Evaluating the autonomy of children with autism spectrum disorder in washing hands: A deep-learning approach

Monitoring children with Autism Spectrum Dis-order (ASD) during the execution of the Applied Behaviour Analysis (ABA) program is crucial to assess the progresses while performing actions. Despite its importance, this monitoring procedure still relies on ABA operators' visual observation and manual annotation of the significant events. In this work a deep learning (DL) based approach has been proposed to evaluate the autonomy of children with ASD while performing the hand-washing task. The goal of the algorithm is the automatic detection of RGB frames in which the ASD child washes his/her hands autonomously (no-aid frames) or is supported by the operator (aid frames). The proposed approach relies on a pre-trained VGG16 convolutional network (CNN) modified to fulfill the binary classification task. The performance of the fine-tuned VGG16 was compared against that of other CNN architectures. The fine-tuned VGG16 achieved the best performance with a recall of 0.92 and 0.89 for the no-aid and aid class, respectively. These results prompt the possibility of translating the presented methodology into the actual monitoring practice. The integration of the presented tool with other computer-aided monitoring systems into a single framework, will provide fully support to ABA operators during the therapy session

po 040 development of a tunable form of interferon alpha for in vivo cancer gene therapy

Introduction The immune system is a double-edge sword in cancer. On the one hand, it exerts immunosurveillance to eradicate transformed cells that occasionally appear in the body; on the other hand, cancer cells can recruit immune cells endowed with pro-tumorigenic activity. Our lab previously developed a strategy for targeted gene-based delivery of interferon alpha (IFNa) to tumours by tumour infiltrating monocytes/macrophages, which induces robust anti-cancer responses in several experimental models without inducing strong IFN responses in normal tissues as compared to systemic administration of recombinant IFNa. Whereas a sustained output could ensure long-term protection from tumour recurrence, it may raise concerns for long-term side effects, especially in case of cancer eradication.To overcome this issue, we are developing inducible strategies to control the amount of IFNa secreted in the tumour microenvironment. Material and methods By fusing a destabilising domain (DD) to a protein of interest (POI) the former can confer its instability to the latter. This destabilisation can be rescued in a reversible and dose dependent manner with the addition of a small molecule specifically binding to the DD. To apply this technology to our strategy we have designed and in vitro tested different fusion proteins of IFNa (DD-IFNa). We also developed improved DD-IFNa with the addition of flexible and/or cleavable linkers and selected them for their capacity to be stabilised in a dose dependent manner in presence of their specific ligand in vitro . Results and discussions Through this approach, we have identified effective fusion proteins with low basal activity and high fold induction upon ligand treatment. These novel tunable forms of IFNa are functional and their specific activity are comparable to the wild type cytokine in inducing IFN responsive genes. Based on these promising in vitro results we are now translating these new platforms in vivo to test their efficacy in inducing anti-tumour responses in melanoma, colon and glioma models of cancer. Conclusion In the perspective of clinical translation our approach can be used in the future to switch on/off the levels of IFNa in a tunable and personalised fashion for cancer eradication

Pericellular activation of hepatocyte growth factor by the transmembrane serine proteases matriptase and hepsin, but not by the membrane-associated protease uPA

HGF (hepatocyte growth factor) is a pleiotropic cytokine homologous to the serine protease zymogen plasminogen that requires canonical proteolytic cleavage to gain functional activity. The activating proteases are key components of its regulation, but controversy surrounds their identity. Using quantitative analysis we found no evidence for activation by uPA (urokinase plasminogen activator), despite reports that this is a principal activator of pro-HGF. This was unaffected by a wide range of experimental conditions, including the use of various molecular forms of both HGF and uPA, and the presence of uPAR (uPA receptor) or heparin. In contrast the catalytic domains of the TTSPs (type-II transmembrane serine proteases) matriptase and hepsin were highly efficient activators (50% activation at 0.1 and 3.4 nM respectively), at least four orders of magnitude more efficient than uPA. PS-SCL (positional-scanning synthetic combinatorial peptide libraries) were used to identify consensus sequences for the TTSPs, which in the case of hepsin corresponded to the pro-HGF activation sequence, demonstrating a high specificity for this reaction. Both TTSPs were also found to be efficient activators at the cell surface. Activation of pro-HGF by PC3 prostate carcinoma cells was abolished by both protease inhibition and matriptase-targeting siRNA (small interfering RNA), and scattering of MDCK (Madin–Darby canine kidney) cells in the presence of pro-HGF was abolished by inhibition of matriptase. Hepsin-transfected HEK (human embryonic kidney)-293 cells also activated pro-HGF. These observations demonstrate that, in contrast with the uPA/uPAR system, the TTSPs matriptase and hepsin are direct pericellular activators of pro-HGF, and that together these proteins may form a pathway contributing to their involvement in pathological situations, including cancer

Design of a regulated lentiviral vector for hematopoietic stem cell gene therapy of globoid cell leukodystrophy

Globoid cell leukodystrophy (GLD) is a demyelinating lysosomal storage disease due to the deficiency of the galactocerebrosidase (GALC) enzyme. The favorable outcome of hematopoietic stem and progenitor cell (HSPC)-based approaches in GLD and other similar diseases suggests HSPC gene therapy as a promising therapeutic option for patients. The path to clinical development of this strategy was hampered by a selective toxicity of the overexpressed GALC in the HSPC compartment. Here, we presented the optimization of a lentiviral vector (LV) in which miR-126 regulation was coupled to codon optimization of the human GALC cDNA to obtain a selective and enhanced enzymatic activity only upon transduced HSPCs differentiation. The safety of human GALC overexpression driven by this LV was extensively demonstrated in vitro and in vivo on human HSPCs from healthy donors. No perturbation in the content of proapoptotic sphingolipids, gene expression profile, and capability of engraftment and mutlilineage differentiation in chimeric mice was observed. The therapeutic potential of this LV was then assessed in a severe GLD murine model that benefited from transplantation of corrected HSPCs with longer survival and ameliorated phenotype as compared to untreated siblings. This construct has thus been selected as a candidate for clinical translatio

Lentiviral Vector Delivery of Human Interleukin-7 (hIL-7) to Human Immune System (HIS) Mice Expands T Lymphocyte Populations

Genetically modified mice carrying engrafted human tissues provide useful models to study human cell biology in physiologically relevant contexts. However, there remain several obstacles limiting the compatibility of human cells within their mouse hosts. Among these is inadequate cross-reactvitiy between certain mouse cytokines and human cellular receptors, depriving the graft of important survival and growth signals. To circumvent this problem, we utilized a lentivirus-based delivery system to express physiologically relevant levels of human interleukin-7 (hIL-7) in Rag2-/-γc-/- mice following a single intravenous injection. hIL-7 promoted homeostatic proliferation of both adoptively transferred and endogenously generated T-cells in Rag2-/-γc-/- Human Immune System (HIS) mice. Interestingly, we found that hIL-7 increased T lymphocyte numbers in the spleens of HIV infected HIS mice without affecting viral load. Taken together, our study unveils a versatile approach to deliver human cytokines to HIS mice, to both improve engraftment and determine the impact of cytokines on human diseases

Analisi dei cambiamenti di uso del suolo nell’area montuosa e collinare dell’Emilia-Romagna nel periodo 1954-1994

Lo scopo di questo studio è stato la realizzazione di una banca dati multitemporale di uso del suolo per i territori montuosi e collinari della Regione Emilia-Romagna, relativa al periodo 1954-1994. Tale banca dati riporta gli attributi relativi all’uso del suolo del 1954-55 e 1994 e consente di analizzare i cambiamenti di estensione, tipologia e localizzazione delle classi di uso del suolo. I dati più recenti sono quelli relativi alla banca dati vettoriale di uso del suolo del 1994 già pubblicata dalla Regione Emilia-Romagna. I dati del 1954 sono stati ottenuti dall’interpretazione e delineazione visuale di ortofoto ottenute da foto aeree pancromatiche del volo G.A.I. del 1954- 1955. È stata utilizzata una nomenclatura di uso del suolo a priori gerarchica a due livelli comparabile con quella della banca dati del 1994. I risultati dimostrano che in quaranta anni, l’area indagata ha subito una significativa evoluzione di uso del suolo. Le aree a seminativo ed a foresta sono aumentate, a scapito delle aree a coltivazione parcellizzata e specializzata e di cespuglieto

TOM40 Mediates Mitochondrial Dysfunction Induced by α-Synuclein Accumulation in Parkinson's Disease.

Alpha-synuclein (α-Syn) accumulation/aggregation and mitochondrial dysfunction play prominent roles in the pathology of Parkinson's disease. We have previously shown that postmortem human dopaminergic neurons from PD brains accumulate high levels of mitochondrial DNA (mtDNA) deletions. We now addressed the question, whether alterations in a component of the mitochondrial import machinery -TOM40- might contribute to the mitochondrial dysfunction and damage in PD. For this purpose, we studied levels of TOM40, mtDNA deletions, oxidative damage, energy production, and complexes of the respiratory chain in brain homogenates as well as in single neurons, using laser-capture-microdissection in transgenic mice overexpressing human wildtype α-Syn. Additionally, we used lentivirus-mediated stereotactic delivery of a component of this import machinery into mouse brain as a novel therapeutic strategy. We report here that TOM40 is significantly reduced in the brain of PD patients and in α-Syn transgenic mice. TOM40 deficits were associated with increased mtDNA deletions and oxidative DNA damage, and with decreased energy production and altered levels of complex I proteins in α-Syn transgenic mice. Lentiviral-mediated overexpression of Tom40 in α-Syn-transgenic mice brains ameliorated energy deficits as well as oxidative burden. Our results suggest that alterations in the mitochondrial protein transport machinery might contribute to mitochondrial impairment in α-Synucleinopathies