Histone H4 asymmetrically dimethylated at arginine 3 (H4R3me2a), a mark of super-enhancers.

Histone H4 asymmetrically dimethylated at arginine 3 (H4R3me2a) is an active histone mark catalyzed by protein arginine methyltransferase 1 (PRMT1), a major arginine methyltransferase in vertebrates catalyzing asymmetric dimethylation of arginine. H4R3me2a stimulates the activity of lysine acetyltransferases such as CBP/p300, which catalyze the acetylation of H3K27, a mark of active enhancers, super-enhancers, and promoters. There are a few studies on the genomic location of H4R3me2a. In chicken polychromatic erythrocytes, H4R3me2a is found in introns and intergenic regions and binds to the globin locus control region (a super-enhancer) and globin regulatory regions. In this report, we analyzed chromatin immunoprecipitation sequencing data for the genomic location of H4R3me2a in the breast cancer cell line MCF7. As in avian cells, MCF7 H4R3me2a is present in intronic and intergenic regions. Nucleosomes with H4R3me2a and H3K27ac next to nucleosome-free regions are found at super-enhancers, enhancers, and promoter regions of expressed genes. Genes with critical roles in breast cancer cells have broad domains of nucleosomes with H4R3me2a, H3K27ac, and H3K4me3. Our results are consistent with PRMT1-mediated H4R3me2a playing a key role in the chromatin organization of regulatory regions of vertebrate genomes.

Effect of Antimicrobial Use in Conventional Versus Natural Cattle Feedlots on the Microbiome and Resistome.

Antimicrobial use (AMU) in the livestock industry has been associated with increased levels of antimicrobial resistance. Recently, there has been an increase in the number of "natural" feedlots in the beef cattle sector that raise cattle without antibiotics. Shotgun metagenomics was employed to characterize the impact of AMU in feedlot cattle on the microbiome, resistome, and mobilome. Sequenced fecal samples identified a decline (q < 0.01) in the genera Methanobrevibacter and Treponema in the microbiome of naturally vs. conventionally raised feedlot cattle, but this difference was not (q > 0.05) observed in catch basin samples. No differences (q > 0.05) were found in the class-level resistome between feedlot practices. In fecal samples, decreases from conventional to natural (q < 0.05) were noted in reads for the antimicrobial-resistant genes (ARGs) mefA, tet40, tetO, tetQ, and tetW. Plasmid-associated ARGs were more common in feces from conventional than natural feedlot cattle. Interestingly, more chromosomal- than plasmid-associated macrolide resistance genes were observed in both natural and conventional feedlots, suggesting that they were more stably conserved than the predominately plasmid-associated tetracycline resistance genes. This study suggests that generationally selected resistomes through decades of AMU persist even after AMU ceases in natural production systems.

Quantum analysis of squiggle data.

Squiggle data is the numerical output of DNA and RNA sequencing by the Nanopore next generation sequencing platform. Nanopore sequencing offers expanded applications compared to previous sequencing techniques but produces a large amount of data in the form of current measurements over time. The analysis of these segments of current measurements require more complex and computationally intensive algorithms than previous sequencing technologies. The purpose of this study is to investigate in principle the potential of using quantum computers to speed up Nanopore data analysis. Quantum circuits are designed to extract major features of squiggle current measurements. The circuits are analyzed theoretically in terms of size and performance. Practical experiments on IBM QX show the limitations of the state of the art quantum computer to tackle real life squiggle data problems. Nevertheless, pre-processing of the squiggle data using the inverse wavelet transform, as experimented and analyzed in this paper as well, reduces the dimensionality of the problem in order to fit a reasonable size quantum computer in the hopefully near future.

Evaluation of metagenomic assembly methods for the detection and characterization of antimicrobial resistance determinants and associated mobilizable elements.

Antimicrobial resistance genes (ARGs) can be transferred between members of a bacterial population by mobile genetic elements (MGE). Understanding the risk of these transfer events is important in monitoring and predicting antimicrobial resistance (AMR), especially in the context of a One Health Continuum. However, there is no universally accepted method for detection of ARGs and MGEs, and especially for determining their linkages. This study used publicly available shotgun metagenomic DNA short-read (Illumina, 100 bp paired-end) sequence data from samples across the One Health Continuum (including beef cattle composite feces from feedlots, catch basin water at feedlots, agricultural soil from feedlot manured surrounding fields, and urban/municipal sewage influent from two municipal wastewater treatment plants) to develop a workflow to identify and associate ARGs and MGEs. ARG- and MGE-based targeted-assemblies with available short-read data were unable to meet this analysis goal. In contrast, de novo assembly of contigs provided enough sequence context to associate ARGs and MGEs, without compromising discovery rate. However, to estimate the relative abundance of these elements, unassembled sequence data must still be used.

Genomic Characterization of Carbapenem-Resistant Bacteria from Beef Cattle Feedlots.

Carbapenems are considered a last resort for the treatment of multi-drug-resistant bacterial infections in humans. In this study, we investigated the occurrence of carbapenem-resistant bacteria in feedlots in Alberta, Canada. The presumptive carbapenem-resistant isolates (n = 116) recovered after ertapenem enrichment were subjected to antimicrobial susceptibility testing against 12 different antibiotics, including four carbapenems. Of these, 72% of the isolates (n = 84) showed resistance to ertapenem, while 27% of the isolates (n = 31) were resistant to at least one other carbapenem, with all except one isolate being resistant to at least two other drug classes. Of these 31 isolates, 90% were carbapenemase positive, while a subset of 36 ertapenem-only resistant isolates were carbapenemase negative. The positive isolates belonged to three genera; Pseudomonas, Acinetobacter, and Stenotrophomonas, with the majority being Pseudomonas aeruginosa (n = 20) as identified by 16S rRNA gene sequencing. Whole genome sequencing identified intrinsic carbapenem resistance genes, including blaOXA-50 and its variants (P. aeruginosa), blaOXA-265 (A. haemolyticus), blaOXA-648 (A. lwoffii), blaOXA-278 (A. junii), and blaL1 and blaL2 (S. maltophilia). The acquired carbapenem resistance gene (blaPST-2) was identified in P. saudiphocaensis and P. stutzeri. In a comparative genomic analysis, clinical P. aeruginosa clustered separately from those recovered from bovine feces. In conclusion, despite the use of selective enrichment methods, finding carbapenem-resistant bacteria within a feedlot environment was a rarity.

Comparative Genomic Analysis of Enterococci across Sectors of the One Health Continuum.

Enterococci are Gram-positive bacteria that can be isolated from a variety of environments including soil, water, plants, and the intestinal tract of humans and animals. Although they are considered commensals in humans, Enterococcus spp. are important opportunistic pathogens. Due to their presence and persistence in diverse environments, Enterococcus spp. are ideal for studying antimicrobial resistance (AMR) from the One Health perspective. We undertook a comparative genomic analysis of the virulome, resistome, mobilome, and the association between the resistome and mobilome of 246 E. faecium and 376 E. faecalis recovered from livestock (swine, beef cattle, poultry, dairy cattle), human clinical samples, municipal wastewater, and environmental sources. Comparative genomics of E. faecium and E. faecalis identified 31 and 34 different antimicrobial resistance genes (ARGs), with 62% and 68% of the isolates having plasmid-associated ARGs, respectively. Across the One Health continuum, tetracycline (tetL and tetM) and macrolide resistance (ermB) were commonly identified in E. faecium and E. faecalis. These ARGs were frequently associated with mobile genetic elements along with other ARGs conferring resistance against aminoglycosides [ant(6)-la, aph(3')-IIIa], lincosamides [lnuG, lsaE], and streptogramins (sat4). Study of the core E. faecium genome identified two main clades, clade 'A' and 'B', with clade A isolates primarily originating from humans and municipal wastewater and carrying more virulence genes and ARGs related to category I antimicrobials. Overall, despite differences in antimicrobial usage across the continuum, tetracycline and macrolide resistance genes persisted in all sectors.

Genomic Characterization of Enterococcus hirae From Beef Cattle Feedlots and Associated Environmental Continuum.

Enterococci are commensal bacteria of the gastrointestinal tract of humans, animals, and insects. They are also found in soil, water, and plant ecosystems. The presence of enterococci in human, animal, and environmental settings makes these bacteria ideal candidates to study antimicrobial resistance in the One-Health continuum. This study focused on Enterococcus hirae isolates (n = 4,601) predominantly isolated from beef production systems including bovine feces (n = 4,117, 89.5%), catch-basin water (n = 306, 66.5%), stockpiled bovine manure (n = 24, 0.5%), and natural water sources near feedlots (n = 145, 32%), and a few isolates from urban wastewater (n = 9, 0.2%) denoted as human-associated environmental samples. Antimicrobial susceptibility profiling of a subset (n = 1,319) of E. hirae isolates originating from beef production systems (n = 1,308) showed high resistance to tetracycline (65%) and erythromycin (57%) with 50.4% isolates harboring multi-drug resistance, whereas urban wastewater isolates (n = 9) were resistant to nitrofurantoin (44.5%) and tigecycline (44.5%) followed by linezolid (33.3%). Genes for tetracycline (tetL, M, S/M, and O/32/O) and macrolide resistance erm(B) were frequently found in beef production isolates. Antimicrobial resistance profiles of E. hirae isolates recovered from different environmental settings appeared to reflect the kind of antimicrobial usage in beef and human sectors. Comparative genomic analysis of E. hirae isolates showed an open pan-genome that consisted of 1,427 core genes, 358 soft core genes, 1701 shell genes, and 7,969 cloud genes. Across species comparative genomic analysis conducted on E. hirae, Enterococcus faecalis and Enterococcus faecium genomes revealed that E. hirae had unique genes associated with vitamin production, cellulose, and pectin degradation, traits which may support its adaptation to the bovine digestive tract. E. faecium and E. faecalis more frequently harbored virulence genes associated with biofilm formation, iron transport, and cell adhesion, suggesting niche specificity within these species.

Mechanism of copper nanoparticle toxicity in rainbow trout olfactory mucosa.

Chemosensory perception is crucial for fish reproduction and survival. Direct contact of olfactory neuroepithelium to the surrounding environment makes it vulnerable to contaminants in aquatic ecosystems. Copper nanoparticles (CuNPs), which are increasingly used in commercial and domestic applications due their exceptional properties, can impair fish olfactory function. However, the molecular events underlying olfactory toxicity of CuNPs are largely unexplored. Our results suggested that CuNPs were bioavailable to olfactory mucosal cells. Using RNA-seq, we compared the effect of CuNPs and copper ions (Cu2+) on gene transcript profiles of rainbow trout (Oncorhynchus mykiss) olfactory mucosa. The narrow overlap in differential gene expression between the CuNP- and Cu2+-exposed fish revealed that these two contaminants exert their effects through distinct mechanisms. We propose a transcript-based conceptual model that shows that olfactory signal transduction, calcium homeostasis, and synaptic vesicular signaling were affected by CuNPs in the olfactory sensory neurons (OSNs). Neuroregenerative pathways were also impaired by CuNPs. In contrast, Cu2+ did not induce toxicity pathways and rather upregulated regeneration pathways. Both Cu treatments reduced immune system pathway transcripts. However, suppression of transcripts that were associated with inflammatory signaling was only observed with CuNPs. Neither oxidative stress nor apoptosis were triggered by Cu2+ or CuNPs in mucosal cells. Dysregulation of transcripts that regulate function, maintenance, and reestablishment of damaged olfactory mucosa represents critical mechanisms of toxicity of CuNPs. The loss of olfaction by CuNPs may impact survival of rainbow trout and impose an ecological risk to fish populations in contaminated environments.

Increased Alu RNA processing in Alzheimer brains is linked to gene expression changes.

Despite significant steps in our understanding of Alzheimer's disease (AD), many of the molecular processes underlying its pathogenesis remain largely unknown. Here, we focus on the role of non-coding RNAs produced by small interspersed nuclear elements (SINEs). RNAs from SINE B2 repeats in mouse and SINE Alu repeats in humans, long regarded as "junk" DNA, control gene expression by binding RNA polymerase II and suppressing transcription. They also possess self-cleaving activity that is accelerated through their interaction with certain proteins disabling this suppression. Here, we show that similar to mouse SINE RNAs, human Alu RNAs, are processed, and the processing rate is increased in brains of AD patients. This increased processing correlates with the activation of genes up-regulated in AD patients, while increased intact Alu RNA levels correlate with down-regulated gene expression in AD. In vitro assays show that processing of Alu RNAs is accelerated by HSF1. Overall, our data show that RNAs from SINE elements in the human brain show a similar pattern of deregulation during amyloid beta pathology as in mouse.

Depletion of eukaryotic initiation factor 5B (eIF5B) reprograms the cellular transcriptome and leads to activation of endoplasmic reticulum (ER) stress and c-Jun N-terminal kinase (JNK).

During the integrated stress response (ISR), global translation initiation is attenuated; however, noncanonical mechanisms allow for the continued translation of specific transcripts. Eukaryotic initiation factor 5B (eIF5B) has been shown to play a critical role in canonical translation as well as in noncanonical mechanisms involving internal ribosome entry site (IRES) and upstream open reading frame (uORF) elements. The uORF-mediated translation regulation of activating transcription factor 4 (ATF4) mRNA plays a pivotal role in the cellular ISR. Our recent study confirmed that eIF5B depletion removes uORF2-mediated repression of ATF4 translation, which results in the upregulation of growth arrest and DNA damage-inducible protein 34 (GADD34) transcription. Accordingly, we hypothesized that eIF5B depletion may reprogram the transcriptome profile of the cell. Here, we employed genome-wide transcriptional analysis on eIF5B-depleted cells. Further, we validate the up- and downregulation of several transcripts from our RNA-seq data using RT-qPCR. We identified upregulated pathways including cellular response to endoplasmic reticulum (ER) stress, and mucin-type O-glycan biosynthesis, as well as downregulated pathways of transcriptional misregulation in cancer and T cell receptor signaling. We also confirm that depletion of eIF5B leads to activation of the c-Jun N-terminal kinase (JNK) arm of the mitogen-activated protein kinase (MAPK) pathway. This data suggests that depletion of eIF5B reprograms the cellular transcriptome and influences critical cellular processes such as ER stress and ISR.

Increased processing of SINE B2 ncRNAs unveils a novel type of transcriptome deregulation in amyloid beta neuropathology.

The functional importance of many non-coding RNAs (ncRNAs) generated by repetitive elements and their connection with pathologic processes remains elusive. B2 RNAs, a class of ncRNAs of the B2 family of SINE repeats, mediate through their processing the transcriptional activation of various genes in response to stress. Here, we show that this response is dysfunctional during amyloid beta toxicity and pathology in the mouse hippocampus due to increased levels of B2 RNA processing, leading to constitutively elevated B2 RNA target gene expression and high Trp53 levels. Evidence indicates that Hsf1, a master regulator of stress response, mediates B2 RNA processing in hippocampal cells and is activated during amyloid toxicity, accelerating the processing of SINE RNAs and gene hyper-activation. Our study reveals that in mouse, SINE RNAs constitute a novel pathway deregulated in amyloid beta pathology, with potential implications for similar cases in the human brain, such as Alzheimer's disease (AD).

Application of artificial intelligence to the in silico assessment of antimicrobial resistance and risks to human and animal health presented by priority enteric bacterial pathogens.

Each year, approximately one in eight Canadians are affected by foodborne illness, either through outbreaks or sporadic illness, with animals being the major reservoir for the pathogens. Whole genome sequence analyses are now routinely implemented by public and animal health laboratories to define epidemiological disease clusters and to identify potential sources of infection. Similarly, a number of bioinformatics tools can be used to identify virulence and antimicrobial resistance (AMR) determinants in the genomes of pathogenic strains. Many important clinical and phenotypic characteristics of these pathogens can now be predicted using machine learning algorithms applied to whole genome sequence data. In this overview, we compare the ability of support vector machines, gradient-boosted decision trees and artificial neural networks to predict the levels of AMR within Salmonella enterica and extended-spectrum ß-lactamase (ESBL) producing Escherichia coli. We show that minimum inhibitory concentrations (MIC) for each of 13 antimicrobials for S. enterica strains can be accurately determined, and that ESBL-producing E. coli strains can be accurately classified as susceptible, intermediate or resistant for each of seven antimicrobials. In addition to AMR and bacterial populations of greatest risk to human health, artificial intelligence algorithms hold promise as tools to predict other clinically and epidemiologically important phenotypes of enteric pathogens.

B2 and ALU retrotransposons are self-cleaving ribozymes whose activity is enhanced by EZH2.

Transposable elements make up half of the mammalian genome. One of the most abundant is the short interspersed nuclear element (SINE). Among their million copies, B2 accounts for ∼350,000 in the mouse genome and has garnered special interest because of emerging roles in epigenetic regulation. Our recent work demonstrated that B2 RNA binds stress genes to retard transcription elongation. Although epigenetically silenced, B2s become massively up-regulated during thermal and other types of stress. Specifically, an interaction between B2 RNA and the Polycomb protein, EZH2, results in cleavage of B2 RNA, release of B2 RNA from chromatin, and activation of thermal stress genes. Although an established RNA-binding protein and histone methyltransferase, EZH2 is not known to be a nuclease. Here, we provide evidence for the surprising conclusion that B2 is a self-cleaving ribozyme. Ribozyme activity depends on Mg+2 and monovalent cations but is resistant to protease treatment. However, contact with EZH2 accelerates cleavage rate by >100-fold, suggesting that EZH2 promotes a cleavage-competent RNA conformation. B2 modification-interference analysis demonstrates that phosphorothioate changes at A and C nucleotides can substitute for EZH2. B2 nucleotides 45 to 55 and 100 to 101 are essential for activity. Finally, another family of SINEs, the human ALU element, also produces a self-cleaving RNA and is cleaved during T-cell activation as well as thermal and endoplasmic reticulum (ER) stress. Thus, B2/ALU SINEs may be classified as "epigenetic ribozymes" that function as transcriptional switches during stress. Given their high copy numbers, B2 and ALU may represent the predominant ribozyme activity in mammalian cells.

Non-coding RNAs in virology: an RNA genomics approach.

Advances in sequencing technologies and bioinformatic analysis techniques have greatly improved our understanding of various classes of RNAs and their functions. Despite not coding for proteins, non-coding RNAs (ncRNAs) are emerging as essential biomolecules fundamental for cellular functions and cell survival. Interestingly, ncRNAs produced by viruses not only control the expression of viral genes, but also influence host cell regulation and circumvent host innate immune response. Correspondingly, ncRNAs produced by the host genome can play a key role in host-virus interactions. In this article, we will first discuss a number of types of viral and mammalian ncRNAs associated with viral infections. Subsequently, we also describe the new possibilities and opportunities that RNA genomics and next-generation sequencing technologies provide for studying ncRNAs in virology.

Destabilization of B2 RNA by EZH2 Activates the Stress Response.

More than 98% of the mammalian genome is noncoding, and interspersed transposable elements account for ∼50% of noncoding space. Here, we demonstrate that a specific interaction between the polycomb protein EZH2 and RNA made from B2 SINE retrotransposons controls stress-responsive genes in mouse cells. In the heat-shock model, B2 RNA binds stress genes and suppresses their transcription. Upon stress, EZH2 is recruited and triggers cleavage of B2 RNA. B2 degradation in turn upregulates stress genes. Evidence indicates that B2 RNA operates as a "speed bump" against advancement of RNA polymerase II, and temperature stress releases the brakes on transcriptional elongation. These data attribute a new function to EZH2 that is independent of its histone methyltransferase activity and reconcile how EZH2 can be associated with both gene repression and activation. Our study reveals that EZH2 and B2 together control activation of a large network of genes involved in thermal stress.

The expression level of small non-coding RNAs derived from the first exon of protein-coding genes is predictive of cancer status.

Small non-coding RNAs (smRNAs) are known to be significantly enriched near the transcriptional start sites of genes. However, the functional relevance of these smRNAs remains unclear, and they have not been associated with human disease. Within the cancer genome atlas project (TCGA), we have generated small RNA datasets for many tumor types. In prior cancer studies, these RNAs have been regarded as transcriptional "noise," due to their apparent chaotic distribution. In contrast, we demonstrate their striking potential to distinguish efficiently between cancer and normal tissues and classify patients with cancer to subgroups of distinct survival outcomes. This potential to predict cancer status is restricted to a subset of these smRNAs, which is encoded within the first exon of genes, highly enriched within CpG islands and negatively correlated with DNA methylation levels. Thus, our data show that genome-wide changes in the expression levels of small non-coding RNAs within first exons are associated with cancer.

Vector-free methods for manipulating miRNA activity in vitro and in vivo.

Vector-based methods for manipulating microRNA (miRNA) activity in vivo and in vitro face a number of disadvantages regarding biosafety, workload, and potential for therapeutic use in patients. Use of miRNA mimics and inhibitors provides an alternative for enhancing or inhibiting translational repression of target genes that could be easier applied to elucidate miRNA function. Here, we describe in detail application of miRNA mimics and inhibitors in vitro using adherent cell lines. In addition, we describe a method how they could be applied in vivo in a possible therapeutic context and provide a protocol for validation of this intervention using miRNA target protectors. We present these methods within a standard experimental design workflow that could be followed and we discuss the technical challenges that have to be taken into account.

microRNA-34c is a novel target to treat dementias.

MicroRNAs are key regulators of transcriptome plasticity and have been implicated with the pathogenesis of brain diseases. Here, we employed massive parallel sequencing and provide, at an unprecedented depth, the complete and quantitative miRNAome of the mouse hippocampus, the prime target of neurodegenerative diseases such as Alzheimer's disease (AD). Using integrative genetics, we identify miR-34c as a negative constraint of memory consolidation and show that miR-34c levels are elevated in the hippocampus of AD patients and corresponding mouse models. In line with this, targeting miR-34 seed rescues learning ability in these mouse models. Our data suggest that miR-34c could be a marker for the onset of cognitive disturbances linked to AD and indicate that targeting miR-34c could be a suitable therapy.

A hippocampal insulin-growth factor 2 pathway regulates the extinction of fear memories.

Extinction learning refers to the phenomenon that a previously learned response to an environmental stimulus, for example, the expression of an aversive behaviour upon exposure to a specific context, is reduced when the stimulus is repeatedly presented in the absence of a previously paired aversive event. Extinction of fear memories has been implicated with the treatment of anxiety disease but the molecular processes that underlie fear extinction are only beginning to emerge. Here, we show that fear extinction initiates upregulation of hippocampal insulin-growth factor 2 (Igf2) and downregulation of insulin-growth factor binding protein 7 (Igfbp7). In line with this observation, we demonstrate that IGF2 facilitates fear extinction, while IGFBP7 impairs fear extinction in an IGF2-dependent manner. Furthermore, we identify one cellular substrate of altered IGF2 signalling during fear extinction. To this end, we show that fear extinction-induced IGF2/IGFBP7 signalling promotes the survival of 17-19-day-old newborn hippocampal neurons. In conclusion, our data suggest that therapeutic strategies that enhance IGF2 signalling and adult neurogenesis might be suitable to treat disease linked to excessive fear memory.

MicroRNA cluster 302-367 enhances somatic cell reprogramming by accelerating a mesenchymal-to-epithelial transition.

MicroRNAs (miRNAs) are emerging critical regulators of cell function that frequently reside in clusters throughout the genome. They influence a myriad of cell functions, including the generation of induced pluripotent stem cells, also termed reprogramming. Here, we have successfully delivered entire miRNA clusters into reprogramming fibroblasts using retroviral vectors. This strategy avoids caveats associated with transient transfection of chemically synthesized miRNA mimics. Overexpression of 2 miRNA clusters, 106a-363 and in particular 302-367, allowed potent increases in induced pluripotent stem cell generation efficiency in mouse fibroblasts using 3 exogenous factors (Sox2, Klf4, and Oct4). Pathway analysis highlighted potential relevant effectors, including mesenchymal-to-epithelial transition, cell cycle, and epigenetic regulators. Further study showed that miRNA cluster 302-367 targeted TGFß receptor 2, promoted increased E-cadherin expression, and accelerated mesenchymal-to-epithelial changes necessary for colony formation. Our work thus provides an interesting alternative for improving reprogramming using miRNAs and adds new evidence for the emerging relationship between pluripotency and the epithelial phenotype.