Intersectin goes nuclear: secret life of an endocytic protein.

Intersectin 1-short (ITSN1-s) is a 1220 amino acid ubiquitously expressed scaffold protein presenting a multidomain structure that allows to spatiotemporally regulate the functional interaction of a plethora of proteins. Besides its well-established role in endocytosis, ITSN1-s is involved in the regulation of cell signaling and is implicated in tumorigenesis processes, although the signaling pathways involved are still poorly understood. Here, we identify ITSN1-s as a nucleocytoplasmic trafficking protein. We show that, by binding to importin (IMP)α, a small fraction of ITSN1-s localizes in the cell nucleus at the steady state, where it preferentially associates with the nuclear envelope and interacts with lamin A/C. However, upon pharmacological ablation of chromosome region maintenance 1 (CRM-1)-dependent nuclear export pathway, the protein accumulates into the nucleus, thus revealing its moonlighting nature. Analysis of deletion mutants revealed that the coiled coil (CC) and Src homology (SH3) regions play the major role in its nucleocytoplasmic shuttling. While no evidence of nuclear localization signal (NLS) was detected in the CC region, a functional bipartite NLS was identified within the SH3D region of ITSN1-s (RKKNPGGWWEGELQARGKKRQIGW-1127), capable of conferring energy-dependent nuclear accumulation to reporter proteins and whose mutational ablation affects nuclear import of the whole SH3 region. Thus, ITSN1-s is an endocytic protein, which shuttles between the nucleus and the cytoplasm in a CRM-1- and IMPα-dependent fashion.

Clinical Laboratory Automation: A Case Study.

BACKGROUND: This paper presents a case study of an automated clinical laboratory in a large urban academic teaching hospital in the North of Italy, the Spedali Civili in Brescia, where four laboratories were merged in a unique laboratory through the introduction of laboratory automation. MATERIALS AND METHODS: The analysis compares the preautomation situation and the new setting from a cost perspective, by considering direct and indirect costs. It also presents an analysis of the turnaround time (TAT). The study considers equipment, staff and indirect costs. RESULTS: The introduction of automation led to a slight increase in equipment costs which is highly compensated by a remarkable decrease in staff costs. Consequently, total costs decreased by 12.55%. The analysis of the TAT shows an improvement of nonemergency exams while emergency exams are still validated within the maximum time imposed by the hospital. CONCLUSIONS: The strategy adopted by the management, which was based on re-using the available equipment and staff when merging the pre-existing laboratories, has reached its goal: introducing automation while minimizing the costs.

A PCR-based protocol to accurately size C9orf72 intermediate-length alleles.

Although large expansions of the non-coding GGGGCC repeat in C9orf72 gene are clearly defined as pathogenic for Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Lobar Degeneration (FTLD), intermediate-length expansions have also been associated with those and other neurodegenerative diseases. Intermediate-length allele sizing is complicated by intrinsic properties of current PCR-based methodologies, in that somatic mosaicism could be suspected. We designed a protocol that allows the exact sizing of intermediate-length alleles, as well as the identification of large expansions.

Prediction of abdominal aortic aneurysm calcification by means of variation of high-sensitivity C-reactive protein.

OBJECTIVE: Abdominal aortic aneurysms are a major cause of death in developed countries, and thrombus and calcification of the aneurysm have been linked to increased complications. This study was conducted in order to identify the biochemical marker associated to the presence of intraluminal thrombus or calcification progression of the aneurysm. DESIGN: Several clinical laboratory parameters were measured in patients with abdominal aortic aneurysms, in particular those already demonstrated to be related to the pathology, such as lipoprotein (a), white blood cell count, fibrinogen and high-sensitivity C-reactive protein. Most of the patients were analysed for the presence of thrombus or aorta calcification using CT angiography. RESULTS: Unlike previous findings, we found no association between intraluminal thrombus formation and lipoprotein (a), but we evidenced that patients with lower grade of calcification tend to have higher plasma high-sensitivity C-reactive protein values compared with patients with a higher degree of calcification. Instead, no association was found with either white blood cell count or fibrinogen level. CONCLUSIONS: This study suggests that high-sensitivity C-reactive protein is a useful biomarker to assess the evolution of calcification and could be used in triaging patients to identify those who should undergo a rapid imaging, thus allowing prompt initiation of treatment or rule-out suspicious patients from non-essential imaging repetition.

VKORC1 and CYP2C9 polymorphisms related to adverse events in case-control cohort of anticoagulated patients.

Vitamin K antagonists (VKAs) are highly effective but have a narrow therapeutic index and require routine monitoring of the INR. The primary aim of pharmacogenetics (PGx) is to optimize patient care, achieving drug treatments that are personalized according to the genetic profile of each patient. The best-characterized genes involved in VKA PGx involve pharmacokinetics (VKORC1) and pharmacodynamics (CYP2C9) of VKA metabolism. The role of these genes in clinical outcomes (bleeding and thrombosis) during oral anticoagulant (OAC) therapy is controversial. The aim of the present study was to evaluate any potential association between genotype VKORC1 and CYP2C9 and adverse events (hemorrhagic and/or thrombotic), during initiation and long-term VKA treatment, in Caucasian patients. Furthermore, we aimed to determine if the concomitant prescription of other selected drugs affected the association between genotype and adverse events.We performed a retrospective, matched case-control study to determine associations between multiple gene variants, drug intake, and any major adverse effects in anticoagulated patients, monitored in 2 Italian anticoagulation clinics.Our results show that anticoagulated patients have a high risk of adverse events if they are carriers of 1 or more genetic polymorphisms in the VKORC1 (rs9923231) and CYP2C9 (rs1799853 and rs1057910) genes.Information on CYP2C9 and VKORC1 variants may be useful to identify individualized oral anticoagulant treatment for each patient, improve management and quality of VKA anticoagulation control, and monitor drug surveillance in pharmacovigilance programs.

B- and T-lymphocyte number and function in HIV+/HIV- lymphoma patients treated with high-dose chemotherapy and autologous bone marrow transplantation.

Combination of anti-retroviral therapy, high-dose chemotherapy (HCT) and autologous stem cell transplantation (ASCT) has led to an improved survival of HIV+ non-Hodgkin lymphoma (NHL) patients. We compared T- and B-cell subset recovery and related capability to respond to in-vitro stimulation, as well as T-cell repertoire modifications of HIV+ and HIV- NHL patients undergoing HCT and ASCT as first-line consolidation or salvage treatment, using sequential blood samples obtained before and at 3, 6, 12 and 24 months after ASCT. B lymphocyte recovery occurred earlier, reaching higher levels in HIV+ patients as compared to HIV- patients and healthy controls; in particular, immature and naïve B cells were significantly higher in HIV+ patients who had received rituximab in the pre-ASCT period. These lymphocytes equally responded to in-vitro stimulation. Newly produced T cells similarly increased in HIV+ and HIV- NHL patients, but their levels remained constantly lower than in healthy controls. T lymphocytes showed a reduced proliferative capacity, but their repertoire was reassorted by the treatment. The functional and numeric B-cell recovery and the qualitative modifications of T-cell receptor repertoire may explain, at least in part, the success of this aggressive therapeutic approach in HIV+ patients.

Caveolin-1, Caveolin-2 and Cavin-1 are strong predictors of adipogenic differentiation in human tumors and cell lines of liposarcoma.

Caveolins (Cav-1, -2 and -3) and Cavins (Cavin-1, -2, -3 and -4) are two protein families controlling the biogenesis and function of caveolae, plasma membrane omega-like invaginations representing the primary site of important cellular processes like endocytosis, cholesterol homeostasis and signal transduction. Caveolae are especially abundant in fat tissue, playing a consistent role in a number of processes, such as the insulin-dependent glucose uptake and transmembrane transport of lipids underlying differentiation, maintenance and adaptive hypertrophy of adipocytes. Based on this premise, in this work we have investigated the expression of caveolar protein components in liposarcoma (LPS), an adipocytic soft tissue sarcoma affecting adults categorized in well-differentiated, dedifferentiated, myxoid and pleomorphic histotypes. By performing an extensive microarray data analysis followed by immunohistochemistry on human LPS tumors, we demonstrated that Cav-1, Cav-2 and Cavin-1 always cluster in all the histotypes, reaching the highest expression in well-differentiated LPS, the least aggressive of the malignant forms composed by tumor cells with a morphology resembling mature adipocytes. In vitro experiments carried out using two human LPS cell lines showed that the expression levels of Cav-1, Cav-2 and Cavin-1 proteins were faintly detectable during cell growth, becoming consistently increased during the accumulation of intracellular lipid droplets characterizing the adipogenic differentiation. Moreover, in differentiated LPS cells the three proteins were also found to co-localize and form molecular aggregates at the plasma membrane, as shown via immunofluorescence and immunoprecipitation analysis. Overall, these data indicate that Cav-1, Cav-2 and Cavin-1 may be considered as reliable markers for identification of LPS tumors characterized by consistent adipogenic differentiation.

Effect of antihypertensive treatment with lercanidipine on endothelial progenitor cells and inflammation in patients with mild to moderate essential hypertension.

BACKGROUND: It has been demonstrated that circulating endothelial progenitor cells (EPCs) number reflects the endogenous vascular repair ability, with the EPCs pool declining in presence of cardiovascular risk factors. Several drugs, including dihydropyridine calcium channel blockers, have been reported to elicit antioxidant and anti-inflammatory properties, as well as to improve vascular remodeling and dysfunction. However, no data are available about the effects of lercanidipine on EPCs. The aim of the present study was therefore to investigate the effects of short-term treatment with lercanidipine on circulating EPCs, as well as on indices of inflammation and oxidative stress. PATIENTS AND METHODS: Twenty essential hypertensive patients were included in the study and treated for 4 weeks with lercanidipine 20 mg per day orally. Investigations were performed in basal condition, after appropriate wash out of previous treatments, and after 4 weeks of lercanidipine treatment. Inflammatory and oxidative stress markers were assessed by ELISA technique. Lin-/7AAD-/CD34+/CD133+/VEGFR-2 + and Lin-/7AAD-/CD34+/VEGFR-2 + cells were identified by flow cytometry and considered as EPCs. EPCs cells were expressed as number of cells per million Lin-mononuclear cells. RESULTS: Circulating EPCs were significantly increased after lercanidipine treatment (CD34+/CD133+/VEGFR-2 + cells: 78.3 ± 64.5 vs 46.6 ± 32.8; CD34+/VEGFR-2+: 87996 ± 165116 vs 1026 ± 1559, respectively, p < 0.05). A modest reduction in circulating indices of inflammation was also observed. CONCLUSIONS: In conclusion, lercanidipine is able to increase the number of circulating EPCs, possibly through a reduction of low-grade inflammation.

Municipal wastewater affects adipose deposition in male mice and increases 3T3-L1 cell differentiation.

Trace concentration of EDs (endocrine disrupting compounds) in water bodies caused by wastewater treatment plant effluents is a recognized problem for the health of aquatic organisms and their potential to affect human health. In this paper we show that continuous exposure of male mice from early development to the adult life (140 days) to unrestricted drinking of wastewater collected from a municipal sewage treatment plant, is associated with an increased adipose deposition and weight gain during adulthood because of altered body homeostasis. In parallel, bisphenol A (BPA) at the administration dose of 5 µg/kg/body weight, shows an increasing effect on total body weight and fat mass. In vitro, a solid phase extract (SPE) of the wastewater (eTW), caused stimulation of 3T3-L1 adipocyte differentiation at dilutions of 0.4 and 1 % in the final culture medium which contained a concentration of BPA of 40 nM and 90 nM respectively. Pure BPA also promoted adipocytes differentiation at the concentration of 50 and 80 µM. BPA effect in 3T3-L1 cells was associated to the specific activation of the estrogen receptor alpha (ERα) in undifferentiated cells and the estrogen receptor beta (ERß) in differentiated cells. BPA also activated the Peroxisome Proliferator Activated Receptor gamma (PPARγ) upregulating a minimal 3XPPARE luciferase reporter and the PPARγ-target promoter of the aP2 gene in adipose cells, while it was not effective in preadipocytes. The pure estrogen receptor agonist diethylstilbestrol (DES) played an opposite action to that of BPA inhibiting PPARγ activity in adipocytes, preventing cell differentiation, activating ERα in preadipocytes and inhibiting ERα and ERß regulation in adipocytes. The results of this work show that the drinking of chemically-contaminated wastewater promotes fat deposition in male mice and that EDs present in sewage are likely responsible for this effect through a nuclear receptor-mediated mechanism.

Merging colloidal nanoplasmonics and surface plasmon resonance spectroscopy for enhanced profiling of multiple myeloma-derived exosomes.

A novel approach for sorting exosomes from multiple myeloma (MM), monoclonal gammopathy of undetermined significance (MGUS) and healthy individuals is presented. The method is based on the combination of colloidal gold nanoplasmonics and surface plasmon resonance (SPR) biosensing and probes distinctive colloidal properties of MM-derived exosomes, such as molar concentration and cell membrane binding preferences. It allowed to discover that MM patients produce about four folds more exosomes than MGUS and healthy individuals. In addition, it showed that among the analyzed exosomes, only the MM-derived ones bind heparin - a structural analog of heparan sulfate proteoglycans known to mediate exosome endocytosis - with an apparent dissociation constant (Kd) equal to about 1 nM, indicating a high affinity binding. This plasmonic method complements the classical biochemical profiling approach to exosomes, expanding the MM biomarker panel and adding biosensors to the toolbox to diagnose MM. It may find applications for other diseases and has wider interest for fundamental and translational research involving exosomes.

The strange case of the [13N]NH3: validation of the production process for human use.

OBJECTIVE: PET radiopharmaceuticals are often injected in patients before all quality controls are performed and before sterility results are available. We propose a process validation to produce very safe and pure [N]NH3 for human use. METHODS: [N]NH3 was produced in the cyclotron target. Online purification was performed by anionic exchange resin. All the production steps were subjected to a sterility test. Some additional controls were added to those required by the monograph. RESULTS: The radiochemical yield of the syntheses was 26.3 and 61.5% corrected for decay, with a radiochemical purity of 100%. In addition to quality controls requested by the European Pharmacopeia monograph, we carefully analyzed the product for the presence of possible contaminants. Some elements, mainly metals, were found in very low amounts at concentrations in the range of ppb. The radionuclidic purity was verified. The achievement of the parameters of osmolality, by addition of saline solution to the preparation, made the analysis of chemical purity difficult and worsened the measurement of radiochemical purity by high performance liquid chromatography. Only pH control is necessary before administration to patients and therefore a safe production process was set up to prevent microbiological contamination. All phases were carefully standardized, starting from in-target production of [N]NH3, to final splitting in the syringes. Sterility tests showed no bacterial growth, indicating the safety of the production process. CONCLUSION: All our syntheses followed the monograph indications and were optimal to obtain PET imaging of a patient's myocardium.

Which sample tube should be used for routine glucose determination?

BACKGROUND: Glucose is one of the most frequently requested analytes in clinical laboratory. Blood glucose analysis is affected from in vitro glycolysis. In order to determine the most suitable blood collection tube for this purpose we have compared different tubes: sodium fluoride, lithium heparin, sodium fluoride/citrate buffer containing tubes and serum with clot activator tube for the measurement of glucose when the tube has been kept at room temperature (RT) for up to 4h. METHODS: Venous blood was collected from 49 healthy volunteers into Sarstedt S-Monovettes for glucose analysis. Reference plasma glucose was determined in a lithium heparin tube and immediately placed in an ice/water slurry. Within 10min it was centrifuged at 4°C and plasma was separated from the blood cells. Samples have been preserved at RT for 1, 2 and 4h after drawing. Glucose has been determined using a hexokinase method. RESULTS: Glucose levels tested in a serum with clot activator tube, in lithium heparin and in sodium fluoride/sodium EDTA tubes when compared with lithium-heparin reference plasma did not meet the desirable bias for glucose (±1.8%) when kept at RT for up to 4h. GlucoEXACT tubes, when corrected by the Sarsted recommended factor of 1.16, showed a mean (95% CI) bias of +0.96% (0.45-1.47) at 1h, +1.40% (0.88-1.93) at 2h and +0.95% (0.44-1.46) at 4h, reaching the analytical goal for the desirable bias. CONCLUSIONS: Samples collected into GlucoEXACT tubes containing sodium fluoride/citrate buffer liquid mixture are equivalent to those collected in reference plasma tubes avoiding glycolysis completely and within a 4h delay in plasma separation.

Postremission sequential monitoring of minimal residual disease by WT1 Q-PCR and multiparametric flow cytometry assessment predicts relapse and may help to address risk-adapted therapy in acute myeloid leukemia patients.

Risk stratification in acute myeloid leukemia (AML) patients using prognostic parameters at diagnosis is effective, but may be significantly improved by the use of on treatment parameters which better define the actual sensitivity to therapy in the single patient. Minimal residual disease (MRD) monitoring has been demonstrated crucial for the identification of AML patients at high risk of relapse, but the best method and timing of MRD detection are still discussed. Thus, we retrospectively analyzed 104 newly diagnosed AML patients, consecutively treated and monitored by quantitative polymerase chain reactions (Q-PCR) on WT1 and by multiparametric flow cytometry (MFC) on leukemia-associated immunophenotypes (LAIPs) at baseline, after induction, after 1st consolidation and after 1st intensification. By multivariate analysis, the factors independently associated with adverse relapse-free survival (RFS) were: bone marrow (BM)-WT1 ≥ 121/10(4) ABL copies (P = 0.02) and LAIP ≥ 0.2% (P = 0.0001) (after 1st consolidation) (RFS at the median follow up of 12.5 months: 51% vs. 82% [P < 0.0001] and 57% vs. 81%, respectively [P = 0.0003]) and PB-WT1 ≥ 16/10(4) ABL copies (P = 0.0001) (after 1st intensification) (RFS 43% vs. 95% [P < 0.0001]) Our data confirm the benefits of sequential MRD monitoring with both Q-PCR and MFC. If confirmed by further prospective trials, they may significantly improve the possibility of a risk-adapted, postinduction therapy of AML.

Serum posaconazole levels during acute myeloid leukaemia induction therapy: correlations with breakthrough invasive fungal infections.

The usefulness of posaconazole therapeutic drug monitoring (TDM) is still a matter of debate. A correlation between posaconazole serum levels and breakthrough invasive fungal infections (IFI) has not been clearly demonstrated so far. We analysed posaconazole serum levels in patients with acute myeloid leukaemia (AML) during induction therapy and correlated them with the incidence of breakthrough IFI and the need of systemic antifungal therapy. Overall, 77 AML patients receiving posaconazole were evaluated for serum levels; breakthrough IFI were observed in five with at least one posaconazole TDM (6.5%). Median serum level was 534 ng ml(-1) (IQ range: 298.5-750.5 ng ml(-1) ) and did not change significantly over time. Four of the 40 patients with median posaconazole levels <500 ng ml(-1) developed IFI, as compared with only 1 of the 37 patients with median levels ≥500 (10% vs. 2.7%, P = 0.19). Median posaconazole levels on day 7 were 384.5 ng ml(-1) (IQ range: 207-659 ng ml(-1) ) and 560.5 ng ml(-1) (IQ range: 395-756 ng ml(-1) ) in patients requiring or not systemic antifungal treatment respectively (P = 0.067). These results seem to confirm that higher median serum levels of posaconazole correlate with higher prophylactic efficacy against proven/probable IFI and with lesser need of systemic antifungal therapy.

Polyclonal versus monoclonal immunoglobulin-free light chains quantification.

BACKGROUND: The clinical usefulness of the serum-free light chain assays has expanded since their first description, and further applications other than plasma cell dyscrasia are emerging. Currently, we have the ability to perform the measurements with two certified methods: the Freelite™ assay (The Binding Site Ltd, Birmingham, UK) and the new N Latex free-light chain assay (Siemens, Germany). In the present study, we investigated the impact of free light chain concentrations and structures on their quantification, performed with both tests. METHODS: A total of 524 serum samples from 497 patients from our routine laboratory were analysed with the Freelite™ and the N Latex free light chain assay. The results were compared in two subgroups: with or without monoclonal component. Twenty-four samples were subsequently investigated for the presence of dimeric and monomeric free light chain with sodium dodecyl sulphate polyacrylamide gel electrophoresis and densitometric quantification. RESULTS: Methods comparison showed that the Pearson rank correlation coefficients were 0.90 for polyclonal k and 0.91 for polyclonal λ free light chain. Conversely for monoclonal immunoglobulins, the Pearson rank correlation coefficient was lower with 0.82 for kM >500 mg/L and 0.56 for λM >500 mg/L. Furthermore, densitometric quantification of the involved monoclonal free light chains showed that both assays do not reflect the Coomassie-stained protein mass. CONCLUSION: Samples containing high amounts of a single pathologic free light chain may not be considered like a sample containing a sum of different polyclonal free light chains. Indeed, free light chain dimerization leads to different scatter efficiency of macromolecular complexes.

Soy and the soy isoflavone genistein promote adipose tissue development in male mice on a low-fat diet.

PURPOSE: Several nutrients act as phytoestrogens, being anti-adipogenic when consumed with a fat-rich diet. Their effect on a low-fat diet (LFD) background is unknown. We tested soy and genistein effects on adipose tissue in LFD-fed mice and genistein activity in the 3T3-L1 adipogenesis model. METHODS: C57BL/6 J male mice were fed an 8.5% soy-supplemented LFD (SS-LFD) or a soy-free LFD (SF-LFD) for 147 days. Groups of 3-week-old (pubertal) and 6-week-old (adult) mice on the SF-LFD were also treated with 17ß-estradiol (E2, 5 µg/kg/day) ip or pure genistein (5 mg/kg/day) by gavage for 15 days. Body fat deposition and gene expression profiles were evaluated. E2 and genistein effects on ERα, ERß and PPARγ transcriptional activities were characterized in ERα- or ERß-transfected 3T3L1 cells during differentiation, by the use of reporter plasmids. RESULTS: The SS-LFD group increased fat mass compared with the SF-LFD group. Genistein alone increased while E2 decreased fat pads in the 15-day-treated mice. In visceral fat, genistein differentially regulated 13 metabolic pathways compared to E2. PPARγ-controlled genes were downregulated by E2, while they were upregulated by genistein. In 3T3-L1 cells, genistein activated ERß-driven transcription, differentiation and lipid accumulation, while inhibited ERα-driven transcription, without effects on lipid accumulation. E2 activated both ERs only in preadipocytes. In differentiated untransfected cells, genistein inhibited PPARγ, while activated PPARγ in the presence of ERß. CONCLUSIONS: Soy and genistein at nutritional doses induce fat development in LFD-fed mice and adipogenesis in 3T3-L1 cells, with a mechanism that involves, at least in vitro, ERß and is dependent on cell differentiation stage.

Immunological biomarkers identifying natalizumab-treated multiple sclerosis patients at risk of progressive multifocal leukoencephalopathy.

Natalizumab-induced progressive multifocal leukoencephalopathy appears to be unleashed by complex interactions between viral and immunological host factors leading the latent form of JC virus to become pathogenic. Positive anti-JC virus antibody status, prior use of immunosuppressants, and increasing duration of natalizumab treatment have been proposed as risk factors for progressive multifocal leukoencephalopathy in multiple sclerosis patients, but while they may help to identify the most appropriate patients for natalizumab, their use have some limitations. Therefore, a large body of studies is ongoing to identify alternative, reliable immunological markers capable to improve the safety and efficacy of therapy, and to guide tailored clinical decisions.

Immunoglobulin Free Light Chains and GAGs Mediate Multiple Myeloma Extracellular Vesicles Uptake and Secondary NfκB Nuclear Translocation.

Multiple myeloma (MM) is a hematological malignancy caused by a microenviromentally aided persistence of plasma cells in the bone marrow. Monoclonal plasma cells often secrete high amounts of immunoglobulin free light chains (FLCs) that could induce tissue damage. Recently, we showed that FLCs are internalized in endothelial and myocardial cell lines and secreted in extracellular vesicles (EVs). MM serum derived EVs presented phenotypic differences if compared with monoclonal gammopathy of undetermined significance (MGUS) serum derived EVs suggesting their involvement in MM pathogenesis or progression. To investigate the effect of circulating EVs on endothelial and myocardial cells, we purified MM and MGUS serum derived EVs with differential ultracentrifugation protocols and tested their biological activity. We found that MM and MGUS EVs induced different proliferation and internalization rates in endothelial and myocardial cells, thus we tried to find specific targets in MM EVs docking and processing. Pre-treatment of EVs with anti-FLCs antibodies or heparin blocked the MM EVs uptake, highlighting that FLCs and glycosaminoglycans are involved. Indeed, only MM EVs exposure induced a strong nuclear factor kappa B nuclear translocation that was completely abolished after anti-FLCs antibodies and heparin pre-treatment. The protein tyrosine kinase c-src is present on MM circulating EVs and redistributes to the cell plasma membrane after MM EVs exposure. The anti-FLCs antibodies and heparin pre-treatments were able to block the intracellular re-distribution of the c-src kinase and the subsequent c-src kinase containing EVs production. Our results open new insights in EVs cellular biology and in MM therapeutic and diagnostic approaches.

Fibroblasts maintained in 3 dimensions show a better differentiation state and higher sensitivity to estrogens.

Cell differentiation and response to hormonal signals were studied in a 3D environment on an in-house generated mouse fibroblast cell line expressing a reporter gene under the control of estrogen responsive sequences (EREs). 3D cell culture conditions were obtained in a Rotary Cell Culture System; (RCCS™), a microgravity based bioreactor that promotes the aggregation of cells into multicellular spheroids (MCS). In this bioreactor the cells maintained a better differentiated phenotype and more closely resembled in vivo tissue. The RCCS™ cultured fibroblasts showed higher expression of genes regulating cell assembly, differentiation and hormonal functions. Microarray analysis showed that genes related to cell cycle, proliferation, cytoskeleton, migration, adhesion and motility were all down-regulated in 3D as compared to 2D conditions, as well as oncogene expression and inflammatory cytokines. Controlled remodeling of ECM, which is an essential aspect of cell organization, homeostasis and tissue was affected by the culture method as assessed by immunolocalization of ß-tubulin. Markers of cell organization, homeostasis and tissue repair, metalloproteinase 2 (MMP2) and its physiological inhibitor (TIMP4) changed expression in association with the relative formation of cell aggregates. The fibroblasts cultured in the RCCS™ maintain a better responsiveness to estrogens, measured as expression of ERα and regulation of an ERE-dependent reporter and of the endogenous target genes CBP, Rarb, MMP1 and Dbp. Our data highlight the interest of this 3D culture model for its potential application in the field of cell response to hormonal signals and the pharmaco-toxicological analyses of chemicals and natural molecules endowed of estrogenic potential.