Climate and landscape composition explain agronomic practices, pesticide use and grape yield in vineyards across Italy

Context Worldwide, organic farming is being promoted as one of the main alternatives to intensive conventional farming. However, the benefits of organic agriculture are still controversial and need to be tested across wide environmental gradients. Objective Here, we carried out an observational study to test how agronomic practices, pest management, environmental impact and yield of conventional and organic vineyards changed along wide climatic and landscape gradients across Italy. Methods We used a block design with 38 pairs of conventional and organic vineyards across Italy. Results and conclusions Most agronomic practices did not differ between conventional and organic vineyards. By contrast, landscape composition and climate were strong predictors of management in both systems. First, increasing semi-natural areas around the vineyards reduced pesticide pressure and related environmental impacts, but was also associated with lower yield. Second, irrespective of the farming system, a warm and dry climate was associated with reduced fungicide pressure. Conventional farming had a yield gain of 40% in cold and wet climate compared to organic but the yield gap disappeared in the warmest regions. Significance In both farming systems, we observed a large variability in management practices that was mainly explained by climate and landscape composition. This large variability should be considered when evaluating the benefits and drawbacks of different farming systems under contrasting environmental contexts

TrkAIII Promotes Microtubule Nucleation and Assembly at the Centrosome in SH-SY5Y Neuroblastoma Cells, Contributing to an Undifferentiated Anaplastic Phenotype

The alternative TrkAIII splice variant is expressed by advanced stage human neuroblastomas (NBs) and exhibits oncogenic activity in NB models. In the present study, employing stable transfected cell lines and assays of indirect immunofluorescence, immunoprecipitation, Western blotting, microtubule regrowth, tubulin kinase, and tubulin polymerisation, we report that TrkAIII binds α-tubulin and promotes MT nucleation and assembly at the centrosome. This effect depends upon spontaneous TrkAIII activity, TrkAIII localisation to the centrosome and pericentrosomal area, and the capacity of TrkAIII to bind, phosphorylate, and polymerise tubulin. We propose that this novel role for TrkAIII contributes to MT involvement in the promotion and maintenance of an undifferentiated anaplastic NB cell morphology by restricting and augmenting MT nucleation and assembly at the centrosomal MTOC

The TrkAIII Oncoprotein Inhibits Mitochondrial Free Radical ROS-Induced Death of SH-SY5Y Neuroblastoma Cells by Augmenting SOD2 Expression and Activity at the Mitochondria, within the Context of a Tumour Stem Cell-like Phenotype

The developmental and stress-regulated alternative TrkAIII splice variant of the NGF receptor TrkA is expressed by advanced stage human neuroblastomas (NBs), correlates with worse outcome in high TrkA expressing unfavourable tumours and exhibits oncogenic activity in NB models. In the present study, we report that constitutive TrkAIII expression in human SH-SY5Y NB cells inhibits Rotenone, Paraquat and LY83583-induced mitochondrial free radical reactive oxygen species (ROS)-mediated death by stimulating SOD2 expression, increasing mitochondrial SOD2 activity and attenuating mitochondrial free radical ROS production, in association with increased mitochondrial capacity to produce H2O2, within the context of a more tumour stem cell-like phenotype. This effect can be reversed by the specific TrkA tyrosine kinase inhibitor GW441756, by the multi-kinase TrkA inhibitors K252a, CEP-701 and Gö6976, which inhibit SOD2 expression, and by siRNA knockdown of SOD2 expression, which restores the sensitivity of TrkAIII expressing SH-SY5Y cells to Rotenone, Paraquat and LY83583-induced mitochondrial free radical ROS production and ROS-mediated death. The data implicate the novel TrkAIII/SOD2 axis in promoting NB resistance to mitochondrial free radical-mediated death and staminality, and suggest that the combined use of TrkAIII and/or SOD2 inhibitors together with agents that induce mitochondrial free radical ROS-mediated death could provide a therapeutic advantage that may also target the stem cell niche in high TrkA expressing unfavourable NB

The TrkAIII oncoprotein inhibits mitochondrial free radical ROS-induced death of SH-SY5Y neuroblastoma cells by augmenting SOD2 expression and activity at the mitochondria, within the context of a tumour stem cell-like phenotype.

The developmental and stress-regulated alternative TrkAIII splice variant of the NGF receptor TrkA is expressed by advanced stage human neuroblastomas (NBs), correlates with worse outcome in high TrkA expressing unfavourable tumours and exhibits oncogenic activity in NB models. In the present study, we report that constitutive TrkAIII expression in human SH-SY5Y NB cells inhibits Rotenone, Paraquat and LY83583-induced mitochondrial free radical reactive oxygen species (ROS)-mediated death by stimulating SOD2 expression, increasing mitochondrial SOD2 activity and attenuating mitochondrial free radical ROS production, in association with increased mitochondrial capacity to produce H2O2, within the context of a more tumour stem cell-like phenotype. This effect can be reversed by the specific TrkA tyrosine kinase inhibitor GW441756, by the multi-kinase TrkA inhibitors K252a, CEP-701 and Gö6976, which inhibit SOD2 expression, and by siRNA knockdown of SOD2 expression, which restores the sensitivity of TrkAIII expressing SH-SY5Y cells to Rotenone, Paraquat and LY83583-induced mitochondrial free radical ROS production and ROS-mediated death. The data implicate the novel TrkAIII/SOD2 axis in promoting NB resistance to mitochondrial free radical-mediated death and staminality, and suggest that the combined use of TrkAIII and/or SOD2 inhibitors together with agents that induce mitochondrial free radical ROS-mediated death could provide a therapeutic advantage that may also target the stem cell niche in high TrkA expressing unfavourable NB

Alendronate promotes plasmin-mediated MMP-9 inactivation by exposing cryptic plasmin degradation sites within the MMP-9 catalytic domain

Irreversible MMP-9 inhibition is considered a significant therapeutic goal in inflammatory, vascular and tumour pathology. We report that divalent cation chelators Alendronate and EDTA not only directly inhibited MMP-9 but also promoted irreversible plasmin-mediated MMP-9 inactivation by exposing cryptic plasmin-degradation sites within the MMP-9 catalytic-domain and producing an inhibitory hemopexin-domain fragment. This effect was also observed using MDA-MB-231 breast cancer cells, which activated exogenous plasminogen to degrade endogenous proMMP-9 in the presence of Alendronate or EDTA. Degradation-mediated inactivation of proMMP-9 occurred in the absence of transient activation, attesting to the incapacity of plasmin to directly activate proMMP-9 and direct MMP-9 inhibition by Alendronate and EDTA. Our study provides a novel rational for therapeutic Alendronate use in MMP-9-dependent pathology characterised by plasminogen activation. Crown Copyright © 2012 Published by Elsevier B.V. on behalf of Federation of European Biochemical society. All rights reserved

Constitutive autotaxin transcription by Nmyc-amplified and non-amplified neuroblastoma cells is regulated by a novel AP-1 and SP-mediated mechanism and abrogated by curcumin

The motility, angiogenesis and metastasis-stimulating factor Autotaxin (Atx), over expressed by human neuroblastomas (NB), is constitutively expressed by human Nmyc-amplified SK-N-BE and non-Nmyc-amplified SH-SY5Y NB cells. Here, we characterise a novel Atx transcriptional mechanism, utilised by both cell lines, that is restricted to the first 285 bp of the Atx promoter and involves AP-1 and SP transcription factors, acting through a CRE/AP-1-like element at position -142 to -149 and a GAbox at position -227 to -235 relative to the Atx translational start site. This novel transcriptional mechanism can be inhibited by internally initiated SP-3 and the natural phenol curcumin. © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved

TrkAIII promotion of SH-SY5Y spheroid growth is inhibited by GW441756 and associates with stem cell marker expression.

<p>A) Representative digital phase-contrast micrographs, demonstrating differences in the relative size of 20-day pcDNA, TrkA and TrkAIII SH-SY5Y tumour spheroids (bar = 100 µm), plus a histogram showing the difference in 20-day pcDNA, TrkA and TrkAIII SH-SY5Y tumour spheroid volumes displayed as the mean+s.d. volume (mm³) of 50 individual tumour spheres per cell line, in three independent experiments. Mean values are provided above each histogram column and asterisks denote statistical significance by t-test comparison of TrkAIII SH-SY5Y cells to either pcDNA or TrkA SH-SY5Y cells. B) Digital phase-contrast micrographs demonstrating differences in the relative tumour spheroid sizes in TrkA and TrkAIII SH-SY5Y cells cultured for 20 days in the absence (Control) or presence of 100 nM or 1 µM GW441756 (bar = 100 µm), plus a histogram showing the difference in TrkA and TrkAIII SH-SY5Y tumour spheroid volumes, cultured as described above, displayed as the mean+s.d. volume (mm³) of 50 individual tumour spheres per cell line, in three independent experiments. Mean values are provided above each histogram column and asterisks denote statistical significance by t-test comparison of GW441756-treated and untreated TrkAIII SH-SY5Y cells. C) Ethidium bromide stained agarose gels demonstrating the relative levels of 4.2 kb SOD2, 1.5 kb SOD2 and GAPDH RT-PCR products in 20-day pcDNA, TrkA and TrkAIII SH-SY5Y spheroids, plus a histogram depicting the relative densitometric differences in 4.2 kb SOD2 and 1.5 kb SOD2 mRNA expression. Results are displayed as mean+s.d. densitometric ratio to GAPDH, in three independent experiments. Mean values are provided above each histogram column and asterisks denote statistical significance by t-test comparison of TrkAIII to both pcDNA and TrkA SH-SY5Y cells. D) Ethidium bromide stained agarose gels demonstrating differences in CD133, CD117, SOX-2, Nestin, Nanog and GAPDH RT-PCR products in 20-day pcDNA, TrkA and TrkAIII SH-SY5Y spheroids, plus histograms depicting fold change in (I), CD117, (II) SOX-2, (III) Nestin, (IV) Nanog and (V) CD133 expression in 20-day tumour spheroids. Results are displayed as mean±s.d. fold change compared to pcDNA SH-SY5Y cells, given the arbitrary value of 1 as a densitometric ratio to GAPDH expression, in three independent experiments. Mean values are provided above each histogram column and asterisks denote statistical significance by t-test comparison of TrkAIII to pcDNA SH-SY5Y cells.</p

SOD2 expression in TrkAIII SH-SY5Y cells is reduced by TrkA, IP3K and NF-κB inhibitors.

<p>A) Western blots demonstrating relative differences in TrkA, TrkAIII, phosphorylated TrkA (pY674/675-TrkA), SOD2, Trx-2 and α-tubulin levels in equal protein concentrations (20 µg) of total extracts from TrkA and TrkAIII SH-SY5Y cell lines, treated without (Con) or with 1 µM CEP-701 (CEP), 1 µM K252a (K2); 25 µM LY294002 (LY294) or 1 µM Gö6976 (Gö69) for 24 hours, at 37°C. B) Representative Western blots demonstrating relative differences in SOD2 and α-tubulin levels in equal protein concentrations (20 µg) of total extracts from TrkA and TrkAIII SH-SY5Y cell lines treated without (Con) or with: 100 nM GW441756 (GW44), 500 µM PDTC (PDTC), 100 µM Bay 11–7082 (BAY11) or 30 µM PD098059 (PD098). C) Representative Western blot of total cell extracts (20 µg), demonstrating inhibition of constitutive TrkAIII Y674/675 phosphorylation (Con) following 3 hours treatment of TrkAIII transfectants with 100 nM GW441756 (GW44). D) Histograms showing the densitometric differences in the SOD2 and Trx-2 levels, as a ratio to α-tubulin, in equal concentrations (20 µg) of total cell extracts from TrkA and TrkAIII SH-SY5Y cells treated without (Con) or with: 1 µM CEP-701 (CEP); 1 µM K252a (K2); 25 µM LY294002 (LY294); 1 µM Gö6976 (Gö69); 100 nM GW441756 (GW44); 500 µM PDTC (PDTC); 100 µM Bay 11–7082 (BAY11) or 30 µM PD098059 (PD098), for 24 hours at 37°C. Results are displayed as mean±s.d fold change compared to untreated controls, adjusted to the arbitrary value of 1±s.d., in 3 independent experiments. Mean values are presented above each column and asterisks denote statistical significance by t-test comparison of treated and untreated TrkAIII SH-SY5Y cells. E) Representative Western blots demonstrating differences in TrkAIII, phosphorylated TrkAIII (pTrkAIII), SOD2 and α-tubulin levels in equal concentrations (20 µg) of total cell extracts from wt-TrkAIII and kd-TrkAIII SH-SY5Y cell lines plus a histogram depicting the densitometric difference in the SOD2 levels as a ratio to α-tubulin, in total cell extracts from wt-TrkAIII and kd-TrkAIII SH-SY5Y cells, displayed as mean±s.d fold difference in densitometric ratio with respect to wt-TrkAIII transfectants adjusted to the arbitrary value of 1±s.d., in three independent experiments. Mean values are presented above each column and asterisks denote statistical significance by t-test comparison of wt and kd TrkAIII transfectants. F) Representative Western blots demonstrating levels of TrkA, tyrosine phosphorylated TrkA (pTrkA), SOD2 and α-tubulin in equal concentrations (20 µg) of total cell extracts from untreated TrkA SH-SY5Y cells (Con) and TrkA SH-SY5Y cells treated for 30, 60 and 180 minutes with 100 ng/ml NGF, plus a histogram showing densitometric differences in the SOD2: α-tubulin ratio in total cell extracts from untreated (Con) and NGF-treated TrkA SH-SY5Y cells (100 ng/ml for 30, 60 and 180 minutes), displayed as mean±s.d. fold difference in SOD2 levels with respect to untreated controls adjusted to the arbitrary value of 1±s.d., in 3 independent experiments. Mean values are provided above each column and asterisks denote statistical significance by t-test comparison between NGF-treated and untreated TrkA SH-SY5Y controls.</p