient endoderm transplant technique, we generated viable chimeric adults that provide the large numbers of insulin mutant larvae required for our screening platform. Using glucose as a disease-relevant readout, we screened 2233 molecules and identified 3 that consistently reduced glucose levels in insulin mutants. Most significantly, we uncovered an insulin-independent beneficial role for androgen receptor antagonism in hyperglycemia, mostly by reducing fasting glucose levels. Our study proposes therapeutic roles for androgen signaling in diabetes and, more broadly, offers a novel in vivo model for rapid screening and decoupling of insulin-dependent and -independent mechanisms.
The structural basis for forkhead box family specificity revealed by the crystal structure of human FOXN1 in complex with DNA
Authors: Newman Joseph A. , Aitkenhead Hazel , Gravard Angeline , Rota Ioanna A. , Handel Adam E. , Hollander Georg A. , Gileadi Opher 
Published: September 2018

SignificanceFOXN1 is a transcription factor that is essential for the development of the thymus and the production of T-lymphocytes. It is a member of a large family of transcription factors which recognize DNA sequences through the conserved Forkhead (FH) domain. FOXN1 recognizes a DNA sequence that is different from the common consensus binding sequence of FH domains, although key binding residues are identical. We present crystal structures of the FH domain of FOXN1, free and DNA-bound, which shed light on the different binding specificities; the structure also revelas the basis of the immunocompromisednudemutation, as well as a preferential binding to non-methylated CpG motifs.AbstractFOXN1 is a member of the forkhead box (FOX) family of transcription factors, and plays an important role in thymic epithelial cell differentiation and function. FOXN1 mutations in humans and mice give rise to the "nude" phenotype which is marked by athymia. FOXN1 belongs to a subset of the FOX family that recognize an alternate consensus sequence (GACGC), which is different from the more widely-recognized canonical sequence consensus RYAAAYA. Here, we present the structure of FOXN1 in complex with DNA at 1.6 Å resolution, in which the DNA sequence is recognised by a mixture of direct and water-mediated contacts provided by residues in an a-helix inserted in the DNA major groove (the recognition helix). Comparisons with other FOX family structures reveal that the canonical and alternate DNA sequences are bound in two distinct modes, with partially different registers for the protein DNA contacts. We identify a single alternate rotamer within the recognition helix itself as an important determinant of DNA specificity, and indicate sequence features in the recognition helix that could be used to predict the specificity of other FOX family members. Finally we demonstrate that FOXN1 has a significantly reduced affinity for DNA containing 5-methylcytosine, which may have implications for the role of FOXN1 in thymic senescence.
Genome-wide analyses supported by RNA-Seq reveal non-canonical splice sites in plant genomes
Authors: Pucker Boas , Brockington Samuel F. 
Published: September 2018

ABSTRACTMost eukaryotic genes comprise exons and introns thus requiring the precise removal of introns from pre-mRNAs to enable protein biosynthesis. U2 and U12 spliceosomes catalyze this step by recognizing motifs on the transcript in order to remove the introns. A process which is dependent on precise definition of exon-intron borders by splice sites, which are consequently highly conserved across species. Only very few combinations of terminal dinucleotides are frequently observed at intron ends, dominated by the canonical GT-AG splice sites on the DNA level.Here we investigate the occurrence of diverse combinations of dinucleotides at predicted splice sites. Analyzing 121 plant genome sequences based on their annotation revealed strong splice site conservation across species, annotation errors, and true biological divergence from canonical splice sites. The frequency of non-canonical splice sites clearly correlates with their divergence from canonical ones indicating either an accumulation of probably neutral mutations, or evolution towards canonical splice sites. Strong conservation across multiple species and non-random accumulation of substitutions in splice sites indicate a functional relevance of non-canonical splice sites. The average composition of splice sites across all investigated species is 98.7% for GT-AG, 1.2% for GC-AG, 0.06% for AT-AC, and 0.09% for minor non-canonical splice sites. RNA-Seq data sets of 35 species were incorporated to validate non-canonical splice site predictions through gaps in sequencing reads alignments and to demonstrate the expression of affected genes. We conclude thatbona fidenon-canonical splice sites are present and appear to be functionally relevant in most plant genomes, if at low abundance.
Uncovering hidden members and functions of the soil microbiome using de novo metaproteomics
Authors: Lee Joon-Yong , Mitchell Hugh D. , Burnet Meagan C. , Wu Ruonan , Jenson Sarah C. , Merkley Eric D. , Nakayasu Ernesto S. , Nicora Carrie D. , Jansson Janet K. , Burnum-Johnson Kristin E. , Payne Samuel H. 
Published: September 2018

AbstractMetaproteomics has been increasingly utilized for high-throughput molecular characterization in complex environments and has been demonstrated to provide insights into microbial composition and functional roles in soil systems. Despite its potential for the study of microbiomes, significant challenges remain in data analysis, including the creation of a sample-specific protein sequence database as the taxonomic composition of soil is often unknown. Almost all metaproteome analysis tools require this database and their accuracy and sensitivity suffer when the database is incomplete or contains extraneous sequences from organisms which are not present. Here, we leverage a de novo peptide sequencing approach to identify sample composition directly from metaproteomic data. First, we created a deep learning model, Kaiko, to predict the peptide sequences from mass spectrometry data, and trained it on 5 million peptide-spectrum matches from 55 phylogenetically diverse bacteria. After training, Kaiko successfully identified unsequenced soil isolates directly from proteomics data. Finally, we created a pipeline for metaproteome database generation using Kaiko. We tested the pipeline on native soils collected in Kansas, showing that the de novo sequencing model can be employed to construct the sample-specific protein database instead of relying on (un)matched metagenomes. Our pipeline identified all highly abundant taxa from 16S ribosomal RNA sequencing of the soil samples and also uncovered several additional species which were strongly represented only in proteomic data. Our pipeline offers an alternative and complementary method for metaproteomic data analysis by creating a protein database directly from proteomic data, thus removing the need for metagenomic sequencing.Significance StatementProteomic characterization of environmental samples, or metaproteomics, reveals microbial activity critical to our understanding of climate, nutrient cycling and human health. Metaproteomic samples originate from diverse environs, such as soil and oceans. One option for data analysis is a de novo interpretation of the mass spectra. Unfortunately, the current generation of de novo algorithms were primarily trained on data originating from human proteins. Therefore, these algorithms struggle with data from environmental samples, limiting our ability to analyze metaproteomics data. To address this challenge, we trained a new algorithm with data from dozens of diverse environmental bacteria and achieved significant improvements in accuracy across a broad range of organisms. This generality opens proteomics to the world of natural isolates and microbiomes.
DNA replication-mediated error correction of ectopic CENP-A deposition maintains centromere identity
Authors: Nechemia-Arbely Yael , Miga Karen H. , Shoshani Ofer , Aslanian Aaron , McMahon Moira A. , Lee Ah Young , Fachinetti Daniele , Yates John R. , Ren Bing , Cleveland Don W. 
Published: September 2018

AbstractChromatin assembled with the histone H3 variant CENP-A is the heritable epigenetic determinant of human centromere identity. Using genome-wide mapping and reference models for 23 human centromeres, CENP-A is shown in early G1 to be assembled into nucleosomes within megabase, repetitive α-satellite DNAs at each centromere and onto 11,390 transcriptionally active sites on the chromosome arms. Here we identify that DNA replication acts as an error correction mechanism to sustain centromere identity through the removal of the sites of CENP-A loading on the chromosome arms, while maintaining centromere-bound CENP-A with the same DNA sequence preferences as in its initial loading.
Evaluating single-subject study methods for personal transcriptomic interpretations to advance precision medicine
Authors: Zaim Samir Rachid , Kenost Colleen , Berghout Joanne , Zhang Helen Hao , Lussier Yves A. 
Published: September 2018

AbstractBackgroundGene expression profiling has benefited medicine by providing clinically relevant insights at the molecular candidate and systems levels. However, to adopt a more ‘precision’ approach that integrates individual variability including ‘omics data into risk assessments, diagnoses, and therapeutic decision making, whole transcriptome expression analysis requires methodological advancements. One need is for users to confidently be able to make individual-level inferences from whole transcriptome data. We propose that biological replicates in isogenic conditions can provide a framework for testing differentially expressed genes (DEGs) in a single subject (ss) in absence of an appropriate external reference standard or replicates.MethodsEight ss methods for identifying genes with differential expression (NOISeq, DEGseq, edgeR, mixture model, DESeq, DESeq2, iDEG, and ensemble) were compared inYeast(parental line versus snf2 deletion mutant; n=42/condition) andMCF7breast-cancer cell (baseline and stimulated with estradiol; n=7/condition) RNA-Seq datasets where replicate analysis was used to build reference standards from NOISeq, DEGseq, edgeR, DESeq, DESeq2. Each dataset was randomly partitioned so that approximately two-thirds of the paired samples were used to construct reference standards and the remainder were treated separately as single-subject sample pairs and DEGs were assayed using ss methods. Receiver-operator characteristic (ROC) and precision-recall plots were determined for all ss methods against each RSs in both datasets (525 combinations).ResultsConsistent with prior analyses of these data,~50% and ~15% DEGs were respectively obtained in Yeast and MCF7 reference standard datasets regardless of the analytical method. NOISeq, edgeR and DESeq were the most concordant and robust methods for creating a reference standard. Single-subject versions of NOISeq, DEGseq, and an ensemble learner achieved the best median ROC-area-under-the-curve to compare two transcriptomes without replicates regardless of the type of reference standard (>90% in Yeast, >0.75 in MCF7).ConclusionBetter and more consistent accuracies are obtained by an ensemble method applied to singlesubject studies across different conditions. In addition, distinct specific sing-subject methods perform better according to different proportions of DEGs. Single-subject methods for identifying DEGs from paired samples need improvement, as no method performs with both precision>90% and recall>90%.http://www.lussiergroup.org/publications/EnsembleBiomarker
The bacterial DNA binding protein MatP involved in linking the nucleoid terminal domain to the divisome at midcell interacts with lipid membranes
Authors: Monterroso Begoña , Zorrilla Silvia , Sobrinos-Sanguino Marta , Robles-Ramos Miguel Ángel , Alfonso Carlos , Söderström Bill , Meiresonne Nils Y , Verheul Jolanda , den Blaauwen Tanneke , Rivas Germán 
Published: September 2018

ABSTRACTDivision ring formation at midcell is controlled by various mechanisms inEscherichia coli, one of them being the linkage between the chromosomal Ter macrodomain and the Z-ring mediated by MatP, a DNA binding protein that organizes this macrodomain and contributes to the prevention of premature chromosome segregation. Here we show that, during cell division, just before splitting the daughter cells, MatP seems to localize close to the cytoplasmic membrane, suggesting that this protein might interact with lipids. To test this hypothesis, we investigated MatP interaction with lipidsin vitro. We found that MatP, when encapsulated inside microdroplets generated by microfluidics and giant vesicles, accumulates at phospholipid bilayers and monolayers matching the lipid composition in theE. coliinner membrane. MatP binding to lipids was independently confirmed using lipid coated microbeads and bio-layer interferometry assays. Interaction of MatP with the lipid membranes also occurs in the presence of the DNA sequences specifically targeted by the protein but there is no evidence of ternary membrane/protein/DNA complexes. We propose that the interaction of MatP with lipids may modulate its spatiotemporal localization and its recognition of other ligands.IMPORTANCEThe division of anE. colicell into two daughter cells with equal genomic information and similar size requires duplication and segregation of the chromosome and subsequent scission of the envelope by a protein ring, the Z-ring. MatP is a DNA binding protein that contributes both to the positioning of the Z-ring at midcell and the temporal control of nucleoid segregation. Our integratedin vivoandin vitroanalysis provides evidence that MatP can interact with lipid membranes comprising the phospholipid mixture in theE. coliinner membrane, without concomitant recruitment of the short DNA sequences specifically targeted by MatP. This observation strongly suggests that the membrane may play a role in the regulation of the function and localization of MatP, which could be relevant for the coordination of the two fundamental processes in which this protein participates, nucleoid segregation and cell division.
Single-molecule live cell imaging of Rep reveals the dynamic interplay between an accessory replicative helicase and the replisome
Authors: Syeda Aisha , Wollman Adam J. M. , Hargreaves Alex , Brüning Janny G. , McGlynn Peter , Leake Mark C. 
Published: September 2018

AbstractDNA replication requires strategies to cope with nucleoprotein barriers that impair the efficient translocation of the replisome. Biochemical and genetic studies indicate accessory helicases play essential roles in continuity of replication in the presence of nucleoprotein barriers, but how they operate in the native cellular environment is unclear. With high-speed single-molecule microscopy we determine the dynamic patterns of localization of genomically-encoded fluorescent protein constructs of the bacterial accessory helicase Rep and core replisome protein DnaQ in liveE. colicells. We demonstrate that Rep colocalizes with 70% of replication forks. Colocalisation is dependent upon interaction with replicative helicase DnaB, with an underlying hexameric stoichiometry of Rep indicating maximal occupancy of the single DnaB hexamer within the replisome. We find that Rep associates dynamically with the replisome with an average dwell time of 6.5 ms dependent on ATP hydrolysis, indicating rapid binding then translocation away from the fork. We also imaged the PriC replication restart factor given the known Rep-PriC functional interaction and observe Rep-replisome association is also dependent on the presence of PriC. Our findings suggest two Rep-replisome populationsin vivo:one involving Rep continually associating with DnaB then translocating away to aid nucleoprotein barrier removal ahead of the fork, another assisting PriC-dependent reloading of DnaB if replisome progression fails. These new findings reveal how a single type of helicase is recruited to the replisome to provide two independent ways of underpinning replication of protein-bound DNA, a problem that all organisms face as they replicate their genomes.Significance statementAll organisms face the challenge of proteins bound to DNA acting as barriers to prevent DNA replication. We have performed fluorescence imaging experiments on living bacteria to track the positions of the replication machinery, a protein called Rep which is involved in removing these barriers, and a protein called PriC believed to be involved with reloading the replication machinery if the original replication machinery breaks down. We find that Rep is very dynamic with continual binding and movement away from the replication machinery. Association with the replication machinery depends on both binding to the replication machinery directly and on PriC. Thus Rep can circumvent barriers in two independent ways: a strategy which may be relevant to all organisms.