Aspekte der antineoplastischen Wirkung der PLK4-Inhibitoren CFI-400945 und Centrinone auf Ewing-Sarkom-Zellen

Purpose: Polo-like kinase 4 (PLK4) inhibitors, such as CFI-400945 and centrinone, are emerging as promising antineoplastic agents. However, their effectiveness against Ewing's sarcoma, a highly aggressive childhood cancer, remains to be established. Methods: CFI-400945 and centrinone were tested in three Ewing's sarcoma cell lines with different TP53 status. Effects were assessed by flow-cytometric analyses of cell death, dissipation of the mitochondrial transmembrane potential and cell cycle distribution, by cell viability assay as well as by caspase 3/7 activity measurement, by immunoblotting and by immunofluorescence microscopy. Results: CFI-400945 and centrinone elicited cell death in p53 wild-type and mutant Ewing's sarcoma cells. Both agents induced mitochondrial membrane depolarisation, caspase 3/7 activation, PARP1 cleavage and DNA fragmentation, indicating an apoptotic form of cell death. In addition, the PLK4 inhibitors induced G2/M cell cycle arrest, particularly when cell killing was attenuated by the pan-caspase inhibitor z-VAD-fmk. Moreover, CFI-400945 treatment produced polyploidy. Conclusion: Our findings show that PLK4 inhibitors were effective against Ewing's sarcoma cells in vitro and thus provide a rationale for their evaluation in vivo

Synthesis, thermogravimetric and high temperature X-ray diffraction analyses of zinc-substituted nickel manganites

Stoichiometric spinel phases Mn2.352xNi0.65ZnxO4 were prepared by thermal decomposition of mixed oxalate precursor powders Mn0.782aNi0.22ZnaC2O4znH2O (with 0 # a # 0.53) at 900°C. Cation-deficient phases Mn2.352xNi0.65Znxh3d/4O41d were identified in the temperature range 350–500°C. The nonstoichiometric coefficient d was found to strongly depend on the zinc content and the decomposition temperature. We showed that the introduction of zinc into the spinel phase enlarges the stability domain of the structure and inhibits oxidation at least up to 900°C. A cubic single-phase was observed for x # 1.00. The lattice parameter variation of the oxides in the composition range 0 # x # 0.60 can be explained using Poix’s method, in terms of the distribution of Zn21 cations on the tetrahedral sites. However, for higher zinc content (x . 0.6) a detailed analysis of data showed that a small fraction of Zn21 is located on octahedral sites

Secretome of endothelial progenitor cells from stroke patients promotes endothelial barrier tightness and protects against hypoxia-induced vascular leakage

Enfermedad cardiovascular; Terapia celular; SecretomaMalaltia cardiovascular; Teràpia cel·lular; SecretomaCardiovascular disease; Cell therapy; SecretomeBackground Cell-based therapeutic strategies have been proposed as an alternative for brain repair after stroke, but their clinical application has been hampered by potential adverse effects in the long term. The present study was designed to test the effect of the secretome of endothelial progenitor cells (EPCs) from stroke patients (scCM) on in vitro human models of angiogenesis and vascular barrier. Methods Two different scCM batches were analysed by mass spectrometry and a proteome profiler. Human primary CD34+-derived endothelial cells (CD34+-ECs) were used for designing angiogenesis studies (proliferation, migration, and tubulogenesis) or in vitro models of EC monolayer (confluent monolayer ECs—CMECs) and blood–brain barrier (BBB; brain-like ECs—BLECs). Cells were treated with scCM (5 μg/mL) or protein-free endothelial basal medium (scEBM—control). CMECs or BLECs were exposed (6 h) to oxygen–glucose deprivation (OGD) conditions (1% oxygen and glucose-free medium) or normoxia (control—5% oxygen, 1 g/L of glucose) and treated with scCM or scEBM during reoxygenation (24 h). Results The analysis of different scCM batches showed a good reproducibility in terms of protein yield and composition. scCM increased CD34+-EC proliferation, tubulogenesis, and migration compared to the control (scEBM). The proteomic analysis of scCM revealed the presence of growth factors and molecules modulating cell metabolism and inflammatory pathways. Further, scCM decreased the permeability of CMECs and upregulated the expression of the junctional proteins such as occludin, VE-cadherin, and ZO-1. Such effects were possibly mediated through the activation of the interferon pathway and a moderate downregulation of Wnt signalling. Furthermore, OGD increased the permeability of both CMECs and BLECs, while scCM prevented the OGD-induced vascular leakage in both models. These effects were possibly mediated through the upregulation of junctional proteins and the regulation of MAPK/VEGFR2 activity. Conclusion Our results suggest that scCM promotes angiogenesis and the maturation of newly formed vessels while restoring the BBB function in ischemic conditions. In conclusion, our results highlight the possibility of using EPC-secretome as a therapeutic alternative to promote brain angiogenesis and protect from ischemia-induced vascular leakage.This work has been supported under the Euronanomed 8th Joint Call-MAGGBRIS collaborative project by grants from the Spanish Ministry of Science and Innovation (PCIN-2017-090) the French national agency (ANR-ANR-17-ENM3-0005-01), the AC17/00004 grant from Instituto Carlos III (ISCIII) with FEDR funds, and the National Centre for Research and Development (NCBR 15/EuronanoMed/2018). A part of this study has been also funded in the frame of the NANOSTEM project, a Marie Skłodowska-Curie Innovative Training Network (ITN) by receiving grant from the European Union's Horizon 2020 research and innovation programme under grant agreement No. 764958 and the Expression of Interest (EoI) for Collaborative Projects on Regenerative Medicine 2019 P-CMR[C]), and the programs 2017-SGR-1427 and 2017-SGR-765 from the Generalitat de Cataluny. Alba Grayston is supported by the fellowship FI17/00073 from ISCIII with FEDR funds. Miguel Garcia-Gabilondo is supported by the PERIS grant SLT017/20/000197 from Generalitat de Cataluny. The mass spectrometer of the Spectrométrie de Masse de l’Artois (SMART) facilities used in this study was funded by the European Regional Development Fund (ERDF), the Hauts-de-France regional council, and the Université d’Artois (France)

Micro-confinement of bacteria into w/o emulsion droplets for rapid detection and enumeration

International audienceToday, rapid detection and identification of bacteria in microbiological diagnosis is a major issue. Reference methods usually rely on growth of microorganisms, with the drawback of lengthy time-to-result. The method provides global information on a clonal population that is known to be inhomogeneous relative to metabolic states and activities. Therefore, there may be a significant advantage of methods that allow characterisation of individual bacteria from a large population, both for test time reduction and the clinical value of the characterisation. We report here a method for rapid detection and real-time monitoring of the metabolic activities of single bacteria. Water-in-oil emulsions were used to encapsulate single Escherichia coli cells into picolitre (pL)-sized microreactor droplets. The glucuronidase activity in each droplet was monitored using the fluorogenic reporter molecule MUG (4-methylumbelliferyl- - d-glucuronide) coupled to time-lapse fluorescence imaging of the droplets. Such bacterial confinement provides several major advantages. (1) Enzymatic activities of a large number of single bacterium-containing droplet could be monitored simultaneously, allowing the full characterisation of metabolic heterogeneity in a clonal population. We monitored glucuronidase enzymatic activity and growth over ∼200 single bacteria over a 24-h period. (2) Micro-confinement of cells in small volumes allows rapid accumulation of the fluorescent metabolite, hence decreasing the detection time. Independent of the initial concentration of bacteria in the sample, detection of the presence of bacteria could be achieved in less than 2 h. (3) Considering the random distribution of bacteria in droplets, this method allowed rapid and reliable enumeration of bacteria in the initial sample. Overall, the results of this study showed that confinement of bacterial cells increased the effective concentration of fluorescent metabolites leading to rapid (2 h) detection of the fluorescent metabolites, thus significantly reducing time to numeration

Early neutrophil trajectory following clozapine may predict clozapine response - Results from an observational study using electronic health records

Background: Clozapine has unique effectiveness in treatment-resistant schizophrenia and is known to cause immunological side-effects. A transient spike in neutrophils commonly occurs in the first weeks of clozapine therapy. There is contradictory evidence in the literature as to whether neutrophil changes with clozapine are linked to treatment response. Aims: The current study aims to further examine the neutrophil changes in response to clozapine and explore any association between neutrophil trajectory and treatment response. Methods: A retrospective cohort study of patients undergoing their first treatment with clozapine and continuing for at least 2 years identified 425 patients (69% male/31% female). Neutrophil counts at baseline, 3 weeks and 1 month were obtained predominantly by linkage with data from the clozapine monitoring service. Clinical Global Impression- Severity (CGI-S) was rated from case notes at the time of clozapine initiation and at 2 years. Latent class growth analysis (LCGA) was performed to define distinct trajectories of neutrophil changes during the first month of treatment. Logistic regression was then conducted to investigate for association between the trajectory of neutrophil count changes in month 1 and clinical response at 2 years as well as between baseline neutrophil count and response. Results: Of the original cohort, 397 (93%) patients had useable neutrophil data during the first 6 weeks of clozapine treatment. LCGA revealed significant differences in neutrophil trajectories with a three-class model being the most parsimonious. The classes had similar trajectory profiles but differed primarily on overall neutrophil count: with low, high-normal and high neutrophil classes, comprising 52%, 40% and 8% of the sample respectively. Membership of the high-normal group was associated with significantly increased odds of a positive response to clozapine, as compared to the low neutrophil group [Odds ratio (OR) = 2.10, p-value = 0.002; 95% confidence interval (95% CI) = 1.31–3.36]. Baseline neutrophil count was a predictor of response to clozapine at 2 years, with counts of ≥5 × 109/l significantly associated with positive response (OR = 1.60, p-value = 0.03; 95% CI = 1.03–2.49). Conclusions: Our data are consistent with the hypothesis that patients with low-level inflammation, reflected in a high-normal neutrophil count, are more likely to respond to clozapine, raising the possibility that clozapine exerts its superior efficacy via immune mechanisms.</p

Power laws in microrheology experiments on living cells: comparative analysis and modelling

We compare and synthesize the results of two microrheological experiments on the cytoskeleton of single cells. In the first one, the creep function J(t) of a cell stretched between two glass plates is measured after applying a constant force step. In the second one, a micrometric bead specifically bound to transmembrane receptors is driven by an oscillating optical trap, and the viscoelastic coefficient $G_e(\omega)$ is retrieved. Both $J(t)$ and $G_e(\omega)$ exhibit power law behavior: $J(t)= A(t/t_0)^\alpha$ and $\bar G_e(\omega)\bar = G_0 (\omega/\omega_0)^\alpha$, with the same exponent $\alpha\approx 0.2$. This power law behavior is very robust ; $\alpha$ is distributed over a narrow range, and shows almost no dependance on the cell type, on the nature of the protein complex which transmits the mechanical stress, nor on the typical length scale of the experiment. On the contrary, the prefactors $A_0$ and $G_0$appear very sensitive to these parameters. Whereas the exponents $\alpha$ are normally distributed over the cell population, the prefactors $A_0$ and $G_0$ follow a log-normal repartition. These results are compared with other data published in the litterature. We propose a global interpretation, based on a semi-phenomenological model, which involves a broad distribution of relaxation times in the system. The model predicts the power law behavior and the statistical repartition of the mechanical parameters, as experimentally observed for the cells. Moreover, it leads to an estimate of the largest response time in the cytoskeletal network: $\tau_m \approx 1000$ s.Comment: 47 pages, 14 figures // v2: PDF file is now Acrobat Reader 4 (and up) compatible // v3: Minor typos corrected - The presentation of the model have been substantially rewritten (p. 17-18), in order to give more details - Enhanced description of protocols // v4: Minor corrections in the text : the immersion angles are estimated and not measured // v5: Minor typos corrected. Two references were clarifie

Global standardization and local complexity. A case study of an aquaculture system in Pampanga delta, Philippines

International standards result from global policies formulated primarily to address issues on food safety, traceability, environmental impact as well as social accountability. As in other agro-food industries, these rules increasingly regulate aquaculture, especially since it has started to be the object of many criticisms. The standards are generally designed in a top-down way and do not always consider the local specificities of production systems. Such implementation favors the emergence of similar patterns of production and trade across different locations. Based on a case study, this paper aims to highlight the gap between the vision conveyed by expert-based, simple and replicable policies of standardization,versusthe real complexity and uniqueness of local aquaculture systems. The assumption is that the lack of recognition of this complexity leadsde factoto the reproduction of dominant modes of production based on standards, ignoring some local actors with a capacity for innovation, while favoring a few larger stakeholders. To reveal the gap, the study looks at some agents of an extensive aquaculture system in the Philippines and at their interaction, focusing on gleaning and trading activities. It then reveals the changes that followed the local implementation of an International food safety standard. It finally discusses (i) the links between the global and normative point of view, and the local and unique dynamics and (ii) some bridges able to reconcile both

MyCoast Pilot Communities

Final Project for URSP688L: Recent Developments in Urban Studies: Planning Technology (Fall 2020). University of Maryland, College Park.Maryland’s MyCoast Program is a strategic effort to anticipate and assess the impacts of flooding events across the state. MyCoast is a National program with the mission to document tides, storm damage, beach cleanups, nuisance flooding and more to allow decision makers, emergency managers, and others to use public reports to make better decisions. MyCoast: Maryland, “is a portal to collect and analyze pictures and data relating to flooding caused by precipitation or coastal events. In Maryland, this is a project supervised by the Chesapeake & Coastal Service and Maryland Department of Natural Resources (MDNR). The MyCoast: Maryland website can be found here: https://mycoast.org/md. To enhance this project’s reach in relevant locales, we set out to discern which communities in the state are most at risk for negative flooding effects. Our partner, Maryland Department of Natural Resources, asked that the project inform their roll-out of the MyCoast program in Maryland, and make sure that they reached the communities that are most at risk for flooding and might be the least able to reach those in power for assistance.Maryland Department of Natural Resourceshttps://storymaps.arcgis.com/stories/24a58f32bd82437f81f80bf3abfa7e9

Electronic energy migration in Microtubules

The repeating arrangement of tubulin dimers confers great mechanical strength to microtubules, which are used as scaffolds for intracellular macromolecular transport in cells and exploited in biohybrid devices. The crystalline order in a microtubule, with lattice constants short enough to allow energy transfer between amino acid chromophores, is similar to synthetic structures designed for light harvesting. After photoexcitation, can these amino acid chromophores transfer excitation energy along the microtubule like a natural or artificial light-harvesting system? Here, we use tryptophan autofluorescence lifetimes to probe energy hopping between aromatic residues in tubulin and microtubules. By studying how the quencher concentration alters tryptophan autofluorescence lifetimes, we demonstrate that electronic energy can diffuse over 6.6 nm in microtubules. We discover that while diffusion lengths are influenced by tubulin polymerization state (free tubulin versus tubulin in the microtubule lattice), they are not significantly altered by the average number of protofilaments (13 versus 14). We also demonstrate that the presence of the anesthetics etomidate and isoflurane reduce exciton diffusion. Energy transport as explained by conventional Förster theory (accommodating for interactions between tryptophan and tyrosine residues) does not sufficiently explain our observations. Our studies indicate that microtubules are, unexpectedly, effective light harvesters

Update on the Serum Biomarkers and Genetic Factors Associated with Safety and Efficacy of rt-PA Treatment in Acute Stroke Patients

An accurate understanding of the mechanisms underlying an individual's response to rt-PA treatment is critical to improve stroke patients' management. We thus reviewed the literature in order to identify biochemical and genetic factors that have been associated with safety and efficacy of rt-PA administration after stroke