Genomic stability of self-inactivating rabies.

Transsynaptic viral vectors provide means to gain genetic access to neurons based on synaptic connectivity and are essential tools for the dissection of neural circuit function. Among them, the retrograde monosynaptic ΔG-Rabies has been widely used in neuroscience research. A recently developed engineered version of the ΔG-Rabies, the non-toxic self-inactivating (SiR) virus, allows the long term genetic manipulation of neural circuits. However, the high mutational rate of the rabies virus poses a risk that mutations targeting the key genetic regulatory element in the SiR genome could emerge and revert it to a canonical ΔG-Rabies. Such revertant mutations have recently been identified in a SiR batch. To address the origin, incidence and relevance of these mutations, we investigated the genomic stability of SiR in vitro and in vivo. We found that "revertant" mutations are rare and accumulate only when SiR is extensively amplified in vitro, particularly in suboptimal production cell lines that have insufficient levels of TEV protease activity. Moreover, we confirmed that SiR-CRE, unlike canonical ΔG-Rab-CRE or revertant-SiR-CRE, is non-toxic and that revertant mutations do not emerge in vivo during long-term experiments.

Combining long-term circuit mapping and network transcriptomics with SiR-N2c.

An exciting frontier in circuit neuroscience lies at the intersection between neural network mapping and single-cell genomics. Monosynaptic rabies viruses provide a promising platform for the merger of circuit mapping methods with -omics approaches. However, three key limitations have hindered the extraction of physiologically meaningful gene expression profiles from rabies-mapped circuits: inherent viral cytotoxicity, high viral immunogenicity and virus-induced alteration of cellular transcriptional regulation. These factors alter the transcriptional and translational profiles of infected neurons and their neighboring cells. To overcome these limitations we applied a self-inactivating genomic modification to the less immunogenic rabies strain, CVS-N2c, to generate a self-inactivating CVS-N2c rabies virus (SiR-N2c). SiR-N2c not only eliminates undesired cytotoxic effects but also substantially reduces gene expression alterations in infected neurons and dampens the recruitment of innate and acquired immune responses, thus enabling open-ended interventions on neural networks and their genetic characterization using single-cell genomic approaches.

Proximity interactions of the ubiquitin ligase Mind bomb 1 reveal a role in regulation of epithelial polarity complex proteins.

MIB1 belongs to the RING domain containing family of E3 ubiquitin ligases. In vertebrates, MIB1 plays an essential role in activation of Notch signaling during development, through the ubiquitination and endocytosis of Notch ligands. More recently, Notch independent functions for MIB1 have been described in centriole homeostasis, dendritic spine outgrowth and directional cell migration. Here we use proximity-dependent biotin identification (BioID) to define the MIB1 interactome that included 163 high confidence interactions with polypeptides linked to centrosomes and cilia, endosomal trafficking, RNA and DNA processing, the ubiquitin system, and cell adhesion. Biochemical analysis identified several proteins within these groups including CCDC14 and EPS15 that were ubiquitinated but not degraded when co-expressed with MIB1. The MIB1 interactome included the epithelial cell polarity protein, EPB41L5. MIB1 binds to and ubiquitinates EPB41L5 resulting in its degradation. Furthermore, MIB1 ubiquitinates the EPB41L5-associated polarity protein CRB1, an important determinant of the apical membrane. In polarized cells, MIB1 localized to the lateral membrane with EPB41L5 and to the tight junction with CRB1, CRB3 and ZO1. Furthermore, over expression of MIB1 resulted in altered epithelial cell morphology and apical membrane expansion. These results support a role for MIB1 in regulation of polarized epithelial cell morphology.

Life-Long Genetic and Functional Access to Neural Circuits Using Self-Inactivating Rabies Virus.

Neural networks are emerging as the fundamental computational unit of the brain and it is becoming progressively clearer that network dysfunction is at the core of a number of psychiatric and neurodegenerative disorders. Yet, our ability to target specific networks for functional or genetic manipulations remains limited. Monosynaptically restricted rabies virus facilitates the anatomical investigation of neural circuits. However, the inherent cytotoxicity of the rabies largely prevents its implementation in long-term functional studies and the genetic manipulation of neural networks. To overcome this limitation, we developed a self-inactivating ΔG-rabies virus (SiR) that transcriptionally disappears from the infected neurons while leaving permanent genetic access to the traced network. SiR provides a virtually unlimited temporal window for the study of network dynamics and for the genetic and functional manipulation of neural circuits in vivo without adverse effects on neuronal physiology and circuit function.

Oral and maxillofacial manifestations of Gardner's syndrome associated with growth hormone deficiency: case report and literature review.

Gardner's syndrome (GS) is a hereditary disorder inherited as autosomal dominant with complete penetrance and variable expression. GS is a variant of familial adenomatous polyposis characterized by extracolonic manifestations including osteomas, dental anomalies, and epidermoid cysts. The association between GS and endocrine abnormalities has been well documented but a direct pituitary involvement has never been reported. We present a case of oral and maxillofacial manifestations in an adult patient affected by GS associated with growth hormone deficiency, a hitherto unreported association. The possible pathogenic mechanisms are discussed.

Odontogenic keratocyst and uterus bicornis in nevoid basal cell carcinoma syndrome: case report and literature review.

Nevoid basal cell carcinoma syndrome (NBCCS), an autosomal dominant disorder with a high degree of penetrance and variable expressivity, is characterized by basal cell carcinomas, odontogenic keratocysts, palmar and/or plantar pits, and ectopic calcifications of the falx cerebri. More than 100 minor criteria have been described, but 2 major and 1 minor criteria or 1 major and 3 minor criteria are necessary for the diagnosis. In this report we present an 8-year-old girl affected by NBCCS showing a uterus bicornis, a hitherto unreported association. However, further research is needed to confirm the association between NBCCS and mullerian fusion defects and to assess the hypothesis that focuses on chromosome 9 the mutant gene for NBCCS and fusion defects of female genital tract.

Profilometric and standard error of the mean analysis of rough implant surfaces treated with different instrumentations.

PURPOSE: This study evaluated, in vitro, the effects of different instrumentations used in the treatment of peri-implantitis on implant surfaces coated with hydroxyapatite or titanium plasma spray (TPS). MATERIALS AND METHODS: There were 14 cylindrical rough implants used, including 7 hydroxyapatite and 7 TPS coated. Split in 2 parts for a total of 24 experimental surfaces, implants were treated with a stainless-steel curette, plastic curette, ultrasonic scaler tip, and air-powder-water spray. There was 1 hydroxyapatite and 1 TPS implant used as controls. Profilometry and scanning electron microscopy were used to examine instrumented surfaces for variations in surface topography. RESULTS: All experimental procedures determined changes on tested rough implant surfaces. Such alterations were related to the implant coating material, and the procedure consisting in coating removal and/or leveling of surface roughness. CONCLUSION: Although a plastic curette and air-powder-water spray induced less implant surface alterations, these instrumentations left deposits on the surface that may affect, in vivo, the tissue healing process.