Short-term soil amendment by sewage sludge anaerobic digestate in a tomato monoculture suppresses Fusarium vascular wilt disease by changing the taxonomic characteristics of soil microbiota

Digestate incorporation into topsoil determines soil fertility improvement by changing composition and structure of soil microbiota. However, how microbiota responds to short-term soil amendment by sewage sludge anaerobic digestate (SSD) for suppressing Fusarium vascular wilt disease is unknown. This study compares the effects of three SSD-based treatments to suppress Fusarium oxysporum f. sp. lycopersici (Fol) in a long-term cherry tomato monoculture under field condition in a 3-year trial. Three sampling sites with two application times (two bulk soils at 3–12 weeks after amendment and one tomato rhizosphere soil at 12 weeks post-amendment) were chosen. Three digestate typologies (liquid, centrifuged, and dried) having physicochemical features and heavy metals content below the legal limits were tested at 50 l, 3.5 kg and 2.5 kg m−2y−1 dose, respectively. Fusarium wilt disease was measured for three consecutive years by severity index and Fol abundance in tomato vascular tissue was assessed by ITS rDNA gene sequencing. Fol abundance and taxonomic structure of Fusarium community in the rhizosphere were determined at specie level at the end of the trial by ITS rDNA and EF1α rDNA genes sequencing, respectively. The taxonomic structure (α-, β-diversity) of soil bacterial community (SBC) was characterized from phylum up to genus level at the end of the trial in all the sites by 16S rRNA gene sequencing. The results showed that dry SSD reduced disease severity in field up to 18 % and Fol abundance in tomato up to 0.9 ITS copies g−1 tissue. Fol abundance was decreased in the rhizosphere up to 0.0027 ng μl−1 DNA, while the Fusarium community shifted between treated-soils and un-amended. The SBC composition (α-diversity) changed in the rhizosphere by applying dry digestate, while the SBC structure (β-diversity) shifted either among treatments or sites. Bacterial members related to Fol suppression (Bacillus, Chitinophaga, Flavihumibacter, Flavobacterium, Pseudomonas and Terrimonas) increased in the rhizosphere (P < 0.01, P < 0.001) more than in the bulk soils by applying both dewatered-SSDs. Thus, digestate soil amendment carried out for three consecutive years has affected tomato Fusarium wilt severity by changing the taxonomic characteristics of fusaria and bacteria communities of the amended soil

Development of personalized thrombogenesis and thrombin generation assays to assess endothelial dysfunction in cardiovascular diseases

The study of endothelial dysfunction (ED) is crucial to identify the pathogenetic mechanism(s) and provide indications for patient management in cardiovascular diseases. It is currently hindered by the limited availability of patient-specific primary endothelial cells (ECs). Endothelial colony-forming cells (ECFCs) represent an optimal non-invasive tool to overcome this issue. Therefore, we investigated the use of ECFCs as a substrate in thrombogenesis and thrombin generation assay (TGA) to assess ED. Both assays were set up on human umbilical vein endothelial cells (HUVECs) and then tested on ECFCs obtained from healthy donors. To prove the ability of the assays to detect endothelial activation, ECs stimulated with TNFα were compared with unstimulated ECs. EC activation was confirmed by the upregulation of VCAM-1 and Tissue Factor expression. Both assays discriminated between unstimulated and activated HUVECs and ECFCs, as significantly higher platelet deposition and fibrin formation in thrombogenesis assay, and thrombin generation in TGA, were observed when TNFα-activated ECs were used as a substrate. The amount of fibrin and thrombin measured in the two assays were directly correlated. Our results support the combined use of a thrombogenesis assay and TGA performed on patient-derived ECFCs to provide a personalized global assessment of ED relevant to the patient’s hemostatic profile

Transcriptomic profile of TNF high MAIT cells is linked to B cell response following SARS-CoV-2 vaccination

Introduction: Higher frequencies of mucosal-associated invariant T (MAIT) cells were associated with an increased adaptive response to mRNA BNT162b2 SARS- CoV-2 vaccine, however, the mechanistic insights into this relationship are unknown. In the present study, we hypothesized that the TNF response of MAIT cells supports B cell activation following SARS-CoV-2 immunization. Methods: To investigate the effects of repeated SARS-CoV-2 vaccinations on the peripheral blood mononuclear cells (PBMCs), we performed a longitudinal single cell (sc)RNA-seq and scTCR-seq analysis of SARS-CoV-2 vaccinated healthy adults with two doses of the Pfizer-BioNTech BNT162b2 mRNA vaccine. Collection of PBMCs was performed 1 day before, 3 and 17 days after prime vaccination, and 3 days and 3 months following vaccine boost. Based on scRNA/ TCR-seq data related to regulatory signals induced by the vaccine, we used computational approaches for the functional pathway enrichment analysis (Reactome), dynamics of the effector cell-polarization (RNA Velocity and CellRank), and cell-cell communication (NicheNet). Results: We identified MAIT cells as an important source of TNF across circulating lymphocytes in response to repeated SARS-CoV-2 BNT162b2 vaccination. The TNFhigh signature of MAIT cells was induced by the second administration of the vaccine. Notably, the increased TNF expression was associated with MAIT cell proliferation and efficient anti-SARS-CoV-2 antibody production. Finally, by decoding the ligand-receptor interactions and incorporating intracellular signaling, we predicted TNFhigh MAIT cell interplay with different B cell subsets. In specific, predicted TNF-mediated activation was selectively directed to conventional switched memory B cells, which are deputed to high-affinity long-term memory. Discussion: Overall, our results indicate that SARS-CoV-2 BNT162b2 vaccination influences MAIT cell frequencies and their transcriptional effector profile with the potential to promote B cell activation. This research also provides a blueprint for the promising use of MAIT cells as cellular adjuvants in mRNA-based vaccines

Microgenomic Analysis in Skeletal Muscle: Expression Signatures of Individual Fast and Slow Myofibers

BACKGROUND: Skeletal muscle is a complex, versatile tissue composed of a variety of functionally diverse fiber types. Although the biochemical, structural and functional properties of myofibers have been the subject of intense investigation for the last decades, understanding molecular processes regulating fiber type diversity is still complicated by the heterogeneity of cell types present in the whole muscle organ. METHODOLOGY/PRINCIPAL FINDINGS: We have produced a first catalogue of genes expressed in mouse slow-oxidative (type 1) and fast-glycolytic (type 2B) fibers through transcriptome analysis at the single fiber level (microgenomics). Individual fibers were obtained from murine soleus and EDL muscles and initially classified by myosin heavy chain isoform content. Gene expression profiling on high density DNA oligonucleotide microarrays showed that both qualitative and quantitative improvements were achieved, compared to results with standard muscle homogenate. First, myofiber profiles were virtually free from non-muscle transcriptional activity. Second, thousands of muscle-specific genes were identified, leading to a better definition of gene signatures in the two fiber types as well as the detection of metabolic and signaling pathways that are differentially activated in specific fiber types. Several regulatory proteins showed preferential expression in slow myofibers. Discriminant analysis revealed novel genes that could be useful for fiber type functional classification. CONCLUSIONS/SIGNIFICANCE: As gene expression analyses at the single fiber level significantly increased the resolution power, this innovative approach would allow a better understanding of the adaptive transcriptomic transitions occurring in myofibers under physiological and pathological condition