Results of Polish Adult Leukemia Study Group (PALG) project assessing TP53 mutations with next-generation sequencing technology in relapsed and refractory chronic lymphocytic leukemia patients — an 18-month update

Indtroduction and methods: In chronic lymphocytic leukemia (CLL), molecular and cytogenetic diagnostics are crucial for the determination of accurate prognosis and treatment choice. Among different genetic aberrations, del(17p13) or TP53 mutations constitute high-risk factors, and early identification of such defects is a high priority for CLL patients. While cytogenetic diagnostics is well-established and accessible for the majority of CLL patients in Poland, molecular diagnostics of TP53 mutations is performed only in a few ERIC-certified centers (eight as of September 2020), and only two of these employ next-generation sequencing (NGS) for routine analysis of TP53 status in CLL patients. Here we report the interim results of a project assessing TP53 mutations with NGS technology in relapsed or refractory CLL patients with confirmed negative del(17p13) status. 249 patients from 32 clinical centers were included in the study. Results: NGS analysis revealed TP53 mutations in 42/249 (17%) patients, half of whom (21/249, 8.5%) had subclonal mutations (VAF ≤10%). These results are in line with published data in relapsed/refractory CLL patients. Conclusions: The results of the project demonstrated the feasibility and accuracy of NGS testing in CLL patients despite several initial logistical and technical obstacles. Our study also proved that, with appropriate funding, CLL patients from any hematological center in Poland can have access to state-of-the-art molecular diagnostic

Global quieting of high-frequency seismic noise due to COVID-19 pandemic lockdown measures

Human activity causes vibrations that propagate into the ground as high-frequency seismic waves. Measures to mitigate the COVID-19 pandemic caused widespread changes in human activity, leading to a months-long reduction in seismic noise of up to 50%. The 2020 seismic noise quiet period is the longest and most prominent global anthropogenic seismic noise reduction on record. While the reduction is strongest at surface seismometers in populated areas, this seismic quiescence extends for many kilometers radially and hundreds of meters in depth. This provides an opportunity to detect subtle signals from subsurface seismic sources that would have been concealed in noisier times and to benchmark sources of anthropogenic noise. A strong correlation between seismic noise and independent measurements of human mobility suggests that seismology provides an absolute, real-time estimate of population dynamics

Identification of New Rat Bone Marrow-Derived Population of Very Small Stem Cell with Oct-4A and Nanog Expression by Flow Cytometric Platforms

Very small embryonic-like stem cells (VSELs) represent a unique rare population of adult stem cells (SCs) sharing several structural, genetic, biochemical, and functional properties with embryonic SCs and have been identified in several adult murine and human tissues. However, rat bone marrow- (BM-) derived SCs closely resembling murine or human VSELs have not been described. Thus, we employed multi-instrumental flow cytometric approach including classical and imaging cytometry and we established that newly identified population of nonhematopoietic cells expressing CD106 (VCAM-I) antigen contains SCs with very small size, expressing markers of pluripotency (Oct-4A and Nanog) on both mRNA and protein levels that indicate VSEL population. Based on our experience in both murine and human VSEL isolation procedures by fluorescence-activated cell sorting (FACS), we also optimized sorting protocol for separation of CD45−/Lin−/CD106+ rat BM-derived VSELs from wild type and eGFP-expressing rats, which are often used as donor animals for cell transplantations in regenerative studies in vivo. Thus, this is a first study identifying multiantigenic phenotype and providing sorting protocols for isolation VSELs from rat BM tissue for further examining of their functional properties in vitro as well as regenerative capacity in distinct in vivo rat models of tissue injury

Diverse impact of xeno-free conditions on biological and regenerative properties of hUC-MSCs and their extracellular vesicles

ABSTRACT: Growing evidence indicates that intracellular signaling mediated by extracellular vesicles (EVs) released by stem cells plays a considerable role in triggering the regenerative program upon transplantation. EVs from umbilical cord mesenchymal stem cells (UC-MSC-EVs) have been shown to enhance tissue repair in animal models. However, translating such results into clinical practice requires optimized EV collection procedures devoid of animal-originating agents. Thus, in this study, we analyzed the influence of xeno-free expansion media on biological properties of UC-MSCs and UC-MSC-EVs for future applications in cardiac repair in humans. Our results show that proliferation, differentiation, phenotype stability, and cytokine secretion by UC-MSCs vary depending on the type of xeno-free media. Importantly, we found distinct molecular and functional properties of xeno-free UC-MSC-EVs including enhanced cardiomyogenic and angiogenic potential impacting on target cells, which may be explained by elevated concentration of several pro-cardiogenic and pro-angiogenic microRNA (miRNAs) present in the EVs. Our data also suggest predominantly low immunogenic capacity of certain xeno-free UC-MSC-EVs reflected by their inhibitory effect on proliferation of immune cells in vitro. Summarizing, conscious selection of cell culture conditions is required to harvest UC-MSC-EVs with the optimal desired properties including enhanced cardiac and angiogenic capacity, suitable for tissue regeneration. KEY MESSAGE: Type of xeno-free media influences biological properties of UC-MSCs in vitro. Certain xeno-free media promote proliferation and differentiation ability of UC-MSCs. EVs collected from xeno-free cultures of UC-MSCs are biologically active. Xeno-free UC-MSC-EVs enhance cardiac and angiogenic potential of target cells. Type of xeno-free media determines immunomodulatory effects mediated by UC-MSC-EVs. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00109-016-1471-7) contains supplementary material, which is available to authorized users

Semiquantitative analysis of angiogenesis-related proteins secreted by MSCs after 10 days of endothelial culture by Western blotting.

<p><b>(A)</b> Representative nitrocellulose membranes incubated with conditioned culture medium harvested from cultures of all three experimental groups of MSC (MCPIP1-overexpressing MSCs, empty vector- treated (Puro) MSCs and untreated (Control) MSCs). Pairs of duplicate spots represent each angiogenesis- related protein. Pair of duplicate spots with upregulated expression when compared with control cells were included in brackets. <b>(B)</b> Semiquantitative assessment of selected protein concentrations based on pixel density analysis with Quantity One software. All results are presented as means ± SD. Statistically significant differences (P<0.05) are shown when compared with Puro (*) and Control (#). The analysis was conducted using a mixture of conditioned media collected under cells prepared from three independent experiments. Control—untreated MSCs; Puro—empty vector-treated MSCs; MCPIP1- MSCs overexpressing MCPIP1.</p

Semiquantitative analysis of angiogenesis-related proteins secreted by MSCs after 10 days of endothelial culture by Western blotting.

<p><b>(A)</b> Representative nitrocellulose membranes incubated with conditioned culture medium harvested from cultures of all three experimental groups of MSC (MCPIP1-overexpressing MSCs, empty vector- treated (Puro) MSCs and untreated (Control) MSCs). Pairs of duplicate spots represent each angiogenesis- related protein. Pair of duplicate spots with upregulated expression when compared with control cells were included in brackets. <b>(B)</b> Semiquantitative assessment of selected protein concentrations based on pixel density analysis with Quantity One software. All results are presented as means ± SD. Statistically significant differences (P<0.05) are shown when compared with Puro (*) and Control (#). The analysis was conducted using a mixture of conditioned media collected under cells prepared from three independent experiments. Control—untreated MSCs; Puro—empty vector-treated MSCs; MCPIP1- MSCs overexpressing MCPIP1.</p