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

Pyrethroid resistance in Italian populations of the mite Varroa destructor: a focus on the Lombardy region

Varroa destructor Anderson et Trueman (Acari Varroidae) is a major pest of honey bees and synthetic acaricide treatments remain the most effective tool to contain its infestations. In 1991, pyrethroid resistance was first reported in Lombardy, and is now spread worldwide. Recently, three different mutations (L925V/I/M) occurring in the voltage-gated sodium channel have been associated with tau-fluvalinate resistance. Furthermore, in the literature, indirect evidence from laboratory bioassays have indicated that high levels of esterases may be involved in mites resistant to tau-fluvalinate. This study provides an update on the actual spread of target-site resistance to tau-fluvalinate in V. destructor samples collected in the Lombardy region. TaqMan assays showed that mutation L925V is present in this area, however only low frequencies of this resistant allele were detected. The majority of resistant mites were found in the homozygous form (11%), and only a small fraction possessed the heterozygous genotype (2%). Additionally, a protocol was set up to detect esterase activity directly in single mites. Slight variability was observed among different populations collected in Lombardy. Additional studies are needed to confirm the involvement of esterases in resistance to pyrethroids in V. destructor and whether this can be correlated to changes in enzyme activity

Characterization of marble weathering through pore structure quantitative analysis

Y Stone weathering is strongly controlled by the intrinsic properties of the stone and by its use. Previous studies demonstrate that the response to natural or artificial ageing processes of the rocks seems to be strongly influenced by the pore structure of the stone. A better understanding of this phenomenon is provided by the study and characterization of porosity and of the pore structure at different degrees of alteration. The analysis of the evolution of the decay leads to the evaluation of the durability of marble in facades, and more generally in buildings, as well as for the protection and recovery of artistic and architectural heritage.In this paper, we apply a methodology for the geometrical characterization of the pore structure to quantify alteration induced by natural weathering on marble slabs. The approach is based on the application of a path-finding algorithm to 2D binary images representative of thin sections of marble at different degrees of alteration. Through the identification of the paths within the porous domain, the methodology allows the characterization of the pore structure in terms of pore radius distribution along the identified paths. Analysis of the results demonstrate a good agreement between the degree of alteration of the pore structure and the corresponding variation of the physical and mechanical properties of the rock samples under investigation

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

Isolation of a Somatic Cell Mutant Resistant to the Induction of Apoptosis by Oxidized Low Density Lipoprotein

Oxidized low density lipoprotein (oxLDL) induces apoptosis in macrophages, smooth muscle cells, and endothelial cells. To elucidate the molecular mechanism of oxLDL-induced cytotoxicity and determine its tissue specificity, we have used Chinese hamster ovary (CHO)-K1 cells expressing human CD36 (CHO/CD36). Expression of CD36 rendered these cells susceptible to killing by oxLDL. This cytotoxicity was due to the induction of apoptosis. Therefore, CD36 expression is the only requirement for oxLDL-induced apoptosis. Oxysterols apparently mediate the cytotoxicity of oxLDL in macrophage foam cells and endothelial cells. 25-Hydroxycholesterol, at concentrations higher than 1 μg/ml, killed CHO-K1 cells, by apoptosis, in medium supplemented with serum as a source of cholesterol. These effects were not seen in a 25-hydroxycholesterol-resistant CHO/CD36 mutant (OX(R)), which was otherwise capable of undergoing apoptosis in response to staurosporine. This mutant was also resistant to killing by oxLDL, suggesting that oxysterols are at least partially responsible for the toxic effects of oxLDL. Oxysterol-induced apoptosis did not involve regulation of sterol regulatory element-binding protein proteolysis or the cholesterol biosynthetic pathway. 25-Hydroxycholesterol stimulated calcium uptake by CHO-K1 cells within 2 min after addition. Treatment of CHO or THP-1 (macrophage) cells with the calcium channel blocker nifedipine prevented 25-hydroxycholesterol induction of apoptosis. OX(R) showed no enhanced calcium uptake in response to 25- hydroxycholesterol

Composition of sands in cores along the high-speed rail (TAV): Preliminary indications on the sedimentary evolution of the Modena plain

The Modena alluvial plain has been geologically investigated in great detail and is characterized by a spectacular abundance of archaeological sites of various age. For this reasons the area may be considered a natural laboratory for the reconstruction of the recent sedimentary evolution of the Po Plain. The alluvial plain area examined for this study has an approximate extent of 150 km2 and is located at the northern side of the Northern Apennines thrust- and fold-belt, where streams draining the chain flow toward the northeast into the Po river. Detailed modal analyses by point-counting of thin sections show that modern stream sands in the Modena plain show similar overall compositional fields, but can be discriminated on the base of key-components, such as quartz, feldspar, carbonate and lithic fragments. The study of sand sediments indicates that the compositional fields have not varied significantly since the Neolithic. The only major diagenetic process is the formation of carbonate concretions (caliche), which can be easily recognized as secondary particles during point counting of thin sections. These results indicate that the reconstruction of the recent evolution of the local drainage system is possible by comparing ancient with modern fluvial sand compositions. The drilling of numerous wells along the new high speed train tract (TAV) provided us with new insight on the sedimentary evolution of the plain through time. The samples recovered from 6 wells reaching depths up to -50 m show that the sand sediments older than 10-12 kyr, have a significant shift in composition from the modern ones. This compositional change consists in a marked overall increase of quartz and feldspar components. The compositional variations can be explained by the combination of various factors: a) significant change in the bedrock lithology through time induced by tectonics and/or change in the local drainage pattern, and b) recycling of older fluvial sediments enriched in feldspar. Because the sands older than 10-12 kyr compared to the overlaying olocenic and modern sediments are enriched in feldspar, which is considered a particularly alterable component, the compositional differentiation can be related to effects induced to glacial-interglacial climate changes but not to post-depositional diagenetic processes. The considerable change in sand composition and the stratigraphy of the deposits suggest dramatic geographical, environmental and depositional variations supporting the observation that a regional unconformity separates the Late Pleistocene from the Olocene sediments

Transtornos Neuropsiquiátricos em Idosos Institucionalizados no Sul de Santa Catarina (Brasil)

Contexto: O processo de envelhecimento está associado às doenças que podem comprometer o bom funcionamento do sistema nervoso central. Objetivo: Avaliar característica de declínio cognitivo e prevalência de sintomas depressivos e sua associação com dados sociodemográficos de idosos institucionalizados em três instituições de longa permanência do sul de Santa Catarina. Métodos: Estudo transversal, avaliando todos idosos institucionalizados em Tubarão e Laguna/SC. Participaram da presente pesquisa 66 idosos, com idade maior ou igual a sessenta anos, de ambos os gêneros. Para coleta de dados utilizou-se um questionário padronizado abordando dados sociodemográficos, a Escala de Depressão Geriátrica reduzida (EDG-15) e o Mini-Exame do Estado Mental. Resultados: A população estudada era predominantemente feminina, viúva e média de idade 77,3±9,9anos. Sintomas depressivos estiveram presentes em 62,1% dos idosos. Possível déficit cognitivo esteve presente em 37,87% da população. Conclusão: Alta prevalência de sintomas depressivos e déficit cognitivo estiveram presentes nos idosos residentes nas instituições de longa permanência pesquisadas. Diante de tais fatos, o serviço de saúde do Brasil deve estar preparado para avaliar e tratar tais pacientes, a fim de melhorar a qualidade de vida dos idosos institucionalizados.Background: The process of aging is associated with the diseases that may compromise the proper functioning of the central nervous system. Objectives: To evaluate the prevalence of cognitive decline and depressive symptoms and their relationship with sociodemographic characteristics of institutionalized elderly in three long-stay institutions in south of Santa Catarina. Methods: cross-sectional study was carried out with all institutionalized elderly in Tubarão and Laguna (SC). In this study were interviewed 66 individuals, 60 years of age or older, male and female. The data collection was performed through a standardized questionnaire with sociodemographic characteristics, the Geriatric Depression Scale in the reduced form (GDS-15) and Mini-Mental State Examination. Results: The study population was predominantly female, widowed, mean age 77.3±9.9 years old. Depressive symptoms were present in 62.1% of institutionalized elderly. The cognitive decline was present in 37.87% of the institutionalized elderly. Conclusion: High prevalence of depressive symptoms and cognitive deficits were present in the elderly living in long-stay institutions surveyed. Given these facts, the health service in Brazil should be prepared to evaluate and treat institutionalized elderly, improving the quality of life of elderly patients

Biochemical evaluation of interactions between synergistic molecules and phase I enzymes involved in insecticide resistance in B- and Q-type Bemisia tabaci (Hemiptera: Aleyrodidae)

BACKGROUND Metabolic resistance is an important consideration in the whitefly Bemisia tabaci, where an esterase‐based mechanism has been attributed to pyrethroid resistance and over‐expression of the cytochrome P450, CYP6CM1, has been correlated to resistance to imidacloprid and other neonicotinoids. RESULTS In vitro interactions between putative synergists and CYP6CM1, B and Q‐type esterases were investigated, and structure–activity relationship analyses allowed the identification of chemical structures capable of acting as inhibitors of esterase and oxidase activities. Specifically, methylenedioxyphenyl (MDP) moieties with a polyether chain were preferable for optimum inhibition of B‐type esterase, whilst corresponding dihydrobenzofuran structures were potent for the Q‐esterase variation. Potent inhibition of CYP6CM1 resulted from structures which contained an alkynyl chain with a terminal methyl group. CONCLUSIONS Synergist candidates could be considered for field control of B. tabaci, especially to abrogate neonicotinoid resistance. © 2017 Society of Chemical Industr

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

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