bluuebunny/crossref_metadata_2025_split huggingface dataset:
Optimized design of antisense oligomers for targeted rRNA depletion
Authors: Phelps Wesley A. , Carlson Anne E. , Lee Miler T. 
Published: June 2020

ABSTRACTRNA sequencing (RNA-seq) has become a standard method for quantifying gene expression transcriptome-wide. Although RNA-seq is often paired with polyadenylate (poly(A)) selection to enrich for messenger RNA (mRNA), many applications require alternate approaches to counteract the high proportion of ribosomal RNA (rRNA) in total RNA. Recently, selective rRNA digestion, using RNaseH and antisense DNA oligomers that tile the entire length of target RNAs, has emerged as an alternative to commercial rRNA depletion kits. Here, we present a streamlined, more economical RNaseH-mediated rRNA depletion method with substantially lower up-front costs, using shorter antisense oligos only sparsely tiled along the target RNA, in a digestion reaction of only 5 minutes. We introduce a novel Web tool, Oligo-ASST, that simplifies oligo design to target regions with optimal thermodynamic properties, and additionally can generate compact, common oligo pools that simultaneously target divergent RNAs, e.g. across different species. We demonstrate the efficacy of these strategies by designing oligo sets to deplete rRNA inXenopus laevisand in zebrafish, which expresses two distinct versions of rRNAs during embryogenesis. The resulting RNA-seq libraries reduce rRNA to <5% of aligned reads, on par with poly(A) selection, and also reveal expression of many non-adenylated RNA species. Oligo-ASST is freely available athttps://mtleelab.pitt.edu/oligoto design antisense oligos for any taxon or to target any abundant RNA for depletion.Abstract Figure
An insight into transcriptome ofLlaNACGene Over-expressing Tobacco Plants
Authors: Singh Sadhana , Grover Atul 
Published: June 2020

AbstractHere, we report a whole transcriptome analysis ofLlaNACgene (fromLepidium latifolium) containing transgenic tobacco line (NC10) and wild type (WT), to attain deeper knowledge into the downstream genes activated by the over-expressing transgene. Transcriptome sequencing of NC10 and WT samples generated huge data using Illumina platform. The maximum number of unigenes GO annotated were of Biological process (8988, 3209) followed by molecular function (5155, 2577) and cellular components (3826, 1583) for WT and NC10 samples respectively. KEGG Pathway analysis revealed the unigenes were enriched in different functional pathway categories. The unigenes whose products involved in carbohydrate metabolism, glycan metabolism, and secondary metabolites synthesis were more for NC10 library in comparison to WT. Greater variety of transcription factors were involved in transgenic than wild-type plants. Genes like, Copia-like retrotransposable element, Peroxidase 64-like, Peptidyl-prolyl cis-trans isomerise, Cytochrome P450, Lipoyl synthase, CBL-interacting serine/threonine-protein kinase 5-like etc. were found differentially expressed in both the samples. Promoter analysis of these differentially expressed genes have elements for defence and stress response, abscisic acid response, shoot specific expression and light response, etc. In summary this study reports the involvement of the overexpressed genes in the dual action of cold tolerance and biomass accumulation, as sugars participate in both of these activities of the cell.
Efficient precisein vivobase editing in adult dystrophic mice
Authors: Xu Li , Zhang Chen , Li Haiwen , Wang Peipei , Gao Yandi , Mohler Peter J. , Mokadam Nahush A. , Ma Jianjie , Arnold William D. , Han Renzhi 
Published: June 2020

ABSTRACTBackgroundRecent advances in the base editing technology have created an exciting opportunity to precisely correct disease-causing mutations. However, the large size of base editors and their inherited off-target activities pose challenges forin vivobase editing. Moreover, the requirement of a protospacer adjacent motif (PAM) sequence within a suitable window near the mutation site further limits the targeting feasibility. In this work, we rationally improved the adenine base editor (ABE) to overcome these challenges and demonstrated the exceptionally high efficiency to precisely edit the Duchenne muscular dystrophy (DMD) mutation in adult mice.MethodsWe employed a fluorescence reporter assay to assess the feasibility of ABE to correct the dystrophin mutation inmdx4cvmice. The intein protein trans-splicing (PTS) was used to split the oversized ABE into two halves for efficient packaging into adeno-associated virus 9 (AAV9). The ABE with broadened PAM recognition (ABE-NG) was rationally re-designed for improved off-target RNA editing activity and on-target DNA editing efficiency. Themdx4cvmice at the 5 weeks of age receiving intramuscular or intravenous injections of AAV9 carrying the improved ABE-NG were analyzed at 10 weeks or 10 months of age. The editing outcomes were analyzed by Sanger and deep sequencing of the amplicons, immunofluorescence staining, Western blot and contractile function measurements. The off-target activities, host immune response and long-term toxicity were analyzed by deep sequencing, ELISA and serological assays, respectively.ResultsWe showed efficientin vitrobase correction of the dystrophin mutation carried inmdx4cvmice using ABE-NG. The super-fast intein-splits of ABE-NG enabled the expression of full-length ABE-NG and efficient AAV9 packaging. We rationally improved ABE-NG with eliminated off-target RNA editing activity and minimal PAM requirement, and packaged into AAV9 (AAV9-iNG). Intramuscular and intravenous administration of AAV9-iNG resulted in dystrophin restoration and functional improvement. At 10 months after AAV9-iNG treatment, a near complete rescue of dystrophin was measured inmdx4cvmouse hearts. The off-target activities remained low and no obvious toxicity was detected.ConclusionsThis study highlights the promise of permanent base editing using iABE-NG for the treatment of monogenic diseases, in particular, the genetic cardiomyopathies.
Topoisomerase IB interacts with genome segregation proteins and is involved in multipartite genome maintenance inDeinococcus radiodurans
Authors: Kota Swathi , Chaudhary Reema , Mishra Shruti , Misra Hari S. 
Published: June 2020

AbstractDeinococcus radiodurans, an extremophile, resistant to many abiotic stresses including ionizing radiation, has 2 type I topoisomerases (drTopo IA and drTopo IB) and one type II topoisomerase (DNA gyrase). The role of drTopo IB in guanine quadruplex DNA (G4 DNA) metabolism was shownin vitro. Here we report thatD. radioduranscells lacking drTopo IB (ΔtopoIB) show sensitivity to G4 DNA binding drug (NMM) under normal growth conditions. The activity of G4 motif containing promoters likemutLandrecQwas reduced in the presence of NMM in mutant cells. In mutant cells, the percentage of anucleated cells was more while the ploidy numbers of genome elements were less as compared to wild type. Protein-protein interaction studies showed that drTopo IB interacts with genome segregation and DNA replication initiation (DnaA) proteins. The typical patterns of cellular localization of GFP-PprA were affected in the mutant cells. Microscopic examination ofD. radioduranscells expressing drTopo IB-RFP showed its localization on nucleoid forming a streak parallel to the old division septum and perpendicular to newly formed septum. These results together suggest the role of drTopo IB in genome maintenance in this bacterium.
Acquisition of hypoxia inducibility by oxygen sensing N-terminal cysteine oxidase in spermatophytes
Authors: Weits Daan A. , Zhou Lina , Giuntoli Beatrice , Carbonare Laura Dalle , Iacopino Sergio , Piccinini Luca , Shukla Vinay , Bui Liem T. , Novi Giacomo , van Dongen Joost T. , Licausi Francesco 
Published: June 2020

AbstractN-terminal cysteine oxidases (NCOs) are enzymes that use molecular oxygen to oxidize the amino-terminal cysteine of specific proteins, thereby initiating the proteolytic N-degron pathway and thus conferring them oxygen-dependent instability. To expand the characterization of the plant family of NCOs (PCOs), we performed a phylogenetic analysis across different plant taxa in terms of sequence similarity and transcriptional regulation. Based on this survey, we propose a distinction of PCOs into two main groups: A-type and B-type sequences. A-type PCOs are conserved across all plant species and are generally unaffected at the mRNA level by oxygen availability. Instead, B-type PCOs differentiated in spermatophytes to acquire specific amino acid features and transcriptional regulation in response to hypoxia. Both groups of PCO proteins possess the ability to destabilize Cys-initiating proteins. Indeed, the inactivation of two A-type PCOs inArabidopsis thaliana, PCO4 and PCO5, is sufficient to activate, at least partially, the anaerobic response in young seedlings, whereas the additional removal of B-type PCOs leads to a stronger induction of anaerobic genes and impairs plant growth and development. Our results show that both PCO types are required to regulate the anaerobic response in angiosperm. Therefore, while it is possible to distinguish two clades within the PCO family, separated by both amino acid features and transcriptional regulation, we conclude that they both contribute to restrain the anaerobic transcriptional program in normoxic conditions and together generate a molecular switch to toggle the hypoxic response in Arabidopsis.One sentence summaryHypoxic induction of Plant Cysteine Oxidases has been acquired and fixed in seed plants by ancestor proteins able to initiate the proteolysis of Cys-initiating protein substrates by the Arg/N-degron pathway.
Increased baseline RASGRP1 signals Enhance Stem Cell Fitness during Native Hematopoiesis
Authors: Karra Laila , Romero-Moya Damia , Ksionda Olga , Krush Milana , Gu Zhaohui , Mues Marsilius , Depeille Philippe , Mullighan Charles , Roose Jeroen P. 
Published: June 2020

AbstractOncogenic mutations inRASgenes, likeKRASG12DorNRASG12D, trap Ras in the active state and cause myeloproliferative disorder and T cell leukemia (T-ALL) when induced in the bone marrow viaMx1CRE. The RAS exchange factor RASGRP1 is frequently overexpressed in T-ALL patients. In T-ALL cell lines overexpression of RASGRP1 increases flux through the RASGTP/RasGDP cycle. Here we expandedRASGRP1expression surveys in pediatric T-ALL and generated aRoLoRiGmouse model crossed toMx1CREto determine the consequences of induced RASGRP1 overexpression in primary hematopoietic cells. RASGRP1-overexpressing, GFP-positive cells outcompeted wild type cells and dominated the peripheral blood compartment over time. RASGRP1 overexpression bestows gain-of-function colony formation properties to bone marrow progenitors in medium containing limited growth factors. RASGRP1 overexpression enhances baseline mTOR-S6 signaling in the bone marrow, but notin vitrocytokine-induced signals. In agreement with these mechanistic findings, hRASGRP1-ires-EGFP enhances fitness of stem- and progenitor- cells, but only in the context of native hematopoiesis. RASGRP1 overexpression is distinct fromKRASG12DorNRASG12D, does not cause acute leukemia on its own, and leukemia virus insertion frequencies predict that RASGRP1 overexpression can effectively cooperate with lesions in many other genes to cause acute T cell leukemia.
Liftoff: an accurate gene annotation mapping tool
Authors: Shumate Alaina , Salzberg Steven L. 
Published: June 2020

AbstractImprovements in DNA sequencing technology and computational methods have led to a substantial increase in the creation of high-quality genome assemblies of many species. To understand the biology of these genomes, annotation of gene features and other functional elements is essential; however for most species, only the reference genome is well-annotated. One strategy to annotate new or improved genome assemblies is to map or ‘lift over’ the genes from a previously-annotated reference genome. Here we describe Liftoff, a new genome annotation lift-over tool capable of mapping genes between two assemblies of the same or closely-related species. Liftoff aligns genes from a reference genome to a target genome and finds the mapping that maximizes sequence identity while preserving the structure of each exon, transcript, and gene. We show that Liftoff can accurately map 99.9% of genes between two versions of the human reference genome with an average sequence identity >99.9%. We also show that Liftoff can map genes across species by successfully lifting over 98.4% of human protein-coding genes to a chimpanzee genome assembly with 98.7% sequence identity.AvailabilityThe source code for Liftoff is available athttps://github.com/agshumate/Liftoff
A Synthetic Single-Stranded Dual-Template Oligonucleotide as a Reference Standard for Monochrome Multiplex qPCR Measurements of Average Telomere Length
Authors: Cawthon Richard M. 
Published: June 2020

ABSTRACTQuantitative PCR is frequently used to measure average telomere length (TL) relative to the TL of a reference DNA sample of the investigator’s choosing. This makes comparisons of TLs across studies and laboratories difficult. Here we demonstrate that a single synthetic single-stranded dual-template oligonucleotide (DTO) containing both a telomere repeat sequence (T) and a segment of the human beta-globin (HBB) single copy gene (S) can be used as a universal reference standard for monochrome multiplex quantitative PCR (MMqPCR) measurements of average TL using SYBR Green I as the only fluorescent reporter dye. A set of twelve concentrations of the DTO is prepared by serial 3-fold dilutions, to a lowest concentration of ~20 copies per μl. The 5 highest concentrations are used for the T standard curve, and the 5 lowest concentrations are used for the S standard curve. For each reaction 5 μl containing approximately 3 ng of genomic DNA (or one of the DTO dilutions) is mixed with 5 μl of a 2x MasterMix containing the primers for T and S amplification, and MMqPCR is performed. The design of the primers and thermal cycling profile allows all T amplification signals to be collected before exponential amplification of the S signal begins. Exponential amplification from S is then carried out in a temperature range that keeps the telomere product fully melted and therefore unable to influence the S amplification signal. The T value for each DNA sample is the Standard Curve DTO dilution that contains the same number of copies of the telomere sequence as the experimental sample, and the S value is the DTO dilution that contains the same number of copies of the single copy gene sequence as the experimental sample. Dividing the first dilution by the second dilution yields an absolute T/S ratio, since it is expressed relative to the fixed 1:1 T/S ratio that is built into the DTO by design. Absolute T/S ratios for average TL in 48 human DNA samples determined by this method correlated strongly with mean Terminal Restriction Fragment (mTRF) lengths for the same DNA samples determined by the Southern Blot method (R-squared = 0.801). This DTO and the accompanying protocol may facilitate the standardization of average telomere length measurements and analyses across laboratories.
Amino acid oxidation constrained protein sequences during evolution: an analysis of a model bacterium
Authors: González-Tortuero Enrique , Rodriguez-Rojas Alexandro 
Published: June 2020

AbstractWithin the cell, there are compartments and local conditions that may constrain amino acid sequence. Life emerged in an anoxic world but releasing a photosynthesis by-product, the molecular oxygen, forced adaptive changes to counteract its toxicity. Several mechanisms evolved to balance the intracellular redox state and maintain a reductive environment more compatible with many essential biological functions. Here, we statistically interrogate the aminoacidic composition ofE. coliproteins and investigate how the proneness or susceptibility to oxidation of amino acid biased their sequences. By sorting the proteins in five different compartments: cytoplasm, internal membrane, periplasm, outer membrane and extracellular; we found that various oxidative lesions constrain protein composition, that there is a dependency of the cellular compartments and there is an impact on the evenness distribution or frequency. This analysis is necessary from an evolutionary point of view to reflect how the oxidative atmosphere could restrict protein composition and probably imposed a trend in the codon bias.
IsoMaTrix: a framework to visualize the isoclines of matrix games and quantify uncertainty in structured populations
Authors: West Jeffrey , Ma Yongqian , Kaznatcheev Artem , Anderson Alexander R. A. 
Published: June 2020

AbstractSummaryEvolutionary game theory describes frequency-dependent selection for fixed, heritable strategies in a population of competing individuals using a payoff matrix, typically described using well-mixed assumptions (replicator dynamics). IsoMaTrix is an open-source package which computes the isoclines (lines of zero growth) of matrix games, and facilitates direct comparison of well-mixed dynamics to structured populations in two or three dimensions. IsoMaTrix is coupled with a Hybrid Automata Library module to simulate structured matrix games on-lattice. IsoMaTrix can also compute fixed points, phase flow, trajectories, velocities (and subvelocities), delineated “region plots” of positive/negative strategy velocity, and uncertainty quantification for stochastic effects in structured matrix games. We describe a result obtained via IsoMaTrix’s spatial games functionality, which shows that the timing of competitive release in a cancer model (under continuous treatment) critically depends on the initial spatial configuration of the tumor.Availability and implementationThe code is available at:https://github.com/mathonco/isomatrix.
ANALYSIS OF MICROBIAL DIVERSITY IN RAW FISH CEVICHE
Authors: Ramírez-Martínez M. E. , Rodríguez-Castillejos G. C. , Hernández-Jiménez M. C. , Ramírez-Quintanilla L. Y. , Siller-López F. , Acosta-Cruz E. , Martínez-Montoya H. 
Published: June 2020

ABSTRACTCeviche is a traditional dish made from raw fish meat marinated in lime juice without any heat cooking step throughout its preparation process. Although the use of organic acids as antibacterial agents is well known; recent research indicates that lime juice actually can reduce the risk ofV. parahemolyticusinfections but it is ineffective against other potential pathogens. Despite the fact that fresh fish meat is safe; exposed organs including skin, gills and guts represent a potential source of bacterial contamination. In Mexico, diarrheal diseases are caused mainly by contaminated food; it is estimated that almost 67% of infections are due the presence of bacterial agents mainly in frozen and fresh fish. The main objective of this study was to estimate the taxonomic diversity of microbial species present in ready-to-eat ceviche using a metagenomic approach. Six samples of commercially available ceviche were subjected to DNA high throughput sequencing and bioinformatics analyses, we identified between 65,000 and 131,000 reads per sample. The predominant phyla identified through the samples wereProteobacteria,BacteroidetesandFirmicutes. We discuss the factors involved in the microbiological quality of this kind of raw foods and how they influence the bacterial diversity within the analyzed samples.
Evaluation of ExoU inhibitors in aPseudomonas aeruginosascratch infection assay
Authors: Foulkes Daniel M. , McLean Keri , Harris Joscelyn , Haneef Atikah S. , Fernig David G. , Winstanley Craig , Berry Neil , Kaye Stephen B. 
Published: June 2020

AbstractPseudomonas aeruginosahas recently been highlighted by the World Health Organisation (WHO) as a major threat with high priority for the development of new therapies. The type III secretion system ofP. aeruginosadelivers the toxin ExoU into the cytosol of target host cells, where its plasma membrane directed phospholipase activity induces rapid cell lysis. Therefore, inhibition of the phospholipase activity of ExoU would be an important treatment strategy inP. aeruginosainfections. We evaluated a panel of ExoU small molecule inhibitors, previously identified from high throughput cellular based assays, and analysed their inhibition of ExoU phospholipase activityin vitro. A corneal epithelial (HCE-T) scratch and infection model using florescence microscopy, and cell viability assays, were used to test the efficacy of compounds to inhibit ExoU fromP. aeruginosa. Compounds Pseudolipasin A, compound A and compound B were effective at mitigating ExoU mediated cytotoxicity after infection at concentrations as low as 0.5 μM. Importantly, by using the antimicrobials moxifloxacin and tobramycin to control bacterial load, these assays were extended from 6 h to 24 h.P. aeruginosaremained cytotoxic to HCE-T cells with moxifloxacin, present at the minimal inhibitory concentration (MIC) for 24 h, but, when used in combination with either PSA, compound A or compound B, partial scratch healing was observed. These results provide evidence that ExoU inhibitors could be used in combination with certain antimicrobials as a novel means to treat clinical infections of ExoU producingP. aeruginosa.
A Decision Analytic Approach for Social Distancing Policies During COVID-19 Pandemic
Authors: Ertem Zeynep , Araz Ozgur M. , Cruz-Aponte Mayteé 
Published: June 2020

AbstractThe COVID-19 pandemic has become a crucial public health issue in many countries including the United States. In the absence of the right vaccine strain and sufficient antiviral stockpiles on hand, non-pharmaceutical interventions have become valuable public health tools at the early stages of the pandemic and they are employed by many countries across the globe. These interventions are designed to increase social distancing between individuals to reduce the transmission of the virus and eventually dampen the burden on the healthcare system. The virus transmissibility is a function of the average number of contacts individuals have in their communities and it is highly dependent on population density and daily mobility patterns, along with other social factors. These show significant variation across the United States. In this article, we study the effectiveness of social distancing measures in communities with different population density. Specifically, first we show how the empirical estimation of reproduction number differs for two completely different states, thus the experience of the COVID-19 outbreak is drastically different, suggesting different outbreak growth rates in practice. Second, we develop an age-structured compartmental model for simulating the disease spread in order to demonstrate the variation in the observed outbreak characteristics. We find that early trigger and late trigger options present a trade-off between the peak magnitude and the overall death toll of the outbreak which may also vary across different populations.