Replicative senescence and high glucose induce the accrual of self-derived cytosolic nucleic acids in human endothelial cells.

Recent literature shows that loss of replicative ability and acquisition of a proinflammatory secretory phenotype in senescent cells is coupled with the build-in of nucleic acids in the cytoplasm. Its implication in human age-related diseases is under scrutiny. In human endothelial cells (ECs), we assessed the accumulation of intracellular nucleic acids during in vitro replicative senescence and after exposure to high glucose concentrations, which mimic an in vivo condition of hyperglycemia. We showed that exposure to high glucose induces senescent-like features in ECs, including telomere shortening and proinflammatory cytokine release, coupled with the accrual in the cytoplasm of telomeres, double-stranded DNA and RNA (dsDNA, dsRNA), as well as RNA:DNA hybrid molecules. Senescent ECs showed an activation of the dsRNA sensors RIG-I and MDA5 and of the DNA sensor TLR9, which was not paralleled by the involvement of the canonical (cGAS) and non-canonical (IFI16) activation of the STING pathway. Under high glucose conditions, only a sustained activation of TLR9 was observed. Notably, senescent cells exhibit increased proinflammatory cytokine (IL-1ß, IL-6, IL-8) production without a detectable secretion of type I interferon (IFN), a phenomenon that can be explained, at least in part, by the accumulation of methyl-adenosine containing RNAs. At variance, exposure to exogenous nucleic acids enhances both IL-6 and IFN-ß1 expression in senescent cells. This study highlights the accrual of cytoplasmic nucleic acids as a marker of senescence-related endothelial dysfunction, that may play a role in dysmetabolic age-related diseases.

Early downregulation of hsa-miR-144-3p in serum from drug-naïve Parkinson's disease patients.

Advanced age represents one of the major risk factors for Parkinson's Disease. Recent biomedical studies posit a role for microRNAs, also known to be remodelled during ageing. However, the relationship between microRNA remodelling and ageing in Parkinson's Disease, has not been fully elucidated. Therefore, the aim of the present study is to unravel the relevance of microRNAs as biomarkers of Parkinson's Disease within the ageing framework. We employed Next Generation Sequencing to profile serum microRNAs from samples informative for Parkinson's Disease (recently diagnosed, drug-naïve) and healthy ageing (centenarians) plus healthy controls, age-matched with Parkinson's Disease patients. Potential microRNA candidates markers, emerging from the combination of differential expression and network analyses, were further validated in an independent cohort including both drug-naïve and advanced Parkinson's Disease patients, and healthy siblings of Parkinson's Disease patients at higher genetic risk for developing the disease. While we did not find evidences of microRNAs co-regulated in Parkinson's Disease and ageing, we report that hsa-miR-144-3p is consistently down-regulated in early Parkinson's Disease patients. Moreover, interestingly, functional analysis revealed that hsa-miR-144-3p is involved in the regulation of coagulation, a process known to be altered in Parkinson's Disease. Our results consistently show the down-regulation of hsa-mir144-3p in early Parkinson's Disease, robustly confirmed across a variety of analytical and experimental analyses. These promising results ask for further research to unveil the functional details of the involvement of hsa-mir144-3p in Parkinson's Disease.

Clinical, Radiometabolic and Immunologic Effects of Olaparib in Locally Advanced Triple Negative Breast Cancer: The OLTRE Window of Opportunity Trial.

INTRODUCTION: Olaparib is effective in metastatic triple negative breast cancer (TNBC) carrying germline mutations in DNA damage repair (DDR) genes BRCA1/2 (gBRCA-mut). The OLTRE window-of-opportunity trial preliminarily investigated potential pathologic, radiometabolic and immune biomarkers of early-response to olaparib in gBRCA-wild-type (wt) TNBC and, as proof-of-concept in gBRCA-mut HER2-negative BC. METHODS: Patients received olaparib for 3 weeks (3w) before standard neoadjuvant chemotherapy and underwent multiple FDG18-PET/CT scan (basal, after olaparib), clinical assessments (basal, every 3w), tumor biopsies and blood samplings (baseline, after olaparib). Clinical and radiometabolic responses were evaluated according to RECIST1.1 and PERCIST criteria. RESULTS: 27 patients with gBRCA-wt TNBC and 8 with gBRCA-mut BC (6 TNBC, 2 HR+/HER2-negative) were enrolled. Three (11.1%) patients showed mutations in non-BRCA1/2 DDR genes and 4 (14.8%) in other genes. 3w olaparib induced 16/35 and 15/27 partial clinical and radiometabolic responses, including in 40.7% and 50.0% gBRCA-wt patients. gBRCA-mut tumors presented numerically higher tumor-infiltrating lymphocytes (TILs) levels and PD-L1 positive tumors. Clinical responders experienced a reduction in T-regs/T-eff ratio (p=0.05), B and NK lymphocytes (p=0.003 both), with an average increase in T-helpers rate (p<0.001) and CD4/CD8 ratio (p=0.02). Ki67% and TILs did not vary significantly (p=0.67 and p=0.77). A numerical increase in PD-L1 positive cases after olaparib was observed, though non-significant (p=0.134). No differences were observed according to gBRCA status and type of response. CONCLUSIONS: Early-stage TNBC might be a target population for olaparib, irrespective of gBRCA mutations. Future trials should combine TILs, PD-L1 and gBRCA status to better identify candidates for escalated/de-escalated treatment strategies including olaparib.

A geroscience approach for Parkinson's disease: Conceptual framework and design of PROPAG-AGEING project.

Advanced age is the major risk factor for idiopathic Parkinson's disease (PD), but to date the biological relationship between PD and ageing remains elusive. Here we describe the rationale and the design of the H2020 funded project "PROPAG-AGEING", whose aim is to characterize the contribution of the ageing process to PD development. We summarize current evidences that support the existence of a continuum between ageing and PD and justify the use of a Geroscience approach to study PD. We focus in particular on the role of inflammaging, the chronic, low-grade inflammation characteristic of elderly physiology, which can propagate and transmit both locally and systemically. We then describe PROPAG-AGEING design, which is based on the multi-omic characterization of peripheral samples from clinically characterized drug-naïve and advanced PD, PD discordant twins, healthy controls and "super-controls", i.e. centenarians, who never showed clinical signs of motor disability, and their offspring. Omic results are then validated in a large number of samples, including in vitro models of dopaminergic neurons and healthy siblings of PD patients, who are at higher risk of developing PD, with the final aim of identifying the molecular perturbations that can deviate the trajectories of healthy ageing towards PD development.

Whole-Transcriptome Analysis Unveils the Synchronized Activities of Genes for Fructans in Developing Tubers of the Jerusalem Artichoke.

Helianthus tuberosus L., known as the Jerusalem artichoke, is a hexaploid plant species, adapted to low-nutrient soils, that accumulates high levels of inulin in its tubers. Inulin is a fructose-based polysaccharide used either as dietary fiber or for the production of bioethanol. Key enzymes involved in inulin biosynthesis are well known. However, the gene networks underpinning tuber development and inulin accumulation in H. tuberous remain elusive. To fill this gap, we selected 6,365 expressed sequence tags (ESTs) from an H. tuberosus library to set up a microarray platform and record their expression across three tuber developmental stages, when rhizomes start enlarging (T0), at maximum tuber elongation rate (T3), and at tuber physiological maturity (Tm), in "VR" and "K8-HS142"clones. The former was selected as an early tuberizing and the latter as a late-tuberizing clone. We quantified inulin and starch levels, and qRT-PCR confirmed the expression of critical genes accounting for inulin biosynthesis. The microarray analysis revealed that the differences in morphological and physiological traits between tubers of the two clones are genetically determined since T0 and that is relatively low the number of differentially expressed ESTs across the stages shared between the clones (93). The expression of ESTs for sucrose:sucrose 1-fructosyltransferase (1-SST) and fructan:fructan 1-fructosyltransferase (1-FFT), the two critical genes for fructans polymerization, resulted to be temporarily synchronized and mirror the progress of inulin accumulation and stretching. The expression of ESTs for starch biosynthesis was insignificant throughout the developmental stages of the clones in line with the negligible level of starch into their mature tubers, where inulin was the dominant polysaccharide. Overall, our study disclosed candidate genes underpinning the development and storage of carbohydrates in the tubers of two H. tuberosus clones. A model according to which the steady-state levels of 1-SST and 1-FFT transcripts are developmentally controlled and might represent a limiting factor for inulin accumulation has been provided. Our finding may have significant repercussions for breeding clones with improved levels of inulin for food and chemical industry.

A chromosome-anchored eggplant genome sequence reveals key events in Solanaceae evolution.

With approximately 450 species, spiny Solanum species constitute the largest monophyletic group in the Solanaceae family, but a high-quality genome assembly from this group is presently missing. We obtained a chromosome-anchored genome assembly of eggplant (Solanum melongena), containing 34,916 genes, confirming that the diploid gene number in the Solanaceae is around 35,000. Comparative genomic studies with tomato (S. lycopersicum), potato (S. tuberosum) and pepper (Capsicum annuum) highlighted the rapid evolution of miRNA:mRNA regulatory pairs and R-type defense genes in the Solanaceae, and provided a genomic basis for the lack of steroidal glycoalkaloid compounds in the Capsicum genus. Using parsimony methods, we reconstructed the putative chromosomal complements of the key founders of the main Solanaceae clades and the rearrangements that led to the karyotypes of extant species and their ancestors. From 10% to 15% of the genes present in the four genomes were syntenic paralogs (ohnologs) generated by the pre-γ, γ and T paleopolyploidy events, and were enriched in transcription factors. Our data suggest that the basic gene network controlling fruit ripening is conserved in different Solanaceae clades, and that climacteric fruit ripening involves a differential regulation of relatively few components of this network, including CNR and ethylene biosynthetic genes.

Apple fruit superficial scald resistance mediated by ethylene inhibition is associated with diverse metabolic processes.

Fruits stored at low temperature can exhibit different types of chilling injury. In apple, one of the most serious physiological disorders is superficial scald, which is characterized by discoloration and brown necrotic patches on the fruit exocarp. Although this phenomenon is widely ascribed to the oxidation of α-farnesene, its physiology is not yet fully understood. To elucidate the mechanism of superficial scald development and possible means of prevention, we performed an integrated metabolite screen, including an analysis of volatiles, phenols and lipids, together with a large-scale transcriptome study. We also determined that prevention of superficial scald, through the use of an ethylene action inhibitor, is associated with the triggering of cold acclimation-related processes. Specifically, the inhibition of ethylene perception stimulated the production of antioxidant compounds to scavenge reactive oxygen species, the synthesis of fatty acids to stabilize plastid and vacuole membranes against cold temperature, and the accumulation of the sorbitol, which can act as a cryoprotectant. The pattern of sorbitol accumulation was consistent with the expression profile of a sorbitol 6-phosphate dehydrogenase, MdS6PDH, the overexpression of which in transgenic Arabidopsis thaliana plants confirmed its involvement in the cold acclimation and freezing tolerance.

Sexual Polyploidization in Medicago sativa L.: Impact on the Phenotype, Gene Transcription, and Genome Methylation.

Polyploidization as the consequence of 2n gamete formation is a prominent mechanism in plant evolution. Studying its effects on the genome, and on genome expression, has both basic and applied interest. We crossed two diploid (2n = 2x = 16) Medicago sativa plants, a subsp. falcata seed parent, and a coerulea × falcata pollen parent that form a mixture of n and 2n eggs and pollen, respectively. Such a cross produced full-sib diploid and tetraploid (2n = 4x = 32) hybrids, the latter being the result of bilateral sexual polyploidization (BSP). These unique materials allowed us to investigate the effects of BSP, and to separate the effect of intraspecific hybridization from those of polyploidization by comparing 2x with 4x full sib progeny plants. Simple sequence repeat marker segregation demonstrated tetrasomic inheritance for all chromosomes but one, demonstrating that these neotetraploids are true autotetraploids. BSP brought about increased biomass, earlier flowering, higher seed set and weight, and larger leaves with larger cells. Microarray analyses with M. truncatula gene chips showed that several hundred genes, related to diverse metabolic functions, changed their expression level as a consequence of polyploidization. In addition, cytosine methylation increased in 2x, but not in 4x, hybrids. Our results indicate that sexual polyploidization induces significant transcriptional novelty, possibly mediated in part by DNA methylation, and phenotypic novelty that could underpin improved adaptation and reproductive success of tetraploid M. sativa with respect to its diploid progenitor. These polyploidy-induced changes may have promoted the adoption of tetraploid alfalfa in agriculture.

Characterization of a genetic mouse model of lung cancer: a promise to identify Non-Small Cell Lung Cancer therapeutic targets and biomarkers.

BACKGROUND: Non-small cell lung cancer (NSCLC) accounts for 81% of all cases of lung cancer and they are often fatal because 60% of the patients are diagnosed at an advanced stage. Besides the need for earlier diagnosis, there is a high need for additional effective therapies. In this work, we investigated the feasibility of a lung cancer progression mouse model, mimicking features of human aggressive NSCLC, as biological reservoir for potential therapeutic targets and biomarkers. RESULTS: We performed RNA-seq profiling on total RNA extracted from lungs of a 30 week-old K-ras(LA1)/p53(R172HΔg) and wild type (WT) mice to detect fusion genes and gene/exon-level differential expression associated to the increase of tumor mass. Fusion events were not detected in K-ras(LA1)/p53(R172HΔg) tumors. Differential expression at exon-level detected 33 genes with differential exon usage. Among them nine, i.e. those secreted or expressed on the plasma membrane, were used for a meta-analysis of more than 500 NSCLC RNA-seq transcriptomes. None of the genes showed a significant correlation between exon-level expression and disease prognosis. Differential expression at gene-level allowed the identification of 1513 genes with a significant increase in expression associated to tumor mass increase. 74 genes, i.e. those secreted or expressed on the plasma membrane, were used for a meta-analysis of two transcriptomics datasets of human NSCLC samples, encompassing more than 900 samples. SPP1 was the only molecule whose over-expression resulted statistically related to poor outcome regarding both survival and metastasis formation. Two other molecules showed over-expression associated to poor outcome due to metastasis formation: GM-CSF and ADORA3. GM-CSF is a secreted protein, and we confirmed its expression in the supernatant of a cell line derived by a K-ras(LA1)/p53(R172HΔg) mouse tumor. ADORA3 is instead involved in the induction of p53-mediated apoptosis in lung cancer cell lines. Since in our model p53 is inactivated, ADORA3 does not negatively affect tumor growth but remains expressed on tumor cells. Thus, it could represent an interesting target for the development of antibody-targeted therapy on a subset of NSCLC, which are p53 null and ADORA3 positive. CONCLUSIONS: Our study provided a complete transcription overview of the K-ras(LA1)/p53(R172HΔg) mouse NSCLC model. This approach allowed the detection of ADORA3 as a potential target for antibody-based therapy in p53 mutated tumors.

Patchwork sequencing of tomato San Marzano and Vesuviano varieties highlights genome-wide variations.

BACKGROUND: Investigation of tomato genetic resources is a crucial issue for better straight evolution and genetic studies as well as tomato breeding strategies. Traditional Vesuviano and San Marzano varieties grown in Campania region (Southern Italy) are famous for their remarkable fruit quality. Owing to their economic and social importance is crucial to understand the genetic basis of their unique traits. RESULTS: Here, we present the draft genome sequences of tomato Vesuviano and San Marzano genome. A 40x genome coverage was obtained from a hybrid Illumina paired-end reads assembling that combines de novo assembly with iterative mapping to the reference S. lycopersicum genome (SL2.40). Insertions, deletions and SNP variants were carefully measured. When assessed on the basis of the reference annotation, 30% of protein-coding genes are predicted to have variants in both varieties. Copy genes number and gene location were assessed by mRNA transcripts mapping, showing a closer relationship of San Marzano with reference genome. Distinctive variations in key genes and transcription/regulation factors related to fruit quality have been revealed for both cultivars. CONCLUSIONS: The effort performed highlighted varieties relationships and important variants in fruit key processes useful to dissect the path from sequence variant to phenotype.

The high polyphenol content of grapevine cultivar tannat berries is conferred primarily by genes that are not shared with the reference genome.

The grapevine (Vitis vinifera) cultivar Tannat is cultivated mainly in Uruguay for the production of high-quality red wines. Tannat berries have unusually high levels of polyphenolic compounds, producing wines with an intense purple color and remarkable antioxidant properties. We investigated the genetic basis of these important characteristics by sequencing the genome of the Uruguayan Tannat clone UY11 using Illumina technology, followed by a mixture of de novo assembly and iterative mapping onto the PN40024 reference genome. RNA sequencing data for genome reannotation were processed using a combination of reference-guided annotation and de novo transcript assembly, allowing 5901 previously unannotated or unassembled genes to be defined and resulting in the discovery of 1873 genes that were not shared with PN40024. Expression analysis showed that these cultivar-specific genes contributed substantially (up to 81.24%) to the overall expression of enzymes involved in the synthesis of phenolic and polyphenolic compounds that contribute to the unique characteristics of the Tannat berries. The characterization of the Tannat genome therefore indicated that the grapevine reference genome lacks many genes that appear to be relevant for the varietal phenotype.

De novo transcriptome characterization of Vitis vinifera cv. Corvina unveils varietal diversity.

BACKGROUND: Plants such as grapevine (Vitis spp.) display significant inter-cultivar genetic and phenotypic variation. The genetic components underlying phenotypic diversity in grapevine must be understood in order to disentangle genetic and environmental factors. RESULTS: We have shown that cDNA sequencing by RNA-seq is a robust approach for the characterization of varietal diversity between a local grapevine cultivar (Corvina) and the PN40024 reference genome. We detected 15,161 known genes including 9463 with novel splice isoforms, and identified 2321 potentially novel protein-coding genes in non-annotated or unassembled regions of the reference genome. We also discovered 180 apparent private genes in the Corvina genome which were missing from the reference genome. CONCLUSIONS: The de novo assembly approach allowed a substantial amount of the Corvina transcriptome to be reconstructed, improving known gene annotations by robustly defining gene structures, annotating splice isoforms and detecting genes without annotations. The private genes we discovered are likely to be nonessential but could influence certain cultivar-specific characteristics. Therefore, the application of de novo transcriptome assembly should not be restricted to species lacking a reference genome because it can also improve existing reference genome annotations and identify novel, cultivar-specific genes.

Molecular analysis of post-harvest withering in grape by AFLP transcriptional profiling.

Post-harvest withering of grape berries is used in the production of dessert and fortified wines to alter must quality characteristics and increase the concentration of simple sugars. The molecular processes that occur during withering are poorly understood, so a detailed transcriptomic analysis of post-harvest grape berries was carried out by AFLP-transcriptional profiling analysis. This will help to elucidate the molecular mechanisms of berry withering and will provide an opportunity to select markers that can be used to follow the drying process and evaluate different drying techniques. AFLP-TP identified 699 withering-specific genes, 167 and 86 of which were unique to off-plant and on-plant withering, respectively. Although similar molecular events were revealed in both withering processes, it was apparent that off-plant withering induced a stronger dehydration stress response resulting in the high level expression of genes involved in stress protection mechanisms, such as dehydrin and osmolite accumulation. Genes involved in hexose metabolism and transport, cell wall composition, and secondary metabolism (particularly the phenolic and terpene compound pathways) were similarly regulated in both processes. This work provides the first comprehensive analysis of the molecular events underpinning post-harvest withering and could help to define markers for different withering processes.

Nitric oxide- and hydrogen peroxide-responsive gene regulation during cell death induction in tobacco.

Nitric oxide (NO) and hydrogen peroxide (H(2)O(2)) are regulatory molecules in various developmental processes and stress responses. Tobacco (Nicotiana tabacum) leaves exposed to moderate high light dramatically potentiated NO-mediated cell death in catalase-deficient (CAT1AS) but not in wild-type plants, providing genetic evidence for a partnership between NO and H(2)O(2) during the induction of programmed cell death. With this experimental model system, the specific impact on gene expression was characterized by either NO or H(2)O(2) alone or both molecules combined. By means of genome-wide cDNA-amplified fragment length polymorphism analysis, transcriptional changes were compared in high light-treated CAT1AS and wild-type leaves treated with or without the NO donor sodium nitroprusside. Differential gene expression was detected for 214 of the approximately 8,000 transcript fragments examined. For 108 fragments, sequence analysis revealed homology to genes with a role in signal transduction, defense response, hormone interplay, proteolysis, transport, and metabolism. Surprisingly, only 16 genes were specifically induced by the combined action of NO and H(2)O(2), whereas the majority were regulated by either of them alone. At least seven transcription factors were mutually up-regulated, indicating significant overlap between NO and H(2)O(2) signaling pathways. These results consolidate significant cross-talk between NO and H(2)O(2), provide new insight into the early transcriptional response of plants to increased NO and H(2)O(2) levels, and identify target genes of the combined action of NO and H(2)O(2) during the induction of plant cell death.

Arabidopsis nonsymbiotic hemoglobin AHb1 modulates nitric oxide bioactivity.

Nitric oxide (NO) is a widespread signaling molecule, and numerous targets of its action exist in plants. Whereas the activity of NO in erythrocytes, microorganisms, and invertebrates has been shown to be regulated by several hemoglobins, the function of plant hemoglobins in NO detoxification has not yet been elucidated. Here, we show that Arabidopsis thaliana nonsymbiotic hemoglobin AHb1 scavenges NO through production of S-nitrosohemoglobin and reduces NO emission under hypoxic stress, indicating its role in NO detoxification. However, AHb1 does not affect NO-mediated hypersensitive cell death in response to avirulent Pseudomonas syringae, suggesting that it is not involved in the removal of NO bursts originated from acute responses when NO mediates crucial defense signaling functions.

Nitric oxide signalling functions in plant-pathogen interactions.

Nitric oxide (NO) is a highly reactive molecule that rapidly diffuses and permeates cell membranes. During the last few years NO has been detected in several plant species, and the increasing number of reports on its function in plants have implicated NO as a key molecular signal that participates in the regulation of several physiological processes; in particular, it has a significant role in plant resistance to pathogens by triggering resistance-associated cell death and by contributing to the local and systemic induction of defence genes. NO stimulates signal transduction pathways through protein kinases, cytosolic Ca2+ mobilization and protein modification (i.e. nitrosylation and nitration). In this review we will examine the synthesis of NO, its effects, functions and signalling giving rise to the hypersensitive response and systemic acquired resistance during plant-pathogen interactions.