Boosting the synthesis of pharmaceutically active abietane diterpenes in S. sclarea hairy roots by engineering the GGPPS and CPPS genes

Abietane diterpenoids (ADs), synthesized in the roots of different Salvia species, such as aethiopinone, 1-oxoaethiopinone, salvipisone, and ferruginol, have a variety of known biological activities. We have shown that aethiopinone has promising cytotoxic activity against several human tumor cell lines, including the breast adenocarcinoma MCF7, HeLa, epithelial carcinoma, prostate adenocarcinoma PC3, and human melanoma A375. The low content of these compounds in natural sources, and the limited possibility to synthesize them chemically at low cost, prompted us to optimize the production of abietane diterpenoids by targeting genes of the methylerythritol phosphate (MEP) pathway, from which they are derived. Here, we report our current and ongoing efforts to boost the metabolic flux towards this interesting class of compounds in Salvia sclarea hairy roots (HRs). Silencing the gene encoding the ent-copalyl-diphosphate synthase gene (entCPPS), acting at the lateral geranylgeranyl pyrophosphate (GGPP) competitive gibberellin route, enhanced the content of aethiopinone and other ADs in S. sclarea HRs, indicating indirectly that the GGPP pool is a metabolic constraint to the accumulation of ADs. This was confirmed by overexpressing the GGPPS gene (geranyl-geranyl diphosphate synthase) which triggered also a significant 8-fold increase of abietane diterpene content above the basal constitutive level, with a major boosting effect on aethiopinone accumulation in S. sclarea HRs. A significant accumulation of aethiopinone and other AD compounds was also achieved by overexpressing the CPPS gene (copalyl diphosphate synthase) pointing to this biosynthetic step as another potential metabolic target for optimizing the biosynthesis of this class of compounds. However, by co-expressing of GGPPS and CPPS genes, albeit significant, the increase of abietane diterpenoids was less effective than that obtained by overexpressing the two genes individually. Taken together, the results presented here add novel and instrumental knowledge to a rational design of a hairy root-based platform to yield reliable amounts of aethiopinone and other ADs for a deeper understanding of their molecular pharmacological targets and potential future commercialization

Survival trends over 20 years in patients with advanced cholangiocarcinoma: Results from a national retrospective analysis of 922 cases in Italy

: Cholangiocarcinoma is a rare group of tumors that involve the hepatic biliary tree. Prognosis for patients with cholangiocarcinoma remains dismal. Herein, we present survival trends over a long time period spanning almost 20 years in patients with advanced cholangiocarcinoma receiving systemic chemotherapy. We retrospectively analyzed a large multicenter dataset of cholangiocarcinoma outpatients evaluated in 14 centers within the Cholangiocarcinoma Italian Group Onlus (Gruppo Italiano Colangiocarcinoma Onlus, G.I.C.O.) between 2000 and 2017 (first-line), and 2002 and 2017 (second-line). Three time periods were considered: 2000-2009, 2010-2013, and 2014-2017. A total of 922 patients (51.19% male) with cholangiocarcinoma undergoing first-line therapy were evaluated. The median durations of follow-up for progression-free survival (PFS) and overall survival (OS) were 37 and 57 months, respectively. PFS at 12 months in the three periods of starting first-line therapy was similar, ranging from 11.71% to 15.25%. OS at 12 months progressively improved (38.30%, 44.61% and 49.52%, respectively), although the differences were not statistically significant after adjusting for age, disease status, and primary tumor site. A total of 410 patients (48.5% male) underwent second-line chemotherapy. The median durations of follow-up for PFS and OS were 47.6 and 41.90 months, respectively. An OS of 24.3%, 32.3%, and 33.1% was observed in 2002-2009, 2010-2013, and 2014-2017, respectively. Despite incremental benefits across years, our clinical experience confirms that modest overall advances have been achieved with first- and second-line chemotherapy in advanced cholangiocarcinoma. Efforts should focus on the identification of patients who derive the greatest benefit from treatment

Monitoring Ancient Buildings: Real Deployment of an IoT System Enhanced by UAVs and Virtual Reality

The historical buildings of a nation are the tangible signs of its history and culture. Their preservation deserves considerable attention, being of primary importance from a historical, cultural, and economic point of view. Having a scalable and reliable monitoring system plays an important role in the Structural Health Monitoring (SHM): therefore, this paper proposes an Internet Of Things (IoT) architecture for a remote monitoring system that is able to integrate, through the Virtual Reality (VR) paradigm, the environmental and mechanical data acquired by a wireless sensor network set on three ancient buildings with the images and context information acquired by an Unmanned Aerial Vehicle (UAV). Moreover, the information provided by the UAV allows to promptly inspect the critical structural damage, such as the patterns of cracks in the structural components of the building being monitored. Our approach opens new scenarios to support SHM activities, because an operator can interact with real-time data retrieved from a Wireless Sensor Network (WSN) by means of the VR environment

Prognostic and predictive role of EGFR pathway alterations in biliary cancer patients treated with chemotherapy and anti-EGFR

The association of anti-EGFR to gemcitabine and oxaliplatin (GEMOX) chemotherapy did not improve survival in biliary tract carcinoma (BTC) patients. Multiple mechanisms might be involved in the resistance to anti-EGFR. Here, we explored the mutation profile of EGFR extracellular domain (ECD), of tyrosine kinase domain (TKD), and its amplification status. EGFR mutational status of exons 12, 18-21 was analyzed in 57 tumors by Sanger sequencing. EGFR amplification was evaluated in 37 tumors by Fluorescent In Situ Hybridization (FISH). Kaplan-Meier curves were calculated using the log-rank test. Six patients had mutations in exon 12 of EGFR ECD and 7 in EGFR TKD. Neither EGFR ECD nor TKD mutations affected progression free survival (PFS) or overall survival (OS) in the entire population. In the panitumumab plus GEMOX (P-GEMOX) arm, ECD mutated patients had a worse OS, while EGFR TKD mutated patients had a trend towards shorter PFS and OS. Overall, the presence of mutations in EGFR or in its transducers did not affect PFS or OS, while the extrahepatic cholangiocarcinoma (ECC) mutated patients had a worse prognosis compared to WT. Nineteen out of 37 tumors were EGFR amplified, but the amplification did not correlate with survival. ECC EGFR amplified patients had improved OS, whereas the amplification significantly correlated with poor PFS (p = 0.03) in gallbladder carcinoma patients. The high molecular heterogeneity is a predominant feature of BTC: the alterations found in this work seem to have a prognostic impact rather than a predictive role towards anti-EGFR therapy

Response to low soil water potential in pea genotypes (Pisum sativum L.) with different leaf morphology

Genetic modifications of canopy structure may result in enhanced yield when water is a limiting factor. Three near-isogenic pea genotypes carrying genes which determine a striking effect on leaf morphology were grown in lysimeters under two extreme water regimes. Pea genotypes of normal and reduced leaf and stipule morphology were cultivated at a plant density of 72 m−2 in drainage lysimeters, where a well watered regime was established by irrigating whenever tensiometric values reached (0.05 MPa). After 70 days from emergence, a second regime was created by withholding water until wilting symptoms were visible; the control plants were irrigated 12 times, whereas the stressed plants only once. Dramatic differences in dry matter accumulation were found between the two irrigation regimes. The physiological response varied accordingly. Under stressed conditions, genotype af, which has the leaflets transformed into tendrils, showed a faster CO2 exchange rate (CER), a lower stomatal resistance, and a lower canopy temperature than the other two genotypes. This allowed a reduced soil water consumption, but a higher pod dry matter accumulation, which doubled in the last 2 weeks. This behaviour was also favoured by a leaf dry matter accumulation, in terms of quantity and duration, significantly higher than that shown by the other two genotypes. Additionally, af plants showed a higher leaf/stem ratio and negligible senescence

Masonry vaults made by calcareous stone under fire conditions

The analysis of the resistance to fire of masonry vaults represents an open issue due to the lack of analytical and/or experimental investigations in the technical literature as well as to the absence of national and international standard treating the specific topic. However, the Italian building heritage is often characterized by vaulted structures that need to be analysed also from a fire resistance point of view. To this scope in the present work, a first approach to study the behaviour of masonry vaults under thermal actions is reported taking into account the effect of the main influencing parameters