Occurrence and diversity of stem nodulation in Aeschynomene and Sesbania legumes from wetlands of Madagascar.

Legumes have the ability to establish a nitrogen-fixing symbiosis with soil rhizobia that they house in specific organs, the nodules. In most rhizobium-legume interactions, nodulation occurs on the root. However, certain tropical legumes growing in wetlands possess a unique trait: the capacity to form rhizobia-harbouring nodules on the stem. Despite the originality of the stem nodulation process, its occurrence and diversity in waterlogging-tolerant legumes remains underexplored, impeding a comprehensive analysis of its genetics and biology. Here, we aimed at filling this gap by surveying stem nodulation in legume species-rich wetlands of Madagascar. Stem nodulation was readily observed in eight hydrophytic species of the legume genera, Aeschynomene and Sesbania, for which significant variations in stem nodule density and morphology was documented. Among these species, A. evenia, which is used as genetic model to study the rhizobial symbiosis, was found to be frequently stem-nodulated. Two other Aeschynomene species, A. cristata and A. uniflora, were evidenced to display a profuse stem-nodulation as occurs in S. rostrata. These findings extend our knowledge on legumes species that are endowed with stem nodulation and further indicate that A. evenia, A. cristata, A. uniflora and S. rostrata are of special interest for the study of stem nodulation. As such, these legume species represent opportunities to investigate different modalities of the nitrogen-fixing symbiosis and this knowledge could provide cues for the engineering of nitrogen-fixation in non-legume crops.

Differential early response of monocyte/macrophage subsets to intra-operative corticosteroid administration in lung transplantation.

Introduction: Lung transplantation often results in primary and/or chronic dysfunctions that are related to early perioperative innate allo-responses where myeloid subsets play a major role. Corticosteroids are administered upon surgery as a standard-of-care but their action on the different myeloid cell subsets in that context is not known. Methods: To address this issue, we used a cross-circulatory platform perfusing an extracorporeal lung coupled to cell mapping in the pig model, that enabled us to study the recruited cells in the allogeneic lung over 10 hours. Results: Myeloid cells, i.e. granulocytes and monocytic cells including classical CD14pos and non-classical/intermediate CD16pos cells, were the dominantly recruited subsets, with the latter upregulating the membrane expression of MHC class II and CD80/86 molecules. Whereas corticosteroids did not reduce the different cell subset recruitment, they potently dampened the MHC class II and CD80/86 expression on monocytic cells and not on alveolar macrophages. Besides, corticosteroids induced a temporary and partial anti-inflammatory gene profile depending on cytokines and monocyte/macrophage subsets. Discussion: This work documents the baseline effects of the standard-of-care corticosteroid treatment for early innate allo-responses. These insights will enable further optimization and improvement of lung transplantation outcomes.

The Medicago truncatula nodule-specific cysteine-rich peptides, NCR343 and NCR-new35 are required for the maintenance of rhizobia in nitrogen-fixing nodules.

In the nodules of IRLC legumes, including Medicago truncatula, nitrogen-fixing rhizobia undergo terminal differentiation resulting in elongated and endoreduplicated bacteroids specialized for nitrogen fixation. This irreversible transition of rhizobia is mediated by host produced nodule-specific cysteine-rich (NCR) peptides, of which c. 700 are encoded in the M. truncatula genome but only few of them have been proved to be essential for nitrogen fixation. We carried out the characterization of the nodulation phenotype of three ineffective nitrogen-fixing M. truncatula mutants using confocal and electron microscopy, monitored the expression of defence and senescence-related marker genes, and analysed the bacteroid differentiation with flow cytometry. Genetic mapping combined with microarray- or transcriptome-based cloning was used to identify the impaired genes. Mtsym19 and Mtsym20 mutants are defective in the same peptide NCR-new35 and the lack of NCR343 is responsible for the ineffective symbiosis of NF-FN9363. We found that the expression of NCR-new35 is significantly lower and limited to the transition zone of the nodule compared with other crucial NCRs. The fluorescent protein-tagged version of NCR343 and NCR-new35 localized to the symbiotic compartment. Our discovery added two additional members to the group of NCR genes essential for nitrogen-fixing symbiosis in M. truncatula.

Proton exchange by the vacuolar nitrate transporter CLCa is required for plant growth and nitrogen use efficiency.

Nitrate is a major nutrient and osmoticum for plants. To deal with fluctuating nitrate availability in soils, plants store this nutrient in their vacuoles. Chloride channel a (CLCa), a 2NO3-/1H+ exchanger localized to the vacuole in Arabidopsis (Arabidopsis thaliana), ensures this storage process. CLCa belongs to the CLC family, which includes anion/proton exchangers and anion channels. A mutation in a glutamate residue conserved across CLC exchangers is likely responsible for the conversion of exchangers to channels. Here, we show that CLCa with a mutation in glutamate 203 (E203) behaves as an anion channel in its native membrane. We introduced the CLCaE203A point mutation to investigate its physiological importance into the Arabidopsis clca knockout mutant. These CLCaE203A mutants displayed a growth deficit linked to the disruption of water homeostasis. Additionally, CLCaE203A expression failed to complement the defect in nitrate accumulation of clca and favored higher N-assimilation at the vegetative stage. Further analyses at the post-flowering stages indicated that CLCaE203A expression results in an increase in N uptake allocation to seeds, leading to a higher nitrogen use efficiency compared to the wild-type. Altogether, these results point to the critical function of the CLCa exchanger on the vacuole for plant metabolism and development.

Developmental timing of programmed DNA elimination in Paramecium tetraurelia recapitulates germline transposon evolutionary dynamics.

With its nuclear dualism, the ciliate Paramecium constitutes a unique model to study how host genomes cope with transposable elements (TEs). P. tetraurelia harbors two germline micronuclei (MICs) and a polyploid somatic macronucleus (MAC) that develops from one MIC at each sexual cycle. Throughout evolution, the MIC genome has been continuously colonized by TEs and related sequences that are removed from the somatic genome during MAC development. Whereas TE elimination is generally imprecise, excision of approximately 45,000 TE-derived internal eliminated sequences (IESs) is precise, allowing for functional gene assembly. Programmed DNA elimination is concomitant with genome amplification. It is guided by noncoding RNAs and repressive chromatin marks. A subset of IESs is excised independently of this epigenetic control, raising the question of how IESs are targeted for elimination. To gain insight into the determinants of IES excision, we established the developmental timing of DNA elimination genome-wide by combining fluorescence-assisted nuclear sorting with high-throughput sequencing. Essentially all IESs are excised within only one endoreplication round (32C to 64C), whereas TEs are eliminated at a later stage. We show that DNA elimination proceeds independently of replication. We defined four IES classes according to excision timing. The earliest excised IESs tend to be independent of epigenetic factors, display strong sequence signals at their ends, and originate from the most ancient integration events. We conclude that old IESs have been optimized during evolution for early and accurate excision by acquiring stronger sequence determinants and escaping epigenetic control.

Molecular and Cellular Analysis of the Repair of Zebrafish Optic Tectum Meninges Following Laser Injury.

We studied cell recruitment following optic tectum (OT) injury in zebrafish (Danio rerio), which has a remarkable ability to regenerate many of its organs, including the brain. The OT is the largest dorsal layered structure in the zebrafish brain. In juveniles, it is an ideal structure for imaging and dissection. We investigated the recruited cells within the juvenile OT during regeneration in a Pdgfrß-Gal4:UAS-EGFP line in which pericytes, vascular, circulating, and meningeal cells are labeled, together with neurons and progenitors. We first performed high-resolution confocal microscopy and single-cell RNA-sequencing (scRNAseq) on EGFP-positive cells. We then tested three types of injury with very different outcomes (needle (mean depth in the OT of 200 µm); deep-laser (depth: 100 to 200 µm depth); surface-laser (depth: 0 to 100 µm)). Laser had the additional advantage of better mimicking of ischemic cerebral accidents. No massive recruitment of EGFP-positive cells was observed following laser injury deep in the OT. This type of injury does not perturb the meninx/brain-blood barrier (BBB). We also performed laser injuries at the surface of the OT, which in contrast create a breach in the meninges. Surprisingly, one day after such injury, we observed the migration to the injury site of various EGFP-positive cell types at the surface of the OT. The migrating cells included midline roof cells, which activated the PI3K-AKT pathway; fibroblast-like cells expressing numerous collagen genes and most prominently in 3D imaging; and a large number of arachnoid cells that probably migrate to the injury site through the activation of cilia motility genes, most likely being direct targets of the FOXJ1a gene. This study, combining high-content imaging and scRNAseq in physiological and pathological conditions, sheds light on meninges repair mechanisms in zebrafish that probably also operate in mammalian meninges.

A chromosome-level, haplotype-phased Vanilla planifolia genome highlights the challenge of partial endoreplication for accurate whole-genome assembly.

Vanilla planifolia, the species cultivated to produce one of the world's most popular flavors, is highly prone to partial genome endoreplication, which leads to highly unbalanced DNA content in cells. We report here the first molecular evidence of partial endoreplication at the chromosome scale by the assembly and annotation of an accurate haplotype-phased genome of V. planifolia. Cytogenetic data demonstrated that the diploid genome size is 4.09 Gb, with 16 chromosome pairs, although aneuploid cells are frequently observed. Using PacBio HiFi and optical mapping, we assembled and phased a diploid genome of 3.4 Gb with a scaffold N50 of 1.2 Mb and 59 128 predicted protein-coding genes. The atypical k-mer frequencies and the uneven sequencing depth observed agreed with our expectation of unbalanced genome representation. Sixty-seven percent of the genes were scattered over only 30% of the genome, putatively linking gene-rich regions and the endoreplication phenomenon. By contrast, low-coverage regions (non-endoreplicated) were rich in repeated elements but also contained 33% of the annotated genes. Furthermore, this assembly showed distinct haplotype-specific sequencing depth variation patterns, suggesting complex molecular regulation of endoreplication along the chromosomes. This high-quality, anchored assembly represents 83% of the estimated V. planifolia genome. It provides a significant step toward the elucidation of this complex genome. To support post-genomics efforts, we developed the Vanilla Genome Hub, a user-friendly integrated web portal that enables centralized access to high-throughput genomic and other omics data and interoperable use of bioinformatics tools.

Genome Size, Cytotype Diversity and Reproductive Mode Variation of Cotoneaster integerrimus (Rosaceae) from the Balkans.

Cotoneaster integerrimus represents a multiploid and facultative apomictic system of widely distributed mountain populations. We used flow cytometry to determine genome size, ploidy level, and reproduction mode variation of the Balkan populations, supplemented by analysis of nuclear microsatellites in order to address: (i) geographic distribution and variation of cytotypes among the populations; (ii) variation of reproduction mode and the frequency of sexuality; (iii) pathways of endosperm formation among the sampled polyploids and their endosperm balance requirements; (iv) genotypic diversity and geographic distribution of clonal lineages of polyploids. The prevalence of apomictic tetraploid cytotype followed by sexual diploids and extremely rare triploids was demonstrated. This prevalence of tetraploids affected the populations' structure composed from clonal genotypes with varying proportions. The co-occurrence of diploids and tetraploids generated higher cytotype, reproductive mode, and genotypic diversity, but mixed-ploidy sites were extremely rare. The endosperm imbalance facilitates the development and the occurrence of intermediate triploids in mixed-ploidy populations, but also different tetraploid lineages elsewhere with unbalanced endosperm. All these results showed that the South European populations of C. integerrimus have higher levels of cytotype and reproductive diversity compared to the Central European ones. Therefore, the South European populations can be considered as a potential reservoir of regional and global diversity for this species.

Sinorhizobium meliloti Functions Required for Resistance to Antimicrobial NCR Peptides and Bacteroid Differentiation.

Legumes of the Medicago genus have a symbiotic relationship with the bacterium Sinorhizobium meliloti and develop root nodules housing large numbers of intracellular symbionts. Members of the nodule-specific cysteine-rich peptide (NCR) family induce the endosymbionts into a terminal differentiated state. Individual cationic NCRs are antimicrobial peptides that have the capacity to kill the symbiont, but the nodule cell environment prevents killing. Moreover, the bacterial broad-specificity peptide uptake transporter BacA and exopolysaccharides contribute to protect the endosymbionts against the toxic activity of NCRs. Here, we show that other S. meliloti functions participate in the protection of the endosymbionts; these include an additional broad-specificity peptide uptake transporter encoded by the yejABEF genes and lipopolysaccharide modifications mediated by lpsB and lpxXL, as well as rpoH1, encoding a stress sigma factor. Strains with mutations in these genes show a strain-specific increased sensitivity profile against a panel of NCRs and form nodules in which bacteroid differentiation is affected. The lpsB mutant nodule bacteria do not differentiate, the lpxXL and rpoH1 mutants form some seemingly fully differentiated bacteroids, although most of the nodule bacteria are undifferentiated, while the yejABEF mutants form hypertrophied but nitrogen-fixing bacteroids. The nodule bacteria of all the mutants have a strongly enhanced membrane permeability, which is dependent on the transport of NCRs to the endosymbionts. Our results suggest that S. meliloti relies on a suite of functions, including peptide transporters, the bacterial envelope structures, and stress response regulators, to resist the aggressive assault of NCR peptides in the nodule cells. IMPORTANCE The nitrogen-fixing symbiosis of legumes with rhizobium bacteria has a predominant ecological role in the nitrogen cycle and has the potential to provide the nitrogen required for plant growth in agriculture. The host plants allow the rhizobia to colonize specific symbiotic organs, the nodules, in large numbers in order to produce sufficient reduced nitrogen for the plants' needs. Some legumes, including Medicago spp., produce massively antimicrobial peptides to keep this large bacterial population in check. These peptides, known as NCRs, have the potential to kill the rhizobia, but in nodules, they rather inhibit the division of the bacteria, which maintain a high nitrogen-fixing activity. In this study, we show that the tempering of the antimicrobial activity of the NCR peptides in the Medicago symbiont Sinorhizobium meliloti is multifactorial and requires the YejABEF peptide transporter, the lipopolysaccharide outer membrane, and the stress response regulator RpoH1.

Bradyrhizobium diazoefficiens USDA110 Nodulation of Aeschynomene afraspera Is Associated with Atypical Terminal Bacteroid Differentiation and Suboptimal Symbiotic Efficiency.

Legume plants can form root organs called nodules where they house intracellular symbiotic rhizobium bacteria. Within nodule cells, rhizobia differentiate into bacteroids, which fix nitrogen for the benefit of the plant. Depending on the combination of host plants and rhizobial strains, the output of rhizobium-legume interactions varies from nonfixing associations to symbioses that are highly beneficial for the plant. Bradyrhizobium diazoefficiens USDA110 was isolated as a soybean symbiont, but it can also establish a functional symbiotic interaction with Aeschynomene afraspera In contrast to soybean, A. afraspera triggers terminal bacteroid differentiation, a process involving bacterial cell elongation, polyploidy, and increased membrane permeability, leading to a loss of bacterial viability while plants increase their symbiotic benefit. A combination of plant metabolomics, bacterial proteomics, and transcriptomics along with cytological analyses were used to study the physiology of USDA110 bacteroids in these two host plants. We show that USDA110 establishes a poorly efficient symbiosis with A. afraspera despite the full activation of the bacterial symbiotic program. We found molecular signatures of high levels of stress in A. afraspera bacteroids, whereas those of terminal bacteroid differentiation were only partially activated. Finally, we show that in A. afraspera, USDA110 bacteroids undergo atypical terminal differentiation hallmarked by the disconnection of the canonical features of this process. This study pinpoints how a rhizobium strain can adapt its physiology to a new host and cope with terminal differentiation when it did not coevolve with such a host.IMPORTANCE Legume-rhizobium symbiosis is a major ecological process in the nitrogen cycle, responsible for the main input of fixed nitrogen into the biosphere. The efficiency of this symbiosis relies on the coevolution of the partners. Some, but not all, legume plants optimize their return on investment in the symbiosis by imposing on their microsymbionts a terminal differentiation program that increases their symbiotic efficiency but imposes a high level of stress and drastically reduces their viability. We combined multi-omics with physiological analyses to show that the symbiotic couple formed by Bradyrhizobium diazoefficiens USDA110 and Aeschynomene afraspera, in which the host and symbiont did not evolve together, is functional but displays a low symbiotic efficiency associated with a disconnection of terminal bacteroid differentiation features.

Genetics of nodulation in Aeschynomene evenia uncovers mechanisms of the rhizobium-legume symbiosis.

Among legumes (Fabaceae) capable of nitrogen-fixing nodulation, several Aeschynomene spp. use a unique symbiotic process that is independent of Nod factors and infection threads. They are also distinctive in developing root and stem nodules with photosynthetic bradyrhizobia. Despite the significance of these symbiotic features, their understanding remains limited. To overcome such limitations, we conduct genetic studies of nodulation in Aeschynomene evenia, supported by the development of a genome sequence for A. evenia and transcriptomic resources for 10 additional Aeschynomene spp. Comparative analysis of symbiotic genes substantiates singular mechanisms in the early and late nodulation steps. A forward genetic screen also shows that AeCRK, coding a receptor-like kinase, and the symbiotic signaling genes AePOLLUX, AeCCamK, AeCYCLOPS, AeNSP2, and AeNIN are required to trigger both root and stem nodulation. This work demonstrates the utility of the A. evenia model and provides a cornerstone to unravel mechanisms underlying the rhizobium-legume symbiosis.

Distinctive Cellular and Metabolic Reprogramming in Porcine Lung Mononuclear Phagocytes Infected With Type 1 PRRSV Strains.

Porcine reproductive and respiratory syndrome (PRRS) has an extensive impact on pig production. The causative virus (PRRSV) is divided into two species, PRRSV-1 (European origin) and PRRSV-2 (North American origin). Within PRRSV-1, PRRSV-1.3 strains, such as Lena, are more pathogenic than PRRSV-1.1 strains, such as Flanders 13 (FL13). To date, the molecular interactions of PRRSV with primary lung mononuclear phagocyte (MNP) subtypes, including conventional dendritic cells types 1 (cDC1) and 2 (cDC2), monocyte-derived DCs (moDC), and pulmonary intravascular macrophages (PIM), have not been thoroughly investigated. Here, we analyze the transcriptome profiles of in vivo FL13-infected parenchymal MNP subpopulations and of in vitro FL13- and Lena-infected parenchymal MNP. The cell-specific expression profiles of in vivo sorted cells correlated with their murine counterparts (AM, cDC1, cDC2, moDC) with the exception of PIM. Both in vivo and in vitro, FL13 infection altered the expression of a low number of host genes, and in vitro infection with Lena confirmed the higher ability of this strain to modulate host response. Machine learning (ML) and gene set enrichment analysis (GSEA) unraveled additional relevant genes and pathways modulated by FL13 infection that were not identified by conventional analyses. GSEA increased the cellular pathways enriched in the FL13 data set, but ML allowed a more complete comprehension of functional profiles during FL13 in vitro infection. Data indicates that cellular reprogramming differs upon Lena and FL13 infection and that the latter might keep antiviral and inflammatory macrophage/DC functions silent. Although the slow replication kinetics of FL13 likely contribute to differences in cellular gene expression, the data suggest distinct mechanisms of interaction of the two viruses with the innate immune system during early infection.

Fluorescent Aminoglycoside Antibiotics and Methods for Accurately Monitoring Uptake by Bacteria.

Characterizing how multidrug-resistant bacteria circumvent the action of clinically used or novel antibiotics requires a detailed understanding of how the antibiotics interact with and cross bacterial membranes to accumulate in the cells and exert their action. When monitoring the interactions of drugs with bacteria, it remains challenging to differentiate functionally relevant internalized drug levels from nonspecific binding. Fluorescence is a method of choice for observing dynamics of biomolecules. In order to facilitate studies involving aminoglycoside antibiotics, we have generated fluorescently labeled aminoglycoside derivatives with uptake and bactericidal activities similar, albeit with a moderate loss, to those of the parent drug. The method combines fluorescence microscopy with fluorescence-activated cell sorting (FACS) using neomycin coupled to nonpermeable cyanine dyes. Fluorescence imaging allowed membrane-bound antibiotic to be distinguished from molecules in the cytoplasm. Patterns of uptake were assigned to different populations in the FACS analysis. Our study illustrates how fluorescent derivatives of an aminoglycoside enable a robust characterization of the three components of uptake: membrane binding, EDPI, and EDPII. Because EDPI levels are weak compared to the two other types of accumulation and critical for the action of these drugs, the three components of uptake must be taken into account separately when drawing conclusions about aminoglycoside function.

Human Peripheral Blood Eosinophils Express High Levels of the Purinergic Receptor P2X4.

Extracellular nucleotides are important mediators of cell activation and trigger multiple responses via membrane receptors known as purinergic receptors (P2). P2X receptors are ligand-gated ion channels, activated by extracellular ATP. P2X4 is one of the most sensitive purinergic receptors, that is typically expressed by neurons, microglia, and some epithelial and endothelial cells. P2X4 mediates neuropathic pain via brain-derived neurotrophic factor and is also involved in inflammation in response to high ATP release. It is therefore involved in multiple inflammatory pathologies as well as neurodegenerative diseases. We have produced monoclonal antibodies (mAb) directed against this important human P2X4 receptor. Focusing on two mAbs, we showed that they also recognize mouse and rat P2X4. We demonstrated that these mAbs can be used in flow cytometry, immunoprecipitation, and immunohistochemistry, but not in Western blot assays, indicating that they target conformational epitopes. We also characterized the expression of P2X4 receptor on mouse and human peripheral blood lymphocytes (PBL). We showed that P2X4 is expressed at the surface of several leukocyte cell types, with the highest expression level on eosinophils, making them potentially sensitive to adenosine triphosphate (ATP). P2X4 is expressed by leucocytes, in human and mouse, with a significant gender difference, males having higher surface expression levels than females. Our findings reveal that PBL express significant levels of P2X4 receptor, and suggest an important role of this receptor in leukocyte activation by ATP, particularly in P2X4high expressing eosinophils.

Macrophage-B Cell Interactions in the Inverted Porcine Lymph Node and Their Response to Porcine Reproductive and Respiratory Syndrome Virus.

Swine lymph nodes (LN) present an inverted structure compared to mouse and human, with the afferent lymph diffusing from the center to the periphery. This structure, also observed in close and distant species such as dolphins, hippopotamus, rhinoceros, and elephants, is poorly described, nor are the LN macrophage populations and their relationship with B cell follicles. B cell maturation occurs mainly in LN B cell follicles with the help of LN macrophage populations endowed with different antigen delivery capacities. We identified three macrophage populations that we localized in the inverted LN spatial organization. This allowed us to ascribe porcine LN MΦ to their murine counterparts: subcapsular sinus MΦ, medullary cord MΦ and medullary sinus MΦ. We identified the different intra and extrafollicular stages of LN B cells maturation and explored the interaction of MΦ, drained antigen and follicular B cells. The porcine reproductive and respiratory syndrome virus (PRRSV) is a major porcine pathogen that infects tissue macrophages (MΦ). PRRSV is persistent in the secondary lymphoid tissues and induces a delay in neutralizing antibodies appearance. We observed PRRSV interaction with two LN MΦ populations, of which one interacts closely with centroblasts. We observed BCL6 up-regulation in centroblast upon PRRSV infection, leading to new hypothesis on PRRSV inhibition of B cell maturation. This seminal study of porcine LN will permit fruitful comparison with murine and human LN for a better understanding of normal and inverted LN development and functioning.

Role of Cis, Trans, and Inbreeding Effects on Meiotic Recombination in Saccharomyces cerevisiae.

Meiotic recombination is a major driver of genome evolution by creating new genetic combinations. To probe the factors driving variability of meiotic recombination, we used a high-throughput method to measure recombination rates in hybrids between SK1 and a total of 26 Saccharomyces cerevisiae strains from different geographic origins and habitats. Fourteen intervals were monitored for each strain, covering chromosomes VI and XI entirely, and part of chromosome I. We found an average number of crossovers per chromosome ranging between 1.0 and 9.5 across strains ("domesticated" or not), which is higher than the average between 0.5 and 1.5 found in most organisms. In the different intervals analyzed, recombination showed up to ninefold variation across strains but global recombination landscapes along chromosomes varied less. We also built an incomplete diallel experiment to measure recombination rates in one region of chromosome XI in 10 different crosses involving five parental strains. Our overall results indicate that recombination rate is increasingly positively correlated with sequence similarity between homologs (i) in DNA double-strand-break-rich regions within intervals, (ii) in entire intervals, and (iii) at the whole genome scale. Therefore, these correlations cannot be explained by cis effects only. We also estimated that cis and trans effects explained 38 and 17%, respectively, of the variance of recombination rate. In addition, by using a quantitative genetics analysis, we identified an inbreeding effect that reduces recombination rate in homozygous genotypes, while other interaction effects (specific combining ability) or additive effects (general combining ability) are found to be weak. Finally, we measured significant crossover interference in some strains, and interference intensity was positively correlated with crossover number.

Porcine Reproductive and Respiratory Syndrome Virus Type 1.3 Lena Triggers Conventional Dendritic Cells 1 Activation and T Helper 1 Immune Response Without Infecting Dendritic Cells.

Porcine Reproductive and Respiratory Syndrome virus (PRRSV) is an arterivirus responsible for highly contagious infection and huge economic losses in pig industry. Two species, PRRSV-1 and PRRSV-2 are distinguished, PRRSV-1 being more prevalent in Europe. PRRSV-1 can further be divided in subtypes. PRRSV-1.3 such as Lena are more pathogenic than PRRSV-1.1 such as Lelystad or Flanders13. PRRSV-1.3 viruses trigger a higher Th1 response than PRRSV-1.1, although the role of the cellular immune response in PRRSV clearance remains ill defined. The pathogenicity as well as the T cell response inductions may be differentially impacted according to the capacity of the virus strain to infect and/or activate DCs. However, the interactions of PRRSV with in vivo-differentiated-DC subtypes such as conventional DC1 (cDC1), cDC2, and monocyte-derived DCs (moDC) have not been thoroughly investigated. Here, DC subpopulations from Lena in vivo infected pigs were analyzed for viral genome detection. This experiment demonstrates that cDC1, cDC2, and moDC are not infected in vivo by Lena. Analysis of DC cytokines production revealed that cDC1 are clearly activated in vivo by Lena. In vitro comparison of 3 Europeans strains revealed no infection of the cDC1 and cDC2 and no or little infection of moDC with Lena, whereas the two PRRSV-1.1 strains infect none of the 3 DC subtypes. In vitro investigation of T helper polarization and cytokines production demonstrate that Lena induces a higher Th1 polarization and IFNγ secretion than FL13 and LV. Altogether, this work suggests an activation of cDC1 by Lena associated with a Th1 immune response polarization.

Surface-dependent endocytosis of poly(isobutylcyanoacrylate) nanoparticles by Trichomonas vaginalis.

Previous data from our research group showed that chitosan-coated poly(isobutylcyanoacrylate) nanoparticles (NPs) (denoted PIBCA/Chito20) exhibited intrinsic anti-Trichomonas vaginalis activity, while PIBCA/pluronic® F68 without chitosan (PIBCA/F68) were inactive. However, the mechanism of anti-T. vaginalis activity of chitosan-coated PIBCA NPs is still unknown. Our hypothesis is that chitosan-coated NPs are internalized by the parasite, contrarily to PIBCA/F68. In this investigation, the impact of NP surface on their internalization by the protozoan was studied using flow cytometry and parasite morphological changes after different incubation times with PIBCA/Chito20 NPs were monitored by electron microscopy. Flow-cytometry revealed that PIBCA/Chito20 NPs were uptaken by T. vaginalis as early as 10-min-incubation. Drastic cell morphological transformations were observed from scanning electron microscopy and transmission electron microscopy after incubation with PIBCA/Chito20 NPs. Numerous pits were seen on cell membrane since 10 min. Gradual increase in contact time increased NP endocytosis and induced proportional damages to T. vaginalis membrane. Then, investigation of whether PIBCA/Chito20 NPs can improve MTZ anti-T. vaginalis activity was studied using checkerboard experiment. Calculation of fractional inhibitory concentration index (FICI = 3.53) showed an additive effect between NPs and MTZ.

High-throughput measurement of recombination rates and genetic interference in Saccharomyces cerevisiae.

Allelic recombination owing to meiotic crossovers is a major driver of genome evolution, as well as a key player for the selection of high-performing genotypes in economically important species. Therefore, we developed a high-throughput and low-cost method to measure recombination rates and crossover patterning (including interference) in large populations of the budding yeast Saccharomyces cerevisiae. Recombination and interference were analysed by flow cytometry, which allows time-consuming steps such as tetrad microdissection or spore growth to be avoided. Moreover, our method can also be used to compare recombination in wild-type vs. mutant individuals or in different environmental conditions, even if the changes in recombination rates are small. Furthermore, meiotic mutants often present recombination and/or pairing defects affecting spore viability but our method does not involve growth steps and thus avoids filtering out non-viable spores.

Transcriptome profiling of sorted endoreduplicated nuclei from tomato fruits: how the global shift in expression ascribed to DNA ploidy influences RNA-Seq data normalization and interpretation.

As part of normal development most eukaryotic organisms, ranging from insects and mammals to plants, display variations in nuclear ploidy levels resulting from somatic endopolyploidy. Endoreduplication is the major source of endopolyploidy in higher plants. Endoreduplication is a remarkable characteristic of the fleshy pericarp tissue of developing tomato fruits, where it establishes a highly integrated cellular system that acts as a morphogenetic factor supporting cell growth. However, the functional significance of endoreduplication is not fully understood. Although endoreduplication is thought to increase metabolic activity due to a global increase in transcription, the issue of gene-specific ploidy-regulated transcription remains open. To investigate the influence of endoreduplication on transcription in tomato fruit, we tested the feasibility of a RNA sequencing (RNA-Seq) approach using total nuclear RNA extracted from purified populations of flow cytometry-sorted nuclei based on their DNA content. Here we show that cell-based approaches to the study of RNA-Seq profiles need to take into account the putative global shift in expression between samples for correct analysis and interpretation of the data. From ploidy-specific expression profiles we found that the activity of cells inside the pericarp is related both to the ploidy level and their tissue location.