Absence of Caspase 8 and High Expression of PED Protect Primitive Neural Cells from Cell Death

The mechanisms that control neural stem and progenitor cell survival are unknown. In several pathological conditions, death receptor (DR) ligands and inflammatory cytokines exert a deleterious effect on neurons, whereas primitive neural cells migrate and survive in the site of lesion. Here, we show that even in the presence of inflammatory cytokines, DRs are unable to generate death signals in primitive neural cells. Neural stem and progenitor cells did not express caspase 8, the presence of which is required for initiating the caspase cascade. However, exogenous or cytokine-mediated expression of caspase 8 was not sufficient to restore their DR sensitivity. Searching for molecules potentially able to block DR death-inducing signaling complex (DISC), we found that primitive neural cells expressed high levels of the death effector domain-containing protein PED (also known as PEA-15). PED localized in the DISC and prevented caspase 8 recruitment and activation. Moreover, lentiviral-mediated delivery of PED antisense DNA resulted in dramatic down-regulation of the endogenous gene expression and sensitization of primitive neural cells to apoptosis mediated by inflammatory cytokines and DRs. Thus, absence of caspase 8 and high expression of PED constitute two levels of protection from apoptosis induced by DRs and inflammatory cytokines in neural stem and progenitor cells

CD95 death-inducing signaling complex formation and internalization occur in lipid rafts of type I and type II cells

We investigated the membrane localization of CD95 in type I and type II cells, which differ in their ability to recruit and activate caspase-8. We found that CD95 was preferentially located in lipid rafts of type I cells, while it was present both in raft and non-raft plasma membrane sub-domains of type II cells. After stimulation, CD95 located in phospholipid-rich plasma membrane was recruited to lipid rafts in both types of cells. Similarly, CD95 cross-linking resulted in caspase-independent translocation of FADD/MORT1 and caspase-8 to the lipid rafts, which was prevented by a death domain-defective receptor. CD95 internalization was then rapid in type I and delayed in type II cells and showed a substantial correlation with the kinetics of Fas-associated death domain (FADD) and caspase-8 recruitment to lipid rafts. Finally, electron microscopy analysis showed that after CD95 stimulation lipid rafts aggregated in large clusters that were internalized in endosomal vesicles, where caspase-8 underwent massive processing. Taken together, our data demonstrate that CD95 death-inducing signaling complex formation and internalization in type I and type II cells occur in lipid rafts, which are a major site of caspase-8 activation. © 2004 WILEY-VCH Verlag GmbH & Co. KGaA

Cheetah:a computational toolkit for cybergenetic control

Abstract Advances in microscopy, microfluidics, and optogenetics enable single-cell monitoring and environmental regulation and offer the means to control cellular phenotypes. The development of such systems is challenging and often results in bespoke setups that hinder reproducibility. To address this, we introduce Cheetah, a flexible computational toolkit that simplifies the integration of real-time microscopy analysis with algorithms for cellular control. Central to the platform is an image segmentation system based on the versatile U-Net convolutional neural network. This is supplemented with functionality to robustly count, characterize, and control cells over time. We demonstrate Cheetah’s core capabilities by analyzing long-term bacterial and mammalian cell growth and by dynamically controlling protein expression in mammalian cells. In all cases, Cheetah’s segmentation accuracy exceeds that of a commonly used thresholding-based method, allowing for more accurate control signals to be generated. Availability of this easy-to-use platform will make control engineering techniques more accessible and offer new ways to probe and manipulate living cells

Selective sparing of bladder and rectum sub-regions in radiotherapy of prostate cancer combining knowledge-based automatic planning and multicriteria optimization

Background and Purpose: The association between dose to selected bladder and rectum symptom-related sub- regions (SRS) and late toxicity after prostate cancer radiotherapy has been evidenced by voxel-wise analyses. The aim of the current study was to explore the feasibility of combining knowledge-based (KB) and multi-criteria optimization (MCO) to spare SRSs without compromising planning target volume (PTV) dose delivery, including pelvic-node irradiation. Materials and Methods: Forty-five previously treated patients (74.2 Gy/28fr) were selected and SRSs (in the bladder, associated with late dysuria/hematuria/retention; in the rectum, associated with bleeding) were generated using deformable registration. A KB model was used to obtain clinically suitable plans (KB-plan). KB- plans were further optimized using MCO, aiming to reduce dose to the SRSs while safeguarding target dose coverage, homogeneity and avoiding worsening dose volume histograms of the whole bladder, rectum and other organs at risk. The resulting MCO-generated plans were examined to identify the best-compromise plan (KB + MCO-plan). Results: The mean SRS dose decreased in almost all patients for each SRS. D1% also decreased in the large majority, less frequently for dysuria/bleeding SRS. Mean differences were statistically significant (p < 0.05) and ranged between 1.3 and 2.2 Gy with maximum reduction of mean dose up to 3&#8211;5 Gy for the four SRSs. The better sparing of SRSs was obtained without compromising PTVs coverage. Conclusions: Selectively sparing SRSs without compromising PTV coverage is feasible and has the potential to reduce toxicities in prostate cancer radiotherapy. Further investigation to better quantify the expected risk reduction of late toxicities is warranted.This work has been supported by Fondazione Regionale per la Ricerca Biomedica, project nr. 110 - JTC PerPlanRT ERA PerMed, GA 779282.Publicad

In vitro and in vivo study of a novel biodegradable synthetic conduit for injured peripheral nerves

In case of peripheral nerve injury (PNI) with wide substance-loss, surgical reconstruction is still a challenge. Bridging the gap by autologous sensory nerves as grafts is the current standard; nevertheless, the related issues have prompted the research towards the development of effective artificial synthetic/biological nerve conduits (NCs). Here, we manufactured a novel NC using oxidized polyvinyl alcohol (OxPVA) that is a biodegradable cryogel recently patented by our group [1]. Thus, its characteristics were compared with neat polyvinyl alcohol (PVA) and silk-fibroin (SF) NCs through in vitro/in vivo analysis. Considering in vitro studies, a morphological characterization was performed by Scanning Electron Microscopy (SEM). Thereafter, cell adhesion and proliferation of a Schwann-cell line (SH-SY5Y) were evaluated by SEM and MTT assay. Regarding in vivo tests, the NCs were implanted into the surgical injured sciatic nerve (gap: 5 mm) of Sprague-Dawley rats, and the functional recovery was assessed after 12-weeks. The NCs were then processed for histological, immunohistochemical (anti-CD3; -β-tubulin; -S100) and Transmission Electron Microscopy (TEM) analyses. In particular, morphometric analyses (section area, total number and density of nerve fibers) were performed at the level of proximal, central and distal portions with respect to NC. In vitro results by SEM showed that PVA and SF supports have a smoother surface than OxPVA scaffolds. Moreover, unlike SF scaffolds, PVA-based ones do not support SH-SY5Y adhesion and proliferation. Regarding the in vivo study, all animals showed a functional recovery with normal walk, even though only animals implanted with PVA and SF NCs sometimes showed spasms while walking. On the contrary, animals implanted with OxPVA NCs exhibited a normal movement. Anti-CD3 immunohistochemistry assessed the absence of severe inflammatory reactions in all the grafts. A strong positive immunoreaction for β-tubulin and S100 demonstrated the good regeneration of nervous fibers. TEM highlighted regeneration of myelinated/un-myelinated axons and Schwann cells in all the grafts. However, morphometric analysis demonstrated that OxPVA assure a better outcome in nerve regeneration in terms of total number of nerve fibers. Our results sustain the potential of OxPVA for the development of NCs useful for PNI with substance loss with the advantage of biodegradation

Measurement of {\eta} meson production in {\gamma}{\gamma} interactions and {\Gamma}({\eta}-->{\gamma}{\gamma}) with the KLOE detector

We present a measurement of {\eta} meson production in photon-photon interactions produced by electron-positron beams colliding with \sqrt{s}=1 GeV. The measurement is done with the KLOE detector at the \phi-factory DA{\Phi}NE with an integrated luminosity of 0.24 fb^{-1}. The e^+e^- --> e^+e^-{\eta} cross section is measured without detecting the outgoing electron and positron, selecting the decays {\eta}-->{\pi}^+{\pi}^-{\pi}^0 and {\eta}-->{\pi}^0{\pi}^0{\pi}^0. The most relevant background is due to e^+e^- --> {\eta}{\gamma} when the monochromatic photon escapes detection. The cross section for this process is measured as {\sigma}(e^+e^- -->{\eta}{\gamma}) = (856 \pm 8_{stat} \pm 16_{syst}) pb. The combined result for the e^+e^- -->e^+e^-{\eta} cross section is {\sigma}(e^+e^- -->e^+e^-{\eta}) = (32.72 \pm 1.27_{stat} \pm 0.70_{syst}) pb. From this we derive the partial width {\Gamma}({\eta}-->{\gamma}{\gamma}) = (520 \pm 20_{stat} \pm 13_{syst}) eV. This is in agreement with the world average and is the most precise measurement to date.Comment: Version accepted by JHE

A new limit on the CP violating decay KS -> 3pi0 with the KLOE experiment

We have carried out a new direct search for the CP violating decay KS -> 3pi0 with 1.7 fb^-1 of e+e- collisions collected by the KLOE detector at the phi-factory DAFNE. We have searched for this decay in a sample of about 5.9 x 10^8 KS KL events tagging the KS by means of the KL interaction in the calorimeter and requiring six prompt photons. With respect to our previous search, the analysis has been improved by increasing of a factor four the tagged sample and by a more effective background rejection of fake KS tags and spurious clusters. We find no candidates in data and simulated background samples, while we expect 0.12 standard model events. Normalizing to the number of KS -> 2pi0 events in the same sample, we set the upper limit on BR(KS -> 3pi0 < 2.6 x 10^-8 at 90% C.L., five times lower than the previous limit. We also set the upper limit on the eta_000 parameter, |eta_000 | < 0.0088 at 90% C.L., improving by a factor two the latest direct measurement.Comment: Accepted for publication in Physics Letters B (15 pages, 13 figures

Resistome and virulome diversity of foodborne pathogens isolated from artisanal food production chain of animal origin in the Mediterranean region

The aim of the present study was to investigate the resistome and virulome diversity of 43 isolates of Listeria monocytogenes, Salmonella enterica and S. aureus collected from artisanal fermented meat and dairy products and their production environments in Portugal, Spain, Italy and Morocco. After DNA extraction, genomes were sequenced, and de novo assembled. Genetic relationships among genomes were investigated by SNP calling and in silico 7- loci MLST. Genomes of the same species belonged to different ST-types demonstrating the circulation of different clones in in the same artisanal production plant. One specific clone included genomes of S. Paratyphi B belonging to ST43 and repeatedly isolated for more than a year in an artisanal sausage production plant. No genomes but three (belonging to Salmonella enterica), were predicted as multiresistant to different antimicrobials classes. Regarding virulence, genomes of L. monocytogenes belonging to ST1, ST3 and ST489, as well as genomes of S.enterica enterica (ST43, ST33, ST314, ST3667, ST1818, ST198) and ST121 S. aureus were predicted as virulent and hypervirulent. The occurrence of virulent and hypervirulent L. monocytogenes, Salmonella enterica and S. aureus strains in artisanal fermented meat and dairy productions as well as in their finished products suggests the need for a specific focus on prevention and control measures able to reduce the risk of these biological hazards in artisanal food productions

Inhibition of DNA methylation sensitizes glioblastoma for tumor necrosis factor-related apoptosis-inducing ligand-mediated destruction

Life expectancy of patients affected by glioblastoma multiforme is extremely low. The therapeutic use of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been proposed to treat this disease based on its ability to kill glioma cell lines in vitro and in vivo. Here, we show that, differently from glioma cell lines, glioblastoma multiforme tumors were resistant to TRAIL stimulation because they expressed low levels of caspase-8 and high levels of the death receptor inhibitor PED/PEA-15. Inhibition of methyltransferases by decitabine resulted in considerable up-regulation of TRAIL receptor-1 and caspase-8, down-regulation of PED/PEA-15, inhibition of cell growth, and sensitization of primary glioblastoma cells to TRAIL-induced apoptosis. Exogenous caspase-8 expression was the main event able to restore TRAIL sensitivity in primary glioblastoma cells. The antitumor activity of decitabine and TRAIL was confirmed in vivo in a mouse model of glioblastoma multiforme. Evaluation of tumor size, apoptosis, and caspase activation in nude mouse glioblastoma multiforme xenografts showed dramatic synergy of decitabine and TRAIL in the treatment of glioblastoma, whereas the single agents were scarcely effective in terms of reduction of tumor mass, apoptosis induction, and caspase activation. Thus, the combination of TRAIL and demethylating agents may provide a key tool to overcome glioblastoma resistance to therapeutic treatments. ©2005 American Association for Cancer Research