End Of Sequence Research Articles

Impact of xylan on field productivity and wood saccharification properties in aspen.

Xylan that comprises roughly 25% of hardwood biomass is undesirable in biorefinery applications involving saccharification and fermentation. Efforts to reduce xylan levels have therefore been made in many species, usually resulting in improved saccharification. However, such modified plants have not yet been tested under field conditions. Here we evaluate the field performance of transgenic hybrid aspen lines with reduced xylan levels and assess their usefulness as short-rotation feedstocks for biorefineries. Three types of transgenic lines were tested in four-year field tests with RNAi constructs targeting either Populus GT43 clades B and C (GT43BC) corresponding to Arabidopsis clades IRX9 and IRX14, respectively, involved in xylan backbone biosynthesis, GATL1.1 corresponding to AtGALT1 involved in xylan reducing end sequence biosynthesis, or ASPR1 encoding an atypical aspartate protease. Their productivity, wood quality traits, and saccharification efficiency were analyzed. The only lines differing significantly from the wild type with respect to growth and biotic stress resistance were the ASPR1 lines, whose stems were roughly 10% shorter and narrower and leaves showed increased arthropod damage. GT43BC lines exhibited no growth advantage in the field despite their superior growth in greenhouse experiments. Wood from the ASPR1 and GT43BC lines had slightly reduced density due to thinner cell walls and, in the case of ASPR1, larger cell diameters. The xylan was less extractable by alkali but more hydrolysable by acid, had increased glucuronosylation, and its content was reduced in all three types of transgenic lines. The hemicellulose size distribution in the GALT1.1 and ASPR1 lines was skewed towards higher molecular mass compared to the wild type. These results provide experimental evidence that GATL1.1 functions in xylan biosynthesis and suggest that ASPR1 may regulate this process. In saccharification without pretreatment, lines of all three constructs provided 8-11% higher average glucose yields than wild-type plants. In saccharification with acid pretreatment, the GT43BC construct provided a 10% yield increase on average. The best transgenic lines of each construct are thus predicted to modestly outperform the wild type in terms of glucose yields per hectare. The field evaluation of transgenic xylan-reduced aspen represents an important step towards more productive feedstocks for biorefineries.

DNA Barcodes of Mansonia (Mansonia) Blanchard, 1901 (Diptera, Culicidae).

Females of the genus Mansonia feed on the blood of humans, livestock, and other vertebrates to develop their eggs. The females' biting behavior may cause severe disturbance to blood hosts, with a negative impact on public health and economics. Certain species have been identified as potential or effective disease vectors. The accurate species identification of field-collected specimens is of paramount importance for the success of monitoring and control strategies. Mansonia (Mansonia) morphological species boundaries are blurred by patterns of intraspecific heteromorphism and interspecific isomorphism. DNA barcodes can help to solve taxonomic controversies, especially if combined with other molecular tools. We used cytochrome c oxidase subunit I (COI) gene 5' end (DNA barcode) sequences to identify 327 field-collected specimens of Mansonia (Mansonia) spp. The sampling encompassed males and females collected from three Brazilian regions and previously assigned to species based on their morphological characteristics. Eleven GenBank and BOLD sequences were added to the DNA barcode analyses. Initial morphospecies assignments were mostly corroborated by the results of five clustering methods based on Kimura two-parameter distance and maximum likelihood phylogeny. Five to eight molecular operational taxonomic units may represent taxonomically unknown species. The first DNA barcode records for Mansonia fonsecai, Mansonia iguassuensis, and Mansonia pseudotitillans are presented.

One-pot making of sequence-restricted DNA dumbbells

A method to build sequence-restricted DNA dumbbells was developed. 5′-exonuclease converts end sequences of DNA targets into sticky ends. Self-looping oligonucleotides with complementary 3′-overhangs are ligated to form dumbbells by DNA polymerase and ligase in a sequence-restricted manner. These reactions proceed in one pot at one temperature. We demonstrated one use of this method to ‘tunnel’ sequencing libraries into dumbbells for the Pacific Biosciences (PacBio) platform. Readouts of an Illumina P5/P7-ended 16S library from a standard microbial community confirmed successful tunneling. Twelve fecal samples additionally showed significant correlations between standard and tunneled 16S sequence variants on the PacBio platform. We further extended the method at a genomic scale to build a ∼0.45 Mbp giant dumbbell on chromosome 6. Sequences inside the dumbbells were successfully protected from a cocktail of exonucleases. Roughly 11-fold enrichment was achieved for the dumbbell-guarded region compared to the vicinity.

Blocking Abundant RNA Transcripts by High-Affinity Oligonucleotides during Transcriptome Library Preparation

BackgroundRNA sequencing has become the gold standard for transcriptome analysis but has an inherent limitation of challenging quantification of low-abundant transcripts. In contrast to microarray technology, RNA sequencing reads are proportionally divided in function of transcript abundance. Therefore, low-abundant RNAs compete against highly abundant - and sometimes non-informative - RNA species.ResultsWe developed an easy-to-use strategy based on high-affinity RNA-binding oligonucleotides to block reverse transcription and PCR amplification of specific RNA transcripts, thereby substantially reducing their abundance in the final sequencing library. To demonstrate the broad application potential of our method, we applied it to different transcripts and library preparation strategies, including YRNAs in small RNA sequencing of human blood plasma, mitochondrial rRNAs in both 3′ end sequencing and long-read sequencing, and MALAT1 in single-cell 3′ end sequencing. We demonstrate that the blocking strategy is highly efficient, reproducible, specific, and generally results in better transcriptome coverage and complexity.ConclusionOur method does not require modifications of the library preparation procedure apart from simply adding blocking oligonucleotides to the RT reaction and can thus be easily integrated into virtually any RNA sequencing library preparation protocol.

Reciprocal regulation of flower induction by ELF3α and ELF3β generated via alternative promoter usage.

Flowering is critical for sexual reproduction and fruit production. Several pear (Pyrus sp.) varieties produce few flower buds, but the underlying mechanisms are unknown. The circadian clock regulator EARLY FLOWERING3 (ELF3) serves as a scaffold protein in the evening complex that controls flowering. Here, we report that the absence of a 58-bp sequence in the 2nd intron of PbELF3 is genetically associated with the production of fewer flower buds in pear. From rapid amplification of cDNA ends sequencing results, we identified a short, previously unknown transcript from the PbELF3 locus, which we termed PbELF3β, whose transcript level was significantly lower in pear cultivars that lacked the 58-bp region. The heterologous expression of PbELF3β in Arabidopsis (Arabidopsis thaliana) accelerated flowering, whereas the heterologous expression of the full-length transcript PbELF3α caused late flowering. Notably, ELF3β was functionally conserved in other plants. Deletion of the 2nd intron reduced AtELF3β expression and caused delayed flowering time in Arabidopsis. AtELF3β physically interacted with AtELF3α, disrupting the formation of the evening complex and consequently releasing its repression of flower induction genes such as GIGANTEA (GI). AtELF3β had no effect in the absence of AtELF3α, supporting the idea that AtELF3β promotes flower induction by blocking AtELF3α function. Our findings show that alternative promoter usage at the ELF3 locus allows plants to fine-tune flower induction.

Characterization of the eukaryotic microbial communities in the chicken ileum in cage-free and conventional commercial housing systems

The current shift from conventional cage (CC) hen housing facilities towards cage-free (CF) hen housing in the egg industry has left many questions regarding how level of fecal exposure and exposure to other hens may influence the hen intestinal microbiota. In a previous publication we reported differences in the bacterial ileal communities and ileal morphology between hens in CC and CF production environments at a single commercial site. Here, we present the first 18S rRNA gene amplicon sequencing-based characterization of the eukaryotic ileal microbiota of adult layer hens, and their associations with intestinal health parameters and the bacterial microbiota. DNA was extracted from the ileal digesta of hens (n=32 CC, n=48 CF) using the Qiagen Powerlyzer Powersoil kit, followed by amplification of the V9 region of the 18S rRNA gene. Paired end sequencing was performed with the Illumina MiSeq platform, and the resulting reads were processed according to the Mothur MiSeq protocol in Mothur v1.43.0. De novo operational taxonomic unit (OTU) clustering was performed in mothur with a 99% similarity threshold, and OTUs were taxonomically classified with the SILVA SSU v138 reference database. OTUs classified as vertebrate, plant, or arthropod were removed, resulting in 3,136,400 high quality reads and 1,370 OTUs. Associations between OTUs and intestinal parameters were calculated using PROC GLIMMIX. PERMANOVA over Bray-Curtis distances revealed differences between CC and CF eukaryotic ileal microbiota at the whole community level, but no OTUs were differentially abundant after correcting for false discovery (P > 0.05; q > 0.1). Kazachstania and Saccharomyces, closely related genera of yeast, represented 77.1% and 9.7% of sequences respectively. Two Kazachstania OTUs and 1 Saccharomycetaceae OTU were positively correlated with intestinal permeability (r2 ≥ 0.35). Eimeria accounted for 7.6% of sequences across all samples. Intriguingly, 15 OTUs classified as Eimeria were inversely associated with intestinal permeability (r2 ≤ -0.35), suggesting Eimeria may play a more complex role in the microbiota of healthy birds than has been observed in disease challenges.

Nuclease P1 Digestion for Bottom-Up RNA Sequencing of Modified siRNA Therapeutics.

siRNA therapeutics provide a selective and powerful approach to reduce the expression of disease-causing genes. For regulatory approval, these modalities require sequence confirmation which is typically achieved by intact tandem mass spectrometry sequencing. However, this process produces highly complex spectra which are difficult to interpret and typically results in less than full sequence coverage. We sought to develop a bottom-up siRNA sequencing platform to ease sequencing data analysis and provide full sequence coverage. Analogous to bottom-up proteomics, this process requires chemical or enzymatic digestion to reduce the oligonucleotide length down to analyzable lengths, but siRNAs commonly contain modifications that inhibit the degradation process. We tested six digestion schemes for their feasibility to digest the 2' modified siRNAs and identified that nuclease P1 provides an effective digestion workflow. Using a partial digestion, nuclease P1 provides high 5' and 3' end sequence coverage with multiple overlapping digestion products. Additionally, this enzyme provides high-quality and highly reproducible RNA sequencing no matter the RNA phosphorothioate content, 2'-fluorination status, sequence, or length. Overall, we developed a robust enzymatic digestion scheme for bottom-up siRNA sequencing using nuclease P1, which can be implemented into existing sequence confirmation workflows.

An Arabidopsis family GT106 glycosyltransferase is essential for xylan biosynthesis and secondary wall deposition.

We have demonstrated that the Arabidopsis FRA9 (fragile fiber 9) gene is specifically expressed in secondary wall-forming cells and essential for the synthesis of the unique xylan reducing end sequence. Xylan is made of a linear chain of β-1,4-linked xylosyl (Xyl) residues that are often substituted with (methyl)glucuronic acid [(Me)GlcA] side chains and may be acetylated at O-2 and/or O-3. The reducing end of xylan from gymnosperms and dicots contains a unique tetrasaccharide sequence consisting of β-D-Xylp-(1 → 3)-α-L-Rhap-(1 → 2)-α-D-GalpA-(1 → 4)-D-Xylp, the synthesis of which requires four different glycosyltransferase activities. Genetic analysis in Arabidopsis thaliana has so far implicated three glycosyltransferase genes, FRA8 (fragile fiber 8), IRX8 (irregular xylem 8) and PARVUS, in the synthesis of this unique xylan reducing end sequence. Here, we report the essential role of FRA9, a member of glycosyltransferase family 106 (GT106), in the synthesis of this sequence. The expression of the FRA9 gene was shown to be induced by secondary wall master transcriptional regulators and specifically associated with secondary wall-forming cells, including xylem and fiber cells. T-DNA knockout mutation of the FRA9 gene caused impaired secondary cell wall thickening in leaf veins and a severe arrest of plant growth. RNA interference (RNAi) downregulation of FRA9 led to a significant reduction in secondary wall thickening of fibers, a deformation of xylem vessels and a decrease in xylan content. Structural analysis of xylanase-released xylooligomers revealed that RNAi downregulation of FRA9 resulted in a diminishment of the unique xylan reducing end sequence and complete methylation of xylan GlcA side chains, chemotypes reminiscent of those of the fra8, irx8 and parvus mutants. Furthermore, two FRA9 close homologs from Populus trichocarpa were found to be wood-associated functional orthologs of FRA9. Together, our findings uncover a member of the GT106 family as a new player involved in the synthesis of the unique reducing end sequence of xylan.

DEtail-seq is an ultra-efficient and convenient method for meiotic DNA break profiling in multiple organisms.

Programmed DNA double-strand break (DSB) formation is a crucial step in meiotic recombination, yet techniques for high-efficiency and precise mapping of the 3' ends of DSBs are still in their infancy. Here, we report a novel technique, named DNA End tailing and sequencing (DEtail-seq), which can directly and ultra-efficiently characterize the 3' ends of meiotic DSBs with near single-nucleotide resolution in a variety of species, including yeast, mouse, and human. We find that the 3' ends of meiotic DSBs are stable without significant resection in budding yeast. Meiotic DSBs are strongly enriched in de novo H3K4me3 peaks in the mouse genome at leptotene stage. We also profile meiotic DSBs in human and find DSB hotspots are enriched near the common fragile sites during human meiosis, especially at CCCTC-binding factor (CTCF)-associated enhancers. Therefore, DEtail-seq provides a powerful method to detect DSB ends in various species, and our results provide new insights into the distribution and regulation of meiotic DSB hotspots.

Molecular characterization of a novel gammacarmovirus infecting cucurbits in India

In this study, we describe the identification of a new gammacarmovirus infecting Cucurbita pepo plants showing a range of mosaic, stunting, yellowing, and wilting symptoms. The virus had a narrow host range and mostly produced chlorotic and necrotic local lesions in the majority of the tested plants. However, Nicotiana benthamiana showed systemic symptoms under laboratory conditions. Using a combination of Sanger sequencing and rapid amplification of cDNA ends (RACE), the complete genome sequence of the virus was determined to be 4274 nucleotides (nt) in length. Its genome organization is similar to that of members of the genus Gammacarmovirus in the family Tombusviridae, consisting of five overlapping open reading frames (ORFs) encoding p28, replicase, p7A, p7B, and coat protein (CP), respectively. The genome is flanked by short 5' and 3' non-coding regions (NCR) at either end. In pairwise comparisons of replicase and CP sequences, the virus showed the highest amino acid sequence identity of 71.55% and 54.86%, respectively, to melon necrotic spot virus (MNSV), the type member of the genus Gammacarmovirus. Since the sequence identity values are below the species demarcation threshold suggested by the International Committee on Taxonomy of Viruses (ICTV), the virus from Cucurbita pepo plants, for which the name "cucurbit carmovirus" (CuCV) is proposed, represents a new species. In phylogenetic analysis based on the replicase and CP amino acid sequences, CuCV clustered with MNSV but formed a distinct branch, further confirming that the virus is a distinct member of the genus Gammacarmovirus.

Programmable 3D Hexagonal Geometry of DNA Tensegrity Triangles.

Non-canonical interactions in DNA remain under-explored in DNA nanotechnology. Recently, many structures with non-canonical motifs have been discovered, notably a hexagonal arrangement of typically rhombohedral DNA tensegrity triangles that forms through non-canonical sticky end interactions. Here, we find a series of mechanisms to program a hexagonal arrangement using: the sticky end sequence; triangle edge torsional stress; and crystallization condition. We showcase cross-talking between Watson-Crick and non-canonical sticky ends in which the ratio between the two dictates segregation by crystal forms or combination into composite crystals. Finally, we develop a method for reconfiguring the long-range geometry of formed crystals from rhombohedral to hexagonal and vice versa. These data demonstrate fine control over non-canonical motifs and their topological self-assembly. This will vastly increase the programmability, functionality, and versatility of rationally designed DNA constructs.

Programmable 3D Hexagonal Geometry of DNA Tensegrity Triangles

AbstractNon‐canonical interactions in DNA remain under‐explored in DNA nanotechnology. Recently, many structures with non‐canonical motifs have been discovered, notably a hexagonal arrangement of typically rhombohedral DNA tensegrity triangles that forms through non‐canonical sticky end interactions. Here, we find a series of mechanisms to program a hexagonal arrangement using: the sticky end sequence; triangle edge torsional stress; and crystallization condition. We showcase cross‐talking between Watson–Crick and non‐canonical sticky ends in which the ratio between the two dictates segregation by crystal forms or combination into composite crystals. Finally, we develop a method for reconfiguring the long‐range geometry of formed crystals from rhombohedral to hexagonal and vice versa. These data demonstrate fine control over non‐canonical motifs and their topological self‐assembly. This will vastly increase the programmability, functionality, and versatility of rationally designed DNA constructs.

First report of Mycobacteroides abscessus subsp. massiliense and Erysipelothrix rhusiopathiae as causative agents of pneumonia and hepatitis in a boa (Boa constrictor)

An adult boa with acute epistaxis, without other clinical signs, was found dead in its terrarium. Macroscopic examination of the specimen revealed multiple yellow foci in the lung and liver. Microscopic findings included severe and multifocal granulomas as well as vasculitis, hemorrhages and thrombosis in liver and lungs, with the presence of intralesional Gram positive and acid-fast bacilli. PCR end point analysis and sequencing using total DNA extracted from formalin-fixed paraffin-embedded samples of liver and lungs identified Mycobacteroides abscessus subsp massiliense and Erysipelothrix rhusiopathiae. Mycobacteria are organisms that can affect a wide range of animals, including reptiles. In snakes, reports are rare and mycobacterial species are highly variable. Erysipelothrix rhusiopathiae is a pathogenic bacillus related to multiple organ disorders, which until now has not been reported in boas. This report describes the first case report of Mycobacteroides abscessus subsp. massiliense and Erysipelothrix rhusiopathiae detection in a boa.

Comparison of Gene Selection Methods for Clustering Single-cell RNA-seq Data

Background: In single-cell RNA-seq data, clustering methods are employed to identify cell types to understand cell-differentiation and development. Because clustering methods are sensitive to the high dimensionality of single-cell RNA-seq data, one effective solution is to select a subset of genes in order to reduce the dimensionality. Numerous methods, with different underlying assumptions, have been proposed for choosing a subset of genes to be used for clustering. Objective: To guide users in selecting suitable gene selection methods, we give an overview of different gene selection methods and compare their performance in terms of the differences between the selected gene sets, clustering performance, running time, and stability. Results: We first review the data preprocessing strategies and gene selection methods in analyzing single-cell RNA-seq data. Then, the overlaps among the gene sets selected by different methods are analyzed and the clustering performance based on different feature gene sets is compared. The analysis reveals that the gene sets selected by the methods based on highly variable genes and high mean genes are most similar, and the highly variable genes play an important role in clustering. Additionally, a small number of selected genes would compromise the clustering performance, such as SCMarker selected fewer genes than other methods, leading to a poorer clustering performance than M3Drop. Conclusion: Different gene selection methods perform differently in different scenarios. HVG works well on the full-transcript sequencing datasets, NBDrop and HMG perform better on the 3’ end sequencing datasets, M3Drop and HMG are more suitable for big datasets, and SCMarker is most consistent in different preprocessing methods.

Enzymatic synthesis of RNA standards for mapping and quantifying RNA modifications in sequencing analysis.

Synthesis of RNA standards that contain an internal site-specific modification is important for mapping and quantification of the modified nucleotide in sequencing analysis. While RNA containing a site-specific modification can be readily synthesized by solid-state coupling for less than 100-mer nucleotides, longer RNA must be synthesized by enzymatic ligation in the presence of a DNA splint. However, long RNAs have structural heterogeneity, and those generated by in vitro transcription have 3'-end sequence heterogeneity, which together substantially reduce the yield of ligation. Here we describe a method of 3-part splint ligation that joins an in vitro transcribed left-arm RNA, an in vitro transcribed right-arm RNA, and a chemically synthesized modification-containing middle RNA, with an efficiency higher than previously reported. We report that the improved efficiency is largely attributed to the inclusion of a pair of DNA disruptors proximal to the ligation sites, and to a lesser extent to the homogeneous processing of the 3'-end of the left-arm RNA. The yields of the ligated long RNA are sufficiently high to afford purification to homogeneity for practical RNA research. We also verify the sequence accuracy at each ligation junction by nanopore sequencing.

Cadherins and catenins as a novel theoretical mechanism in a polyorchid cadaver

Polyorchidism is a rare congenital disorder defined as the presence of more than two testicles for which the underlying etiology is unknown. Prior research in this laboratory suggested a new anatomical- functional description for polyorchidism of Type 3 Subgroup B, or supernumerary testis (SNT) attached to the draining epididymis and vas deferens without reproductive potential and SNT located outside the scrotal sac. The purpose of the current investigation was to examine the mechanism underlying the role(s) of cadherins and catenins in the development of polyorchidism using genomic analysis of a cadaveric polyorchid. Formyl Fix Paraffin Embedded tissue samples of a SNT from a 96-year-old polyorchid were prepared using the Accel-NGS 2S Plus DNA Library Kit for whole exome sequencing (Integrated DNA Technology, Coralville, IA). Paired end sequencing was carried out using the Illumina NovaSeq 6000 system (Illumina, San Diego, CA) using 150 bp reads to an average depth of coverage of 44x. BLAST was used to analyze and compare the SNT sequence to reference genomes in the NCBI database. Sequence analysis of the SNT showed two missense mutations that resulted in single nucleotide variants (SNV) within exons of the N-Cadherin gene (CHD2), NT 23, T to C (Leu 8 to Pro) and NT 2441, A to G (Asn 845 to Ser), respectively. A mutation in the Desmocollin 2 (DSC2) gene was also demonstrated; NT 2393, G to A. The present research suggests a novel biomolecular mechanism based on N-Cad and p120 catenin underlying the development of polyorchidism with application to supernumerary organs in other systems and to metastasis of neoplasms.

BAC-FISH Based Physical Map of Endangered Catfish Clarias magur for Chromosome Cataloguing and Gene Isolation through Positional Cloning.

Construction of a physical chromosome map of a species is important for positional cloning, targeted marker development, fine mapping of genes, selection of candidate genes for molecular breeding, as well as understanding the genome organization. The genomic libraries in the form of bacterial artificial chromosome (BAC) clones are also a very useful resource for physical mapping and identification and isolation of full-length genes and the related cis acting elements. Some BAC-FISH based studies reported in the past were gene based physical chromosome maps of Clarias magur (magur) to understand the genome organization of the species and to establish the relationships with other species in respect to genes' organization and evolution in the past. In the present study, we generated end sequences of the BAC clones and analyzed those end sequences within the scaffolds of the draft genome of magur to identify and map the genes bioinformatically for each clone. A total of 36 clones mostly possessing genes were identified and used in probe construction and their subsequent hybridization on the metaphase chromosomes of magur. This study successfully mapped all 36 specific clones on 16 chromosome pairs, out of 25 pairs of magur chromosomes. These clones are now recognized as chromosome-specific makers, which are an aid in individual chromosome identification and fine assembly of the genome sequence, and will ultimately help in developing anchored genes' map on the chromosomes of C. magur for understanding their organization, inheritance of important fishery traits and evolution of magur with respect to channel catfish, zebrafish and other species.

Cell-type specific profiling of histone post-translational modifications in the adult mouse striatum

Epigenetic gene regulation in the heterogeneous brain remains challenging to decipher with current strategies. Bulk tissue analysis from pooled subjects reflects the average of cell-type specific changes across cell-types and individuals, which obscures causal relationships between epigenetic modifications, regulation of gene expression, and complex pathology. To address these limitations, we optimized a hybrid protocol, ICuRuS, for the isolation of nuclei tagged in specific cell-types and histone post translational modification profiling from the striatum of a single mouse. We combined affinity-based isolation of the medium spiny neuron subtypes, Adenosine 2a Receptor or Dopamine Receptor D1, with cleavage of histone-DNA complexes using an antibody-targeted micrococcal nuclease to release DNA complexes for paired end sequencing. Unlike fluorescence activated cell sorting paired with chromatin immunoprecipitation, ICuRuS allowed for robust epigenetic profiling at cell-type specific resolution. Our analysis provides a framework to understand combinatorial relationships between neuronal-subtype-specific epigenetic modifications and gene expression.

Whole Genome Sequencing (WGS) Analysis of Virulence and AMR Genes in Extended-Spectrum β-Lactamase (ESBL)-Producing Escherichia coli from Animal and Environmental Samples in Four Italian Swine Farms.

Whole genome sequencing (WGS) is a powerful tool to analyze bacterial genomes rapidly, and can be useful to study and detect AMR genes. We carried out WGS on a group of Escherichia coli (n = 30), sampled from healthy animals and farm environment in four pigsties in northern Italy. Two × 250bp paired end sequencing strategy on Illumina MiSeq™ was used. We performed in silico characterization of E. coli isolates through the web tools provided by the Center for Genomic Epidemiology (cge.cbs.dtu.dk/services/) to study AMR and virulence genes. Bacterial strains were further analyzed to detect phenotypic antimicrobial susceptibility against several antimicrobials. Data obtained from WGS were compared to phenotypic results. All 30 strains were MDR, and they were positive for the genes blaCTX-M and blaTEM as verified by PCR. We observed a good concordance between phenotypic and genomic results. Different AMR determinants were identified (e.g., qnrS, sul, tet). Potential pathogenicity of these strains was also assessed, and virulence genes were detected (e.g., etsC, gad, hlyF, iroN, iss), mostly related to extraintestinal E. coli pathotypes (UPEC/APEC). However, enterotoxin genes, such as astA, ltcA and stb were also identified, indicating a possible hybrid pathogenic nature. Various replicons associated to plasmids, previously recovered in pathogenic bacteria, were identified (e.g., IncN and IncR plasmid), supporting the hypothesis that our strains were pathogenic. Eventually, through WGS it was possible to confirm the phenotypic antibiotic resistance results and to appreciate the virulence side of our ESBL-producing E. coli. These findings highlight the need to monitor commensal E. coli sampled from healthy pigs considering a One Health perspective.

Human histone pre-mRNA assembles histone or canonical mRNA-processing complexes by overlapping 3'-end sequence elements.

Human pre-mRNA processing relies on multi-subunit macromolecularcomplexes, which recognize specific RNA sequence elements essential for assembly and activity. Canonical pre-mRNA processing proceeds via the recognition of a polyadenylation signal (PAS) and a downstream sequence element (DSE), and produces polyadenylated mature mRNAs, while replication-dependent (RD)histone pre-mRNA processingrequires association with a stem-loop (SL) motif and a histone downstream element (HDE), and produces cleaved but non-polyadenylated mature mRNAs. H2AC18 mRNA, a specific H2A RD histone pre-mRNA, can be processed to give either a non-polyadenylated mRNA, ending at the histone SL, or a polyadenylated mRNA. Here, we reveal how H2AC18 captures the two human pre-mRNA processing complexes in a mutually exclusive mode by overlapping a canonical PAS (AAUAAA) sequence element with a HDE. Disruption of the PAS sequence on H2AC18 pre-mRNA prevents recruitment of the canonical complex in vitro, without affecting the histone machinery. This shows how the relative position of cis-acting elements in histone pre-mRNAs allows the selective recruitment of distinct human pre-mRNA complexes, thereby expanding the capability to regulate 3' processing and polyadenylation.