Nano-particle characterization by using Exposure Time Dependent Spectrum and scattering in the near field methods: how to get fast dynamics with low-speed CCD camera

Light scattering detection in the near field, a rapidly expanding family of scattering techniques, has recently proved to be an appropriate procedure for performing dynamic measurements. Here we report an innovative algorithm, based on the evaluation of the Exposure Time Dependent Spectrum (ETDS), which makes it possible to measure the fast dynamics of a colloidal suspension with the aid of a simple near field scattering apparatus and a CCD camera. Our algorithm consists in acquiring static spectra in the near field at different exposure times, so that the measured decay times are limited only by the exposure time of the camera and not by its frame rate. The experimental set-up is based on a modified microscope, where the light scattered in the near field is collected by a commercial objective, but (unlike in standard microscopes) the light source is a He-Ne laser which increases the instrument sensitivity. The apparatus and the algorithm have been validated by considering model systems of standard spherical nano-particle

The Nanomechanical Properties of CLL Cells Are Linked to the Actin Cytoskeleton and Are a Potential Target of BTK Inhibitors.

Chronic lymphocytic leukemia (CLL) is an incurable disease characterized by an intense trafficking of the leukemic cells between the peripheral blood and lymphoid tissues. It is known that the ability of lymphocytes to recirculate strongly depends on their capability to rapidly rearrange their cytoskeleton and adapt to external cues; however, little is known about the differences occurring between CLL and healthy B cells during these processes. To investigate this point, we applied a single-cell optical (super resolution microscopy) and nanomechanical approaches (atomic force microscopy, real-time deformability cytometry) to both CLL and healthy B lymphocytes and compared their behavior. We demonstrated that CLL cells have a specific actomyosin complex organization and altered mechanical properties in comparison to their healthy counterpart. To evaluate the clinical relevance of our findings, we treated the cells in vitro with the Bruton's tyrosine kinase inhibitors and we found for the first time that the drug restores the CLL cells mechanical properties to a healthy phenotype and activates the actomyosin complex. We further validated these results in vivo on CLL cells isolated from patients undergoing ibrutinib treatment. Our results suggest that CLL cells' mechanical properties are linked to their actin cytoskeleton organization and might be involved in novel mechanisms of drug resistance, thus becoming a new potential therapeutic target aiming at the normalization of the mechanical fingerprints of the leukemic cells.CS project is supported by Associazione Italiana per la Ricerca sul Cancro AIRC under IG 2018 - ID 21332 project. OO gratefully acknowledges financial support from the German Federal Ministry of Education and Research (ZIK grant to OO under grant agreement no. 03Z22CN11) as well as from the German Center for Cardiovascular Research (Postdoc start-up grant to OO under grant agreement no. 81X3400107). CAM acknowledges financial support from the Italian Ministry of University and Research (MIUR) Department of Excellence project PREMIA (PREcision MedIcine Approach: bringing biomarker research to clinics). STED microscopy was conducted at the Microscopy & Dynamic Imaging Unit, CNIC, ICTS-ReDib, co-funded by MCIN/AEI/10.13039/501100011033, and FEDER “Una manera de hacer Europa” (#ICTS-2018-04-CNIC-16). The CNIC is supported by the Ministerio de Ciencia e Innovación and the Pro CNIC Foundation and is a Severo Ochoa Center of Excellence (CEX2020-001041-S). Schemes in figures 1, 2, 3 and 4 have been generated with BioRender.com. Funding for the project was provided by the European Union’s Seventh Framework Programme (FP7/2007-2013) under grant agreement no 282510 – BLUEPRINT.S

ECMO for COVID-19 patients in Europe and Israel

Since March 15th, 2020, 177 centres from Europe and Israel have joined the study, routinely reporting on the ECMO support they provide to COVID-19 patients. The mean annual number of cases treated with ECMO in the participating centres before the pandemic (2019) was 55. The number of COVID-19 patients has increased rapidly each week reaching 1531 treated patients as of September 14th. The greatest number of cases has been reported from France (n = 385), UK (n = 193), Germany (n = 176), Spain (n = 166), and Italy (n = 136) .The mean age of treated patients was 52.6 years (range 16–80), 79% were male. The ECMO configuration used was VV in 91% of cases, VA in 5% and other in 4%. The mean PaO2 before ECMO implantation was 65 mmHg. The mean duration of ECMO support thus far has been 18 days and the mean ICU length of stay of these patients was 33 days. As of the 14th September, overall 841 patients have been weaned from ECMO support, 601 died during ECMO support, 71 died after withdrawal of ECMO, 79 are still receiving ECMO support and for 10 patients status n.a. . Our preliminary data suggest that patients placed on ECMO with severe refractory respiratory or cardiac failure secondary to COVID-19 have a reasonable (55%) chance of survival. Further extensive data analysis is expected to provide invaluable information on the demographics, severity of illness, indications and different ECMO management strategies in these patients

Epilepsy in NF1: Epidemiologic, Genetic, and Clinical Features. A Monocentric Retrospective Study in a Cohort of 784 Patients

An increased lifetime risk of epilepsy has been reported in neurofibromatosis type 1 (NF1) patients, ranging between 4% and 14%. To further analyze the correlation between NF1 and epilepsy, we retrospectively reviewed the epidemiologic, clinical, radiological, and molecular data of 784 unselected patients diagnosed with NF1 and referred to the neurofibromatosis outpatient clinics at the University Hospital of Padua. A crude prevalence of epilepsy of 4.7% was observed. In about 70% of cases, seizures arose in the context of neuroradiological findings, with the main predisposing factors being cerebral vasculopathies and hydrocephalus. In the absence of structural abnormalities, the prevalence of epilepsy was found to be 1.27%, which is approximately equal to the total prevalence in the general population. NF1 patients with seizures exhibit a higher incidence of intellectual disability and/or developmental delay, as well as of isolated learning disabilities. The comparison of causative NF1 mutations between the two groups did not reveal a specific genotype–phenotype correlation. Our data refine the current knowledge on epileptological manifestations in NF1 patients, arguing against the hypothesis that specific mechanisms, inherent to neurofibromin cellular function, might determine an increased risk of epilepsy in this condition

Dendrogram obtained by performing cluster analysis on Probe1 <<i>τ_D</i>> values, applying Euclidean metrics and the complete-linkage cluster-joining criterion.

<p>Dendrogram obtained by performing cluster analysis on Probe1 <<i>τ_D</i>> values, applying Euclidean metrics and the complete-linkage cluster-joining criterion.</p

Sequences of the eight target oligonucleotides and of the TAMRA-BHQ2 dual-labelled probes 1 and 2.

<p>Mismatch-sites of the target oligonucleotides with respect to Probe1 are black-shaded, those with respect to Probe2 are light-gray shaded. Mismatching sites common to both probes are indicated by dark-grey shading.</p

Cross combination of the <<i>τ_D</i>> values obtained with the two probes allows discrimination of all possible DQB1 genotypes.

<p>Here, each genotype is represented by a point whose abscissa is given by the <<i>τ_D,Probe2</i>> value and whose ordinate is <<i>τ_D,Probe1</i>>. The half-base and half-height of the rectangles surrounding each point represent σ(<<i>τ_D,Probe2</i>>) and σ(<<i>τ_D,Probe1</i>>), respectively. Discrimination is warranted by lack of superposition among different rectangles.</p