Analysis of biological and technical variability in gene expression assays from formalin-fixed paraffin-embedded classical Hodgkin lymphomas

Formalin-fixed paraffin-embedded (FFPE) tissues are invaluable sources of biological material for research and diagnostic purposes. In this study, we aimed to identify biological and technical variability in RT-qPCR TaqMan® assays performed with FFPE-RNA from lymph nodes of classical Hodgkin lymphoma samples. An ANOVA-nested 6-level design was employed to evaluate BCL2, CASP3, IRF4, LYZ and STAT1 gene expression. The most variable genes were CASP3 (low expression) and LYZ (high expression). Total variability decreased after normalization for all genes, except by LYZ. Genes with moderate and low expression were identified and suffered more the effects of the technical manipulation than high-expression genes. Pre-amplification was shown to introduce significant technical variability, which was partially alleviated by lowering to a half the amount of input RNA. Ct and Cy0 quantification methods, based on cycle-threshold and the kinetic of amplification curves, respectively, were compared. Cy0 method resulted in higher quantification values, leading to the decrease of total variability in CASP3 and LYZ genes. The mean individual noise was 0.45 (0.31 to 0.61 SD), indicating a variation of gene expression over ~1.5 folds from one case to another. We showed that total variability in RT-qPCR from FFPE-RNA is not higher than that reported for fresh complex tissues, and identified gene-, and expression level-sources of biological and technical variability, which can allow better strategies for designing RT-qPCR assays from highly degraded and inhibited samples

Bioinformatics and mathematical modelling in the study of receptor-receptor interactions and receptor oligomerization: focus on adenosine receptors.

none8sìThe concept of intra-membrane receptor-receptor interactions (RRIs) between different types of G protein-coupled receptors (GPCRs) and evidence for their existence was introduced by Agnati and Fuxe in 1980/81 through the biochemical analysis of the effects of neuropeptides on the binding characteristics of monoamine receptors in membrane preparations from discrete brain regions and functional studies of the interactions between neuropeptides and monoamines in the control of specific functions such as motor control and arterial blood pressure control in animal models. Whether GPCRs can form high-order structures is still a topic of an intense debate. Increasing evidence, however, suggests that the hypothesis of the existence of high-order receptor oligomers is correct. A fundamental consequence of the view describing GPCRs as interacting structures, with the likely formation at the plasma membrane of receptor aggregates of multiple receptors (Receptor Mosaics) is that it is no longer possible to describe signal transduction simply as the result of the binding of the chemical signal to its receptor, but rather as the result of a filtering/integration of chemical signals by the Receptor Mosaics (RMs) and membrane-associated proteins. Thus, in parallel with experimental research, significant efforts were spent in bioinformatics and mathematical modelling. We review here the main approaches that have been used to assess the interaction interfaces allowing the assembly of GPCRs and to shed some light on the integrative functions emerging from the complex behaviour of these RMs. Particular attention was paid to the RMs generated by adenosine A(2A), dopamine D-2, cannabinoid CB1, and metabotropic glutamate mGlu(5) receptors (A(2A). D-2, CB1, and mGlu(5), respectively), and a possible approach to model the interplay between the D-2-A(2A)-CB1 and D-2-A(2A)-mGlu(5) trimers is proposed. This article is part of a Special Issue entitled: "Adenosine Receptors". (C) 2010 Elsevier B.V. All rights reserved.openD. GUIDOLIN; F. CIRUELA; S. GENEDANI; M. GUESCINI; C. TORTORELLA; G. ALBERTIN; K. FUXE; L.F. AGNATID., Guidolin; F., Ciruela; S., Genedani; Guescini, Michele; C., Tortorella; G., Albertin; K., Fuxe; L. F., Agnat

Proteomics-based investigation in C2C12 myoblast differentiation

Skeletal muscle cell differentiation is a multistage process extensively studied over the years. Even if great improvements have been achieved in defining biological process underlying myogenesis, many molecular mechanisms need still to be clarified

C2C12 MYOBLASTS RELEASE MICRO-VESICLES CONTAINING mtDNA AND PROTEINS INVOLVED IN SIGNAL TRANSDUCTION

none11Micro-vesicles can be released by different cell types and operate as ‘safe containers’ mediatine inter-cellular communication. In this work we investigated whether cultured myoblasts could release exosomes. The reported data demonstrate, for the first time, that C2C12 myoblasts release micro-vesicles as shown by the presence of two exosome markers (Tsg101 and Alix proteins). Using real-time PCR analysis it was shown that these micro-vesicles, like other cell types, carry mtDNA. Proteomic characterization of the released micro-vesicle contents showed the presence of many proteins involved in signal transduction. The bioinformatics assessment of the Disorder Index and Aggregation Index of these proteins suggested that C2C12 micro-vesicles mainly deliver the machinery for signal transduction to target cells rather than key proteins involved in hub functions in molecular networks. The presence of IGFBP-5 in the purified micro-vesicles represents an exception, since this binding protein can play a key role in the modulation of the IGF-1 signalling pathway. In conclusion, the present findings demonstrate that skeletal muscle cells release micro-vesicles, which probably have an important role in the communication processes within skeletal muscles and between skeletal muscles and other organs. In particular, the present findings suggest possibile new diagnostic approaches to skeletal muscle diseases.openM. GUESCINI; D. GUIDOLIN; L. VALLORANI; L. CASADEI; A.M. GIOACCHINI; P. TIBOLLO; M. BATTISTELLI; E. FALCIERI; L. BATTISTIN; L.F. AGNATI; V. STOCCHIGuescini, Michele; D., Guidolin; Vallorani, Luciana; Casadei, Lucia; Gioacchini, ANNA MARIA; P., Tibollo; Battistelli, Michela; Falcieri, Elisabetta; L., Battistin; L. F., Agnati; Stocchi, Vilbert

Detection and investigation of extracellular vesicles in serum and urine supernatant of prostate cancer patients

Prostate Cancer (PCa) is one of the most frequently identified urological cancers. PCa patients are often over-diagnosed due to still not highly specific diagnostic methods. The need for more accurate diagnostic tools to prevent overestimated diagnosis and unnecessary treatment of patients with non-malignant conditions is clear, and new markers and methods are strongly desirable. Extracellular vesicles (EVs) hold great promises as liquid biopsy-based markers. Despite the biological and technical issues present in their detection and study, these particles can be found highly abundantly in the biofluid and encompass a wealth of macromolecules that have been reported to be related to many physiological and pathological processes, including cancer onset, metastasis spreading, and treatment resistance. The present study aims to perform a technical feasibility study to develop a new workflow for investigating EVs from several biological sources. Serum and urinary supernatant EVs of PCa, benign prostatic hyperplasia (BPH) patients, and healthy donors were isolated and investigated by a fast, easily performable, and cost-effective cytofluorimetric approach for a multiplex detection of 37 EV-antigens. We also observed significant alterations in serum and urinary supernatant EVs potentially related to BPH and PCa, suggesting a potential clinical application of this workflow

Dietary fat differentially modulate the mRNA expression levels of oxidative mitochondrial genes in skeletal muscle of healthy subjects.

Background and aims: Different types of dietary fats exert differential effects on glucose and lipid metabolism. Our aim was to evaluate the impact of different dietary fats on the expression of skeletal muscle genes regulating mitochondrial replication and function in healthy subjects. Methods and results: Ten healthy subjects (age 29±3 years; BMI 25.0±3kg/m2) received in a random order a test meal with the same energy content but different composition in macronutrients and quality of fat: Mediterranean (MED) meal, SAFA meal (Lipid 66%, saturated 36%) and MUFA meal (Lipid 63%, monounsaturated 37%). At fast and after 180min, a fine needle aspiration was performed from the vastus lateralis for determination of mitochondrial gene expression by quantitative PCR. No difference in glucose and triglyceride response was observed between the three meals, while NEFA levels were significantly higher following fat-rich meals compared to MED meal (p<0.002-0.0001). MED meal was associated with an increased expression, albeit not statistically significant, of some genes regulating both replication and function. Following MUFA meal, a significant increase in the expression of PGC1β (p=0.02) and a reduction in the transcription factor PPARδ (p=0.006) occurred with no change in the expression of COX and GLUT4 genes. In contrast, SAFA meal was associated with a marked reduction in the expression of COX (p<0.001) PFK (p<0.003), LPL (p=0.002) and GLUT4 (p=0.009) genes. Conclusion: Dietary fats differentially modulate gene transcriptional profile since saturated, but not monounsaturated fat, downregulate the expression of genes regulating muscle glucose transport and oxidation

Prunus spinosa extract loaded in biomimetic nanoparticles evokes in vitro anti-inflammatory and wound healing activities

Prunus spinosa fruits (PSF) contain different phenolic compounds showing antioxidant and anti-inflammatory activities. Innovative drug delivery systems such as biomimetic nanoparti-cles could improve the activity of PSF extract by promoting (i) the protection of payload into the lipidic bilayer, (ii) increased accumulation to the diseased tissue due to specific targeting properties, (iii) improved biocompatibility, (iv) low toxicity and increased bioavailability. Using membrane proteins extracted from human monocyte cell line THP-1 cells and a mixture of phospholipids, we formulated two types of PSF-extract-loaded biomimetic vesicles differing from each other for the presence of either 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) or 1,2-dioleoyl-sn-glycero-3-phospho-(1\u2032-rac-glycerol) (DOPG). The biological activity of free extract (PSF), compared to both types of extract-loaded vesicles (PSF-DOPCs and PSF-DOPGs) and empty vesicles (DOPCs and DOPGs), was evaluated in vitro on HUVEC cells. PSF-DOPCs showed preferential incorporation of the extract. When enriched into the nanovesicles, the extract showed a significantly increased anti-inflammatory activity, and a pronounced wound-healing effect (with PSF-DOPCs more efficient than PSF-DOPG) compared to free PSF. This innovative drug delivery system, combining nutraceuti-cal active ingredients into a biomimetic formulation, represents a possible adjuvant therapy for the treatment of wound healing. This nanoplatform could be useful for the encapsulation/enrichment of other nutraceutical products with short stability and low bioavailability

Microvesicle and tunneling nanotube mediated intercellular transfer of g-protein coupled receptors in cell cultures.

none12Recent evidence shows that cells exchange collections of signals via microvesicles (MVs) and tunneling nano-tubes (TNTs). In this paper we have investigated whether in cell cultures GPCRs can be transferred by means of MVs and TNTs from a source cell to target cells. Western blot, transmission electron microscopy and gene expression analyses demonstrate that A(2A) and D(2) receptors are present in released MVs. In order to further demonstrate the involvement of MVs in cell-to-cell communication we created two populations of cells (HEK293T and COS-7) transiently transfected with D(2)R-CFP or A(2A)R-YFP. These two types of cells were co-cultured, and FRET analysis demonstrated simultaneously positive cells to the D(2)R-CFP and A(2A)R-YFP. Fluorescence microscopy analysis also showed that GPCRs can move from one cell to another also by means of TNTs. Finally, recipient cells pre-incubated for 24 h with A(2A)R positive MVs were treated with the adenosine A(2A) receptor agonist CGS-21680. The significant increase in cAMP accumulation clearly demonstrated that A(2A)Rs were functionally competent in target cells. These findings demonstrate that A(2A) receptors capable of recognizing and decoding extracellular signals can be safely transferred via MVs from source to target cells.openM. Guescini; G. Leo; S. Genedani; C. Carone; F. Pederzoli; F. Ciruela; D. Guidolin; V. Stocchi; M. Mantuano; D.O. Borroto-Escuela; K. Fuxe; L.F. AgnatiGuescini, Michele; G., Leo; S., Genedani; C., Carone; F., Pederzoli; F., Ciruela; D., Guidolin; Stocchi, Vilberto; Mantuano, Michela; D. O., Borroto Escuela; K., Fuxe; L. F., Agnat

Measurement of the cross-section and charge asymmetry of WW bosons produced in proton-proton collisions at s=8\sqrt{s}=8 TeV with the ATLAS detector

This paper presents measurements of the $W^+ \rightarrow \mu^+\nu$ and $W^- \rightarrow \mu^-\nu$ cross-sections and the associated charge asymmetry as a function of the absolute pseudorapidity of the decay muon. The data were collected in proton--proton collisions at a centre-of-mass energy of 8 TeV with the ATLAS experiment at the LHC and correspond to a total integrated luminosity of 20.2~\mbox{fb^{-1}}. The precision of the cross-section measurements varies between 0.8% to 1.5% as a function of the pseudorapidity, excluding the 1.9% uncertainty on the integrated luminosity. The charge asymmetry is measured with an uncertainty between 0.002 and 0.003. The results are compared with predictions based on next-to-next-to-leading-order calculations with various parton distribution functions and have the sensitivity to discriminate between them.Comment: 38 pages in total, author list starting page 22, 5 figures, 4 tables, submitted to EPJC. All figures including auxiliary figures are available at https://atlas.web.cern.ch/Atlas/GROUPS/PHYSICS/PAPERS/STDM-2017-13

Standalone vertex finding in the ATLAS muon spectrometer

A dedicated reconstruction algorithm to find decay vertices in the ATLAS muon spectrometer is presented. The algorithm searches the region just upstream of or inside the muon spectrometer volume for multi-particle vertices that originate from the decay of particles with long decay paths. The performance of the algorithm is evaluated using both a sample of simulated Higgs boson events, in which the Higgs boson decays to long-lived neutral particles that in turn decay to bbar b final states, and pp collision data at √s = 7 TeV collected with the ATLAS detector at the LHC during 2011