The iBuddy experience: A digital simulation-based approach to enhance secondary school students' privacy awareness.

Privacy is a central issue in the digitalization of society and directly concerns all Internet users. Privacy education is part of the picture of a more just digital society: it aims at making users more aware of the importance of their data and of the technical and financial tools and processes that involve their personal data. Nonetheless, privacy education is confronted with a paradox: while people perceive the importance of privacy, they seldom take action to actually protect their personal data. iBuddy is a narrative simulation-based session inspired by research evidence about the privacy paradox and aims to (a) enhancing awareness and (b) promoting the uptake of privacy-safe behaviors for secondary and higher students (age range 11-20). The paper presents the design and development of the simulation and of the following modular debriefing, as a case study in evidence-based collaborative instructional design and in the instructional used of digital technology. The evaluation of iBuddy, which combined a post-session satisfaction and perceived learning survey (N = 978) and a follow-up survey (N = 124), provides insights in the novel domain of privacy education. Results suggests that iBuddy sessions are engaging, effective and conducive to medium-term behavioral change, thus indirectly confirming the design assumptions about how to tackle the privacy paradox through a simulation-based approach.

Long-term effects of amyloid-beta deposits in human iPSC-derived astrocytes.

Growing evidence indicates that astrocytes are tightly connected to Alzheimer's disease (AD) pathogenesis. However, the way in which astrocytes participate in AD initiation and progression remains to be clarified. Our previous data show that astrocytes engulf large amounts of aggregated amyloid-beta (Aß) but are unable to successfully degrade the material. In this study, we aimed to evaluate how intracellular Aß-accumulation affects the astrocytes over time. For this purpose, human induced pluripotent cell (hiPSC)-derived astrocytes were exposed to sonicated Aß-fibrils and then cultured further for one week or ten weeks in Aß-free medium. Cells from both time points were analyzed for lysosomal proteins and astrocyte reactivity markers and the media were screened for inflammatory cytokines. In addition, the overall health of cytoplasmic organelles was investigated by immunocytochemistry and electron microscopy. Our data demonstrate that long-term astrocytes retained frequent Aß-inclusions that were enclosed within LAMP1-positive organelles and sustained markers associated with reactivity. Furthermore, Aß-accumulation resulted in endoplasmic reticulum and mitochondrial swelling, increased secretion of the cytokine CCL2/MCP-1 and formation of pathological lipid structures. Taken together, our results provide valuable information of how intracellular Aß-deposits affect astrocytes, and thereby contribute to the understanding of the role of astrocytes in AD progression.

Amyloid-ß accumulation in human astrocytes induces mitochondrial disruption and changed energy metabolism.

BACKGROUND: Astrocytes play a central role in maintaining brain energy metabolism, but are also tightly connected to the pathogenesis of Alzheimer's disease (AD). Our previous studies demonstrate that inflammatory astrocytes accumulate large amounts of aggregated amyloid-beta (Aß). However, in which way these Aß deposits influence their energy production remain unclear. METHODS: The aim of the present study was to investigate how Aß pathology in astrocytes affects their mitochondria functionality and overall energy metabolism. For this purpose, human induced pluripotent cell (hiPSC)-derived astrocytes were exposed to sonicated Aß42 fibrils for 7 days and analyzed over time using different experimental approaches. RESULTS: Our results show that to maintain stable energy production, the astrocytes initially increased their mitochondrial fusion, but eventually the Aß-mediated stress led to abnormal mitochondrial swelling and excessive fission. Moreover, we detected increased levels of phosphorylated DRP-1 in the Aß-exposed astrocytes, which co-localized with lipid droplets. Analysis of ATP levels, when blocking certain stages of the energy pathways, indicated a metabolic shift to peroxisomal-based fatty acid ß-oxidation and glycolysis. CONCLUSIONS: Taken together, our data conclude that Aß pathology profoundly affects human astrocytes and changes their entire energy metabolism, which could result in disturbed brain homeostasis and aggravated disease progression.

Intracellular deposits of amyloid-beta influence the ability of human iPSC-derived astrocytes to support neuronal function.

BACKGROUND: Astrocytes are crucial for maintaining brain homeostasis and synaptic function, but are also tightly connected to the pathogenesis of Alzheimer's disease (AD). Our previous data demonstrate that astrocytes ingest large amounts of aggregated amyloid-beta (Aß), but then store, rather than degrade the ingested material, which leads to severe cellular stress. However, the involvement of pathological astrocytes in AD-related synaptic dysfunction remains to be elucidated. METHODS: In this study, we aimed to investigate how intracellular deposits of Aß in astrocytes affect their interplay with neurons, focusing on neuronal function and viability. For this purpose, human induced pluripotent stem cell (hiPSC)-derived astrocytes were exposed to sonicated Αß42 fibrils. The direct and indirect effects of the Αß-exposed astrocytes on hiPSC-derived neurons were analyzed by performing astrocyte-neuron co-cultures as well as additions of conditioned media or extracellular vesicles to pure neuronal cultures. RESULTS: Electrophysiological recordings revealed significantly decreased frequency of excitatory post-synaptic currents in neurons co-cultured with Aß-exposed astrocytes, while conditioned media from Aß-exposed astrocytes had the opposite effect and resulted in hyperactivation of the synapses. Clearly, factors secreted from control, but not from Aß-exposed astrocytes, benefited the wellbeing of neuronal cultures. Moreover, reactive astrocytes with Aß deposits led to an elevated clearance of dead cells in the co-cultures. CONCLUSIONS: Taken together, our results demonstrate that inclusions of aggregated Aß affect the reactive state of the astrocytes, as well as their ability to support neuronal function.

Synthesis and automated fluorine-18 radiolabeling of new PSMA-617 derivatives with a CuAAC radiosynthetic approach.

In the last decade, the development of new radiopharmaceuticals for the imaging and therapy of prostate cancer has been a highly active and important area of research, especially focusing on the prostate-specific membrane antigen (PSMA), an antigen which is upregulated in prostate, as well as in other tumor cells. A large variety of PSMA ligands have been radiolabeled, to date. Among the various derivatives, PSMA-617 resulted to be one of the most interesting in terms of interaction with the antigen and clinical properties, and its lutetium-177 labeled version has recently been approved by regulatory agencies for therapeutic purposes. For this reasons, the radiolabeling with fluorine-18 of a PSMA-617 derivative might be of interest. Beside other methodologies to radiolabel macromolecules with fluorine-18, the "click-chemistry" approach resulted to be very useful, and the copper-catalyzed azide-alkyne cycloaddition (CuAAC) is considered one of most efficient and reliable. This paper proposes the synthesis of a suitable precursor for the radiolabeling with fluorine-18 of a new PSMA-617 derivative. The whole radiosynthetic procedure has been fully automated, and the final product, which proved to be stable in plasma, has been obtained with radiochemical yield and purity suitable for subsequent preclinical studies.

Traumatic brain injury in the presence of Aß pathology affects neuronal survival, glial activation and autophagy.

Traumatic brain injury (TBI) presents a widespread health problem in the elderly population. In addition to the acute injury, epidemiological studies have observed an increased probability and earlier onset of dementias in the elderly following TBI. However, the underlying mechanisms of the connection between TBI and Alzheimer's disease in the aged brain and potential exacerbating factors is still evolving. The aim of this study was to investigate cellular injury-induced processes in the presence of amyloid ß (Aß) pathology. For this purpose, a co-culture system of cortical stem-cell derived astrocytes, neurons and oligodendrocytes were exposed to Aß42 protofibrils prior to a mechanically induced scratch injury. Cellular responses, including neurodegeneration, glial activation and autophagy was assessed by immunoblotting, immunocytochemistry, ELISA and transmission electron microscopy. Our results demonstrate that the combined burden of Aß exposure and experimental TBI causes a decline in the number of neurons, the differential expression of the key astrocytic markers glial fibrillary acidic protein and S100 calcium-binding protein beta, mitochondrial alterations and prevents the upregulation of autophagy. Our study provides valuable information about the impact of TBI sustained in the presence of Aß deposits and helps to advance the understanding of geriatric TBI on the cellular level.

Enhanced neprilysin-mediated degradation of hippocampal Aß42 with a somatostatin peptide that enters the brain.

Background: Aggregation of the amyloid-beta (Aß) peptide is one of the main neuropathological events in Alzheimer's disease (AD). Neprilysin is the major enzyme degrading Aß, with its activity enhanced by the neuropeptide somatostatin (SST). SST levels are decreased in the brains of AD patients. The poor delivery of SST over the blood-brain barrier (BBB) and its extremely short half-life of only 3 min limit its therapeutic significance. Methods: We recombinantly fused SST to a BBB transporter binding to the transferrin receptor. Using primary neuronal cultures and neuroblastoma cell lines, the ability of the formed fusion protein to activate neprilysin was studied. SST-scFv8D3 was administered to mice overexpressing the Aß-precursor protein (AßPP) with the Swedish mutation (APPswe) as a single injection or as a course of three injections over a 72 h period. Levels of neprilysin and Aß were quantified using an Enzyme-linked immunosorbent assay (ELISA). Distribution of SST-scFv8D3 in the brain, blood and peripheral organs was studied by radiolabeling with iodine-125. Results: The construct, SST-scFv8D3, exhibited 120 times longer half-life than SST alone, reached the brain in high amounts when injected intravenously and significantly increased the brain concentration of neprilysin in APPswe mice. A significant decrease in the levels of membrane-bound Aß42 was detected in the hippocampus and the adjacent cortical area after only three injections. Conclusion: With intravenous injections of our BBB permeable SST peptide, we were able to significantly increase the levels neprilysin, an effect that was followed by a significant and selective degradation of membrane-bound Aß42 in the hippocampus. Being that membrane-bound Aß triggers neuronal toxicity and the hippocampus is the central brain area in the progression of AD, the study has illuminated a new potential treatment paradigm with a promising safety profile targeting only the disease affected areas.

Extracellular vesicles from amyloid-ß exposed cell cultures induce severe dysfunction in cortical neurons.

Alzheimer's disease (AD) is characterized by a substantial loss of neurons and synapses throughout the brain. The exact mechanism behind the neurodegeneration is still unclear, but recent data suggests that spreading of amyloid-ß (Aß) pathology via extracellular vesicles (EVs) may contribute to disease progression. We have previously shown that an incomplete degradation of Aß42 protofibrils by astrocytes results in the release of EVs containing neurotoxic Aß. Here, we describe the cellular mechanisms behind EV-associated neurotoxicity in detail. EVs were isolated from untreated and Aß42 protofibril exposed neuroglial co-cultures, consisting mainly of astrocytes. The EVs were added to cortical neurons for 2 or 4 days and the neurodegenerative processes were followed with immunocytochemistry, time-lapse imaging and transmission electron microscopy (TEM). Addition of EVs from Aß42 protofibril exposed co-cultures resulted in synaptic loss, severe mitochondrial impairment and apoptosis. TEM analysis demonstrated that the EVs induced axonal swelling and vacuolization of the neuronal cell bodies. Interestingly, EV exposed neurons also displayed pathological lamellar bodies of cholesterol deposits in lysosomal compartments. Taken together, our data show that the secretion of EVs from Aß exposed cells induces neuronal dysfunction in several ways, indicating a central role for EVs in the progression of Aß-induced pathology.

Incidence of bacteremias and invasive mycoses in children with acute non-lymphoblastic leukemia: results from a multi-center Italian study.

BACKGROUND: Data on the epidemiology of bacteremias and invasive fungal diseases (IFD) in children with acute myeloid leukemia (AML) are scarce. DESIGN AND METHODS: In a multi-center, retrospective study, we analyzed proportion, rate per 1,000 person-days at risk, and cumulative risk of bacteremias and IFD in children with AML. RESULTS: Between January 1998 and December 2005, 240 children were treated for AML at 8 Italian Centers, for a total of 521 treatment courses and 63,232 person-days at risk. Bacteremia was observed in 32% of treatment courses and IFD was seen in 10% (P < 0.0001), with rates of 2.62 and 0.84, respectively (P < 0.001). There was a significantly higher frequency of IFD during relapse treatment: proportion 15% versus 9% (P = 0.05), rate 2.10 versus 0.64 (P = 0.008) and cumulative risk 32% versus 12% (P = 0.007), while there were no differences in the proportion, rate and cumulative risk of bacteremia during front-line or relapse treatment. The epidemiology of bacteremias and IFD was different during front-line therapy for M3 as compared to other types of AML, but the differences were not statistically significant. CONCLUSIONS: Severe infectious complications are frequent during the treatment of pediatric AML, especially during relapse treatment, and bacteremias are more frequent than IFD.

Multidrug resistant Pseudomonas aeruginosa infection in children undergoing chemotherapy and hematopoietic stem cell transplantation.

Pseudomonas aeruginosa is one leading gram-negative organism associated with nosocomial infections. Bacteremia is life-threatening in the immunocompromised host. Increasing frequency of multi-drug-resistant (MDRPA) strains is concerning. We started a retrospective survey in the pediatric hematology oncology Italian network. Between 2000 and 2008, 127 patients with Pseudomonas aeruginosa bacteremia were reported from 12 centers; 31.4% of isolates were MDRPA. Death within 30 days of a positive blood culture occurred in 19.6% (25/127) of total patients; in patients with MDRPA infection it occurred in 35.8% (14/39). In the multivariate analysis, only MDRPA had significant association with infection-related death. This is the largest series of Pseudomonas aeruginosa bacteremia cases from pediatric hematology oncology centers. Monitoring local bacterial isolates epidemiology is mandatory and will allow empiric antibiotic therapy to be tailored to reduce fatalities.

Prolonged extracorporeal membrane oxygenation therapy for severe acute respiratory distress syndrome in a child affected by rituximab-resistant autoimmune hemolytic anemia: a case report.

INTRODUCTION: Autoimmune hemolytic anemia in children younger than 2 years of age is usually characterized by a severe course, with a mortality rate of approximately 10%. The prolonged immunosuppression following specific treatment may be associated with a high risk of developing severe infections. Recently, the use of monoclonal antibodies (rituximab) has allowed sustained remissions to be obtained in the majority of pediatric patients with refractory autoimmune hemolytic anemia. CASE PRESENTATION: We describe the case of an 8-month-old Caucasian girl affected by a severe form of autoimmune hemolytic anemia, which required continuous steroid treatment for 16 months. Thereafter, she received 4 weekly doses of rituximab (375 mg/m(2)/dose) associated with steroid therapy, which was then tapered over the subsequent 2 weeks. One month after the last dose of rrituximab, she presented with recurrence of severe hemolysis and received two more doses of rrituximab. The patient remained in clinical remission for 7 months, before presenting with a further relapse. An alternative heavy immunosuppressive therapy was administered combining cyclophosphamide 10 mg/kg/day for 10 days with methylprednisolone 40 mg/kg/day for 5 days, which was then tapered down over 3 weeks. While still on steroid therapy, the patient developed an interstitial pneumonia with Acute Respiratory Distress Syndrome, which required immediate admission to the intensive care unit where extracorporeal membrane oxygenation therapy was administered continuously for 37 days. At 16-month follow-up, the patient is alive and in good clinical condition, with no organ dysfunction, free from any immunosuppressive treatment and with a normal Hb level. CONCLUSIONS: This case shows that aggressive combined immunosuppressive therapy may lead to a sustained complete remission in children with refractory autoimmune hemolytic anemia. However, the severe life-threatening complication presented by our patient indicates that strict clinical monitoring must be vigilantly performed, that antimicrobial prophylaxis should always be considered and that experienced medical and nursing staff must be available, to deliver highly specialized supportive salvage therapies, if necessary, during intensive care monitoring.

REST selectively represses a subset of RE1-containing neuronal genes in mouse embryonic stem cells.

REST is a transcriptional repressor that targets a group of neuronal genes in non-neuronal cells. In embryonic stem (ES) cells, REST has been implicated in controlling the expression of transcription factor genes that are crucial for lineage determination and for maintaining ES cell potential. Here, we asked whether REST directly regulates neural-specifying genes in mouse ES cells using siRNA-mediated REST knockdown and ES cells that lack functional REST protein as a result of gene targeting. Loss of REST did not affect the expression of any of ten transcription factor genes known to promote neural commitment and did not affect the expression of several microRNAs, including miR-21, a putative REST target in ES cells. REST-deficient ES cells retained the ability to self-renew and to undergo appropriate differentiation towards mesoderm, endoderm and ectoderm lineages upon LIF withdrawal. Genome-wide expression profiling showed that genes that were deregulated in the absence of REST were preferentially expressed in the brain and highly enriched for the presence of canonical REST binding sites (RE1). Chromatin immunoprecipitation studies confirmed these genes as direct targets of REST in ES cells. Collectively, these data show that REST selectively silences a cohort of neuronal genes in ES cells.

Regulation of the cyclin-dependent kinase inhibitor p57Kip2 expression by p63.

The cyclin-dependent kinase (CDK) inhibitor p57Kip2 is a negative regulator of cell proliferation, binding to a variety of cyclin-CDK complexes and inhibiting their kinase activities. The p57Kip2 gene was recognized as a target gene for p73beta, one member of the p53 family. In spite of this, the phenotypes of p73 and p57Kip2 knockout mice do not resemble each other while there is a phenotypic overlap between the p57Kip2 null mice, the p63 null mice and patients affected by p63 associated syndromes, suggesting that p57Kip2 could be indeed a downstream target of p63. By ChIP we determined that in the HaCaT cell line the DeltaNp63alpha protein is associated to three different regions of the p57Kip2 gene. DeltaNp63 can activate both the endogenous p57Kip2 gene and a reporter vector containing a -2191 promoter fragment of the p57Kip2 gene. Natural p63 mutants, associated to the AEC syndrome, show a partial or complete lack of transactivation potential of the p57Kip2 promoter, while three other natural p63 mutants, associated to the EEC, LMS and SHFM-4 syndromes, were less affected. These data suggests that p63 play an important role in the regulation of p57Kip2 expression and that this regulation is subverted in AEC p63 mutants.

FLT3 internal tandem duplication in childhood acute myeloid leukaemia: association with hyperleucocytosis in acute promyelocytic leukaemia.

We evaluated the incidence of FLT3/internal tandem duplication (ITD) mutation in childhood acute myeloid leukaemia (AML) diagnosed over 15 years. FLT3/ITD was found in 10 of 45 (22.2%) non-acute promyelocytic leukaemia (non-APL) patients. The 5-year event-free survival of non-APL patients was higher in FLT3/ITD-negative versus -positive patients (48.9%, SE 8.9, vs 20.0%, SE 16.1, P = 0.03). In childhood APL, FLT3/ITD incidence was higher than in non-APL, although not statistically significant (10 out of 29 patients, 34.5%, P = 0.29). In APL patients, FLT3/ITD was strongly correlated to a higher white blood cell count at diagnosis and the M3 French-American-British subtype.