C. elegans as a model to identify and functionally characterize novel genes causing RASopathies and other developmental diseases

RASopathies are a family of syndromes affecting development and growth, sharing RAS signaling dysregulation as pathogenetic mechanism. Past work of our group and others have significantly contributed to our understanding of the molecular causes of these diseases. However, a large fraction of RASopathy cases remains unexplained molecularly. Here, I used the nematode C. elegans to reveal novel molecular mechanisms underlying RASopathies, as well as to identify new candidate genes for these group of developmental disorders. C. elegans is an excellent model to study RASopathies since the RAS-MAPK pathway is well conserved in worms, where it plays a crucial role in vulval development. Based on a gene candidacy approach, we identified two germline mutations in RRAS, a gene encoding a small monomeric GTPase controlling cell adhesion, spreading and migration, underlying a rare and atypical form of Noonan syndrome (NS), the most common RASopathy. We also identified somatic RRAS mutations in 2 cases of non-syndromic juvenile myelomonocytic leukaemia (JMML), a childhood myeloproliferative/myelodysplastic disease caused by upregulated RAS signaling. Two of the three identified mutations affected known oncogenic hotspots of RAS genes and conferred variably enhanced RRAS function and stimulus-dependent MAPK activation. Expression of an RRAS mutant homolog in C. elegans enhanced RAS signaling causing a multivulva (Muv) phenotype, and engendered protruding vulva (Pvl), a phenotype previously linked to the RASopathy-causing SHOC2S2G mutant. These findings provided evidence of a functional link between RRAS and MAPK signaling and reveal an unpredicted role of enhanced RRAS function in human disease. Epistatic analyses performed on C. elegans transgenic lines allowed us to establish that the RASopathy-causing SHOC2 and RRAS mutants belong to the same pathway. Within this signaling network, both RAS-1/RRAS and RAS-2/MRAS are downstream to constitutively active SHOC2, with the former being epistatic to the latter. By using a reverse genetic approach based on RNA interference experiments, we demonstrated that the Muv phenotype was completely mediated by LET-60/RAS, while the Pvl phenotype was modulated by the RHO-family small GTPases CDC-42 and RAC1. We then confirmed these results in fibroblasts derived from patients with Mazzanti syndrome (NS with loose anagen hair) and transfected cell lines. In these models, we observed constitutive RAC1 activation and aberrant lamellipodia formation in cells expressing SHOC2S2G compared to wild-type cells. These results suggested RHO GTPases as excellent candidate genes to be mutated in RASopathies. To explore this hypothesis, mutation scanning of RAC1, RAC2 and CDC42 genes was performed in RASopathy patients by targeted resequencing and identified seven different germline CDC42 mutations in 11 unrelated subjects with a variable phenotype partially overlapping NS and predisposing to thrombocytopenia. In vitro biochemical characterization demonstrated a variable impact of the mutations on GTPase activity and defective binding to WASP. In vitro and in vivo (C. elegans) functional characterization of these mutants allowed to define their impact on cell migration and proliferation, as well as on vulval induction and morphogenesis. A first class of mutations was shown to have an hypomorphic effect on processes mediating cell polarized migration, with no effect on the RAS-MAPK signaling, while a second class of mutations had a gain-of-function effect on both cell migration/proliferation and LET-60/RAS-mediated vulval induction. Overall, our data highlighted the possible contribution of dysregulated signaling controlling cell spreading and migration to certain features of RASopathies, such as lymphedema, cardiac defects and lymphocytes infiltration in non-hematopoietic tissues in case of JMML

A vision-based system for internal pipeline inspection

The internal inspection of large pipeline infrastructures, such as sewers and waterworks, is a fundamental task for the prevention of possible failures. In particular, visual inspection is typically performed by human operators on the basis of video sequences either acquired on-line or recorded for further off-line analysis. In this work, we propose a vision-based software approach to assist the human operator by conveniently showing the acquired data and by automatically detecting and highlighting the pipeline sections where relevant anomalies could occur

A Rover-based System for Searching Encrypted Targets in Unknown Environments

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C. elegans-based chemosensation strategy for the early detection of cancer metabolites in urine samples

Chemosensory receptors play a crucial role in distinguishing the wide range of volatile/soluble molecules by binding them with high accuracy. Chemosensation is the main sensory modality in organisms lacking long-range sensory mechanisms like vision/hearing. Despite its low number of sensory neurons, the nematode Caenorhabditis elegans possesses several chemosensory receptors, allowing it to detect about as many odorants as mammals. Here, we show that C. elegans displays attraction towards urine samples of women with breast cancer, avoiding control ones. Behavioral assays on animals lacking AWC sensory neurons demonstrate the relevance of these neurons in sensing cancer odorants: calcium imaging on AWC increases the accuracy of the discrimination (97.22%). Also, chemotaxis assays on animals lacking GPCRs expressed in AWC allow to identify receptors involved in binding cancer metabolites, suggesting that an alteration of a few metabolites is sufficient for the cancer discriminating behavior of C. elegans, which may help identify a fundamental fingerprint of breast cancer

The recurrent pathogenic Pro890Leu substitution in CLTC causes a generalized defect in synaptic transmission in Caenorhabditis elegans

De novo CLTC mutations underlie a spectrum of early-onset neurodevelopmental phenotypes having developmental delay/intellectual disability (ID), epilepsy, and movement disorders (MD) as major clinical features. CLTC encodes the widely expressed heavy polypeptide of clathrin, a major component of the coated vesicles mediating endocytosis, intracellular trafficking, and synaptic vesicle recycling. The underlying pathogenic mechanism is largely unknown. Here, we assessed the functional impact of the recurrent c.2669C > T (p.P890L) substitution, which is associated with a relatively mild ID/MD phenotype. Primary fibroblasts endogenously expressing the mutated protein show reduced transferrin uptake compared to fibroblast lines obtained from three unrelated healthy donors, suggesting defective clathrin-mediated endocytosis. In vitro studies also reveal a block in cell cycle transition from G0/G1 to the S phase in patient's cells compared to control cells. To demonstrate the causative role of the p.P890L substitution, the pathogenic missense change was introduced at the orthologous position of the Caenorhabditis elegans gene, chc-1 (p.P892L), via CRISPR/Cas9. The resulting homozygous gene-edited strain displays resistance to aldicarb and hypersensitivity to PTZ, indicating defective release of acetylcholine and GABA by ventral cord motor neurons. Consistently, mutant animals show synaptic vesicle depletion at the sublateral nerve cords, and slightly defective dopamine signaling, highlighting a generalized deficit in synaptic transmission. This defective release of neurotransmitters is associated with their secondary accumulation at the presynaptic membrane. Automated analysis of C. elegans locomotion indicates that chc-1 mutants move slower than their isogenic controls and display defective synaptic plasticity. Phenotypic profiling of chc-1 (+/P892L) heterozygous animals and transgenic overexpression experiments document a mild dominant-negative behavior for the mutant allele. Finally, a more severe phenotype resembling that of chc-1 null mutants is observed in animals harboring the c.3146 T > C substitution (p.L1049P), homologs of the pathogenic c.3140 T > C (p.L1047P) change associated with a severe epileptic phenotype. Overall, our findings provide novel insights into disease mechanisms and genotype-phenotype correlations of CLTC-related disorders

De novo DHDDS variants cause a neurodevelopmental and neurodegenerative disorder with myoclonus

Subcellular membrane systems are highly enriched in dolichol, whose role in organelle homeostasis and endosomal-lysosomal pathway remains largely unclear besides being involved in protein glycosylation. DHDDS encodes for the catalytic subunit (DHDDS) of the enzyme cis-prenyltransferase (cis-PTase), involved in dolichol biosynthesis and dolichol-dependent protein glycosylation in the endoplasmic reticulum. An autosomal recessive form of retinitis pigmentosa (retinitis pigmentosa 59) has been associated with a recurrent DHDDS variant. Moreover, two recurring de novo substitutions were detected in a few cases presenting with neurodevelopmental disorder, epilepsy, and movement disorder. We evaluated a large cohort of patients (n=25) with de novo pathogenic variants in DHDDS and provided the first systematic description of the clinical features and long-term outcome of this new neurodevelopmental and neurodegenerative disorder. The functional impact of the identified variants was explored by yeast complementation system and enzymatic assay. Patients presented during infancy or childhood with a variable association of neurodevelopmental disorder, generalized epilepsy, action myoclonus/cortical tremor, and ataxia. Later in the disease course they experienced a slow neurological decline with the emergence of hyperkinetic and/or hypokinetic movement disorder, cognitive deterioration, and psychiatric disturbances. Storage of lipidic material and altered lysosomes were detected in myelinated fibers and fibroblasts, suggesting a dysfunction of the lysosomal enzymatic scavenger machinery. Serum glycoprotein hypoglycosylation was not detected and, in contrast to retinitis pigmentosa and other congenital disorders of glycosylation involving dolichol metabolism, the urinary dolichol D18/D19 ratio was normal. Mapping the disease-causing variants into the protein structure revealed that most of them clustered around the active site of the DHDDS subunit. Functional studies using yeast complementation assay and in vitro activity measurements confirmed that these changes affected the catalytic activity of the cis-PTase and showed growth defect in yeast complementation system as compared with the wild-type enzyme and retinitis pigmentosa-associated protein. In conclusion, we characterized a distinctive neurodegenerative disorder due to de novo DHDDS variants, which clinically belongs to the spectrum of genetic progressive encephalopathies with myoclonus. Clinical and biochemical data from this cohort depicted a condition at the intersection of congenital disorders of glycosylation and inherited storage diseases with several features akin to of progressive myoclonus epilepsy such as neuronal ceroid lipofuscinosis and other lysosomal disorders

Aberrant function of the C-terminal tail of HIST1H1E Aacelerates cellular senescence and causes premature aging

Histones mediate dynamic packaging of nuclear DNA in chromatin, a process that is precisely controlled to guarantee efficient compaction of the genome and proper chromosomal segregation during cell division and to accomplish DNA replication, transcription, and repair. Due to the important structural and regulatory roles played by histones, it is not surprising that histone functional dysregulation or aberrant levels of histones can have severe consequences for multiple cellular processes and ultimately might affect development or contribute to cell transformation. Recently, germline frameshift mutations involving the C-terminal tail of HIST1H1E, which is a widely expressed member of the linker histone family and facilitates higher-order chromatin folding, have been causally linked to an as-yet poorly defined syndrome that includes intellectual disability. We report that these mutations result in stable proteins that reside in the nucleus, bind to chromatin, disrupt proper compaction of DNA, and are associated with a specific methylation pattern. Cells expressing these mutant proteins have a dramatically reduced proliferation rate and competence, hardly enter into the S phase, and undergo accelerated senescence. Remarkably, clinical assessment of a relatively large cohort of subjects sharing these mutations revealed a premature aging phenotype as a previously unrecognized feature of the disorder. Our findings identify a direct link between aberrant chromatin remodeling, cellular senescence, and accelerated aging

A Practical Framework for the Development of Augmented Reality Applications by using ArUco Markers.

The Augmented Reality (AR) is an expanding field of the Computer Graphics (CG) that merges items of the real-world environment (e.g., places, objects) with digital information (e.g., multimedia files, virtual objects) to provide users with an enhanced interactive multi-sensorial experience of the real-world that surrounding them. Currently, a wide range of devices is used to vehicular AR systems. Common devices (e.g., cameras equipped on smartphones) enable users to receive multimedia information about target objects (non-immersive AR). Advanced devices (e.g., virtual windscreens) provide users with a set of virtual information about points of interest (POIs) or places (semi-immersive AR). Finally, an ever-increasing number of new devices (e.g., HeadMounted Display, HMD) support users to interact with mixed reality environments (immersive AR). This paper presents a practical framework for the development of non-immersive augmented reality applications through which target objects are enriched with multimedia information. On each target object is applied a different ArUco marker. When a specific application hosted inside a device recognizes, via camera, one of these markers, then the related multimedia information are loaded and added to the target object. The paper also reports a complete case study together with some considerations on the framework and future work