Flow cytometric analysis of extracellular vesicles from cell-conditioned media

Flow cytometry (FC) is the method of choice for semi-quantitative measurement of cell-surface antigen markers. Recently, this technique has been used for phenotypic analyses of extracellular vesicles (EV) including exosomes (Exo) in the peripheral blood and other body fluids. The small size of EV mandates the use of dedicated instruments having a detection threshold around 50-100 nm. Alternatively, EV can be bound to latex microbeads that can be detected by FC. Microbeads, conjugated with antibodies that recognize EV-associated markers/Cluster of Differentiation CD63, CD9, and CD81 can be used for EV capture. Exo isolated from CM can be analyzed with or without pre-enrichment by ultracentrifugation. This approach is suitable for EV analyses using conventional FC instruments. Our results demonstrate a linear correlation between Mean Fluorescence Intensity (MFI) values and EV concentration. Disrupting EV through sonication dramatically decreased MFI, indicating that the method does not detect membrane debris. We report an accurate and reliable method for the analysis of EV surface antigens, which can be easily implemented in any laboratory

General purpose readout board {\pi} LUP: overview and results

This work gives an overview of the PCI-Express board $\pi$LUP, focusing on the motivation that led to its development, the technological choices adopted and its performance. The $\pi$LUP card was designed by INFN and University of Bologna as a readout interface candidate to be used after the Phase-II upgrade of the Pixel Detector of the ATLAS and CMS experiments at LHC. The same team in Bologna is also responsible for the design and commissioning of the ReadOut Driver (ROD) board - currently implemented in all the four layers of the ATLAS Pixel Detector (Insertable B-Layer, B-Layer, Layer-1 and Layer-2) - and acquired in the past years expertise on the ATLAS readout chain and the problematics arising in such experiments. Although the $\pi$LUP was designed to fulfill a specific task, it is highly versatile and might fit a wide variety of applications, some of which will be discussed in this work. Two 7$^{th}$-generation Xilinx FPGAs are mounted on the board: a Zynq-7 with an embedded dual core ARM Processor and a Kintex-7. The latter features sixteen 12.5$\,$Gbps transceivers, allowing the board to interface easily to any other electronic board, either electrically and/or optically, at the current bandwidth of the experiments for LHC. Many data-transmission protocols have been tested at different speeds, results will be discussed later in this work. Two batches of $\pi$LUP boards have been fabricated and tested, two boards in the first batch (version 1.0) and four boards in the second batch (version 1.1), encapsulating all the patches and improvements required by the first version.Comment: 6 pages, 10 figures, 21th Real Time Conference, winner of "2018 NPSS Student Paper Award Second Prize

Beneficial effects of long-term treatment with bosentan on the development of pulmonary arterial hypertension in patients with systemic sclerosis

OBJECTIVE: To investigate the effects of long-term treatment with bosentan on pulmonary arterial hypertension (PAH) in patients with systemic sclerosis. METHODS: Patients with systemic sclerosis were followed between 2003 and 2014; those who developed digital ulcers were treated with standard regimens of bosentan. Patients were assessed at baseline and every 12\u2009months using transthoracic Doppler echocardiography, 6-min walking distance test, Borg dyspnoea index and monitoring of plasma levels of 76-amino-acid N-terminal probrain natriuretic peptide. Patients who developed PAH underwent right heart catheterization to confirm the diagnosis. RESULTS: Sixty-nine patients with systemic sclerosis were enrolled in the study. Of these, 25 developed digital ulcers and received treatment with bosentan; the remaining 44 comprised the control group. None of the patients treated with bosentan developed PAH during the follow-up period. Furthermore, in these patients the mean\u2009\ub1\u2009SD systolic pulmonary arterial pressure significantly decreased from 33.64\u2009\ub1\u20092.91\u2009mmHg at baseline to 26.20\u2009\ub1\u20091.78\u2009mmHg at the end of the follow-up period. In contrast, in the control group, seven patients developed PAH during the follow-up period, with the mean\u2009\ub1\u2009SD systolic pulmonary arterial pressure significantly increasing from 33.57\u2009\ub1\u20092.75\u2009mmHg at baseline to 39.41\u2009\ub1\u20094.11\u2009mmHg at the end of the follow-up period. CONCLUSION: Long-term treatment with bosentan reduces the risk of developing PAH in patients with systemic sclerosis

An extracellular vesicle epitope profile is associated with acute myocardial infarction

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General Characteristics and Risk Factors of Cardiovascular Disease among Interstate Bus Drivers

Workers in the transportation industry are at greater risk of an incorrect diet and sedentary behavior. The aim of our study was to characterize a population of professional bus drivers with regard to clinical and demographic variables, lipid profile, and the presence of cardiovascular risk factors. Data from 659 interstate bus drivers collected retrospectively, including anthropometric characteristics, systolic and diastolic blood pressure, lipid profile, fasting blood glucose, meatoscopy, and audiometry. All participants were male, with a mean age of 41.7 ± 6.9 years, weight of 81.4 ± 3.3 kg, and BMI 27.2 ± 3.3 Kg/m2; the mean abdominal and neck circumferences were 94.4 ± 8.6 cm and 38.9 ± 2.2  cm; 38.2% of the sample was considered hypertensive; mean HDL cholesterol was 47.9 ± 9.5 mg/dL, mean triglyceride level was 146.3 ± 87.9 mg/dL, and fasting glucose was above 100 mg/dL in 249 subjects (39.1%). Drivers exhibited reduced audiometric hearing at 4–8 kHz, being all sensorineural hearing loss. The clinical characterization of a young male population of interstate bus drivers revealed a high frequency of cardiovascular risk factors, as obesity, hypertension, hyperlipidemia, and hyperglycemia, as well as contributing functional characteristics, such as a low-intensity activity, sedentary behavior, long duration in a sitting position, and high-calorie diet, which lead to excessive weight gain and associated comorbidities

Risk stratification of patients with SARS-CoV-2 by tissue factor expression in circulating extracellular vesicles

Inflammatory response following SARS-CoV-2 infection results in substantial increase of amounts of intravascular pro-coagulant extracellular vesicles (EVs) expressing tissue factor (CD142) on their surface. CD142-EV turned out to be useful as diagnostic biomarker in COVID-19 patients. Here we aimed at studying the prognostic capacity of CD142-EV in SARS-CoV-2 infection. Expression of CD142-EV was evaluated in 261 subjects admitted to hospital for pneumonia and with a positive molecular test for SARS-CoV-2. The study population consisted of a discovery cohort of selected patients (n = 60) and an independent validation cohort including unselected consecutive enrolled patients (n = 201). CD142-EV levels were correlated with post-hospitalization course of the disease and compared to the clinically available 4C Mortality Score as referral. CD142-EV showed a reliable performance to predict patient prognosis in the discovery cohort (AUC = 0.906) with an accuracy of 81.7%, that was confirmed in the validation cohort (AUC = 0.736). Kaplan-Meier curves highlighted a high discrimination power in unselected subjects with CD142-EV being able to stratify the majority of patients according to their prognosis. We obtained a comparable accuracy, being not inferior in terms of prediction of patients' prognosis and risk of mortality, with 4C Mortality Score. The expression of surface vesicular CD142 and its reliability as prognostic marker was technically validated using different immunocapture strategies and assays. The detection of CD142 on EV surface gains considerable interest as risk stratification tool to support clinical decision making in COVID-19

The Italian National Project of Astrobiology-Life in Space-Origin, Presence, Persistence of Life in Space, from Molecules to Extremophiles

The \u2018\u2018Life in Space\u2019\u2019 project was funded in the wake of the Italian Space Agency\u2019s proposal for the development of a network of institutions and laboratories conceived to implement Italian participation in space astrobiology experiments