Characterization of Canine Peyer's Patches by Multidimensional Analysis: Insights from Immunofluorescence, Flow Cytometry, and Single-Cell RNA Sequencing.

The oral route is effective and convenient for vaccine administration to stimulate a protective immune response. GALT plays a crucial role in mucosal immune responses, with Peyer's patches (PPs) serving as the primary site of induction. A comprehensive understanding of the structures and functions of these structures is crucial for enhancing vaccination strategies and comprehending disease mechanisms; nonetheless, our current knowledge of these structures in dogs remains incomplete. We performed immunofluorescence and flow cytometry studies on canine PPs to identify cell populations and structures. We also performed single-cell RNA sequencing (scRNA-seq) to investigate the immune cell subpopulations present in PPs at steady state in dogs. We generated and validated an Ab specifically targeting canine M cells, which will be a valuable tool for elucidating Ag trafficking into the GALT of dogs. Our findings will pave the way for future studies of canine mucosal immune responses to oral vaccination and enteropathies. Moreover, they add to the growing body of knowledge in canine immunology, further expanding our understanding of the complex immune system of dogs.

RNA helicase-dependent gene looping impacts messenger RNA processing.

DDX5 and DDX17 are DEAD-box RNA helicase paralogs which regulate several aspects of gene expression, especially transcription and splicing, through incompletely understood mechanisms. A transcriptome analysis of DDX5/DDX17-depleted human cells confirmed the large impact of these RNA helicases on splicing and revealed a widespread deregulation of 3' end processing. In silico analyses and experiments in cultured cells showed the binding and functional contribution of the genome organizing factor CTCF to chromatin sites at or near a subset of DDX5/DDX17-dependent exons that are characterized by a high GC content and a high density of RNA Polymerase II. We propose the existence of an RNA helicase-dependent relationship between CTCF and the dynamics of transcription across DNA and/or RNA structured regions, that contributes to the processing of internal and terminal exons. Moreover, local DDX5/DDX17-dependent chromatin loops spatially connect RNA helicase-regulated exons with their cognate promoter, and we provide the first direct evidence that de novo gene looping modifies alternative splicing and polyadenylation. Overall our findings uncover the impact of DDX5/DDX17-dependent chromatin folding on pre-messenger RNA processing.

Regulation of Clostridium tetani Neurotoxin Expression by Culture Conditions.

BACKGROUND: Ensuring consistency of tetanus neurotoxin (TeNT) production by Clostridium tetani could help to ensure consistent product quality in tetanus vaccine manufacturing, ultimately contributing to reduced animal testing. The aim of this study was to identify RNA signatures related to consistent TeNT production using standard and non-standard culture conditions. METHODS: We applied RNA sequencing (RNA-Seq) to study C. tetani gene expression in small-scale batches under several culture conditions. RESULTS: We identified 1381 time-dependent differentially expressed genes (DEGs) reflecting, among others, changes in growth rate and metabolism. Comparing non-standard versus standard culture conditions identified 82 condition-dependent DEGs, most of which were specific for one condition. The tetanus neurotoxin gene (tetX) was highly expressed but showed expression changes over time and between culture conditions. The tetX gene showed significant down-regulation at higher pH levels (pH 7.8), which was confirmed by the quantification data obtained with the recently validated targeted LC-MS/MS approach. CONCLUSIONS: Non-standard culture conditions lead to different gene expression responses. The tetX gene appears to be the best transcriptional biomarker for monitoring TeNT production as part of batch-to-batch consistency testing during tetanus vaccine manufacturing.

Integrative Cell Type-Specific Multi-Omics Approaches Reveal Impaired Programs of Glial Cell Differentiation in Mouse Culture Models of DM1.

Myotonic dystrophy type 1 (DM1) is a neuromuscular disorder caused by a non-coding CTG repeat expansion in the DMPK gene. This mutation generates a toxic CUG RNA that interferes with the RNA processing of target genes in multiple tissues. Despite debilitating neurological impairment, the pathophysiological cascade of molecular and cellular events in the central nervous system (CNS) has been less extensively characterized than the molecular pathogenesis of muscle/cardiac dysfunction. Particularly, the contribution of different cell types to DM1 brain disease is not clearly understood. We first used transcriptomics to compare the impact of expanded CUG RNA on the transcriptome of primary neurons, astrocytes and oligodendrocytes derived from DMSXL mice, a transgenic model of DM1. RNA sequencing revealed more frequent expression and splicing changes in glia than neuronal cells. In particular, primary DMSXL oligodendrocytes showed the highest number of transcripts differentially expressed, while DMSXL astrocytes displayed the most severe splicing dysregulation. Interestingly, the expression and splicing defects of DMSXL glia recreated molecular signatures suggestive of impaired cell differentiation: while DMSXL oligodendrocytes failed to upregulate a subset of genes that are naturally activated during the oligodendroglia differentiation, a significant proportion of missplicing events in DMSXL oligodendrocytes and astrocytes increased the expression of RNA isoforms typical of precursor cell stages. Together these data suggest that expanded CUG RNA in glial cells affects preferentially differentiation-regulated molecular events. This hypothesis was corroborated by gene ontology (GO) analyses, which revealed an enrichment for biological processes and cellular components with critical roles during cell differentiation. Finally, we combined exon ontology with phosphoproteomics and cell imaging to explore the functional impact of CUG-associated spliceopathy on downstream protein metabolism. Changes in phosphorylation, protein isoform expression and intracellular localization in DMSXL astrocytes demonstrate the far-reaching impact of the DM1 repeat expansion on cell metabolism. Our multi-omics approaches provide insight into the mechanisms of CUG RNA toxicity in the CNS with cell type resolution, and support the priority for future research on non-neuronal mechanisms and proteomic changes in DM1 brain disease.

Intragenic recruitment of NF-κB drives splicing modifications upon activation by the oncogene Tax of HTLV-1.

Chronic NF-κB activation in inflammation and cancer has long been linked to persistent activation of NF-κB-responsive gene promoters. However, NF-κB factors also massively bind to gene bodies. Here, we demonstrate that recruitment of the NF-κB factor RELA to intragenic regions regulates alternative splicing upon NF-κB activation by the viral oncogene Tax of HTLV-1. Integrative analyses of RNA splicing and chromatin occupancy, combined with chromatin tethering assays, demonstrate that DNA-bound RELA interacts with and recruits the splicing regulator DDX17, in an NF-κB activation-dependent manner. This leads to alternative splicing of target exons due to the RNA helicase activity of DDX17. Similar results were obtained upon Tax-independent NF-κB activation, indicating that Tax likely exacerbates a physiological process where RELA provides splice target specificity. Collectively, our results demonstrate a physical and direct involvement of NF-κB in alternative splicing regulation, which significantly revisits our knowledge of HTLV-1 pathogenesis and other NF-κB-related diseases.

Characterizing the interplay between gene nucleotide composition bias and splicing.

BACKGROUND: Nucleotide composition bias plays an important role in the 1D and 3D organization of the human genome. Here, we investigate the potential interplay between nucleotide composition bias and the regulation of exon recognition during splicing. RESULTS: By analyzing dozens of RNA-seq datasets, we identify two groups of splicing factors that activate either about 3200 GC-rich exons or about 4000 AT-rich exons. We show that splicing factor-dependent GC-rich exons have predicted RNA secondary structures at 5' ss and are dependent on U1 snRNP-associated proteins. In contrast, splicing factor-dependent AT-rich exons have a large number of decoy branch points, SF1- or U2AF2-binding sites and are dependent on U2 snRNP-associated proteins. Nucleotide composition bias also influences local chromatin organization, with consequences for exon recognition during splicing. Interestingly, the GC content of exons correlates with that of their hosting genes, isochores, and topologically associated domains. CONCLUSIONS: We propose that regional nucleotide composition bias over several dozens of kilobase pairs leaves a local footprint at the exon level and induces constraints during splicing that can be alleviated by local chromatin organization at the DNA level and recruitment of specific splicing factors at the RNA level. Therefore, nucleotide composition bias establishes a direct link between genome organization and local regulatory processes, like alternative splicing.

A genome-wide screen identifies IRF2 as a key regulator of caspase-4 in human cells.

Caspase-4, the cytosolic LPS sensor, and gasdermin D, its downstream effector, constitute the non-canonical inflammasome, which drives inflammatory responses during Gram-negative bacterial infections. It remains unclear whether other proteins regulate cytosolic LPS sensing, particularly in human cells. Here, we conduct a genome-wide CRISPR/Cas9 screen in a human monocyte cell line to identify genes controlling cytosolic LPS-mediated pyroptosis. We find that the transcription factor, IRF2, is required for pyroptosis following cytosolic LPS delivery and functions by directly regulating caspase-4 levels in human monocytes and iPSC-derived monocytes. CASP4, GSDMD, and IRF2 are the only genes identified with high significance in this screen highlighting the simplicity of the non-canonical inflammasome. Upon IFN-γ priming, IRF1 induction compensates IRF2 deficiency, leading to robust caspase-4 expression. Deficiency in IRF2 results in dampened inflammasome responses upon infection with Gram-negative bacteria. This study emphasizes the central role of IRF family members as specific regulators of the non-canonical inflammasome.

Interplay between coding and exonic splicing regulatory sequences.

The inclusion of exons during the splicing process depends on the binding of splicing factors to short low-complexity regulatory sequences. The relationship between exonic splicing regulatory sequences and coding sequences is still poorly understood. We demonstrate that exons that are coregulated by any given splicing factor share a similar nucleotide composition bias and preferentially code for amino acids with similar physicochemical properties because of the nonrandomness of the genetic code. Indeed, amino acids sharing similar physicochemical properties correspond to codons that have the same nucleotide composition bias. In particular, we uncover that the TRA2A and TRA2B splicing factors that bind to adenine-rich motifs promote the inclusion of adenine-rich exons coding preferentially for hydrophilic amino acids that correspond to adenine-rich codons. SRSF2 that binds guanine/cytosine-rich motifs promotes the inclusion of GC-rich exons coding preferentially for small amino acids, whereas SRSF3 that binds cytosine-rich motifs promotes the inclusion of exons coding preferentially for uncharged amino acids, like serine and threonine that can be phosphorylated. Finally, coregulated exons encoding amino acids with similar physicochemical properties correspond to specific protein features. In conclusion, the regulation of an exon by a splicing factor that relies on the affinity of this factor for specific nucleotide(s) is tightly interconnected with the exon-encoded physicochemical properties. We therefore uncover an unanticipated bidirectional interplay between the splicing regulatory process and its biological functional outcome.

Prospective Whole-Genome Sequencing in Tuberculosis Outbreak Investigation, France, 2017-2018.

During June 2017-April 2018, active tuberculosis with Beijing SIT1 isolates was diagnosed in 14 persons living in 4 distant cities in France. Whole-genome sequencing indicated that these patients belonged to a single transmission chain. Whole-genome sequencing-based laboratory investigations enabled prompt tracing of linked cases to improve tuberculosis control.

Efficacy of live attenuated porcine reproductive and respiratory syndrome virus 2 strains to protect pigs from challenge with a heterologous Vietnamese PRRSV 2 field strain.

BACKGROUND: Effective vaccines against porcine reproductive and respiratory syndrome virus (PRRSV), especially against highly pathogenic (HP) PRRSV are still missing. The objective of this study was to evaluate the protective efficacy of an experimental live attenuated PRRSV 2 vaccine, composed of two strains, against heterologous challenge with a Vietnamese HP PRRSV 2 field strain. For this reason, 20 PRRSV negative piglets were divided into two groups. The pigs of group 1 were vaccinated with the experimental vaccine, group 2 remained unvaccinated. All study piglets received an intranasal challenge of the HP PRRSV 2 on day 0 of the study (42 days after vaccination). Blood samples were taken on days 7 and 21 after vaccination and on several days after challenge. On day 28 after challenge, all piglets were euthanized and pathologically examined. RESULTS: On days 7 and 21 after vaccination, a PRRSV 2 viraemia was seen in all piglets of group 1 which remained detectable in seven piglets up to 42 days after vaccination. On day 3 after challenge, all piglets from both groups were positive in PRRSV 2 RT-qPCR. From day 7 onwards, viral load and number of PRRSV 2 positive pigs were lower in group 1 than in group 2. All pigs of group 1 seroconverted after PRRSV 2 vaccination. PRRSV antibodies were detected in serum of all study pigs from both groups from day 14 after challenge onwards. In group 2, moderate respiratory symptoms with occasional coughing were seen following the challenge with HP PRRSV 2. Pigs of group 1 remained clinically unaffected. Interstitial pneumonia was found in four piglets of group 1 and in all ten piglets of group 2. Histopathological findings were more severe in group 2. CONCLUSIONS: It was thus concluded that the used PRRSV 2 live experimental vaccine provided protection from clinical disease and marked reduction of histopathological findings and viral load in pigs challenged with a Vietnamese HP PRRSV 2 field strain.

Complementarity of assembly-first and mapping-first approaches for alternative splicing annotation and differential analysis from RNAseq data.

Genome-wide analyses estimate that more than 90% of multi exonic human genes produce at least two transcripts through alternative splicing (AS). Various bioinformatics methods are available to analyze AS from RNAseq data. Most methods start by mapping the reads to an annotated reference genome, but some start by a de novo assembly of the reads. In this paper, we present a systematic comparison of a mapping-first approach (FARLINE) and an assembly-first approach (KISSPLICE). We applied these methods to two independent RNAseq datasets and found that the predictions of the two pipelines overlapped (70% of exon skipping events were common), but with noticeable differences. The assembly-first approach allowed to find more novel variants, including novel unannotated exons and splice sites. It also predicted AS in recently duplicated genes. The mapping-first approach allowed to find more lowly expressed splicing variants, and splice variants overlapping repeats. This work demonstrates that annotating AS with a single approach leads to missing out a large number of candidates, many of which are differentially regulated across conditions and can be validated experimentally. We therefore advocate for the combined use of both mapping-first and assembly-first approaches for the annotation and differential analysis of AS from RNAseq datasets.

Complete Genome Sequence of a Recombinant Porcine Reproductive and Respiratory Syndrome Virus Strain from Two Genotype 1 Modified Live Virus Vaccine Strains.

This paper provides information on the complete genome sequence of a porcine reproductive and respiratory syndrome virus (PRRSV) strain isolated on a French pig farm which was identified as a recombinant strain from two commercial modified live virus vaccine strains of genotype 1 (VP-046BIS and DV strains).

Ultradeep pyrosequencing of NS3 to predict response to triple therapy with protease inhibitors in previously treated chronic hepatitis C patients.

Despite the gain in sustained virological responses (SVR) provided by protease inhibitors (PIs), failures still occur. The aim of this study was to determine if a baseline analysis of the NS3 region using ultradeep pyrosequencing (UDPS) can help to predict an SVR. Serum samples from 40 patients with previously nonresponding genotype 1 chronic hepatitis C who were retreated with triple therapy, including a PI, were analyzed. Baseline UDPS of the NS3 gene was performed on plasma and peripheral blood mononuclear cells (PBMC). Mutations conferring resistance to PIs were sought. The overall diversity of the quasispecies was evaluated by calculating the Shannon entropy (SE). Resistance mutations were found in plasma and PBMC but were not discriminating enough to predict an SVR. NS3 quasispecies heterogeneity was significantly lower at baseline in patients achieving an SVR than in those not achieving an SVR (SE of 26.98 ± 16.64 × 10(-3) versus 44.93 ± 19.58 × 10(-3), P = 0.0047). With multivariate analysis, the independent predictors of an SVR were fibrosis of stage F ≤2 (odds ratio [OR], 13.3; 95% confidence interval [CI], 1.25 to 141.096; P < 0.03) and SE below the median (OR, 5.4; 95% CI, 1.22 to 23.87; P < 0.03). More than the presence of minor mutations at the baseline in plasma or in PBMC, the NS3 viral heterogeneity determined by UDPS is an independent factor for an SVR in previously treated patients receiving triple therapy that includes a PI.

Characterization of the genome of the dairy Lactobacillus helveticus bacteriophage {Phi}AQ113.

The complete genomic sequence of the dairy Lactobacillus helveticus bacteriophage ΦAQ113 was determined. Phage ΦAQ113 is a Myoviridae bacteriophage with an isometric capsid and a contractile tail. The final assembled consensus sequence revealed a linear, circularly permuted, double-stranded DNA genome with a size of 36,566 bp and a G+C content of 37%. Fifty-six open reading frames (ORFs) were predicted, and a putative function was assigned to approximately 90% of them. The ΦAQ113 genome shows functionally related genes clustered together in a genome structure composed of modules for DNA replication/regulation, DNA packaging, head and tail morphogenesis, cell lysis, and lysogeny. The identification of genes involved in the establishment of lysogeny indicates that it may have originated as a temperate phage, even if it was isolated from natural cheese whey starters as a virulent phage, because it is able to propagate in a sensitive host strain. Additionally, we discovered that the ΦAQ113 phage genome is closely related to Lactobacillus gasseri phage KC5a and Lactobacillus johnsonii phage Lj771 genomes. The phylogenetic similarities between L. helveticus phage ΦAQ113 and two phages that belong to gut species confirm a possible common ancestral origin and support the increasing consideration of L. helveticus as a health-promoting organism.

Broad neutralization of wild-type dengue virus isolates following immunization in monkeys with a tetravalent dengue vaccine based on chimeric yellow fever 17D/dengue viruses.

The objective of the study was to evaluate if the antibodies elicited after immunization with a tetravalent dengue vaccine, based on chimeric yellow fever 17D/dengue viruses, can neutralize a large range of dengue viruses (DENV). A panel of 82 DENVs was developed from viruses collected primarily during the last decade in 30 countries and included the four serotypes and the majority of existing genotypes. Viruses were isolated and minimally amplified before evaluation against a tetravalent polyclonal serum generated during vaccine preclinical evaluation in monkey, a model in which protection efficacy of this vaccine has been previously demonstrated (Guirakhoo et al., 2004). Neutralization was observed across all the DENV serotypes, genotypes, geographical origins and isolation years. These data indicate that antibodies elicited after immunization with this dengue vaccine candidate should widely protect against infection with contemporary DENV lineages circulating in endemic countries.

Stress-induced evolution of Escherichia coli points to original concepts in respiratory cofactor selectivity.

Bacterial metabolism is characterized by a remarkable capacity to rapidly adapt to environmental changes. We restructured the central metabolic network in Escherichia coli to force a higher production of NADPH, and then grew this strain in conditions favoring adaptive evolution. A six-fold increase in growth capacity was attained that could be attributed in multiple clones, after whole genome mutation mapping, to a specific single mutation. Each clone had an evolved NuoF*(E183A) enzyme in the respiratory complex I that can now oxidize both NADH and NADPH. When a further strain was constructed with an even higher degree of NADPH stress such that growth was impossible on glucose mineral medium, a solid-state screening for mutations restoring growth, led to two different types of NuoF mutations in strains having recovered growth capacity. In addition to the previously seen E183A mutation other clones showed a E183G mutation, both having NADH and NADPH oxidizing ability. These results demonstrate the unique solution used by E. coli to overcome the NADPH stress problem. This solution creates a new function for NADPH that is no longer restricted to anabolic synthesis reactions but can now be also used to directly produce catabolic energy.

Basis of recognition between the NarJ chaperone and the N-terminus of the NarG subunit from Escherichia coli nitrate reductase.

A novel class of molecular chaperones co-ordinates the assembly and targeting of complex metalloproteins by binding to an amino-terminal peptide of the cognate substrate. We have previously shown that the NarJ chaperone interacts with the N-terminus of the NarG subunit coming from the nitrate reductase complex, NarGHI. In the present study, NMR structural analysis revealed that the NarG(1-15) peptide adopts an alpha-helical conformation in solution. Moreover, NarJ recognizes and binds the helical NarG(1-15) peptide mostly via hydrophobic interactions as deduced from isothermal titration calorimetry analysis. NMR and differential scanning calorimetry analysis revealed a modification of NarJ conformation during complex formation with the NarG(1-15) peptide. Isothermal titration calorimetry and BIAcore experiments support a model whereby the protonated state of the chaperone controls the time dependence of peptide interaction.

Crystal structure of pyridoxal kinase from the Escherichia coli pdxK gene: implications for the classification of pyridoxal kinases.

The pdxK and pdxY genes have been found to code for pyridoxal kinases, enzymes involved in the pyridoxal phosphate salvage pathway. Two pyridoxal kinase structures have recently been published, including Escherichia coli pyridoxal kinase 2 (ePL kinase 2) and sheep pyridoxal kinase, products of the pdxY and pdxK genes, respectively. We now report the crystal structure of E. coli pyridoxal kinase 1 (ePL kinase 1), encoded by a pdxK gene, and an isoform of ePL kinase 2. The structures were determined in the unliganded and binary complexes with either MgATP or pyridoxal to 2.1-, 2.6-, and 3.2-A resolutions, respectively. The active site of ePL kinase 1 does not show significant conformational change upon binding of either pyridoxal or MgATP. Like sheep PL kinase, ePL kinase 1 exhibits a sequential random mechanism. Unlike sheep pyridoxal kinase, ePL kinase 1 may not tolerate wide variation in the size and chemical nature of the 4' substituent on the substrate. This is the result of differences in a key residue at position 59 on a loop (loop II) that partially forms the active site. Residue 59, which is His in ePL kinase 1, interacts with the formyl group at C-4' of pyridoxal and may also determine if residues from another loop (loop I) can fill the active site in the absence of the substrate. Both loop I and loop II are suggested to play significant roles in the functions of PL kinases.

CaspR: a web server for automated molecular replacement using homology modelling.

Molecular replacement (MR) is the method of choice for X-ray crystallography structure determination when structural homologues are available in the Protein Data Bank (PDB). Although the success rate of MR decreases sharply when the sequence similarity between template and target proteins drops below 35% identical residues, it has been found that screening for MR solutions with a large number of different homology models may still produce a suitable solution where the original template failed. Here we present the web tool CaspR, implementing such a strategy in an automated manner. On input of experimental diffraction data, of the corresponding target sequence and of one or several potential templates, CaspR executes an optimized molecular replacement procedure using a combination of well-established stand-alone software tools. The protocol of model building and screening begins with the generation of multiple structure-sequence alignments produced with T-COFFEE, followed by homology model building using MODELLER, molecular replacement with AMoRe and model refinement based on CNS. As a result, CaspR provides a progress report in the form of hierarchically organized summary sheets that describe the different stages of the computation with an increasing level of detail. For the 10 highest-scoring potential solutions, pre-refined structures are made available for download in PDB format. Results already obtained with CaspR and reported on the web server suggest that such a strategy significantly increases the fraction of protein structures which may be solved by MR. Moreover, even in situations where standard MR yields a solution, pre-refined homology models produced by CaspR significantly reduce the time-consuming refinement process. We expect this automated procedure to have a significant impact on the throughput of large-scale structural genomics projects. CaspR is freely available at http://igs-server.cnrs-mrs.fr/Caspr/.

Structural genomics of highly conserved microbial genes of unknown function in search of new antibacterial targets.

With more than 100 antibacterial drugs at our disposal in the 1980's, the problem of bacterial infection was considered solved. Today, however, most hospital infections are insensitive to several classes of antibacterial drugs, and deadly strains of Staphylococcus aureus resistant to vancomycin--the last resort antibiotic--have recently begin to appear. Other life-threatening microbes, such as Enterococcus faecalis and Mycobacterium tuberculosis are already able to resist every available antibiotic. There is thus an urgent, and continuous need for new, preferably large-spectrum, antibacterial molecules, ideally targeting new biochemical pathways. Here we report on the progress of our structural genomics program aiming at the discovery of new antibacterial gene targets among evolutionary conserved genes of uncharacterized function. A series of bioinformatic and comparative genomics analyses were used to identify a set of 221 candidate genes common to Gram-positive and Gram-negative bacteria. These genes were split between two laboratories. They are now submitted to a systematic 3-D structure determination protocol including cloning, protein expression and purification, crystallization, X-ray diffraction, structure interpretation, and function prediction. We describe here our strategies for the 111 genes processed in our laboratory. Bioinformatics is used at most stages of the production process and out of 111 genes processed--and 17 months into the project--108 have been successfully cloned, 103 have exhibited detectable expression, 84 have led to the production of soluble protein, 46 have been purified, 12 have led to usable crystals, and 7 structures have been determined.