Underutilized Crocus Sativus L. Flowers: A Hidden Source of Sustainable High Value-Added Ingredients.

Crocus sativus L. is used as a spice due to its organoleptic characteristics. Only flower stigmas are used for its production, as the rest of the flower is discarded as waste. This fact represents a lack of sustainability, since around 230,000 flowers are necessary to produce 1 kg of saffron. The main aim of this study was to contribute to the valorization of Crocus sativus L. spice and its floral by-products, through the study of their nutritional value and composition, in terms of hydrophilic and lipophilic compounds, as well as their functional properties. The results showed that saffron stigmas and floral bio-residues presented high contents of fiber, and the most abundant macronutrient were the carbohydrates, followed by proteins, and a low content in fats. All samples had high concentrations of glucose, fructose, lactic and malic acids, and minerals, mainly K, Ca and Mg. Furthermore, the polyunsaturated fatty acids were predominant, being linoleic acid (C18:2n6) the most abundant. Therefore, this research provides more in-depth information about the composition of saffron stigmas and floral by-products, to be considered as promising sources for the development of functional ingredients with new applications in the food industry.

Saffron bioactives crocin, crocetin and safranal: effect on oxidative stress and mechanisms of action.

Saffron (Crocus sativus L.) is used as a spice for its organoleptic characteristics related to its coloring and flavoring properties, and it has been also used in traditional medicine to treat various diseases. The main chemical components responsible for these properties are crocin, crocetin and safranal. These compounds have been shown to have a wide spectrum of biological activities, including several properties as antigenotoxic, antioxidant, anticancer, anti-inflammatory, antiatherosclerotic, antidiabetic, hypotensive, hypoglycemic, antihyperlipidemic, antidegenerative and antidepressant, among others. This review article highlights the antioxidant effects of these bioactive compounds to reduce reactive oxygen species (ROS) and the mechanisms of action involved, since there are a multitude of diseases related to oxidative stress and the generation of free radicals (FRs). Recent studies have shown that the effects of crocin, crocetin and safranal against oxidative stress include the reduction in lipid peroxidation (malondialdehyde [MDA] levels) and nitric oxide (NO) levels, and the increase in the levels of glutathione, antioxidant enzymes (superoxide dismutase [SOD], catalase (CAT) and glutathione peroxidase [GPx]) and thiol content. Therefore, due to the great antioxidant effects of these saffron compounds, it makes saffron a potential source of bioactive extracts for the development of bioactive ingredients, which can be used to produce functional foods.

Probiotic red quinoa drinks for celiacs and lactose intolerant people: study of functional, physicochemical and probiotic properties during fermentation and gastrointestinal digestion.

The consumption of probiotic foods is rather limited for many sectors of the population (vegans, lactose intolerant and celiacs). Therefore, it is necessary to offer functional alternatives for these sectors. Different red quinoa drinks were fermented with L. plantarum (QLPBB) and B. longum (QBLBB) at different times. Results showed that microbial concentration reached high levels (6-8 Log CFU/mL) after 6 h and probiotic viability was very high (6 Log CFU/mL) after gastrointestinal digestion (GD). However, QBLBB reached the best probiotic concentration at 24 h. From 6 h of fermentation, probiotic resistance to some antibiotics, especially B. longum, could have a great potential to restore the intestinal microbiota during and after treatment with certain antibiotics. QLPBB showed the highest levels of total polyphenols and antioxidant capacity (AOC) after GD. Therefore, these red quinoa drinks have potential as functional probiotic beverages for vegans, celiacs and allergic to milk protein and lactose-intolerant people.

mTORC2 Is the Major Second Layer Kinase Negatively Regulating FOXO3 Activity.

Forkhead box O (FOXO) proteins are transcription factors involved in cancer and aging and their pharmacological manipulation could be beneficial for the treatment of cancer and healthy aging. FOXO proteins are mainly regulated by post-translational modifications including phosphorylation, acetylation and ubiquitination. As these modifications are reversible, activation and inactivation of FOXO factors is attainable through pharmacological treatment. One major regulatory input of FOXO signaling is mediated by protein kinases. Here, we use specific inhibitors against different kinases including PI3K, mTOR, MEK and ALK, and other receptor tyrosine kinases (RTKs) to determine their effect on FOXO3 activity. While we show that inhibition of PI3K efficiently drives FOXO3 into the cell nucleus, the dual PI3K/mTOR inhibitors dactolisib and PI-103 induce nuclear FOXO translocation more potently than the PI3Kδ inhibitor idelalisib. Furthermore, specific inhibition of mTOR kinase activity affecting both mTORC1 and mTORC2 potently induced nuclear translocation of FOXO3, while rapamycin, which specifically inhibits the mTORC1, failed to affect FOXO3. Interestingly, inhibition of the MAPK pathway had no effect on the localization of FOXO3 and upstream RTK inhibition only weakly induced nuclear FOXO3. We also measured the effect of the test compounds on the phosphorylation status of AKT, FOXO3 and ERK, on FOXO-dependent transcriptional activity and on the subcellular localization of other FOXO isoforms. We conclude that mTORC2 is the most important second layer kinase negatively regulating FOXO activity.

Pain relief in chronic pancreatitis: endoscopic or surgical treatment? a systematic review with meta-analysis.

BACKGROUND AND AIMS: Pain is one of the consequences of chronic pancreatitis (CP) that has the greatest impact on the quality of life of patients. Endoscopic and surgical interventions, by producing a decrease in intraductal pancreatic pressure, can provide pain relief. This is the first systematic review that includes only randomized clinical trials (RTCs) comparing outcomes in the short-term (less than 2 years) and long-term (more than 2 years) between these two types of interventions. MATERIAL AND METHODS: A comprehensive search of multiple electronic databases to identify RTCs comparing short and long-term pain relief, procedural complications, and days of hospitalization between endoscopic and surgical interventions was performed following the PRISMA guidelines. RESULTS: Three RCTs evaluating a total of 199 patients (99 in the endoscopy group and 100 in the surgery group) were included in this study. Surgical interventions provided complete pain relief, with statistical difference, in the long-term (16,4% vs 35.7%; RD 0.19; 95% CI 0.03-0.35; p = 0.02; I2 = 0%), without significant difference in short-term (17.5% vs 31.2%; RD 0.14; 95% CI -0.01-0.28; p = 0.07; I2 = 0%) when compared to endoscopy. There was no statistical difference in short-term (17.5% vs 28.1%; RD 0.11; 95% CI -0.04-0.25; p = 0.15; I2 = 0%) and long-term (34% vs 41.1%; RD 0.07; 95% CI -0.10-0.24; p = 0.42; I2 0%) in partial relief of pain between both interventions. In the short-term, both complications (34.9% vs 29.7%; RD 0.05; 95% CI -0.10-0.21; p = 0.50; I2 = 48%) and days of hospitalization (MD -1.02; 95% CI -2.61-0.58; p = 0.21; I2 = 0%) showed no significant differences. CONCLUSION: Surgical interventions showed superior results when compared to endoscopy in terms of complete long-term pain relief. The number of complications and length of hospitalization in both groups were similar.

EUS-guided gastroenterostomy versus duodenal stent placement and surgical gastrojejunostomy for the palliation of malignant gastric outlet obstruction: a systematic review and meta-analysis.

PURPOSE: Malignant gastric outlet obstruction (GOO) is associated with significant morbidity and decreased quality of life, thereby necessitating effective and safe palliative treatment. As such, we sought to compare endoscopic ultrasound-guided gastroenterostomy (EUS-GE) versus duodenal stent (DS) placement and surgical gastrojejunostomy (SGJ) for palliation of malignant GOO. METHODS: Searches of electronic databases were performed to identify studies comparing EUS-GE versus DS and/or SGJ for palliative treatment of GOO. Outcomes included technical and clinical success, severe adverse events (SAEs), rate of stent obstruction (including tumor ingrowth), length of hospital stay (LOS), reintervention, and 30-day all-cause mortality. Differences in dichotomous and continuous outcomes were reported as risk difference and mean difference, respectively. RESULTS: Seven studies (n = 513 patients) were included. When compared to DS placement, EUS-GE was associated with a higher clinical success, fewer SAEs, decreased stent obstruction, lower rate of tumor ingrowth, and decreased need for reintervention. Compared to SGJ, EUS-GE was associated with a lower technical success; however, LOS was significantly decreased. All other outcomes including clinical success, SAEs, reintervention rate, and 30-day mortality were not significantly different between an EUS-guided versus surgical approach. CONCLUSIONS: EUS-GE was associated with significantly improved outcomes compared to DS placement for palliative treatment of malignant GOO. Despite SGJ possessing a higher technical success compared to EUS-GE, LOS was significantly longer with no difference in clinical success or rate of adverse events.

Generation of tricyclic imidazo[1,2-a]pyrazines as novel PI3K inhibitors by application of a conformational restriction strategy.

The involvement of the phosphoinositide 3-kinases (PI3Ks) in several diseases, especially in the oncology area, has singled it as one of the most explored therapeutic targets in the last two decades. Many different inhibitor classes have been developed by the industry and academia with a diverse selectivity profile within the PI3K family. In the present manuscript we report a further exploration of our lead PI3K inhibitor ETP-46321 (Martínez González et al., 2012)1 by the application of a conformational restriction strategy. For that purpose we have successfully synthesized novel tricyclic imidazo[1,2-a]pyrazine derivatives as PI3K inhibitors. This new class of compounds had enable the exploration of the solvent-accessible region within PI3K and resulted in the identification of molecule 8q with the best selectivity PI3Kα/δ isoform profile in vitro, and promising in vivo PK data.

Identification of novel PI3K inhibitors through a scaffold hopping strategy.

A scaffold hopping strategy, including intellectual property availability assessment, was successfully applied for the discovery of novel PI3K inhibitors. Compounds were designed based on the chemical structure of the lead compound ETP-46321, a potent PI3K inhibitor, previously reported by our group. The new generated compounds showed good in vitro potency and selectivity, proved to inhibit potently the phosphorylation of AKTSer473 in cells and demonstrated to be orally bioavailable, thus becoming potential back-up candidates for ETP-46321.

Alternative Activation Mechanisms of Protein Kinase B Trigger Distinct Downstream Signaling Responses.

Protein kinase B (PKB/Akt) is an important mediator of signals that control various cellular processes including cell survival, growth, proliferation, and metabolism. PKB promotes these processes by phosphorylating many cellular targets, which trigger distinct downstream signaling events. However, how PKB is able to selectively target its substrates to induce specific cellular functions remains elusive. Here we perform a systematic study to dissect mechanisms that regulate intrinsic kinase activity versus mechanisms that specifically regulate activity toward specific substrates. We demonstrate that activation loop phosphorylation and the C-terminal hydrophobic motif are essential for high PKB activity in general. On the other hand, we identify membrane targeting, which for decades has been regarded as an essential step in PKB activation, as a mechanism mainly affecting substrate selectivity. Further, we show that PKB activity in cells can be triggered independently of PI3K by initial hydrophobic motif phosphorylation, presumably through a mechanism analogous to other AGC kinases. Importantly, different modes of PKB activation result in phosphorylation of distinct downstream targets. Our data indicate that specific mechanisms have evolved for signaling nodes, like PKB, to select between various downstream events. Targeting such mechanisms selectively could facilitate the development of therapeutics that might limit toxic side effects.

Non-genotoxic activation of p53 through the RPL11-dependent ribosomal stress pathway.

Nucleolar disruption has recently emerged as a relevant means to activate p53 through inhibition of HDM2 by ribosome-free RPL11. Most drugs that induce nucleolar disruption also possess important genotoxic activity, which can have lasting mutagenic effects. Therefore, it is of interest to identify compounds that selectively produce nucleolar disruption in the absence of DNA damage. Here, we have performed a high-throughput screening to search for nucleolar disruptors. We have identified an acridine derivative (PubChem CID-765471) previously known for its capacity to activate p53 independently of DNA damage, although the molecular mechanism underlying p53 activation had remained uncharacterized. We report that CID-765471 produces nucleolar disruption by inhibiting ribosomal DNA transcription in a process that includes the selective degradation of the RPA194 subunit of RNA polymerase I. Following nucleolar disruption, CID-765471 activates p53 through the RPL11/HDM2 pathway in the absence of detectable DNA damage. In a secondary screening of compounds approved for medical use, we identify two additional acridine derivatives, aminacrine and ethacridine, that operate in a similar manner as CID-765471. These findings provide the basis for non-genotoxic chemotherapeutic approaches that selectively target the nucleolus.

A novel phosphatidylinositol 3-kinase (PI3K) inhibitor directs a potent FOXO-dependent, p53-independent cell cycle arrest phenotype characterized by the differential induction of a subset of FOXO-regulated genes.

INTRODUCTION: The activation of the phosphoinositide 3-kinase (PI3K)/AKT signalling pathway is one the most frequent genetic events in breast cancer, consequently the development of PI3K inhibitors has attracted much attention. Here we evaluate the effect of PI3K inhibition on global gene expression in breast cancer cells. METHODS: We used a range of methodologies that include in silico compound analysis, in vitro kinase assays, cell invasion assays, proliferation assays, genome-wide transcription studies (Agilent Technologies full genome arrays), gene set enrichment analysis, quantitative real-time PCR, immunoblotting in addition to chromatin immunoprecipitation. RESULTS: We defined the physico-chemical and the biological properties of ETP-45658, a novel potent PI3K inhibitor. We demonstrated that ETP-45658 potently inhibited cell proliferation within a broad range of human cancer cells, most potently suppressing the growth of breast cancer cells via inhibiting cell cycle. We show that this response is Forkhead box O (FOXO) protein dependent and p53 independent. Our genome-wide microarray analysis revealed that the cell cycle was the most affected biological process after exposure to ETP-45658 (or our control PI3K inhibitor PI-103), that despite the multiple transcription factors that are regulated by the PI3K/AKT signalling cascade, only the binding sites for FOXO transcription factors were significantly enriched and only a subset of all FOXO-dependent genes were induced. This disparity in gene transcription was not due to differential FOXO promoter recruitment. CONCLUSIONS: The constitutive activation of PI3Ks and thus the exclusion of FOXO transcription factors from the nucleus is a key feature of breast cancer. Our results presented here highlight that PI3K inhibition activates specific FOXO-dependent genes that mediate cell cycle arrest in breast cancer cells.

SETD8 inhibition targets cancer cells with increased rates of ribosome biogenesis.

SETD8 is a methyltransferase that is overexpressed in several cancers, which monomethylates H4K20 as well as other non-histone targets such as PCNA or p53. We here report novel SETD8 inhibitors, which were discovered while trying to identify chemicals that prevent 53BP1 foci formation, an event mediated by H4K20 methylation. Consistent with previous reports, SETD8 inhibitors induce p53 expression, although they are equally toxic for p53 proficient or deficient cells. Thermal stability proteomics revealed that the compounds had a particular impact on nucleoli, which was confirmed by fluorescent and electron microscopy. Similarly, Setd8 deletion generated nucleolar stress and impaired ribosome biogenesis, supporting that this was an on-target effect of SETD8 inhibitors. Furthermore, a genome-wide CRISPR screen identified an enrichment of nucleolar factors among those modulating the toxicity of SETD8 inhibitors. Accordingly, the toxicity of SETD8 inhibition correlated with MYC or mTOR activity, key regulators of ribosome biogenesis. Together, our study provides a new class of SETD8 inhibitors and a novel biomarker to identify tumors most likely to respond to this therapy.

Biological characterization of ETP-46321 a selective and efficacious inhibitor of phosphoinositide-3-kinases.

Inhibitors of PI3K signaling are of great therapeutic interest in oncology. The phosphoinositide-3-kinase signaling pathway is activated in a variety of solid and non-solid tumors. We have identified an imidazopyrazine derivative, ETP-46321, as a potent inhibitor of PI3Kα [Formula: see text]. The compound was 6 times less potent towards PI3Kδ and more than 200 and 60 times less potent at inhibiting PI3Kß and PI3Kγ and did not significantly inhibit the related phosphoinositide-3-kinase-related protein kinase family kinases mTOR or DNA PK (IC(50)'s > 5 µM), or an additional 287 protein kinases that were screened. ETP-46321 inhibited PI3K signaling in treated tumor cell lines, induced cell cycle arrest and inhibited VEGF-dependent sprouting of HUVEC cells. The compound was anti-proliferative and synergized with both cytotoxic and targeted therapeutics. The compound induced a reduction in the phosphorylation of Akt in U87 MG xenografts after a single treatment. The growth of colon and lung cancinoma HT-29 and A549 xenografts was delayed by once a day treatment with ETP-46321. The compound synergized with Doxotaxel in a model of ovarian cancer.

Influence of Synthetic Fibers on the Flexural Properties of Concrete: Prediction of Toughness as a Function of Volume, Slenderness Ratio and Elastic Modulus of Fibers.

The construction industry requires concrete with adequate post-cracking behavior for applications such as tunnels, bridges, and pavements. For this reason, polypropylene macrofibers are used, which are synthetic fibers that fulfill the function of providing residual strength to concrete. In this study, an experimental plan is carried out to evaluate the bending behavior of concrete reinforced with polypropylene fibers using the four-point bending test according to ASTM C1609. Three fiber dosages (3.6, 7.2 and 10.8 kg/m3) and three fiber lengths (40, 50, and 60 mm) were used. The use of macro polypropylene fibers increased the post-cracking behavior of concrete. In addition, based on the experimentally obtained results and available literature data, a multivariable equation was developed to predict the concrete toughness as a function of the volume, slenderness, and modulus of elasticity of the fibers. A Pearson's correlation coefficient, r of 0.90, showed a strong correlation between the developed equation and the experimental data. From this equation, it was possible to determine the participation of the following parameters in calculating toughness. The participation or weight of the fiber's modulus of elasticity on the concrete's tenacity is 26%, the volume of the fiber is 39%, the slenderness is 19%, and the reinforcement index is 16%.

Hit to lead evaluation of 1,2,3-triazolo[4,5-b]pyridines as PIM kinase inhibitors.

PIM kinases have become targets of interest due to their association with biochemical mechanisms affecting survival, proliferation and cytokine production. 1,2,3-Triazolo[4,5-b]pyridines were identified as PIM inhibitors applying a scaffold hopping approach. Initial exploration around this scaffold and X-ray crystallographic data are hereby described.

Imidazo[1,2-a]pyrazines as novel PI3K inhibitors.

Phosphoinositide-3-kinase (PI3K) is an important target for cancer therapeutics due to the deregulation of its signaling pathway in a wide variety of human cancers. We describe herein a novel series of imidazo[1,2-a]pyrazines as PI3K inhibitors.

Identification of ETP-46321, a potent and orally bioavailable PI3K α, δ inhibitor.

Phosphoinositide-3-kinase (PI3K) is an important target for cancer therapeutics due to the deregulation of this signaling pathway in a wide variety of human cancers. Herein, we describe the optimization of imidazo [1,2-a] pyrazines, which allow us to identify compound 14 (ETP-46321), with potent biochemical and cellular activity and good pharmacokinetic properties (PK) after oral dosing. ETP-46321 PK/PD studies showed time dependent downregulation of AKT(Ser473) phosphorylation, which correlates with compound levels in tumor tissue and demonstrating to be efficacious in a GEMM mouse tumor model driven by a K-Ras(G12V) oncogenic mutation. Treatment with ETP-46321 resulted in significant tumor growth inhibition.

Rapid identification of ETP-46992, orally bioavailable PI3K inhibitor, selective versus mTOR.

Phosphoinositide-3-kinases (PI3K) are a family of lipid kinases mediating numerous cell processes such as proliferation, migration and differentiation. PI3K is an important target for cancer therapeutics due to the deregulation of this signaling pathway in a wide variety of human cancers. Herein, we describe the rapid identification of ETP-46992, within 2-aminocarbonyl imidazo [1,2-a] pyrazine series, with suitable pharmacokinetic (PK) properties that allows the establishment of mechanism of action and efficacy in vivo studies. ETP-46992 showed tumor growth inhibition in a GEMM mouse tumor model driven by a K-Ras(G12V) oncogenic mutation and in tumor xenograft models with PI3K pathway deregulated (BT474).