Partial Activation of SA- and JA-Defensive Pathways in Strawberry upon Colletotrichum acutatum Interaction

[EN] Understanding the nature of pathogen host interaction may help improve strawberry (Fragaria x anahassa) cultivars. Plant resistance to pathogenic agents usually operates through a complex network of defense mechanisms mediated by a diverse array of signaling molecules. In strawberry, resistance to a variety of pathogens has been reported to be mostly polygenic and quantitatively inherited, making it difficult to associate molecular markers with disease resistance genes. Colletotrichum acutaturn spp. is a major strawberry pathogen, and completely resistant cultivars have not been reported. Moreover, strawberry defense network components and mechanisms remain largely unknown and poorly understood. Assessment of the strawberry response to C. acutatum included a global transcript analysis, and acidic hormones SA and JA measurements were analyzed after challenge with the pathogen. Induction of transcripts corresponding to the SA and JA signaling pathways and key genes controlling major steps within these defense pathways was detected. Accordingly, SA and JA accumulated in strawberry after infection. Contrastingly, induction of several important SA, JA, and oxidative stress-responsive defense genes, including FaPR1-1, FaLOX2, FaJAR1, FaPDF1, and FaGST1, was not detected, which suggests that specific branches in these defense pathways (those leading to FaPR1-2, FaPR2-1, FaPR2-2, FaAOS, FaPR5, and FaPR10) were activated. Our results reveal that specific aspects in SA and JA dependent signaling pathways are activated in strawberry upon interaction with C. acutatum. Certain described defense-associated transcripts related to these two known signaling pathways do not increase in abundance following infection. This finding suggests new insight into a specific putative molecular strategy for defense against this pathogen.Authors are grateful to Dr. JM Lopez-Aranda (IFAPA-Centro de Churriana) for providing micropropagated strawberry plants and to Nicolas Garcia-Caparros for technical assistance. Authors also want to thank Kevin M. Folta for his insightful comments on the paper. This work was supported by Junta de Andalucia, Spain [Proyectos de Excelencia P07-AGR-02482/P12-AGR-2174, and grants to Grupo-BIO278].Amil-Ruiz, F.; Garrido-Gala, J.; Gadea Vacas, J.; Blanco-Portales, R.; Munoz-Merida, A.; Trelles, O.; De Los Santos, B.... (2016). Partial Activation of SA- and JA-Defensive Pathways in Strawberry upon Colletotrichum acutatum Interaction. Frontiers in Plant Science. 7(1036). https://doi.org/10.3389/fpls.2016.01036S71036Acosta, I. F., & Farmer, E. E. (2010). Jasmonates. The Arabidopsis Book, 8, e0129. doi:10.1199/tab.0129Al-Shahrour, F., Diaz-Uriarte, R., & Dopazo, J. (2004). 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The existence of strong genetic structure is expected in species with limited ability to disperse and philopatric behaviour. These life-history traits are found in many small benthic elasmobranchs, such as in the small-spotted catshark (Scyliorhinus canicula). However, no evidence of genetic structure was found across its northeastern Atlantic (NEA) range using traditional molecular markers. Here, fine-scale genetic differentiation was detected between the British Isles and southern Iberia using 2674 single nucleotide polymorphism loci generated using 2b-restriction siteassociated DNA (2b-RAD). Geographical distance and historical demography were two major drivers shaping the distribution of genetic diversity of S. canicula along the NEA. Significant positive spatial autocorrelation of allelic frequencies was detected, with genetic differentiation generally increasing with geographical distance. However, marked genetic divergence of the Celtic Sea and South Portugal collections from their closest neighbours resulted in geographically constrained genetic breaks south of the British Isles and off southwestern Iberia. Historical demographic reconstruction of population pairs across these genetic breaks suggested a scenario of historical isolation before secondary contact, probably related to distinct northern and southern glacial refugia. These results provide new insights into the population structure of S. canicula along the NEA and serve as a reference for benthic elasmobranchs with similar distribution ranges

Dysregulation of the repressive H3K27 trimethylation mark in head and neck squamous cell carcinoma contributes to dysregulated squamous differentiation

Recent studies have reported that epigenetic mechanisms may regulate the initiation and progress of squamous differentiation in normal and transformed keratinocytes. In particular, the role of the repressive H3K27me3 mark in the regulation of squamous differentiation has been prominent. However, there is conflicting literature showing that squamous differentiation may be dependent upon or independent of changes in H3K27me3 status. In this study we have examined the binding of trimethylated H3K27 to the promoters of proliferation or differentiation genes in keratinocytes undergoing squamous differentiation in vitro and in vivo. Initially, we examined the expression levels for EZH1, EZH2, and H3K27me3 in differentiating keratinocytes in vitro and in vivo. We extended this to include H3K27me3 chromatin immunoprecipitation sequencing (ChIP-seq). Based on these studies, we could find no evidence for an association between widespread gain or loss of H3K27me3 on the promoters of proliferation-specific or differentiation-specific target genes, respectively, during squamous differentiation in adult human keratinocytes. These data suggest that squamous differentiation may occur independent of regulation by H3K27me3 on proliferation and differentiation genes of normal adult human keratinocytes

Brevi note sul divieto di mutatio libelli (anche) nella fase sommaria del c.d. “Rito Fornero”

Pulmonary metastasis is the major untreatable complication of osteosarcoma (OS) resulting in 10-20% long-term survival. The factors and pathways regulating these processes remain unclear, yet their identification is crucial in order to find new therapeutic targets. In this study we used a multi-omics approach to identify molecules in metastatic and non-metastatic OS cells that may contribute to OS metastasis, followed by validation in vitro and in vivo. We found elevated levels of the urokinase plasminogen activator (uPA) and of the uPA receptor (uPAR) exclusively in metastatic OS cells. uPA was secreted in soluble form and as part of the protein cargo of OS-secreted extracellular vesicles, including exosomes. In addition, in the tumour microenvironment, uPA was expressed and secreted by bone marrow cells (BMC), and OS- and BMC-derived uPA significantly and specifically stimulated migration of metastatic OS cells via uPA-dependent signaling pathways. Silencing of uPAR in metastatic OS cells abrogated the migratory response to uPA in vitro and decreased metastasis in vivo. Finally, a novel small-molecule inhibitor of uPA significantly (P = 0.0004) inhibited metastasis in an orthotopic mouse model of OS. Thus, we show for the first time that malignant conversion of OS cells to a metastatic phenotype is defined by activation of the uPA/uPAR axis in both an autocrine and paracrine fashion. Furthermore, metastasis is driven by changes in OS cells as well as in the microenvironment. Finally, our data show that pharmacological inhibition of the uPA/uPAR axis with a novel small-molecule inhibitor can prevent the emergence of metastatic foci

A novel E2F/sphingosine kinase 1 axis regulates anthracycline response in squamous cell carcinoma

Purpose: Head and neck squamous cell carcinomas (HNSCC) are frequently drug resistant and have a mortality rate of 45%. We have previously shown that E2F7 may contribute to drug resistance in SCC cells. However, the mechanism and pathways involved remain unknown

Bone marrow increases the migration of metastatic OS through uPA/uPAR.

<p><b>(A)</b> Migration of metastatic and non-metastatic OS cell lines in the presence of uPA-rich medium (20–80 U/mL) collected from bone marrow cell cultures (BMC). Percentage migration is normalized against KHOS control. Bars: SEM. ***<i>P</i> <0.0001, **<i>P</i> = 0.0084. Experiments were performed in triplicate at least twice. <b>(B)</b> Immunohistochemistry of the same FFPE section of mouse bone showing expression of uPA in bone marrow cells (right panels). Left panels: negative rabbit IgG (Dako), 2 μg/mL. Right panels: rabbit anti-human uPA (H-140) (Santa Cruz Biotechnology), 1:100 (2 μg/mL). The top panels show strong uPA staining in an area in the proximal epiphysis near the epiphyseal line, an area of rapid bone turnover, rich in osteoblasts and osteoclasts which secrete uPA. The bottom panels show and area in the diaphysis in the border of the medullary cavity and compact bone, where osteoblasts and osteoclasts are not so abundant. Magnification: 10X. <b>(C)</b> Activity of uPA in various batches (A-D) of bone marrow conditioned medium measured by a uPA Activity Assay Kit (Merck Millipore). <b>(D)</b> Migration of KHOS cells in the presence of normal growth medium (control) and each of the batches (A-D) of bone marrow conditioned medium in (C). Percentage migration is normalized against KHOS control. Bars: SEM. *<i>P</i> < 0.02, **<i>P</i> < 0.009. Experiments were performed in triplicate at least twice. <b>(E)</b> Linear regression of uPA activity <i>vs</i>. migration. Slope = 0.7589 ± 0.3693; Y-intercept when X = 0.0 is -65.61 ± -53.78; X-intercept when Y = 0.0 is 86.45.</p

Top hits from (A) transcriptomic, (B, D) proteomic and (C) secretomic analyses.

<p>Top hits from (A) transcriptomic, (B, D) proteomic and (C) secretomic analyses.</p

uPAR silencing inhibits migration and metastasis of OS.

<p><b>(A)</b> Migration of KHOS WT and KHOS uPAR-KD in the absence (control) or presence of 100 nM uPA (5.4 μg/mL). Percentage migration is normalized against KHOS. Bars: SEM. ***<i>P</i> < 0.0002. <b>(B)</b> Migration of KHOS wild-type and uPAR-KD cells in the presence of growth medium (control), 100 nM uPA amino terminal fragment (ATF) or high molecular weight (HMW) uPA. Percentage migration is normalized against KHOS. Migration experiments were performed in triplicate at least twice. Bars: SEM. ***<i>P</i> ≤ 0.0002. <b>(C)</b> Reduction of Erk1/2 phosphorylation in uPAR-KD cells but not in WT after 24 h treatment with 100 nM rh-uPA. <b>(D)</b> Metastatic burden (% area of lung covered by metastatic lesions/% total lung area) in mice (<i>n</i> = 5) injected with KHOS WT, uPAR-KD or uPAR-SCR cells. *<i>P =</i> 0.013. Bars: SEM. <b>(E)</b> Quantitative image analysis of DAB-stained FFPE sections of KHOS WT primary tumour and the corresponding lungs after immunohistochemistry with goat anti-human uPAR (Santa Cruz), 1:100. <b>(F)</b> Western blot showing expression of CD63 in KHOS-secreted extracellular vesicles (EV) but not in KHOS whole cell extract (WCE), and expression of uPA in both EV and WCE.</p

Inhibition of OS metastasis by the uPA inhibitor, WX-340.

<p><b>(A)</b> Metastatic burden (% area covered by metastatic lesions/% total lung area) in animals treated with vehicle (control) (<i>n</i> = 14) or treated with 10 mg/kg WX-340 (<i>n</i> = 15) i.p. 3 times/week. Left: scatter plot. Right: Bars: SEM. ***<i>P</i> = 0.0004. <b>(B)</b> Average tumour growth of mice in (A).</p