Arbuscular Mycorrhizal Symbiosis Differentially Affects the Nutritional Status of Two Durum Wheat Genotypes under Drought Conditions

Durum wheat is one of the most important agricultural crops, currently providing 18% of the daily intake of calories and 20% of daily protein intake for humans. However, being wheat that is cultivated in arid and semiarid areas, its productivity is threatened by drought stress, which is being exacerbated by climate change. Therefore, the identification of drought tolerant wheat genotypes is critical for increasing grain yield and also improving the capability of crops to uptake and assimilate nutrients, which are seriously affected by drought. This work aimed to determine the effect of arbuscular mycorrhizal fungi (AMF) on plant growth under normal and limited water availability in two durum wheat genotypes (Svevo and Etrusco). Furthermore, we investigated how the plant nutritional status responds to drought stress. We found that the response of Svevo and Etrusco to drought stress was differentially affected by AMF. Interestingly, we revealed that AMF positively affected sulfur homeostasis under drought conditions, mainly in the Svevo cultivar. The results provide a valuable indication that the identification of drought tolerant plants cannot ignore their nutrient use efficiency or the impact of other biotic soil components (i.e., AMF)

von Willebrand factor-cleaving protease (ADAMTS13) activity in normal non-pregnant women, pregnant and post-delivery women

ADAMTS13 dysfunction has been involved in the pathogenesis of Thrombotic Thrombocytopenic Purpura. This disorder occurs more frequently in women and, in 13% of them, is associated with pregnancy. However, there is little information on the protease behaviour in normal pregnancy. We studied von Willebrand factor and ADAMTS13 activity changes in normal non-pregnant, pregnant and post-delivery women. Fifty-five non-pregnant women, normal blood bank donors, who were not taking contraceptive pills were included as controls. A prospective cross-sectional study of 270 normal pregnant and post-delivery women was carried out. ADAMTS13 activity decreased progressively as from the period of 12-16 weeks up to the end of early puerperium (mean 52%, range 22-89, p < 0.0001), to increase slightly thereafter. Nulliparous presented mildly lower levels of ADAMTS13 activity than parous women (65% vs. 83%, p=0.0003), and primigravidae than multigravidae between 6-11 weeks up to 17-23 weeks of pregnancy (69% vs. 80%, p=0.005). Although in all women the protease levels were the same by blood groups, the O blood group non-pregnant women showed a higher mean of ADAMTS 13 activity than those non-O (78% vs. 69%, p= 0.064). Our results suggest that the changing levels of protease activity during pregnancy and puerperium, induced by unidentified mechanisms, could render the peripartum time more vulnerable to developed thrombotic microangiopathies.Fil: Sánchez Luceros, Analía Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Farias, Cristina Elena. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Amaral, María Marta. Academia Nacional de Medicina de Buenos Aires. Instituto de Investigaciones Hematológicas "Mariano R. Castex". Departamento de Hemostasia y Trombosis; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Kempfer, Ana Catalina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Votta, Roberto. Ciudad Autónoma de Buenos Aires. Hospital General de Agudos "Dr. C. Argerich"; ArgentinaFil: Marchese, Carlos. Ciudad Autónoma de Buenos Aires. Hospital General de Agudos "Dr. C. Argerich"; ArgentinaFil: Salviú, María J.. Academia Nacional de Medicina de Buenos Aires. Instituto de Investigaciones Hematológicas "Mariano R. Castex". Departamento de Hemostasia y Trombosis; ArgentinaFil: Woods, Adriana Inés. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Meschengieser, Susana S.. Academia Nacional de Medicina de Buenos Aires. Instituto de Investigaciones Hematológicas "Mariano R. Castex". Departamento de Hemostasia y Trombosis; ArgentinaFil: Lazzari, María Ángela. Academia Nacional de Medicina de Buenos Aires. Instituto de Investigaciones Hematológicas "Mariano R. Castex". Departamento de Hemostasia y Trombosis; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Protein Kinase A Activation Promotes Cancer Cell Resistance to Glucose Starvation and Anoikis.

Cancer cells often rely on glycolysis to obtain energy and support anabolic growth. Several studies showed that glycolytic cells are susceptible to cell death when subjected to low glucose availability or to lack of glucose. However, some cancer cells, including glycolytic ones, can efficiently acquire higher tolerance to glucose depletion, leading to their survival and aggressiveness. Although increased resistance to glucose starvation has been shown to be a consequence of signaling pathways and compensatory metabolic routes activation, the full repertoire of the underlying molecular alterations remain elusive. Using omics and computational analyses, we found that cyclic adenosine monophosphate-Protein Kinase A (cAMP-PKA) axis activation is fundamental for cancer cell resistance to glucose starvation and anoikis. Notably, here we show that such a PKA-dependent survival is mediated by parallel activation of autophagy and glutamine utilization that in concert concur to attenuate the endoplasmic reticulum (ER) stress and to sustain cell anabolism. Indeed, the inhibition of PKA-mediated autophagy or glutamine metabolism increased the level of cell death, suggesting that the induction of autophagy and metabolic rewiring by PKA is important for cancer cellular survival under glucose starvation. Importantly, both processes actively participate to cancer cell survival mediated by suspension-activated PKA as well. In addition we identify also a PKA/Src mechanism capable to protect cancer cells from anoikis. Our results reveal for the first time the role of the versatile PKA in cancer cells survival under chronic glucose starvation and anoikis and may be a novel potential target for cancer treatment

MDA-MB-231 cells cultivation in LG results in a strong endogenous activation of PKA pathway with pro-survival effects.

<p>(<b>A)</b> PKA time-dependent activation in HG and LG was evaluated by Western blot analysis of p-(Ser/Thr) PKA substrates and pCREB S133. (<b>B)</b> Percentage of floating and dead cells at indicated time points of culture in LG. (<b>C)</b> At 72h of culture in LG suspended and adherent cells were separately collected to analyze PKA activity by Western blot as well as by ELISA assay. (<b>D-J)</b> All analyses were performed on floating cells collected at 72h of culture in LG. <b>(D)</b> Co-staining with a fluorescent substrate for active caspase 3, a p-PKAs antibody and DAPI. <b>(E)</b> PKA activity was determined by ELISA assay in cells treated or not treated with 10μM H89 for 6h. (<b>F)</b> Western blot analysis of pCREB S133 and p-(Ser/Thr) PKA substrates as well as of active caspase 3 in cells treated or not with H89. (<b>G</b>) Trypan blue exclusion assay was performed in cells treated or not treated with 10μM H89. (<b>H)</b> Colony formation assay was performed with cells treated or not with H89 for 6h. The colony formation was evaluated after 14 days of culture in HG as crystal violet absorbance. (<b>I-J)</b> Viable count by trypan blue was performed in cells treated with 10μM BPTES (I) or 10μM CQ (J) for 6h. All data represent the average of different experiments (n = 3).</p

FSK-mediated attenuation of UPR is accompanied by an increase of membrane protein glycosylation.

<p>(<b>A-B)</b> UPR-related transcriptional data from microarray (A) and proteomic (B) data. (<b>C-G)</b> All the analyses are referred to Transformed cells and were performed at 72h (C-E) or 96h (F-G) of culture. (<b>C)</b> qPCR analysis of UPR mRNA levels in FSK-treated cells with respect to untreated cells. (<b>D)</b> Western blot analysis of UPR-related proteins. (<b>E)</b> Schematic representation of the HBP in which the expression levels of some HBP-related mRNAs have been indicated. The data are the ratio TF/T and are represented in color code (red up-regulated; yellow unchanged; green down-regulated). (<b>F)</b> FACS analysis of live cells stained with fluorochrome-conjugated ConcanavalinA. (<b>G)</b> Western blot analysis using a specific anti O-GlcNAc antibody and densitometric analysis of the film. All data represent the average of different experiments (n>3).</p

FSK mediates a change in glutamine metabolism in human MDA-MB-231 cancer cells.

<p>(<b>A)</b> Glutamine, glutamate and ammonia levels were analyzed in culture media of MDA-MB-231 cells. Glutamine anaplerosis was determined based on glutamine uptake and glutamate secretion. Both glutamine anaplerosis and the released ammonia were calculated as pmol*cell^-1*h^-1. (<b>B-C</b>) MIDs of target metabolites from [U¹³C₅]glutamine were evaluated in MDA-MB-231 cells after 56h of culture. Fraction of glutamine derived isotopologues (B) and contribution of [U¹³C₅]glutamine to target metabolites (C) were determined. In panel B, x-axis represents the mass isotopomer, while the y-axis indicates the relative abundance from [U¹³C₅]glutamine. (<b>D</b>) -/+ FSK cells were treated with BPTES for 24h and counted at 72h of culture. Percentage of reduction after the treatment is shown. All data represent the average of different experiments (n≥3).</p

The inhibition of PKA impacts on c-Src phosphorylation, mitochondrial respiration and intracellular ROS levels in MDA-MB-231 suspended cells.

<p>All analyses were performed in floating cells collected at 72h of culture in LG. <b>(A)</b> After 9h of treatment with DMSO or 10μM H89, Src and CREB phosphorylation was analyzed by Western blot. <b>(B)</b> After treatment with 1μM Saracatinib for 9h, trypan blue exclusion assay was performed. <b>(C)</b> OCR was measured with the Seahorse instrument. After cell treatment with DMSO or 10μM H89, the OCR of -/+H89 cells was measured every 30 minutes (steps 1–4, two measurements at each step). OCR was then measured after addition of 1μM FCCP (step 5, the last three measurements) to evaluate the mitochondrial reserve capacity (right histogram). The total duration of the analysis was 3–3.5h. The data are represented as mean±SEM. <b>(D)</b> Intracellular ATP level was measured in floating cells treated with DMSO or H89 for 3h. <b>(E)</b> Intracellular ROS were measured by using H₂DCFDA in floating cells treated with DMSO or H89 for 3–6h. <b>(F)</b> Viable count by trypan blue was performed in floating cells treated with 10μM H89 and 5mM NAC for 6h. All data represent the average of at least three independent experiments. *p<0.05, **p<0.01, ***p<0.001 Student’s t-test. <b>(G)</b> Schematic representation of the PKA-mediated resistance to glucose deprivation and <i>anoikis</i> in cancer cells.</p