Identification of key regulators for grapevine ripening by integrated genetic approaches

Grapevine is one of the most economically relevant fruit crops in the world. In the past few years climate change and in particular global warming have started to significantly impact agriculture and viticulture as well. Indeed, higher temperatures occurring early during the growing season may advance the date of the phenological stages like bud burst, flowering and veraison. As a consequence, the shift in veraison to earlier dates causes ripening to occur under higher temperatures which impairs wine quality. It is of great interest for viticulture to investigate the timing of veraison and disentangle its genetic control. Genetic variation between cultivars leads to differences in the expression of phenotypic traits such as veraison time. A deeper knowledge of the genes involved in the phenology processes is critical to select varieties that are well adapted for current and future climatic conditions. The aim of this thesis was to identify the most promising candidate genes for the regulation of the transition to grapevine berry ripening through complementary genetic study. The thesis is divided in two chapters: in the first chapter an approach based on the integration of data from QTL studies has been developed, in order to narrow down the number of candidate genes that may be related with berry ripening. Meta-QTL analysis integrated with transcriptomic data led to the identification of 61 candidate genes related to veraison, including well known transcription factors and genes related to carbohydrate metabolism. In the second part, we have characterized a wide germplasm collection of different grapevine accessions (from CREA-VIT Conegliano collection) with the aim to identify the best panels of grapevine varieties, representing the most diverse genotypes and phenotypes for the traits of interest, to be exploited to test the genotype-phenotype association. The Conegliano collection has been previously phenotyped for flowering time and veraison time in the last 50 years; our work focused on the phenotypic data of the last 13 years (data kindly made available by our partners). These phenotypic data have been firstly used to assist the construction of a core collection, including the most genetic diverse accessions and individuals with contrasting phenotypes. Approximately 600 CREA-VIT accessions were previously genotyped (Cipriani et al., 2010) using 45 microsatellite markers. The 6 genotypic data have been used to assess the genetic diversity and population structure of the Conegliano collection. The end of the second part was dedicated to performing the association analysis itself with two different approaches, firstly using the GrapeReSeq 18K Vitis genotyping chip, then with an innovative approach called XP-GWAS (Yang et al., 2015) where pool of individuals displaying extreme phenotypes are whole-genome resequenced and allele frequencies compared to a random pool are screened for enrichment. The findings of this study provide indications of the genetic factors controlling or influencing veraison time in grapevine. The elucidation of the genetic network underlying the beginning of the berry ripening phase is of fundamental importance for the need to breed new grapevine varieties adapted to changing climatic conditions

Selection of candidate genes controlling veraison time in grapevine through integration of meta-QTL and transcriptomic data

Background High temperature during grape berry ripening impairs the quality of fruits and wines. Veraison time, which marks ripening onset, is a key factor for determining climatic conditions during berry ripening. Understanding its genetic control is crucial to successfully breed varieties more adapted to a changing climate. Quantitative trait loci (QTL) studies attempting to elucidate the genetic determinism of developmental stages in grapevine have identified wide genomic regions. Broad scale transcriptomic studies, by identifying sets of genes modulated during berry development and ripening, also highlighted a huge number of putative candidates. Results With the final aim of providing an overview about available information on the genetic control of grapevine veraison time, and prioritizing candidates, we applied a meta-QTL analysis for grapevine phenology-related traits and checked for co-localization of transcriptomic candidates. A consensus genetic map including 3130 markers anchored to the grapevine genome assembly was compiled starting from 39 genetic maps. Two thousand ninety-three QTLs from 47 QTL studies were projected onto the consensus map, providing a comprehensive overview about distribution of available QTLs and revealing extensive co-localization especially across phenology related traits. From 141 phenology related QTLs we generated 4 veraison meta-QTLs located on linkage group (LG) 1 and 2, and 13 additional meta-QTLs connected to the veraison time genetic control, among which the most relevant were located on LG 14, 16 and 18. Functional candidates in these intervals were inspected. Lastly, taking advantage of available transcriptomic datasets, expression data along berry development were integrated, in order to pinpoint among positional candidates, those differentially expressed across the veraison transition. Conclusion Integration of meta-QTLs analysis on available phenology related QTLs and data from transcriptomic dataset allowed to strongly reduce the number of candidate genes for the genetic control of the veraison transition, prioritizing a list of 272 genes, among which 78 involved in regulation of gene expression, signal transduction or development

Genotyping-by-sequencing in an orphan plant species Physocarpus opulifolius helps identify the evolutionary origins of the genus Prunus

Background: The Rosaceae family encompasses numerous genera exhibiting morphological diversification in fruit types and plant habit as well as a wide variety of chromosome numbers. Comparative genomics between various Rosaceous genera has led to the hypothesis that the ancestral genome of the family contained nine chromosomes, however, the synteny studies performed in the Rosaceae to date encompass species with base chromosome numbers x = 7 (Fragaria), x = 8 (Prunus), and x = 17 (Malus), and no study has included species from one of the many Rosaceous genera containing a base chromosome number of x = 9. Results: A genetic linkage map of the species Physocarpus opulifolius (x = 9) was populated with sequence characterised SNP markers using genotyping by sequencing. This allowed for the first time, the extent of the genome diversification of a Rosaceous genus with a base chromosome number of x = 9 to be performed. Orthologous loci distributed throughout the nine chromosomes of Physocarpus and the eight chromosomes of Prunus were identified which permitted a meaningful comparison of the genomes of these two genera to be made. Conclusions: The study revealed a high level of macro-synteny between the two genomes, and relatively few chromosomal rearrangements, as has been observed in studies of other Rosaceous genomes, lending further support for a relatively simple model of genomic evolution in Rosaceae

The last of the large-sized tortoises of the Mediterranean islands

Altres ajuts: CERCA Programme/Generalitat de CatalunyaAltres ajuts: Operational Programme Research, Development and Education Project (CZ.02.2.69/0.0/0.0/16_027/0008360)Archaeological investigations carried out in the cave Zubbio di Cozzo San Pietro, Bagheria, Sicily, revealed the presence of a few skeletal elements of a large-sized tortoise in a funerary area dating to the Copper/Bronze Age. The tortoise has been AMS-dated revealing an age of 12.5 ± 0.5 kyr BP and therefore it pre-dates the funerary activities. The morphology of the retrieved skeletal elements differs from that of the only native tortoise currently living in Sicily, Testudo hermanni. The tortoise's size significantly exceeds the size range of extant Te. hermanni and all Testudo spp., as well as that of their known fossils, and suggests a shell length of 50-60 cm. Repeated efforts to obtain DNA sequences from the tortoise of Zubbio di Cozzo San Pietro failed, but the morphology of the femur is distinct enough to allow us to erect a new taxon, Solitudo sicula gen. et sp. nov., based on a parsimony analysis. It belongs to a hitherto unrecognized clade that includes other large-sized tortoises from Mediterranean islands, like Malta and Menorca. A review of the pertinent taxa indicates that the remains here described represent the geologically youngest large-sized tortoise of the Mediterranean area

FT-IR Transflection Micro-Spectroscopy Study on Normal Human Breast Cells after Exposure to a Proton Beam

Fourier transform infrared micro-spectroscopy (mu-FT-IR) is nowadays considered a valuable tool for investigating the changes occurring in human cells after exposure to ionizing radiation. Recently, considerable attention has been devoted to the use of this optical technique in the study of cells exposed to proton beams, that are being increasingly adopted in cancer therapy. Different experimental configurations are used for proton irradiation and subsequent spectra acquisition. To facilitate the use of mu-FT-IR, it may be useful to investigate new experimental approaches capable of speeding up and simplifying the irradiation and measurements phases. Here, we propose the use of low-e-substrates slides for cell culture, allowing the irradiation and spectra acquisition in transflection mode in a fast and direct way. In recent years, there has been a wide debate about the validity of these supports, but many researchers agree that the artifacts due to the presence of the electromagnetic standing wave effects are negligible in many practical cases. We investigated human normal breast cells (MCF-10 cell line) fixed immediately after the irradiation with graded proton radiation doses (0, 0.5, 2, and 4 Gy). The spectra obtained in transflection geometry showed characteristics very similar to those present in the spectra acquired in transmission geometry and confirm the validity of the chosen approach. The analysis of spectra indicates the occurrence of significant changes in DNA and lipids components of cells. Modifications in protein secondary structure are also evidenced

Intratumoral injection of TLR9 agonist promotes an immunopermissive microenvironment transition and causes cooperative antitumor activity in combination with anti-PD1 in pancreatic cancer

Background: Complex tumor and immune microenvironment render pancreatic ductal adenocarcinoma (PDAC) resistant to immune checkpoint inhibitors (ICIs). Therefore, a strategy to convert the immune hostile into an immunopermissive tumor is required. Recent studies showed that intratumoral injection of Toll-like receptor 9 agonist IMO-2125 primes the adaptive immune response. Phase I and II trials with intratumoral IMO-2125 demonstrated its safety and antitumoral activity. Methods: We generated an array of preclinical models by orthotopically engrafting PDAC-derived cell lines in syngeneic mice and categorized them as high, low and no immunogenic potential, based on the ability of tumor to evoke T lymphocyte or NK cell response. To test the antitumor efficacy of IMO-2125 on locally treated and distant sites, we engrafted cancer cells on both flanks of syngeneic mice and treated them with intratumoral IMO-2125 or vehicle, alone or in combination with anti-PD1 ICI. Tumor tissues and systemic immunity were analyzed by transcriptomic, cytofluorimetric and immunohistochemistry analysis. Results: We demonstrated that intratumoral IMO-2125 as single agent triggers immune system response to kill local and distant tumors in a selected high immunogenic subtype affecting tumor growth and mice survival. Remarkably, intratumoral IMO-2125 in combination with systemic anti-PD1 causes a potent antitumor effect on primary injected and distant sites also in pancreatic cancer models with low immunogenic potential, preceded by a transition toward an immunopermissive microenvironment, with increase in tumor-infiltrating dendritic and T cells in tumor and lymph nodes. Conclusion: We demonstrated a potent antitumor activity of IMO-2125 and anti-PD1 combination in immunotherapy-resistant PDAC models through the modulation of immune microenvironment, providing the rationale to translate this strategy into a clinical setting

Immunoevolution of mouse pancreatic organoid isografts from preinvasive to metastatic disease

Pancreatic ductal adenocarcinoma (PDA) has a highly immunosuppressive microenvironment, which is contributed by the complex interaction between cancer cells and a heterogeneous population of stromal cells. Therefore, facile and trackable models are needed for integrative and dynamic interrogation of cancer-stroma interaction. Here, we tracked the immunoevolution of PDA in a genetically-defined transplantable model of mouse pancreatic tumour organoids that recapitulates the progression of the disease from early preinvasive lesions to metastatic carcinomas. We demonstrated that organoid-derived isografts (ODI) can be used as a biological source of biomarkers (NT5E, TGFB1, FN1, and ITGA5) of aggressive molecular subtypes of human PDA. In ODI, infiltration from leukocytes is an early event during progression of the disease as observed for autochthonous models. Neoplastic progression was associated to accumulation of Maf+ macrophages, which inversely correlated with CD8+ T cells infiltration. Consistently, levels of MAF were enriched in human PDA subtypes characterized by abundance of macrophage-related transcripts and indicated poor patients' survival. Density of MAF+ macrophages was higher in human PDA tissues compared to preinvasive lesions. Our results suggest that ODIs represent a suitable system for genotypic-immunophenotypic studies and support the hypothesis of MAF+ macrophages as a prominent immunosuppressive population in PDA

Extracellular Vesicles Mediate Mesenchymal Stromal Cell-Dependent Regulation of B Cell PI3K-AKT Signaling Pathway and Actin Cytoskeleton

Mesenchymal stromal cells (MSCs) are adult, multipotent cells of mesodermal origin representing the progenitors of all stromal tissues. MSCs possess significant and broad immunomodulatory functions affecting both adaptive and innate immune responses once MSCs are primed by the inflammatory microenvironment. Recently, the role of extracellular vesicles (EVs) in mediating the therapeutic effects of MSCs has been recognized. Nevertheless, the molecular mechanisms responsible for the immunomodulatory properties of MSC-derived EVs (MSC-EVs) are still poorly characterized. Therefore, we carried out a molecular characterization of MSC-EV content by high-throughput approaches. We analyzed miRNA and protein expression profile in cellular and vesicular compartments both in normal and inflammatory conditions. We found several proteins and miRNAs involved in immunological processes, such as MOES, LG3BP, PTX3, and S10A6 proteins, miR-155-5p, and miR-497-5p. Different in silico approaches were also performed to correlate miRNA and protein expression profile and then to evaluate the putative molecules or pathways involved in immunoregulatory properties mediated by MSC-EVs. PI3K-AKT signaling pathway and the regulation of actin cytoskeleton were identified and functionally validated in vitro as key mediators of MSC/B cell communication mediated by MSC-EVs. In conclusion, we identified different molecules and pathways responsible for immunoregulatory properties mediated by MSC-EVs, thus identifying novel therapeutic targets as safer and more useful alternatives to cell or EV-based therapeutic approaches

Long-term organoid culture of a small intestinal neuroendocrine tumor

Gastroenteropancreatic neuroendocrine tumors (GEP-NETs) are rare and highly heterogeneous neoplasms whose incidence has markedly increased over the last decades. A grading system based on the tumor cells’ proliferation index predicts high-risk for G3 NETs. However, low-to-intermediate grade (G1/G2) NETs have an unpredictable clinical course that varies from indolent to highly malignant. Cultures of human cancer cells enable to perform functional perturbation analyses that are instrumental to enhance our understanding of cancer biology. To date, no tractable and reliable long-term culture of G1/G2 NET has been reported to permit disease modeling and pharmacological screens. Here, we report of the first long-term culture of a G2 metastatic small intestinal NET that preserves the main genetic drivers of the tumor and retains expression patterns of the endocrine cell lineage. Replicating the tissue, this long-term culture showed a low proliferation index, and yet it could be propagated continuously without dramatic changes in the karyotype. The model was readily available for pharmacological screens using targeted agents and as expected, showed low tumorigenic capacity in vivo. Overall, this is the first long-term culture of NETs to faithfully recapitulate many aspects of the original neuroendocrine tumor

Axon guidance cue SEMA3A promotes the aggressive phenotype of basal-like PDAC

Objective: The dysregulation of the axon guidance pathway is common in pancreatic ductal adenocarcinoma (PDAC), yet our understanding of its biological relevance is limited. Here, we investigated the functional role of the axon guidance cue SEMA3A in supporting PDAC progression. Design: We integrated bulk and single-cell transcriptomic datasets of human PDAC with in situ hybridisation analyses of patients’ tissues to evaluate SEMA3A expression in molecular subtypes of PDAC. Gain and loss of function experiments in PDAC cell lines and organoids were performed to dissect how SEMA3A contributes to define a biologically aggressive phenotype. Results: In PDAC tissues, SEMA3A is expressed by stromal elements and selectively enriched in basal-like/squamous epithelial cells. Accordingly, expression of SEMA3A in PDAC cells is induced by both cell-intrinsic and cell-extrinsic determinants of the basal-like phenotype. In vitro, SEMA3A promotes cell migration as well as anoikis resistance. At the molecular level, these phenotypes are associated with increased focal adhesion kinase signalling through canonical SEMA3A-NRP1 axis. SEMA3A provides mouse PDAC cells with greater metastatic competence and favours intratumoural infiltration of tumour-associated macrophages and reduced density of T cells. Mechanistically, SEMA3A functions as chemoattractant for macrophages and skews their polarisation towards an M2-like phenotype. In SEMA3Ahigh tumours, depletion of macrophages results in greater intratumour infiltration by CD8+T cells and better control of the disease from antitumour treatment. Conclusions: Here, we show that SEMA3A is a stress-sensitive locus that promotes the malignant phenotype of basal-like PDAC through both cell-intrinsic and cell-extrinsic mechanisms