c-Flip KO fibroblasts display lipid accumulation associated with endoplasmic reticulum stress

c-Flip proteins are well-known apoptosis modulators. They generally contribute to tissue homeostasis maintenance by inhibiting death-receptor-mediated cell death. In the present manuscript, we showthat c-Flip knock-out (KO) mouse embryonic fibroblasts (MEFs) kept in culture under starvation conditions gradually modify their phenotype and accumulate vacuoles, becoming progressively larger according to the duration of starvation. Large vacuoles are present in KO MEFs though not in WT MEFs, and are Oil Red-O positive, which indicates that they represent lipid droplets. Western blot experiments reveal that, unlikeWTMEFs, KOMEFs express high levels of the lipogenic transcription factor PPAR-γ. Lipid droplet accumulation was found to be associated with endoplasmic reticulum (ER) stress activation and autophagic modulation valuated by means of BIP increase, LC3 lipidation and AMP-activated protein kinase (AMPK) phosphorylation, and p62 accumulation. Interestingly, XBP-1, an ER stress-induced lipogenic transcription factor, was found to preferentially localize in the nucleus rather than in the cytoplasm of KO MEFs. These data demonstrate that, upon starvation, c-Flip affects lipid accumulation, ER stress and autophagy, thereby pointing to an important role of c-Flip in the adaptive response and ER stress response programs under both normal and pathological conditions

Cancer microenvironment and endoplasmic reticulum stress response

Different stressful conditions such as hypoxia, nutrient deprivation, pH changes, or reduced vascularization, potentially able to act as growth-limiting factors for tumor cells, activate the unfolded protein response (UPR). UPR is therefore involved in tumor growth and adaptation to severe environments and is generally cytoprotective in cancer. The present review describes the molecular mechanisms underlying UPR and able to promote survival and proliferation in cancer. The critical role of UPR activation in tumor growth promotion is discussed in detail for a few paradigmatic tumors such as prostate cancer and melanoma

Universal newborn hearing screening in the Lazio region, Italy

Background: The introduction of Universal Newborn Hearing Screening (UNHS) programs has drastically contributed to the early diagnosis of hearing loss in children, allowing prompt intervention with significant results on speech and language development in affected children. UNHS in the Lazio region has been initially deliberated in 2012; however, the program has been performed on a universal basis only from 2015. The aim of this retrospective study is to present and discuss the preliminary results of the UNHS program in the Lazio region for the year 2016, highlighting the strengths and weaknesses of the program. Methods: Data from screening facilities in the Lazio region for year 2016 were retrospectively analyzed. Data for Level I centers were supplied by the Lazio regional offices; data for Level II and III centers were provided by units that participated to the study. Results: During 2016, a total of 44,805 babies were born in the Lazio region. First stage screening was performed on 41,821 children in 37 different birth centers, with a coverage rate of 93.3%. Of these, 38.977 (93.2%) obtained a "pass" response; children with a "refer" result in at least one ear were 2844 (6.8%). Data from Level II facilities are incomplete due to missing reporting, one of the key issues in Lazio UNHS. Third stage evaluation was performed on 365 children in the three level III centers of the region, allowing identification of 70 children with unilateral (40%) or bilateral (60%) hearing loss, with a prevalence of 1.6/1000. Conclusions: The analysis of 2016 UNHS in the Lazio region allowed identification of several strengths and weaknesses of the initial phase of the program. The strengths include a correct spread and monitoring of UNHS among Level I facilities, with an adequate coverage rate, and the proper execution of audiological monitoring and diagnosis among Level III facilities. Weakness, instead, mainly consisted in lack of an efficient and automated central process for collecting, monitoring and reporting of data and information

L-Ferritin targets breast cancer stem cells and delivers therapeutic and imaging agents

A growing body of evidence suggests that cancer stem cells (CSC) have the unique biological properties necessary for tumor maintenance and spreading, and function as a reservoir for the relapse and metastatic evolution of the disease by virtue of their resistance to radio- and chemo-therapies. Thus, the efficacy of a therapeutic approach relies on its ability to effectively target and deplete CSC. In this study, we show that CSC-enriched tumorspheres from breast cancer cell lines display an increased L-Ferritin uptake capability compared to their monolayer counterparts as a consequence of the upregulation of the L-Ferritin receptor SCARA5. L-Ferritin internalization was exploited for the simultaneous delivery of Curcumin, a natural therapeutic molecule endowed with antineoplastic action, and the MRI contrast agent Gd-HPDO3A, both entrapped in the L-Ferritin cavity. This theranostic system was able to impair viability and self-renewal of tumorspheres in vitro and to induce the regression of established tumors in mice. In conclusion, here we show that Curcumin-loaded L-Ferritin has a strong therapeutic potential due to the specific targeting of CSC and the improved Curcumin bioavailability, opening up the possibility of its use in a clinical setting

A novel role of c-FLIP protein in regulation of ER stress response

Cellular-Flice-like Inhibitory Protein (c-FLIP) is an apoptosis modulator known to inhibit the extrinsic apoptotic pathway thus blocking Caspase-8 processing in the Death Inducing Signalling Complex (DISC). We previously demonstrated that c-FLIP localizes at the Endoplasmic Reticulum (ER) and that c-FLIP-deficient Mouse Embryonic Fibroblasts (MEFs) display an enlarged ER morphology. In the present study, we have addressed the consequences of c-FLIP ablation in the ER stress response by investigating the effects of pharmacologically-induced ER stress in Wild Type (WT) and c-FLIP-/- MEFs. Surprisingly, c-FLIP-/- MEFs were found to be strikingly more resistant than WT MEFs to ER stress-mediated apoptosis. Analysis of Unfolded Protein Response (UPR) pathways revealed that Pancreatic ER Kinase (PERK) and Inositol-Requiring Enzyme 1 (IRE1) branch signalling is compromised in c-FLIP-/- cells when compared with WT cells. We found that c-FLIP modulates the PERK pathway by interfering with the activity of the serine threonine kinase AKT. Indeed, c-FLIP-/- MEFs display higher levels of active AKT than WT MEFs upon ER stress, while treatment with a specific AKT inhibitor of c-FLIP-/- MEFs subjected to ER stress restores the PERK but not the IRE1 pathway. Importantly, the AKT inhibitor or dominant negative AKT transfection sensitizes c-FLIP-/- cells to ER stress-induced cell death while the expression of a constitutively active AKT reduces WT cells sensitivity to ER stress-induced death. Thus, our results demonstrate that c-FLIP modulation of AKT activity is crucial in controlling PERK signalling and sensitivity to ER stress, and highlight c-FLIP as a novel molecular player in PERK and IRE1-mediated ER stress response

c-FLIPL regulates endoplasmic reticulum morphology and mitochondria-associated membranes functions

Physical and functional interactions between endoplasmic reticulum (ER) and mitochondria take place at the mitochondria-associated membranes (MAMs), ER subdomains at the interface between the two organelles. Protein complexes at MAMs regulate lipid synthesis, Ca2+ signaling and apoptosis 1. Furthermore, they influence both ER and mitochondrial morphology, as their ablation is frequently associated to the dramatic remodeling of these organelles. Here we show that c-FLIPL (cellular FLICE-inhibitory protein, long isoform), mainly known as inhibitor of caspase-8, can be retrieved at the ER and MAMs. c-FLIP ablation in mouse embryonic fibroblasts (MEFs) impairs ER morphology and luminal contiguity, by inducing the proliferation of ER cisternae to the detriment of tubular ER. Furthermore, c-FLIPL controls the ER-mitochondria apposition, as the depletion of this protein physically uncouples the two organelles. Moreover, functionally, c-FLIP ablation lowers the cytosolic Ca2+-increase evoked either by agonists stimulation or by passive ER discharge and increases the resistance of c-FLIP-/- cells to ER stress-induced apoptosis. We also report that c-FLIP-/- cells show an increased caspase-mediated cleavage of the ER-shaping protein Reticulon-4 (which is a well-known regulator of ER biogenesis and morphology), at both basal level and upon TNFα-dependent apoptosis. In agreement with these findings, we finally show that c-FLIP absence enhances basal caspase-8 activation in c-FLIP-/- MEFs and that caspase-8 inhibition reverts morphological alterations in ER shape observed in c-FLIP-/- cells, suggesting a novel role for caspase-8 and c-FLIPL as regulators of ER functions and ER-mitochondria crosstalk

Simultaneous quantification of natural and inducible regulatory T-cell subsets during interferon-\u3b2 therapy of multiple sclerosis patients

The mechanisms underlying the therapeutic activity of interferon-\u3b2 in multiple sclerosis are still not completely understood. In the present study, we evaluated the short and long-term effects of interferon-\u3b2 treatment on different subsets of regulatory T cells in relapsing-remitting multiple sclerosis patients biologically responsive to treatment because of mixovirus resistance protein A inducibility