A versatile ultrasound system for in vitro experiments

Objective One of the most difficult tasks to achieve with the available instrumentations used to study the interaction between ultrasound (US) and cellular model systems is to design an experiment, where only the effects of one physical parameter at a time is evaluated, while all the others are kept constant. The set-ups are usually custom-made, often by means of clinical instrument intended for a different therapeutic purpose. Furthermore, the results are not strictly comparable with others obtained with techniques considered standard in molecular and cellular biology at this time, because there is the need to use non-standard devices to contain biological samples. Sterility, as well as temperature, is not well controlled and reproducibility is usually a major concern. In our study we show the effects of ultrasound treatments on different cellular systems. The experiments are performed with a versatile bench-top US apparatus to be adapted for several in vitro experiments and that allows easy and robust reproducibility using standard set-ups for the cell samples. Methods One main feature of our bench-top US system is that it has been designed in order to use standard plasticware commonly used in molecular biology labs, ensuring the temperature control and sterility conditions needed in the field. We present a set-up where the simultaneous use of a set of transducers operating at different frequencies on the same plate, allows the comparison of the deposition of the same acoustic pressure, whilst evaluating the effect of frequency alone on the readout of the cell experiments. The apparatus modular design also allows the use of a set of transducers operating at the same frequency, in experiments where the throughput is a relevant factor. We demonstrate that it is possible to define the position of the target within all the achievable areas of the acoustic field with sub-millimetric accuracy. Tests for several applications based on biologic effects by ultrasound have been carried out by varying the acoustic parameters such as power, frequency range, sonication time and duty cycle, all controlled within robust protocols executed in automation. Results The resulting data proves that it is possible to perform in vitro experiments for different purposes (i.e. drug delivery, cellular sonoporation, nanoparticles or microbubbles swelling, tissue regeneration, neuronal cell stimulation etc.) keeping the relevant physical parameters of sonication constant, for instance acoustic pressure, but varying the others parameters (i.e. frequency, pulse length or duty cycle etc) one at the time. Conclusions We show that with our apparatus it is possible to obtain robust and reproducible results on cellular experiments, using all the standard devices that are commonly available in biological labs. The improvement on the side of reproducibility and portability of the experiments allows a straightforward comparison between our results and those obtained with other techniques

Microenvironment in neuroblastoma: Isolation and characterization of tumor-derived mesenchymal stromal cells

Background: It has been proposed that mesenchymal stromal cells (MSCs) promote tumor progression by interacting with tumor cells and other stroma cells in the complex network of the tumor microenvironment. We characterized MSCs isolated and expanded from tumor tissues of pediatric patients diagnosed with neuroblastomas (NB-MSCs) to define interactions with the tumor microenvironment. Methods: Specimens were obtained from 7 pediatric patients diagnosed with neuroblastoma (NB). Morphology, immunophenotype, differentiation capacity, proliferative growth, expression of stemness and neural differentiation markers were evaluated. Moreover, the ability of cells to modulate the immune response, i.e. inhibition of phytohemagglutinin (PHA) activated peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cytotoxic function, was examined. Gene expression profiles, known to be related to tumor cell stemness, Wnt pathway activation, epithelial-mesenchymal transition (EMT) and tumor metastasis were also evaluated. Healthy donor bone marrow-derived MSCs (BM-MSC) were employed as controls. Results: NB-MSCs presented the typical MSC morphology and phenotype. They showed a proliferative capacity superimposable to BM-MSCs. Stemness marker expression (Sox2, Nanog, Oct3/4) was comparable to BM-MSCs. NB-MSC in vitro osteogenic and chondrogenic differentiation was similar to BM-MSCs, but NB-MSCs lacked adipogenic differentiation capacity. NB-MSCs reached senescence phases at a median passage of P7 (range, P5-P13). NB-MSCs exhibited greater immunosuppressive capacity on activated T lymphocytes at a 1:2 (MSC: PBMC) ratio compared with BM-MSCs (p = 0.018). NK cytotoxic activity was not influenced by co-culture, either with BM-MSCs or NB-MSCs. Flow-cytometry cell cycle analysis showed that NB-MSCs had an increased number of cells in the G0-G1 phase compared to BM-MSCs. Transcriptomic profiling results indicated that NB-MSCs were enriched with EMT genes compared to BM-MSCs. Conclusions: We characterized the biological features, the immunomodulatory capacity and the gene expression profile of NB-MSCs. The NB-MSC gene expression profile and their functional properties suggest a potential role in promoting tumor escape, invasiveness and metastatic traits of NB cancer cells. A better understanding of the complex mechanisms underlying the interactions between NB cells and NB-derived MSCs should shed new light on potential novel therapeutic approaches

CdO-based nanostructures as novel CO2 gas sensors

Crystalline Cd(OH)2/CdCO3 nanowires, having lengths in the range from 0.3 up to several microns and 5–30 nm in diameter, were synthesized by a microwave-assisted wet chemical route and used as a precursor to obtain CdO nanostructures after a suitable thermal treatment in air. The morphology and microstructure of the as-synthesized and annealed materials have been investigated by scanning electron microscopy, transmission electron microscopy, x-ray diffraction and thermogravimetry–differential scanning calorimetry. The change in morphology and electrical properties with temperature has revealed a wire-to-rod transformation along with a decreases of electrical resistance. Annealed samples were printed on a ceramic substrate with interdigitated contacts to fabricate resistive solid state sensors. Gas sensing properties were explored by monitoring CO2 in synthetic air in the concentration range 0.2–5 v/v% (2000–50 000 ppm). The effect of annealing temperature, working temperature and CO2 concentration on sensing properties (sensitivity, response/recovery time and stability) were investigated. The results obtained demonstrate that CdO-based thick films have good potential as novel CO2 sensors for practical applications

Apoptosis induced by a HIPK2 full-length-specific siRNA is due to off-target effects rather than prevalence of HIPK2-Δe8 isoform.

Small interfering RNAs (siRNAs) are widely used to study gene function and extensively exploited for their potential therapeutic applications. HIPK2 is an evolutionary conserved kinase that binds and phosphorylates several proteins directly or indirectly related to apoptosis. Recently, an alternatively spliced isoform skipping 81 nucleotides of exon 8 (Hipk2-∆e8) has been described. Selective depletion of Hipk2 full-length (Hipk2-FL) with a speci c siRNA that spares the Hipk2-∆e8 isoform has been shown to strongly induce apoptosis, suggesting an unpredicted dominant- negative effect of Hipk2-FL over the ∆e8 isoform. From this observation, we sought to take advantage and assessed the therapeutic potential of generating Hipk2 isoform unbalance in tumor-initiating cells derived from colorectal cancer patients. Strong reduction of cell viability was induced in vitro and in vivo by the originally described exon 8-speci c siRNA, supporting a potential therapeutic application. However, validation analyses performed with additional exon8-speci c siRNAs with different stabilities showed that all exon8-targeting siRNAs can induce comparable Hipk2 isoform unbalance but only the originally reported e8-siRNA promotes cell death. These data show that loss of viability does not depend on the prevalence of Hipk2- ∆e8 isoform but it is rather due to microRNA-like off-target effects

Detection of the Cherenkov light diffused by Sea Water with the ULTRA Experiment

The study of Ultra High Energy Cosmic Rays represents one of the most challenging topic in the Cosmic Rays and in the Astroparticle Physics fields. The interaction of primary particles with atmospheric nuclei produces a huge Extensive Air Shower together with isotropic emission of UV fluorescence light and highly directional Cherenkov photons, that are reflected/diffused isotropically by the impact on the Earth's surface or on high optical depth clouds. For space-based observations, detecting the reflected Cherenkov signal in a delayed coincidence with the fluorescence light improves the accuracy of the shower reconstruction in space and in particular the measurement of the shower maximum, giving a strong signature for discriminating hadrons and neutrinos, and helping to estimate the primary chemical composition. Since the Earth's surface is mostly covered by water, the ULTRA (UV Light Transmission and Reflection in the Atmosphere)experiment has been designed to provide the diffusing properties of sea water, overcoming the lack of information in this specific field. A small EAS array, made up of 5 particle detectors, and an UV optical device, have been coupled to detect in coincidence both electromagnetic and UV components. The detector was in operation from May to December, 2005, in a small private harbor in Capo Granitola (Italy); the results of these measurements in terms of diffusion coefficient and threshold energy are presented here.Comment: 4 pages, 3 figures, PDF format, Proceedings of 30th ICRC, International Cosmic Ray Conference 2007, Merida, Yucatan, Mexico, 3-11 July 200

Distinctive features of tumor-infiltrating gd T lymphocytes in human colorectal cancer

gd T cells usually infiltrate many different types of cancer, but it is unclear whether they inhibit or promote tumor progression. Moreover, properties of tumor-infiltrating gd T cells and those in the corresponding normal tissue remain largely unknown. Here we have studied features of gd T cells in colorectal cancer, normal colon tissue and peripheral blood, and correlated their levels with clinicopathologic hallmarks. Flow cytometry and transcriptome analyses showed that the tumor comprised a highly variable rate of TILs (5-90%) and 4%gd T cells on average, with the majority expressing Vd1. Most Vd1 and Vd2 T cells showed a predominant effector memory phenotype and had reduced production of IFN-g which was likely due to yet unidentified inhibitory molecules present in cancer stem cell secretome. Transcriptome analyses revealed that patients containing abundant gd T cells had significantly longer 5-year disease free survival rate, suggesting their efficacy in controlling tumor at very early stage

Experimental study of a liquid Xenon PET prototype module

A detector using liquid Xenon in the scintillation mode is studied for Positron Emission Tomography (PET). The specific design aims at taking full advantage of the liquid Xenon properties. It does feature a promising insensitive to any parallax effect. This work reports on the performances of the first LXe prototype module, equipped with a position sensitive PMT operating in the VUV range (178 nm).Comment: Proc. of the 7th International Workshops on Radiation Imaging Detectors (IWORID-7), Grenoble, France 4-7 July 200

TEMPERATURE AND LEVEL DENSITY PARAMETER OF EVAPORATION RESIDUES PRODUCED IN THE REACTION 165Ho + 600 MeV 20Ne

Evaporative and preequilibrium neutrons emitted from evaporation residues in the reaction Ho + 600 MeV neon are exploited to deduce the thermal excitation energy E* and temperature T of the residues. From these quantities the level density parameter is deduced at a temperature of 4.1 MeV

Dual targeting of HER3 and MEK may overcome HER3-dependent drug-resistance of colon cancers

Although the medical treatment of colorectal cancer has evolved greatly in the last years, a significant portion of early-stage patients develops recurrence after therapies. The current clinical trials are directed to evaluate new drug combinations and treatment schedules. By the use of patient-derived or established colon cancer cell lines, we found that the tyrosine kinase receptor HER3 is involved in the mechanisms of resistance to therapies. In agreement, the immunohistochemical analysis of total and phospho-HER3 expression in 185 colorectal cancer specimens revealed a significant correlation with lower disease-free survival. Targeting HER3 by the use of the monoclonal antibody patritumab we found induction of growth arrest in all cell lines. Despite the high efficiency of patritumab in abrogating the HER3-dependent activation of PI3K pathway, the HER2 and EGFR-dependent MAPK pathway is activated as a compensatory mechanism. Interestingly, we found that the MEK-inhibitor trametinib inhibits, as expected, the MAPK pathway but induces the HER3-dependent activation of PI3K pathway. The combined treatment results in the abrogation of both PI3K and MAPK pathways and in a significant reduction of cell proliferation and survival. These data suggest a new strategy of therapy for HER3-overexpressing colon cancers