Dorsomorphin reverses the mesenchymal phenotype of breast cancer initiating cells by inhibition of bone morphogenetic protein signaling

Increasing evidence supports the theory that tumor growth, homeostasis, and recurrence are dependent on a small subset of cells with stem cell properties, redefined cancer initiating cells (CICs) or cancer stem cells. Bone morphogenetic proteins (BMPs) are involved in cell-fate specification during embryogenesis, in the maintenance of developmental potency in adult stem cells and may contribute to sustain CIC populations in breast carcinoma. Using the mouse A17 cell model previously related to mesenchymal cancer stem cells and displaying properties of CICs, we investigated the role of BMPs in the control of breast cancer cell plasticity. We showed that an autocrine activation of BMP signaling is crucial for the maintenance of mesenchymal stem cell phenotype and tumorigenic potential of A17 cells. Pharmacological inhibition of BMP signaling cascade by Dorsomorphin resulted in the acquisition of epithelial-like traits by A17 cells, including expression of Citokeratin-18 and E-cadherin, through downregulation of Snail and Slug transcriptional factors and Cyclooxygenase-2 (COX2) expression, and in the loss of their stem-features and self-renewal ability. This phenotypic switch compromised A17 cell motility, invasiveness and in vitro tumor growth. These results reveal that BMPs are key molecules at the crossroad between stemness and cancer

Cholesterol dependent macropinocytosis and endosomal escape control the transfection efficiency of lipoplexes in CHO Living Cells

Here we investigate the cellular uptake mechanism and final intracellular fate of two cationic liposome formulations characterized by similar physicochemical properties but very different lipid composition and efficiency for intracellular delivery of DNA. The first formulation is made of cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and the zwitterionic helper dioleoylphosphocholine (DOPC), while the second one is made of the cationic 3 beta-[N-(N,N-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol) and the zwitterionic lipid dioleoylphosphatidylethanolamine (DOPE). Combining pharmacological and imaging approaches we show that both DOTAP-DOPC/DNA and DC-Chol-DOPE/DNA lipoplexes are taken up in Chinese hamster ovary (CHO) living cells mainly through fluid-phase macropinocytosis. Our results also indicate that lipoplex macropinocytosis is a cholesterol-sensitive uptake mechanism. On the other side, both clathrin-mediated and caveolae-mediated endocytosis play a minor role, if any, in the cell uptake. Colocalization of fluorescently tagged lipoplexes and Lysosensor, a primary lysosome marker, reveals that poorly efficient DOTAP-DOPC/DNA lipoplexes are largely degraded in the lysosomes, while efficient DC-Chol-DOPE/DNA systems can efficiently escape from endosomal compartments

Aspirin modulates LPS-induced nitric oxide release in rat glial cells

Nitric oxide and prostaglandins are among the numerous substances released by activated glial cells. The aim of this study was to evaluate the effect of high-level aspirin on iNOS expression in cultured rat glial cells treated with lipopolysaccharide (LPS) as pathological stimulator. Using Western Blotting, we verified that aspirin enhanced LPS-induced iNOS expression and the presence of 15-deoxy-Delta(12,14)-prostaglandin (15d-PGJ(2)) suppressed this aspirin effect. However, the exposure of LPS-treated glial cells to aspirin resulted in a decrease of NO production. These results suggest that aspirin interferes with the cross-talk of prostaglandins and NO, blocking the endogenous negative control exerted by COX products on iNOS expression. On the other side, aspirin seems to act directly on iNOS reducing its activity, even if it does not completely block NO release by LPS-stimulated glial cells. Then aspirin could maintain homeostatic functions of NO, while it prevents toxic effects, corresponding to high NO concentrations. (c) 2005 Elsevier Ireland Ltd. All rights reserved

Surface area of lipid membranes regulates the DNA-binding capacity of cationic liposomes.

We have applied electrophoresis on agarose gels to investigate the DNA-binding capacity of cationic liposomes made of cationic DC-cholesterol and neutral dioleoylphosphatidylethanolamine as a function of membrane charge density and cationic lipid/DNA charge ratio. While each cationic liposome formulation exhibits a distinctive DNA-protection ability, here we show that such a capacity is universally regulated by surface area of lipid membranes available for binding in an aspecific manner. The relevance of DNA protection for gene transfection is also discussed

Impact of the COVID-19 pandemic on ophthalmologic outpatient care: experience from an Italian Tertiary Medical Center

Purpose: To evaluate the impact the COVID-19-related national lockdown has had on Ophthalmologic Outpatient Care in an Italian Tertiary Medical Centre. Methods: We reviewed all the performances that were carried out as outpatient services at our department between 1 January 2020 and 30 November 2020. We compared data among four different periods: from 1 January 2020 to 17 March 2020 ("Pre-Lockdown"); from 18 March 2020 to 17 May 2020 (Lockdown); from 18 May 2020 to 2 November 2020 (Post-Lockdown) and from 3 November 2020 to 30 November 2020 (Regional Lockdown). Results: The overall number of performed routine outpatient visits per day (ROVs) was 11,871 (Mean \ub1 SD = 35.76 \ub1 17.81), whereas booked appointments (BAs) were 21272 (Mean \ub1 SD = 63.86 \ub1 9.27), meaning a decline in the number of ROVs by 44.01%. (Mean \ub1 SD = 28.10 \ub1 12.11, p<.001). Post-Lockdown and Regional Lockdown clinical activities, dropped respectively by 31 and 25.14% (38.87 \ub1 3.88 vs. 56.34 \ub1 11.06, p<.001 and 6.04 \ub1 4.51 vs. 56.34 \ub1 11.06 p<.001). The number of BAs per day decreased during the pandemic, going from a mean of 77.81 \ub1 2.57 booked appointments per day before the lockdown, to a mean of 53.14 \ub1 4.94, 61.80 \ub1 4.62 and 72.07 \ub1 1.09 appointments per day respectively during the lockdown, the post-lockdown and the regional lockdown periods. Conclusions: During the various lockdown periods, at our institution the volume of outpatient ophthalmological visits drastically dropped. This testifies the dramatic impact the COVID-19 pandemic has had on the supply of ophthalmic care

VirF Relieves the Transcriptional Attenuation of the Virulence Gene icsA of Shigella flexneri Affecting the icsA mRNA-RnaG Complex Formation

VirF is the master activator of virulence genes of Shigella and its expression is required for the invasion of the human intestinal mucosa by pathogenic bacteria. VirF was shown to directly activate the transcription of virB and icsA, which encode two essential proteins involved in the pathogenicity process, by binding their promoter regions. In this study, we demonstrate by band shift, enzymatic probing and cross-linking experiments that VirF, in addition to DNA, can also bind the icsA transcript and RnaG, an antisense non-coding small RNA that promotes the premature termination of icsA mRNA through a transcriptional attenuation mechanism. Furthermore, we show that VirF binds in vitro also other species of RNAs, although with lower specificity. The existence of VirF-RnaG and VirF-icsA mRNA complexes is confirmed in a pulldown assay carried out under experimental conditions that very close reproduce the in vivo conditions and that allows immobilized VirF to "fish" out RnaG and icsA mRNA from a total RNA extract. The VirF binding sites identified on both icsA mRNA and RnaG contain a 13 nucleotides stretch (5'-UUUUaGYcUuUau-3') that is the RNA-converted consensus sequence previously proposed for the VirF-DNA interaction. Band-shift assays with a synthetic RNA molecule whose sequence perfectly matches the consensus indicate that this signature plays a key role also in the VirF-RNA interaction, in particular when exposed in a stem-loop structure. To further explore the icsA-RnaG-VirF regulatory system, we developed an in vitro test (RNA-RNA Pairing Assay) in which pairing between icsA mRNA and synthetic RNAs that reproduce the individual stem-loop motifs of RnaG, was analyzed in the presence of VirF. This assay shows that this protein can prevent the formation of the kissing complex, defined as the initial nucleation points for RNA heteroduplex formation, between RnaG and icsA mRNA. Consistently, VirF alleviates the RnaG-mediated repression of icsA transcription in vitro. Therefore VirF, by hindering the icsA transcript-RnaG interaction, exhibits an activity opposed to that usually displayed by proteins, which generally assist the RNA-RNA interaction; this quite uncommon and new function and the regulatory implications of VirF as a potential RNA-binding protein are discussed