PO-040 Characterisation of cdk12 knocked out ovarian cancer cell lines

Introduction While cyclin-dependent kinases (CDKs) have a key role in promoting/controlling transition between the different phases of the cell cycle, transcriptional kinases, like CDK12, are mainly involved in gene transcription. CDK12 has been shown to regulate the expression of genes involved in DNA damage and to maintain genomic stability. Impairment of CDK12 activity is synergic with PARP inhibitor and cisplatin treatments in different cellular systems. We here aimed to generate ovarian cancer cell lines knocked out (KO) for CDK12 to understand its role in ovarian cancer and in response to chemotherapy. Material and methods A2780 and SKOV3 CDK12 KO clones were generated with CRISPR/Cas9 technology. Cell cycle analysis was evaluated by standard flow cytometric methods and DNA repair genes levels by Real Time PCR. Caspase 3 activity was measured to detect apoptosis with a luminescence-based assay. Cytotoxicity experiments were performed treating cells with different drug concentrations and evaluating cell survival after 72 hours by MTS assay. For in vivo studies 7.5 millions of cells were transplanted subcutaneously in nude mice and animals were monitored for tumour appearance and growth. Results and discussions We obtained 2 CDK12 KO ovarian cancer clones, A2780 KO and SKOV3 KO, out of more than 300 clones screened. The cell growth of both A2780 KO and SKOV3 KO cells is slower than the wild type (WT) cells, they have a less clonogenic ability and a tetraploid DNA content. Both CDK12 KO clones have a higher basal caspase activity than the WT cell lines, indicative of higher basal induction of apoptosis, while no increase in autophagy or senescence is observed. Both CDK12 KO clones show a decreased expression in BRCA1 and FANCD2 DNA repair genes than the WT cells. Cytotoxic experiments with anticancer agents with different mechanism of action show that both KO clones are less sensitive to ATM, CHK1 and WEE1 inhibitors treatment as compared to WT cells, while platinum and PARP inhibitors show similar cytotoxic activity in KO and WT cells. Interestingly enough, when KO clones were transplanted in nude mice, no tumour take was observed. Conclusion We were able to obtain CDK12 KO cells. We think that these models could help in disclosing new roles of CDK12 in ovarian carcinoma and may represent a useful tool to study new combination therapies for tumours with CDK12 mutations

Slug monitoring and impacts on the ground beetle community in the frame of sustainable pest control in conventional and conservation agroecosystems

In conservation agriculture, slugs are considered significant pests and their monitoring is a key option in the integrated pest management framework. Together with molluscicide applications, predators such as ground beetles can offer a tool for slug control in the field. Through the evaluation of slug and ground beetle monitoring strategies, this work compared their presence in conventional and conservation agricultural plots. The invasive Deroceras invadens was the dominant slug species to occur in all sampling periods. Among Carabidae, Poecilus cupreus and Pterostichus melas were the most abundant species, and Bembidion spp., Brachinus spp., and Harpalus spp. were also common. Beer-baited pitfall traps, whatever their alcoholic content, caught more slugs and ground beetles than wooden boards used as shelters. Slugs were more abundant in conventional plots than in conservation plots, possibly due to the lower presence of natural enemies such as ground beetles. Despite possible impacts on Carabidae, beer-baited pitfall traps should be considered a useful tool for slug monitoring and for the planning of molluscicide applications. Soil management such as minimum-or no-tillage and the presence of cover crops are important elements influencing both slug and ground beetle presence, possibly playing a key role in the maintenance of natural enemy populations

Design, Fabrication, and Experimental Validation of Microfluidic Devices for the Investigation of Pore-Scale Phenomena in Underground Gas Storage Systems

The understanding of multiphase flow phenomena occurring in porous media at the pore scale is fundamental in a significant number of fields, from life science to geo and environmental engineering. However, because of the optical opacity and the geometrical complexity of natural porous media, detailed visual characterization is not possible or is limited and requires powerful and expensive imaging techniques. As a consequence, the understanding of micro-scale behavior is based on the interpretation of macro-scale parameters and indirect measurements. Microfluidic devices are transparent and synthetic tools that reproduce the porous network on a 2D plane, enabling the direct visualization of the fluid dynamics. Moreover, microfluidic patterns (also called micromodels) can be specifically designed according to research interests by tuning their geometrical features and surface properties. In this work we design, fabricate and test two different micromodels for the visualization and analysis of the gas-brine fluid flow, occurring during gas injection and withdrawal in underground storage systems. In particular, we compare two different designs: a regular grid and a real rock-like pattern reconstructed from a thin section of a sample of Hostun rock. We characterize the two media in terms of porosity, tortuosity and pore size distribution using the A* algorithm and CFD simulation. We fabricate PDMS-glass devices via soft lithography, and we perform preliminary air-water displacement tests at different capillary numbers to observe the impact of the design on the fluid dynamics. This preliminary work serves as a validation of design and fabrication procedures and opens the way to further investigations

Antitumour activity of trabectedin in myelodysplastic/myeloproliferative neoplasms

Background: Juvenile myelomonocytic leukaemia (JMML) and chronic myelomonocytic leukaemia (CMML) are myelodysplastic myeloproliferative (MDS/MPN) neoplasms with unfavourable prognosis and without effective chemotherapy treatment. Trabectedin is a DNA minor groove binder acting as a modulator of transcription and interfering with DNA repair mechanisms; it causes selective depletion of cells of the myelomonocytic lineage. We hypothesised that trabectedin might have an antitumour effect on MDS/MPN. Methods: Malignant CD14+ monocytes and CD34+ haematopoietic progenitor cells were isolated from peripheral blood/bone marrow mononuclear cells. The inhibition of CFU-GM colonies and the apoptotic effect on CD14+ and CD34+ induced by trabectedin were evaluated. Trabectedin's effects were also investigated in vitro on THP-1, and in vitro and in vivo on MV-4-11 cell lines. Results: On CMML/JMML cells, obtained from 20 patients with CMML and 13 patients with JMML, trabectedin - at concentration pharmacologically reasonable, 1-5 nM - strongly induced apoptosis and inhibition of growth of haematopoietic progenitors (CFU-GM). In these leukaemic cells, trabectedin downregulated the expression of genes belonging to the Rho GTPases pathway (RAS superfamily) having a critical role in cell growth and cytoskeletal dynamics. Its selective activity on myelomonocytic malignant cells was confirmed also on in vitro THP-1 cell line and on in vitro and in vivo MV-4-11 cell line models. Conclusions: Trabectedin could be good candidate for clinical studies in JMML/CMML patients

How underground systems can contribute to meet the challenges of energy transition

The paper provides an overview of the several scientific and technical issues and challenges to be addressed for underground storage of carbon dioxide, hydrogen and mixtures of hydrogen and natural gas. The experience gained on underground energy systems and materials is complemented by new competences to adequately respond to the new needs raised by transition from fossil fuels to renewables. The experimental characterization and modeling of geological formations (including geochemical and microbiological issues), fluids and fluid-flow behavior and mutual interactions of all the systems components at the thermodynamic conditions typical of underground systems as well as the assessment and monitoring of safety conditions of surface facilities and infrastructures require a deeply integrated teamwork and fit-for-purpose laboratories to support theoretical research. The group dealing with large-scale underground energy storage systems of Politecnico di Torino has joined forces with the researchers of the Center for Sustainable Future Technologies of the Italian Institute of Technology, also based in Torino, to meet these new challenges of the energy transition era, and evidence of the ongoing investigations is provided in this paper

Digital twin and IOT technology for secure manufacturing systems

The Digital Twin (DT) method offers new concepts for choosing the context of information technologies and smart production systems. This research focuses on low-price, extremely effective defect diagnosis methods, low-efficiency, high-cost devices to obtain timely feedback and accurate fault detection results, and secure manufacturing systems. The data structure, control plane, and output units are the three components of the manufacturing system that creates a data link between the virtual model using Micro-Electro-Mechanical (MEM) devices and the Zigbee wireless transmission system in the database layer. This study acquired DT information from the control plane using the Internet of Things (IoT) through sensors for secure manufacturing information. It separates and calls the pertinent data by the attribute processor and transfers it to the outcome units. To produce the classification and outcomes of work build features information, the evaluation method analysis the output nodes that split the test set and learning group using a dynamic database. The hybrid IoT with DT technology to examine the consequences of defects detected efficiently predicted and secures the manufacturing system