Human Cancer Cell Radiation Response Investigated through Topological Analysis of 2D Cell Networks

Clonogenic assays are routinely used to evaluate the response of cancer cells to external radiation fields, assess their radioresistance and radiosensitivity, estimate the performance of radiotherapy. However, classic clonogenic tests focus on the number of colonies forming on a substrate upon exposure to ionizing radiation, and disregard other important characteristics of cells such their ability to generate structures with a certain shape. The radioresistance and radiosensitivity of cancer cells may depend less on the number of cells in a colony and more on the way cells interact to form complex networks. In this study, we have examined whether the topology of 2D cancer-cell graphs is influenced by ionizing radiation. We subjected different cancer cell lines, i.e. H4 epithelial neuroglioma cells, H460 lung cancer cells, PC3 bone metastasis of grade IV of prostate cancer and T24 urinary bladder cancer cells, cultured on planar surfaces, to increasing photon radiation levels up to 6 Gy. Fluorescence images of samples were then processed to determine the topological parameters of the cell-graphs developing over time. We found that the larger the dose, the less uniform the distribution of cells on the substrate—evidenced by high values of small-world coefficient (cc), high values of clustering coefficient (cc), and small values of characteristic path length (cpl). For all considered cell lines, >1 for doses higher or equal to 4 Gy, while the sensitivity to the dose varied for different cell lines: T24 cells seem more distinctly affected by the radiation, followed by the H4, H460 and PC3 cells. Results of the work reinforce the view that the characteristics of cancer cells and their response to radiotherapy can be determined by examining their collective behavior—encoded in a few topological parameters—as an alternative to classical clonogenic assays

The New Youth of the In Situ Transmission Electron Microscopy

The idea of in situ transmission electron microscopy (TEM) and its possible ramifications were proposed at the very dawn of electron microscopy, but the translation from theory to practice encountered many technological setbacks, which hindered the feasibility of the most elaborated approaches until recent times. However, the several technological improvements achieved in the last 10–15 years filled this gap, allowing the direct observation of the dynamic response of materials to external stimuli under a vast range of conditions going from vacuum to gaseous or liquid environment. This resulted in a blossoming of the in situ TEM and scanning TEM (STEM) techniques to a new youth for a vast, growing range of applications, which cannot be rightfully detailed in a short span; therefore, this chapter should be intended as a guide highlighting a selection of the most inspiring, recently achieved results

JRC Ispra EMEP-GAW Regional Station for Atmospheric Research - 2007 Report

The aim of the JRC-Ispra station for atmospheric research (45°49'N, 8°38'E) is to monitor atmospheric parameters (pollutant concentrations and fluxes, atmospheric particle chemical composition, number size distribution and optical properties) to contribute in assessing the impact of European policies on air pollution and climate change. The station has been operated continuously since November 1985, with a gap in gas phase data due to a severe breakdown of the data acquisition system in 2003 though. The measurements performed in 2007 led to annual averages of ca. 32 µg m-3 O3, 0.8 µg m-3 SO2, 21 µg m-3 NO2 and 30 µg m-3 PM10. Carbonaceous species (organic matter plus elemental carbon) are the main constituents of PM2.5 (> 55 %) followed by NH4NO3 (20-30 %) and (NH4)2SO4 (10-20 %). The measurements confirmed the seasonal variations observed over the previous years, mainly driven by meteorology rather than by changes in emissions. Aerosol physical and optical properties were also measured in 2007. The average particle number (from 10 nm to 10 µm) was about 9200 cm-3 in 2007. The mean (close to dry) aerosol single scattering albedo (0.79) was low compared to the values generally observed in Europe, which means that the cooling effect of aerosols is reduced in our region compared to others. Long-term trends (over 20 years) show consistent decreases in sulfur concentrations and deposition, PM mass concentration (-0.9 µg m-3 yr-1) and in extreme ozone value occurrence frequency. The decreasing trends in oxidised and reduced nitrogen species are much less pronounced. However, historical minimum in NO3-, NH4+, (and SO42-) wet deposition, as well as in O3 pollution indicators (AOT40 and SOMO35) were observed in 2007.JRC.H.2-Climate chang

Localization and subcellular association of Grapevine Pinot Gris Virus in grapevine leaf tissues

Despite the increasing impact of Grapevine Pinot gris disease (GPG-disease) worldwide, etiology about this disorder is still uncertain. The presence of the putative causal agent, the Grapevine Pinot Gris Virus (GPGV), has been reported in symptomatic grapevines (presenting stunting, chlorotic mottling, and leaf deformation) as well as in symptom-free plants. Moreover, information on virus localization in grapevine tissues and virus-plant interactions at the cytological level is missing at all. Ultrastructural and cytochemical investigations were undertaken to detect virus particles and the associated cytopathic effects in field-grown grapevine showing different symptom severity. Asymptomatic greenhouse-grown grapevines, which tested negative for GPGV by real time RT-PCR, were sampled as controls. Multiplex real-time RT-PCR and ELISA tests excluded the presence of viruses included in the Italian certification program both in field-grown and greenhouse-grown grapevines. Conversely, evidence was found for ubiquitous presence of Grapevine Rupestris Stem Pitting-associated Virus (GRSPaV), Hop Stunt Viroid (HSVd), and Grapevine Yellow Speckle Viroid 1 (GYSVd-1) in both plant groups. Moreover, in every field-grown grapevine, GPGV was detected by real-time RT-PCR. Ultrastructural observations and immunogold labelling assays showed filamentous flexuous viruses in the bundle sheath cells, often located inside membrane-bound organelles. No cytological differences were observed among field-grown grapevine samples showing different symptom severity. GPGV localization and associated ultrastructural modifications are reported and discussed, in the perspective of assisting management and control of the disease. \ua9 2017 The Author(s

Filamentous sieve element proteins are able to limit phloem mass flow, but not phytoplasma spread

In Fabaceae, dispersion of forisomes\u2014highly ordered aggregates of sieve element proteins\u2014in response to phytoplasma infection was proposed to limit phloem mass flow and, hence, prevent pathogen spread. In this study, the involvement of filamentous sieve element proteins in the containment of phytoplasmas was investigated in non-Fabaceae plants. Healthy and infected Arabidopsis plants lacking one or two genes related to sieve element filament formation\u2014AtSEOR1 (At3g01680), AtSEOR2 (At3g01670), and AtPP2-A1 (At4g19840)\u2014were analysed. TEM images revealed that phytoplasma infection induces phloem protein filament formation in both the wild-type and mutant lines. This result suggests that, in contrast to previous hypotheses, sieve element filaments can be produced independently of AtSEOR1 and AtSEOR2 genes. Filament presence was accompanied by a compensatory overexpression of sieve element protein genes in infected mutant lines in comparison with wild-type lines. No correlation was found between phloem mass flow limitation and phytoplasma titre, which suggests that sieve element proteins are involved in defence mechanisms other than mechanical limitation of the pathogen

Agroinoculation of Grapevine Pinot Gris Virus in tobacco and grapevine provides insights on viral pathogenesis

The Grapevine Pinot Gris disease (GPG-d) is a novel disease characterized by symptoms such as leaf mottling and deformation, which has been recently reported in grapevines, and mostly in Pinot gris. Plants show obvious symptoms at the beginning of the growing season, while during summer symptom recovery frequently occurs, manifesting as symptomless leaves. A new Trichovirus, named Grapevine Pinot gris virus (GPGV), which belongs to the family Betaflexiviridae was found in association with infected plants. The detection of the virus in asymptomatic grapevines raised doubts about disease aetiology. Therefore, the primary target of this work was to set up a reliable system for the study of the disease in controlled conditions, avoiding interfering factor(s) that could affect symptom development. To this end, two clones of the virus, pRI::GPGV-vir and pRI::GPGV-lat, were generated from total RNA collected from one symptomatic and one asymptomatic Pinot gris grapevine, respectively. The clones, which encompassed the entire genome of the virus, were used in Agrobacterium-mediated inoculation of Vitis vinifera and Nicotiana benthamiana plants. All inoculated plants developed symptoms regardless of their inoculum source, demonstrating a correlation between the presence of GPGV and symptomatic manifestations. Four months post inoculum, the grapevines inoculated with the pRI::GPGV-lat clone developed asymptomatic leaves that were still positive to GPGV detection. Three to four weeks later (i.e. ca. 5 months post inoculum), the same phenomenon was observed in the grapevines inoculated with pRI::GPGV-vir. This observation perfectly matches symptom progression in infected field-grown grapevines, suggesting a possible role for plant antiviral mechanisms, such as RNA silencing, in the recovery process.</div

Non-Immersive Virtual Reality Telerehabilitation System Improves Postural Balance in People with Chronic Neurological Diseases

Background: People with chronic neurological diseases, such as Parkinson’s Disease (PD) and Multiple Sclerosis (MS), often present postural disorders and a high risk of falling. When difficulties in achieving outpatient rehabilitation services occur, a solution to guarantee the continuity of care may be telerehabilitation. This study intends to expand the scope of our previously published research on the impact of telerehabilitation on quality of life in an MS sample, testing the impact of this type of intervention in a larger sample of neurological patients also including PD individuals on postural balance. Methods: We included 60 participants with MS and 72 with PD. All enrolled subjects were randomized into two groups: 65 in the intervention group and 67 in the control group. Both treatments lasted 30–40 sessions (5 days/week, 6–8 weeks). Motor, cognitive, and participation outcomes were registered before and after the treatments. Results: All participants improved the outcomes at the end of the treatments. The study’s primary outcome (Mini-BESTest) registered a greater significant improvement in the telerehabilitation group than in the control group. Conclusions: Our results demonstrated that non-immersive virtual reality telerehabilitation is well tolerated and positively affects static and dynamic balance and gait in people with PD and MS

Generation and maturation of human iPSC-derived 3D organotypic cardiac microtissues in long-term culture

Cardiovascular diseases remain the leading cause of death worldwide; hence there is an increasing focus on developing physiologically relevant in vitro cardiovascular tissue models suitable for studying personalized medicine and pre-clinical tests. Despite recent advances, models that reproduce both tissue complexity and maturation are still limited. We have established a scaffold-free protocol to generate multicellular, beating human cardiac microtissues in vitro from hiPSCs-namely human organotypic cardiac microtissues (hOCMTs)-that show some degree of self-organization and can be cultured for long term. This is achieved by the differentiation of hiPSC in 2D monolayer culture towards cardiovascular lineage, followed by further aggregation on low-attachment culture dishes in 3D. The generated hOCMTs contain multiple cell types that physiologically compose the heart and beat without external stimuli for more than 100 days. We have shown that 3D hOCMTs display improved cardiac specification, survival and metabolic maturation as compared to standard monolayer cardiac differentiation. We also confirmed the functionality of hOCMTs by their response to cardioactive drugs in long-term culture. Furthermore, we demonstrated that they could be used to study chemotherapy-induced cardiotoxicity. Due to showing a tendency for self-organization, cellular heterogeneity, and functionality in our 3D microtissues over extended culture time, we could also confirm these constructs as human cardiac organoids (hCOs). This study could help to develop more physiologically-relevant cardiac tissue models, and represent a powerful platform for future translational research in cardiovascular biology