Untuned antiviral immunity in COVID-19 revealed by temporal type I/III interferon patterns and flu comparison.

A central paradigm of immunity is that interferon (IFN)-mediated antiviral responses precede pro-inflammatory ones, optimizing host protection and minimizing collateral damage1,2. Here, we report that for coronavirus disease 2019 (COVID-19) this paradigm does not apply. By investigating temporal IFN and inflammatory cytokine patterns in 32 moderate-to-severe patients with COVID-19 hospitalized for pneumonia and longitudinally followed for the development of respiratory failure and death, we reveal that IFN-λ and type I IFN production were both diminished and delayed, induced only in a fraction of patients as they became critically ill. On the contrary, pro-inflammatory cytokines such as tumor necrosis factor (TNF), interleukin (IL)-6 and IL-8 were produced before IFNs in all patients and persisted for a prolonged time. This condition was reflected in blood transcriptomes wherein prominent IFN signatures were only seen in critically ill patients who also exhibited augmented inflammation. By comparison, in 16 patients with influenza (flu) hospitalized for pneumonia with similar clinicopathological characteristics to those of COVID-19 and 24 nonhospitalized patients with flu with milder symptoms, IFN-λ and type I IFN were robustly induced earlier, at higher levels and independently of disease severity, whereas pro-inflammatory cytokines were only acutely produced. Notably, higher IFN-λ concentrations in patients with COVID-19 correlated with lower viral load in bronchial aspirates and faster viral clearance and a higher IFN-λ to type I IFN ratio correlated with improved outcome for critically ill patients. Moreover, altered cytokine patterns in patients with COVID-19 correlated with longer hospitalization and higher incidence of critical disease and mortality compared to flu. These data point to an untuned antiviral response in COVID-19, contributing to persistent viral presence, hyperinflammation and respiratory failure.

Interferon-λ Mediates Non-redundant Front-Line Antiviral Protection against Influenza Virus Infection without Compromising Host Fitness.

Lambda interferons (IFNλs) or type III IFNs share homology, expression patterns, signaling cascades, and antiviral functions with type I IFNs. This has complicated the unwinding of their unique non-redundant roles. Through the systematic study of influenza virus infection in mice, we herein show that IFNλs are the first IFNs produced that act at the epithelial barrier to suppress initial viral spread without activating inflammation. If infection progresses, type I IFNs come into play to enhance viral resistance and induce pro-inflammatory responses essential for confronting infection but causing immunopathology. Central to this are neutrophils which respond to both cytokines to upregulate antimicrobial functions but exhibit pro-inflammatory activation only to type I IFNs. Accordingly, Ifnlr1-/- mice display enhanced type I IFN production, neutrophilia, lung injury, and lethality, while therapeutic administration of PEG-IFNλ potently suppresses these effects. IFNλs therefore constitute the front line of antiviral defense in the lung without compromising host fitness.

Notch signaling in adipose tissue macrophages prevents diet-induced inflammation and metabolic dysregulation.

The importance of macrophages in adipose tissue (AT) homeostasis and inflammation is well established. However, the potential cues that regulate their function remain incompletely understood. To bridge this important gap, we sought to characterize novel pathways involved using a mouse model of diet-induced obesity. By performing transcriptomics analysis of AT macrophages (ATMs), we found that late-stage ATMs from high-fat diet mice presented with perturbed Notch signaling accompanied by robust proinflammatory and metabolic changes. To explore the hypothesis that the deregulated Notch pathway contributes to the development of AT inflammation and diet-induced obesity, we employed a genetic approach to abrogate myeloid Notch1 and Notch2 receptors. Our results revealed that the combined loss of Notch1 and Notch2 worsened obesity-related metabolic dysregulation. Body and AT weight gain was higher, blood glucose levels increased and metabolic parameters were substantially worsened in deficient mice fed high-fat diet. Moreover, serum insulin and leptin were elevated as were triglycerides. Molecular analysis of ATMs showed that deletion of Notch receptors escalated inflammation through the induction of an M1-like pro-inflammatory phenotype. Our findings thus support a protective role of myeloid Notch signaling in adipose tissue inflammation and metabolic dysregulation.

The perennial fruit tree proteogenomics atlas: a spatial map of the sweet cherry proteome and transcriptome.

Genome-wide transcriptome analysis provides systems-level insights into plant biology. Due to the limited depth of quantitative proteomics our understanding of gene-protein-complex stoichiometry is largely unknown in plants. Recently, the complexity of the proteome and its cell-/tissue-specific distribution have boosted the research community to the integration of transcriptomics and proteomics landscapes in a proteogenomic approach. Herein, we generated a quantitative proteome and transcriptome abundance atlas of 15 major sweet cherry (Prunus avium L., cv 'Tragana Edessis') tissues represented by 29 247 genes and 7584 proteins. Additionally, 199 984 alternative splicing events, particularly exon skipping and alternative 3' splicing, were identified in 23 383 transcribed regions of the analyzed tissues. Common signatures as well as differences between mRNA and protein quantities, including genes encoding transcription factors and allergens, within and across the different tissues are reported. Using our integrated dataset, we identified key putative regulators of fruit development, notably genes involved in the biosynthesis of anthocyanins and flavonoids. We also provide proteogenomic-based evidence for the involvement of ethylene signaling and pectin degradation in cherry fruit ripening. Moreover, clusters of genes and proteins with similar and different expression and suppression trends across diverse tissues and developmental stages revealed a relatively low RNA abundance-to-protein correlation. The present proteogenomic analysis allows us to identify 17 novel sweet cherry proteins without prior protein-level annotation evidenced in the currently available databases. To facilitate use by the community, we also developed the Sweet Cherry Atlas Database (https://grcherrydb.com/) for viewing and data mining these resources. This work provides new insights into the proteogenomics workflow in plants and a rich knowledge resource for future investigation of gene and protein functions in Prunus species.

CD103 integrin identifies a high IL-10-producing FoxP3+ regulatory T-cell population suppressing allergic airway inflammation.

BACKGROUND: Although FoxP3+ regulatory T (Treg) cells constitute a highly heterogeneous population, with different regulatory potential depending on the disease context, distinct subsets or phenotypes remain poorly defined. This hampers the development of immunotherapy for allergic and autoimmune disorders. The present study aimed at characterizing distinct FoxP3+ Treg subpopulations involved in the suppression of Th2-mediated allergic inflammation in the lung. METHODS: We used an established mouse model of allergic airway disease based on ovalbumin sensitization and challenge to analyze FoxP3+ Tregs during the induction and resolution of inflammation, and identify markers that distinguish their most suppressive phenotypes. We also developed a new knock-in mouse model (Foxp3cre Cd103dtr ) enabling the specific ablation of CD103+ FoxP3+ Tregs for functional studies. RESULTS: We found that during resolution of allergic airway inflammation in mice >50% of FoxP3+ Treg cells expressed the integrin CD103 which marks FoxP3+ Treg cells of high IL-10 production, increased expression of immunoregulatory molecules such as KLRG1, ICOS and CD127, and enhanced suppressive capacity for Th2-mediated inflammatory responses. CD103+ FoxP3+ Tregs were essential for keeping allergic inflammation under control as their specific depletion in Foxp3cre Cd103dtr mice lead to severe alveocapillary damage, eosinophilic pneumonia, and markedly reduced lifespan of the animals. Conversely, adoptive transfer of CD103+ FoxP3+ Tregs effectively treated disease, attenuating Th2 responses and allergic inflammation in an IL-10-dependent manner. CONCLUSIONS: Our study identifies a novel regulatory T-cell population, defined by CD103 expression, programmed to prevent exuberant type 2 inflammation and keep homeostasis in the respiratory tract under control. This has important therapeutic implications.

Expanding Phaseolus coccineus Genomic Resources: De Novo Transcriptome Assembly and Analysis of Landraces 'Gigantes' and 'Elephantes' Reveals Rich Functional Variation.

Beans are one of the most important staple crops in the world. Runner bean (Phaseolus coccineus L.) is a small-scale agriculture crop compared to common bean (Phaseolusvulgaris). Beans have been introduced to Europe from the Central America to Europe and since then they have been scattered to different geographical regions. This has resulted in the generation of numerous local cultivars and landraces with distinguished characters and adaptive potential. To identify and characterize the underlying genomic variation of two very closely related runner bean cultivars, we performed RNA-Seq with de novo transcriptome assembly in two landraces of P. coccineus, 'Gigantes' and 'Elephantes' phenotypically distinct, differing in seed size and shape. The cleaned reads generated 37,379 and 37,774 transcripts for 'Gigantes' and 'Elephantes,' respectively. A total of 1896 DEGs were identified between the two cultivars, 1248 upregulated in 'Elephantes' and 648 upregulated in 'Gigantes.' A significant upregulation of defense-related genes was observed in 'Elephantes,' among those, numerous members of the AP2-EREBP, WRKY, NAC, and bHLH transcription factor families. In total, 3956 and 4322 SSRs were identified in 'Gigantes' and 'Elephantes,' respectively. Trinucleotide repeats were the most dominant repeat motif, accounting for 41.9% in 'Gigantes' and 40.1% in 'Elephantes' of the SSRs identified, followed by dinucleotide repeats (29.1% in both cultivars). Additionally, 19,281 putative SNPs were identified, among those 3161 were non-synonymous, thus having potential functional implications. High-confidence non-synonymous SNPs were successfully validated with an HRM assay, which can be directly adopted for P. coccineus molecular breeding. These results significantly expand the number of polymorphic markers within P. coccineus genus, enabling the robust identification of runner bean cultivars, the construction of high-resolution genetic maps, potentiating genome-wide association studies. They finally contribute to the genetic reservoir for the improvement of the closely related and intercrossable Phaseolus vulgaris.

De novo transcriptome analysis of petal senescence in Gardenia jasminoides Ellis.

BACKGROUND: The petal senescence of ethylene insensitive species has not been investigated thoroughly while little is known about the temporal and tissue specific expression patterns of transcription factors (TFs) in this developmental process. Even less is known on flower senescence of the ornamental pot plant Gardenia jasminoides, a non climacteric flower with significant commercial value. RESULTS: We initiated a de novo transcriptome study to investigate the petal senescence in four developmental stages of cut gardenia flowers considering that the visible symptoms of senescence appear within 4 days of flower opening. De novo assembly of transcriptome sequencing resulted in 102,263 contigs with mean length of 360 nucleotides that generated 57,503 unigenes. These were further clustered into 20,970 clusters and 36,533 singletons. The comparison of the consecutive developmental stages resulted in 180 common, differentially expressed unigenes. A large number of Simple Sequence Repeats were also identified comprising a large number of dinucleotides and trinucleotides. The prevailing families of differentially expressed TFs comprise the AP2/EREBP, WRKY and the bHLH. There are 81 differentially expressed TFs when the symptoms of flower senescence become visible with the most prevailing being the WRKY family with 19 unigenes. No other WRKY TFs had been identified up to now in petal senescence of ethylene insensitive species. A large number of differentially expressed genes were identified at the initiation of visible symptoms of senescence compared to the open flower stage indicating a significant shift in the expression profiles which might be coordinated by up-regulated and/or down-regulated TFs. The expression of 16 genes that belong to the TF families of WRKY, bHLH and the ethylene sensing pathway was validated using qRT--PCR. CONCLUSION: This de novo transcriptome analysis resulted in the identification of TFs with specific temporal expression patterns such as two WRKYs and one bHLH, which might play the role of senescence progression regulators. Further research is required to investigate their role in gardenia flowers in order to develop tools to delay petal senescence.

Coordinated activation of TGF-ß and BMP pathways promotes autophagy and limits liver injury after acetaminophen intoxication.

Ligands of the transforming growth factor-ß (TGF-ß) superfamily, including TGF-ßs, activins, and bone morphogenetic proteins (BMPs), have been implicated in hepatic development, homeostasis, and pathophysiology. We explored the mechanisms by which hepatocytes decode and integrate injury-induced signaling from TGF-ßs and activins (TGF-ß/Activin) and BMPs. We mapped the spatiotemporal patterns of pathway activation during liver injury induced by acetaminophen (APAP) in dual reporter mice carrying a fluorescent reporter of TGF-ß/Activin signaling and a fluorescent reporter of BMP signaling. APAP intoxication induced the expression of both reporters in a zone of cells near areas of tissue damage, which showed an increase in autophagy and demarcated the borders between healthy and injured tissues. Inhibition of TGF-ß superfamily signaling by overexpressing the inhibitor Smad7 exacerbated acute liver histopathology but eventually accelerated tissue recovery. Transcriptomic analysis identified autophagy as a process stimulated by TGF-ß1 and BMP4 in hepatocytes, with Trp53inp2, which encodes a rate-limiting factor for autophagy initiation, as the most highly induced autophagy-related gene. Collectively, these findings illustrate the functional interconnectivity of the TGF-ß superfamily signaling system, implicate the coordinated activation of TGF-ß/Activin and BMP pathways in balancing tissue reparatory and regenerative processes upon APAP-induced hepatotoxicity, and highlight opportunities and potential risks associated with targeting this signaling system for treating hepatic diseases.

Recurrent Wheeze Exacerbations Following Acute Bronchiolitis-A Machine Learning Approach.

Introduction: Acute bronchiolitis is one of the most common respiratory infections in infancy. Although most infants with bronchiolitis do not get hospitalized, infants with hospitalized bronchiolitis are more likely to develop wheeze exacerbations during the first years of life. The objective of this prospective cohort study was to develop machine learning models to predict incidence and persistence of wheeze exacerbations following the first hospitalized episode of acute bronchiolitis. Methods: One hundred thirty-one otherwise healthy term infants hospitalized with the first episode of bronchiolitis at a tertiary pediatric hospital in Athens, Greece, and 73 age-matched controls were recruited. All patients/controls were followed up for 3 years with 6-monthly telephone reviews. Through principal component analysis (PCA), a cluster model was used to describe main outcomes. Associations between virus type and the clusters and between virus type and other clinical characteristics and demographic data were identified. Through random forest classification, a prediction model with smallest classification error was identified. Primary outcomes included the incidence and the number of caregiver-reported wheeze exacerbations. Results: PCA identified 2 clusters of the outcome measures (Cluster 1 and Cluster 2) that were significantly associated with the number of recurrent wheeze episodes over 3-years of follow-up (Chi-Squared, p < 0.001). Cluster 1 included infants who presented higher number of wheeze exacerbations over follow-up time. Rhinovirus (RV) detection was more common in Cluster 1 and was more strongly associated with clinical severity on admission (p < 0.01). A prediction model based on virus type and clinical severity could predict Cluster 1 with an overall error 0.1145 (sensitivity 75.56% and specificity 91.86%). Conclusion: A prediction model based on virus type and clinical severity of first hospitalized episode of bronchiolitis could predict sensitively the incidence and persistence of wheeze exacerbations during a 3-year follow-up. Virus type (RV) was the strongest predictor.

Whole genome re-sequencing of sweet cherry (Prunus avium L.) yields insights into genomic diversity of a fruit species.

Sweet cherries, Prunus avium L. (Rosaceae), are gaining importance due to their perenniallity and nutritional attributes beneficial for human health. Interestingly, sweet cherry cultivars exhibit a wide range of phenotypic diversity in important agronomic traits, such as flowering time and defense reactions against pathogens. In this study, whole-genome resequencing (WGRS) was employed to characterize genetic variation, population structure and allelic variants in a panel of 20 sweet cherry and one wild cherry genotypes, embodying the majority of cultivated Greek germplasm and a representative of a local wild cherry elite phenotype. The 21 genotypes were sequenced in an average depth of coverage of 33.91×. and effective mapping depth, to the genomic reference sequence of 'Satonishiki' cultivar, between 22.21× to 36.62×. Discriminant analysis of principal components (DAPC) with SNPs revealed two clusters of genotypes. There was a rapid linkage disequilibrium decay, as the majority of SNP pairs with r2 in near complete disequilibrium (>0.8) were found at physical distances less than 10 kb. Functional analysis of the variants showed that the genomic ratio of non-synonymous/synonymous (dN/dS) changes was 1.78. The higher dN frequency in the Greek cohort of sweet cherry could be the result of artificial selection pressure imposed by breeding, in combination with the vegetative propagation of domesticated cultivars through grafting. The majority of SNPs with high impact (e.g., stop codon gaining, frameshift), were identified in genes involved in flowering time, dormancy and defense reactions against pathogens, providing promising resources for future breeding programs. Our study has established the foundation for further large scale characterization of sweet cherry germplasm, enabling breeders to incorporate diverse germplasm and allelic variants to fine tune flowering and maturity time and disease resistance in sweet cherry cultivars.

Systems-Based Approaches to Unravel Networks and Individual Elements Involved in Apple Superficial Scald.

Superficial scald is a major physiological disorder in apple fruit that is induced by cold storage and is mainly expressed as brown necrotic patches on peel tissue. However, a global view of the gene-protein-metabolite interactome underlying scald prevention/sensitivity is currently missing. Herein, we have found for the first time that cold storage in an atmosphere enriched with ozone (O3) induced scald symptoms in 'Granny Smith' apple fruits during subsequent ripening at room temperature. In contrast, treatment with the ethylene perception inhibitor 1-methylcyclopropene (1-MCP) reversed this O3-induced scald effect. Amino acids, including branched-chain amino acids, were the most strongly induced metabolites in peel tissue of 1-MCP treated fruits. Proteins involved in oxidative stress and protein trafficking were differentially accumulated prior to and during scald development. Genes involved in photosynthesis, flavonoid biosynthesis and ethylene signaling displayed significant alterations in response to 1-MCP and O3. Analysis of regulatory module networks identified putative transcription factors (TFs) that could be involved in scald. Subsequently, a transcriptional network of the genes-proteins-metabolites and the connected TFs was constructed. This approach enabled identification of several genes coregulated by TFs, notably encoding glutathione S-transferase (GST) protein(s) with distinct signatures following 1-MCP and O3 treatments. Overall, this study is an important contribution to future functional studies and breeding programs for this fruit, aiding to the development of improved apple cultivars to superficial scald.

Activation of both transforming growth factor-ß and bone morphogenetic protein signalling pathways upon traumatic brain injury restrains pro-inflammatory and boosts tissue reparatory responses of reactive astrocytes and microglia.

Various ligands and receptors of the transforming growth factor-ß superfamily have been found upregulated following traumatic brain injury; however, the role of this signalling system in brain injury pathophysiology is not fully characterized. To address this, we utilized an acute stab wound brain injury model to demonstrate that hallmarks of transforming growth factor-ß superfamily system activation, such as levels of phosphorylated Smads, ligands and target genes for both transforming growth factor-ß and bone morphogenetic protein pathways, were upregulated within injured tissues. Using a bone morphogenetic protein-responsive reporter mouse model, we showed that activation of the bone morphogenetic protein signalling pathway involves primarily astrocytes that demarcate the wound area. Insights regarding the potential role of transforming growth factor-ß superfamily activation in glia cells within the injured tissues were obtained indirectly by treating purified reactive astrocytes and microglia with bone morphogenetic protein-4 or transforming growth factor-ß1 and characterizing changes in their transcriptional profiles. Astrocytes responded to both ligands with considerably overlapping profiles, whereas, microglia responded selectively to transforming growth factor-ß1. Novel pathways, crucial for repair of tissue-injury and blood-brain barrier, such as activation of cholesterol biosynthesis and transport, production of axonal guidance and extracellular matrix components were upregulated by transforming growth factor-ß1 and/or bone morphogenetic protein-4 in astrocytes. Moreover, both ligands in astrocytes and transforming growth factor-ß1 in microglia shifted the phenotype of reactive glia cells towards the anti-inflammatory and tissue reparatory 'A2'-like and 'M0/M2'-like phenotypes, respectively. Increased expression of selected key components of the in vitro modulated pathways and markers of 'A2'-like astrocytes was confirmed within the wound area, suggesting that these processes could also be modulated in situ by the integrated action of transforming growth factor-ß and/or bone morphogenetic protein-mediated signalling. Collectively, our study provides a comprehensive comparative analysis of transforming growth factor-ß superfamily signalling in reactive astrocytes and microglia and points towards a crucial role of both transforming growth factor-ß and bone morphogenetic protein pathways in modulating the inflammatory and brain injury reparatory functions of activated glia cells.

Pyrrolo[2,3-a]carbazoles as potential cyclin dependent kinase 1 (CDK1) Inhibitors. Synthesis, biological evaluation, and binding mode through docking simulations.

Pyrrolo[2,3- a]carbazole derivatives were synthesized, and their effects on CDK1/cyclinB activity were evaluated. The most potent and efficacious inhibitor was found to be ethyl 9-chloro-1H-pyrrolo[2,3-alpha]carbazole-2-carboxylate (1e), exhibiting an IC50 in the low micromolar range and leading to 90% at higher concentrations. Using a computational model for CDK1-1e, binding we have observed that 1e exhibited two likely binding modes in the ATP-binding cleft that involve interactions with Lys130, Thr14, and Asp146 of the enzyme.

De novo comparative transcriptome analysis of genes involved in fruit morphology of pumpkin cultivars with extreme size difference and development of EST-SSR markers.

The genetic basis of fruit size and shape was investigated for the first time in Cucurbita species and genetic loci associated with fruit morphology have been identified. Although extensive genomic resources are available at present for tomato (Solanum lycopersicum), cucumber (Cucumis sativus), melon (Cucumis melo) and watermelon (Citrullus lanatus), genomic databases for Cucurbita species are limited. Recently, our group reported the generation of pumpkin (Cucurbita pepo) transcriptome databases from two contrasting cultivars with extreme fruit sizes. In the current study we used these databases to perform comparative transcriptome analysis in order to identify genes with potential roles in fruit morphology and fruit size. Differential Gene Expression (DGE) analysis between cv. 'Munchkin' (small-fruit) and cv. 'Big Moose' (large-fruit) revealed a variety of candidate genes associated with fruit morphology with significant differences in gene expression between the two cultivars. In addition, we have set the framework for generating EST-SSR markers, which discriminate different C. pepo cultivars and show transferability to related Cucurbitaceae species. The results of the present study will contribute to both further understanding the molecular mechanisms regulating fruit morphology and furthermore identifying the factors that determine fruit size. Moreover, they may lead to the development of molecular marker tools for selecting genotypes with desired morphological traits.

In silico Transcriptional Regulatory Networks Involved in Tomato Fruit Ripening.

Tomato fruit ripening is a complex developmental programme partly mediated by transcriptional regulatory networks. Several transcription factors (TFs) which are members of gene families such as MADS-box and ERF were shown to play a significant role in ripening through interconnections into an intricate network. The accumulation of large datasets of expression profiles corresponding to different stages of tomato fruit ripening and the availability of bioinformatics tools for their analysis provide an opportunity to identify TFs which might regulate gene clusters with similar co-expression patterns. We identified two TFs, a SlWRKY22-like and a SlER24 transcriptional activator which were shown to regulate modules by using the LeMoNe algorithm for the analysis of our microarray datasets representing four stages of fruit ripening, breaker, turning, pink and red ripe. The WRKY22-like module comprised a subgroup of six various calcium sensing transcripts with similar to the TF expression patterns according to real time PCR validation. A promoter motif search identified a cis acting element, the W-box, recognized by WRKY TFs that was present in the promoter region of all six calcium sensing genes. Moreover, publicly available microarray datasets of similar ripening stages were also analyzed with LeMoNe resulting in TFs such as SlERF.E1, SlERF.C1, SlERF.B2, SLERF.A2, SlWRKY24, SLWRKY37, and MADS-box/TM29 which might also play an important role in regulation of ripening. These results suggest that the SlWRKY22-like might be involved in the coordinated regulation of expression of the six calcium sensing genes. Conclusively the LeMoNe tool might lead to the identification of putative TF targets for further physiological analysis as regulators of tomato fruit ripening.

De novo transcriptome assembly of two contrasting pumpkin cultivars.

Cucurbita pepo (squash, pumpkin, gourd), a worldwide-cultivated vegetable of American origin, is extremely variable in fruit characteristics. However, the information associated with genes and genetic markers for pumpkin is very limited. In order to identify new genes and to develop genetic markers, we performed a transcriptome analysis (RNA-Seq) of two contrasting pumpkin cultivars. Leaves and female flowers of cultivars, 'Big Moose' with large round fruits and 'Munchkin' with small round fruits, were harvested for total RNA extraction. We obtained a total of 6 GB (Big Moose; http://www.ncbi.nlm.nih.gov/Traces/sra/?run=SRR3056882) and 5 GB (Munchkin; http://www.ncbi.nlm.nih.gov/Traces/sra/?run=SRR3056883) sequence data (NCBI SRA database SRX1502732 and SRX1502735, respectively), which correspond to 18,055,786 and 14,824,292 150-base reads. After quality assessment, the clean sequences where 17,995,932 and 14,774,486 respectively. The numbers of total transcripts for 'Big Moose' and 'Munchkin' were 84,727 and 68,051, respectively. TransDecoder identified possible coding regions in assembled transcripts. This study provides transcriptome data for two contrasting pumpkin cultivars, which might be useful for genetic marker development and comparative transcriptome analyses.

454 Pyrosequencing of Olive (Olea europaea L.) Transcriptome in Response to Salinity.

Olive (Olea europaea L.) is one of the most important crops in the Mediterranean region. The expansion of cultivation in areas irrigated with low quality and saline water has negative effects on growth and productivity however the investigation of the molecular basis of salt tolerance in olive trees has been only recently initiated. To this end, we investigated the molecular response of cultivar Kalamon to salinity stress using next-generation sequencing technology to explore the transcriptome profile of olive leaves and roots and identify differentially expressed genes that are related to salt tolerance response. Out of 291,958 obtained trimmed reads, 28,270 unique transcripts were identified of which 35% are annotated, a percentage that is comparable to similar reports on non-model plants. Among the 1,624 clusters in roots that comprise more than one read, 24 were differentially expressed comprising 9 down- and 15 up-regulated genes. Respectively, inleaves, among the 2,642 clusters, 70 were identified as differentially expressed, with 14 down- and 56 up-regulated genes. Using next-generation sequencing technology we were able to identify salt-response-related transcripts. Furthermore we provide an annotated transcriptome of olive as well as expression data, which are both significant tools for further molecular studies in olive.

Transcriptional profiling unravels potential metabolic activities of the olive leaf non-glandular trichome.

The olive leaf trichomes are multicellular peltate hairs densely distributed mainly at the lower leaf epidermis. Although, non-glandular, they have gained much attention since they significantly contribute to abiotic and biotic stress tolerance of olive leaves. The exact mechanisms by which olive trichomes achieve these goals are not fully understood. They could act as mechanical barrier but they also accumulate high amounts of flavonoids among other secondary metabolites. However, little is currently known about the exact compounds they produce and the respective metabolic pathways. Here we present the first EST analysis from olive leaf trichomes by using 454-pyrosequencing. A total of 5368 unigenes were identified out of 7258 high quality reads with an average length of 262 bp. Blast search revealed that 27.5% of them had high homologies to known proteins. By using Blast2GO, 1079 unigenes (20.1%) were assigned at least one Gene Ontology (GO) term. Most of the genes were involved in cellular and metabolic processes and in binding functions followed by catalytic activity. A total of 521 transcripts were mapped to 67 KEGG pathways. Olive trichomes represent a tissue of highly unique transcriptome as per the genes involved in developmental processes and the secondary metabolism. The results indicate that mature olive trichomes are trancriptionally active, mainly through the potential production of enzymes that contribute to phenolic compounds with important roles in biotic and abiotic stress responses.

Modeling regulatory cascades using Artificial Neural Networks: the case of transcriptional regulatory networks shaped during the yeast stress response.

Over the last decade, numerous computational methods have been developed in order to infer and model biological networks. Transcriptional networks in particular have attracted significant attention due to their critical role in cell survival. The majority of network inference methods use genome-wide experimental data to search for modules of genes with coherent expression profiles and common regulators, often ignoring the multi-layer structure of transcriptional cascades. Modeling methodologies on the other hand assume a given network structure and vary significantly in their algorithmic approach, ranging from over-simplified representations (e.g., Boolean networks) to detailed -but computationally expensive-network simulations (e.g., with differential equations). In this work we use Artificial Neural Networks (ANNs) to model transcriptional regulatory cascades that emerge during the stress response in Saccharomyces cerevisiae and extend in three layers. We confine the structure of the ANNs to match the structure of the biological networks as determined by gene expression, DNA-protein interaction and experimental evidence provided in publicly available databases. Trained ANNs are able to predict the expression profile of 11 target genes across multiple experimental conditions with a correlation coefficient >0.7. When time-dependent interactions between upstream transcription factors (TFs) and their indirect targets are also included in the ANNs, accurate predictions are achieved for 30/34 target genes. Moreover, heterodimer formation is taken into account. We show that ANNs can be used to (1) accurately predict the expression of downstream genes in a 3-layer transcriptional cascade based on the expression of their indirect regulators and (2) infer the condition- and time-dependent activity of various TFs as well as during heterodimer formation. We show that a three-layer regulatory cascade whose structure is determined by co-expressed gene modules and their regulators can successfully be modeled using ANNs with a similar configuration.

Comparative transcriptome analysis of two olive cultivars in response to NaCl-stress.

BACKGROUND: Olive (Olea europaea L.) cultivation is rapidly expanding and low quality saline water is often used for irrigation. The molecular basis of salt tolerance in olive, though, has not yet been investigated at a system level. In this study a comparative transcriptomics approach was used as a tool to unravel gene regulatory networks underlying salinity response in olive trees by simulating as much as possible olive growing conditions in the field. Specifically, we investigated the genotype-dependent differences in the transcriptome response of two olive cultivars, a salt-tolerant and a salt-sensitive one. METHODOLOGY/PRINCIPAL FINDINGS: A 135-day long salinity experiment was conducted using one-year old trees exposed to NaCl stress for 90 days followed by 45 days of post-stress period during the summer. A cDNA library made of olive seedling mRNAs was sequenced and an olive microarray was constructed. Total RNA was extracted from root samples after 15, 45 and 90 days of NaCl-treatment as well as after 15 and 45 days of post-treatment period and used for microarray hybridizations. SAM analysis between the NaCl-stress and the post-stress time course resulted in the identification of 209 and 36 differentially expressed transcripts in the salt-tolerant and salt-sensitive cultivar, respectively. Hierarchical clustering revealed two major, distinct clusters for each cultivar. Despite the limited number of probe sets, transcriptional regulatory networks were constructed for both cultivars while several hierarchically-clustered interacting transcription factor regulators such as JERF and bZIP homologues were identified. CONCLUSIONS/SIGNIFICANCE: A systems biology approach was used and differentially expressed transcripts as well as regulatory interactions were identified. The comparison of the interactions among transcription factors in olive with those reported for Arabidopsis might indicate similarities in the response of a tree species with Arabidopsis at the transcriptional level under salinity stress.