Constitutive Expression of Pluripotency-Associated Genes in Mesodermal Progenitor Cells (MPCs)

Background: We recently characterized a progenitor of mesodermal lineage (MPCs) from the human bone marrow of adults or umbilical cord blood. These cells are progenitors able to differentiate toward mesenchymal, endothelial and cardiomyogenic lineages. Here we present an extensive molecular characterization of MPCs, from bone marrow samples, including 39 genes involved in stem cell machinery, differentiation and cell cycle regulation. Methodology/Principal Findings: MPCs are cytofluorimetrically characterized and quantitative RT-PCR was performed to evaluate the gene expression profile, comparing it with MSCs and hESCs lines. Immunofluorescence and dot-blot analysis confirm qRT-PCR data. MPCs exhibit an increased expression of OCT4, NANOG, SALL4, FBX15, SPP1 and to a lesser extent c-MYC and KLF4, but lack LIN28 and SOX2. MPCs highly express SOX15. Conclusions/Significance: MPCs express many pluripotency-associated genes and show a peculiar Oct-4 molecular circuit. Understanding this unique molecular mechanism could lead to identifying MPCs as feasible, long telomeres, target cells for reprogramming with no up-regulation of the p53 pathway. Furthermore MPCs are easily and inexpensively harvested fro

SWI/SNF-like chromatin remodeling factor Fun30 supports point centromere function in S. cerevisiae

Budding yeast centromeres are sequence-defined point centromeres and are, unlike in many other organisms, not embedded in heterochromatin. Here we show that Fun30, a poorly understood SWI/SNF-like chromatin remodeling factor conserved in humans, promotes point centromere function through the formation of correct chromatin architecture at centromeres. Our determination of the genome-wide binding and nucleosome positioning properties of Fun30 shows that this enzyme is consistently enriched over centromeres and that a majority of CENs show Fun30-dependent changes in flanking nucleosome position and/or CEN core micrococcal nuclease accessibility. Fun30 deletion leads to defects in histone variant Htz1 occupancy genome-wide, including at and around most centromeres. FUN30 genetically interacts with CSE4, coding for the centromere-specific variant of histone H3, and counteracts the detrimental effect of transcription through centromeres on chromosome segregation and suppresses transcriptional noise over centromere CEN3. Previous work has shown a requirement for fission yeast and mammalian homologs of Fun30 in heterochromatin assembly. As centromeres in budding yeast are not embedded in heterochromatin, our findings indicate a direct role of Fun30 in centromere chromatin by promoting correct chromatin architecture

Italian Society of Anesthesia, Analgesia, Resuscitation, and Intensive Care expert consensus statement on the use of lung ultrasound in critically ill patients with coronavirus disease 2019 (ITACO)

Background To produce statements based on the available evidence and an expert consensus (as members of the Lung Ultrasound Working Group of the Italian Society of Analgesia, Anesthesia, Resuscitation, and Intensive Care, SIAARTI) on the use of lung ultrasound for the management of patients with COVID-19 admitted to the intensive care unit. Methods A modified Delphi method was applied by a panel of anesthesiologists and intensive care physicians expert in the use of lung ultrasound in COVID-19 intensive critically ill patients to reach a consensus on ten clinical questions concerning the role of lung ultrasound in the following: COVID-19 diagnosis and monitoring (with and without invasive mechanical ventilation), positive end expiratory pressure titration, the use of prone position, the early diagnosis of pneumothorax- or ventilator-associated pneumonia, the process of weaning from invasive mechanical ventilation, and the need for radiologic chest imaging. Results A total of 20 statements were produced by the panel. Agreement was reached on 18 out of 20 statements (scoring 7–9; “appropriate”) in the first round of voting, while 2 statements required a second round for agreement to be reached. At the end of the two Delphi rounds, the median score for the 20 statements was 8.5 [IQR 8.9], and the agreement percentage was 100%. Conclusion The Lung Ultrasound Working Group of the Italian Society of Analgesia, Anesthesia, Resuscitation, and Intensive Care produced 20 consensus statements on the use of lung ultrasound in COVID-19 patients admitted to the ICU. This expert consensus strongly suggests integrating lung ultrasound findings in the clinical management of critically ill COVID-19 patients

Mesodermal Progenitor Cells (MPCs) Differentiate into Mesenchymal Stromal Cells (MSCs) by Activation of Wnt5/Calmodulin Signalling Pathway

Mesenchymal Stromal Cells (MSCs) remain poorly characterized because of the absence of manifest physical, phenotypic, and functional properties in cultured cell populations. Despite considerable research on MSCs and their clinical application, the biology of these cells is not fully clarified and data on signalling activation during mesenchymal differentiation and proliferation are controversial. The role of Wnt pathways is still debated, partly due to culture heterogeneity and methodological inconsistencies. Recently, we described a new bone marrow cell population isolated from MSC cultures that we named Mesodermal Progenitor Cells (MPCs) for their mesenchymal and endothelial differentiation potential. An optimized culture method allowed the isolation from human adult bone marrow of a highly pure population of MPCs (more than 97%), that showed the distinctive SSEA-4+CD105+CD90(neg) phenotype and not expressing MSCA-1 antigen. Under these selective culture conditions the percentage of MSCs (SSEA-4(neg)CD105+CD90(bright) and MSCA-1+), in the primary cultures, resulted lower than 2%.We demonstrate that MPCs differentiate to MSCs through an SSEA-4+CD105+CD90(bright) early intermediate precursor. Differentiation paralleled the activation of Wnt5/Calmodulin signalling by autocrine/paracrine intense secretion of Wnt5a and Wnt5b (p<0.05 vs uncondictioned media), which was later silenced in late MSCs (SSEA-4(neg)). We found the inhibition of this pathway by calmidazolium chloride specifically blocked mesenchymal induction (ID₅₀ =  0.5 µM, p<0.01), while endothelial differentiation was unaffected.The present study describes two different putative progenitors (early and late MSCs) that, together with already described MPCs, could be co-isolated and expanded in different percentages depending on the culture conditions. These results suggest that some modifications to the widely accepted MSC nomenclature are required

Impairment of DNA Methylation Maintenance Is the Main Cause of Global Demethylation in Naive Embryonic Stem Cells.

Global demethylation is part of a conserved program of epigenetic reprogramming to naive pluripotency. The transition from primed hypermethylated embryonic stem cells (ESCs) to naive hypomethylated ones (serum-to-2i) is a valuable model system for epigenetic reprogramming. We present a mathematical model, which accurately predicts global DNA demethylation kinetics. Experimentally, we show that the main drivers of global demethylation are neither active mechanisms (Aicda, Tdg, and Tet1-3) nor the reduction of de novo methylation. UHRF1 protein, the essential targeting factor for DNMT1, is reduced upon transition to 2i, and so is recruitment of the maintenance methylation machinery to replication foci. Concurrently, there is global loss of H3K9me2, which is needed for chromatin binding of UHRF1. These mechanisms synergistically enforce global DNA hypomethylation in a replication-coupled fashion. Our observations establish the molecular mechanism for global demethylation in naive ESCs, which has key parallels with those operating in primordial germ cells and early embryos

A somatotopic bidirectional hand prosthesis with transcutaneous electrical nerve stimulation based sensory feedback

According to amputees, sensory feedback is amongst the most important features lacking from commercial prostheses. Although restoration of touch by means of implantable neural interfaces has been achieved, these approaches require surgical interventions, and their long-term usability still needs to be fully investigated. Here, we developed a non-invasive alternative which maintains some of the advantages of invasive approaches, such as a somatotopic sensory restitution scheme. We used transcutaneous electrical nerve stimulation (TENS) to induce referred sensations to the phantom hand of amputees. These sensations were characterized in four amputees over two weeks. Although the induced sensation was often paresthesia, the location corresponded to parts of the innervation regions of the median and ulnar nerves, and electroencephalographic (EEG) recordings confirmed the presence of appropriate responses in relevant cortical areas. Using these sensations as feedback during bidirectional prosthesis control, the patients were able to perform several functional tasks that would not be possible otherwise, such as applying one of three levels of force on an external sensor. Performance during these tasks was high, suggesting that this approach could be a viable alternative to the more invasive solutions, offering a trade-off between the quality of the sensation, and the invasiveness of the intervention