In-vivo repeatability of three intra-oral spectrophotometers.

OBJECTIVE: The objective was to assess the repeatability of three spectrophotometers, based on the CIELCh factors and shadeguide reference measurements. MATERIALS AND METHODS: Color analysis was performed using three devices: Rayplicker, Easyshade 4, and Easyshade V. Five repeated measures were performed by the same operator, on the right central maxillary incisor of 30 patients. The CIELCh factors were retrieved and the intra-class correlation was calculated. The Vita Classical and Vita 3D Master shadeguides were used to evaluate the respective Fleiss' Kappa factors. RESULTS: Rayplicker and Easyshade V demonstrated strong intra-class correlation based on the CIELCh factors: 0.98, 0.99, and 0.91 for Rayplicker, and 0.95, 0.99, and 0.93 for Easyshade V, for the L*, C*, and h* parameters, respectively. Regarding the repeatability of the shadeguide data, while Easyshade 4 had the best repeatability when using the Vita Classical as a reference, Rayplicker and Easyshade V showed better repeatability when using the Vita 3D Master. CONCLUSION: These findings suggest that both Rayplicker and Easyshade V are reliable devices for measuring CIELCh parameters. In terms of shadeguide references, the reliability of spectrophotometers is generally lower compared to the CIELCh measurements. CLINICAL SIGNIFICANCE: Given their good repeatability, both the Rayplicker and the Easyshade V spectrophotometers are reliable tools for prosthetic dental practices.

Intraoral optical impression versus conventional impression for fully edentulous maxilla: an in vivo comparative study.

AIM: The aim of the present in vivo study was to compare the clinical trueness of primary mucostatic impressions obtained either by a classical alginate or an optical intraoral scanner technique in patients with a fully edentulous maxilla. MATERIALS AND METHODS: A total of 30 patients with a fully edentulous maxilla were included in the study and underwent both conventional impressions and intraoral optical impressions (Trios 3). The conventional impressions were casted and the resulting plaster casts were digitized using a desktop scanner (Imetric D104i). These digitized impressions were superimposed over the optical impressions to compare the differences between the two data sets. Statistical analyses were performed to identify relevant deviations. RESULTS: For the 30 intraoral impressions, 80.88% of the surface areas were below the tolerance threshold of 25 µm and were thus considered similar to the areas scanned with the desktop scanner from the reference plaster cast. Interestingly, the differences (19.12% of the surface areas) were localized in depressible areas such as the vestibule, soft palate, incisive papilla, and flabby ridges. These locations were consistent with the mean of positive differences of +22.8 µm, indicating deformation or less compression with the use of the intraoral scanner. CONCLUSIONS: The digital primary impression of the fully edentulous maxilla can be considered similar to the conventional alginate impression except in the depressible areas. Considering the mucostatic objective of such a primary impression, one may consider the optical impression to be more accurate than the conventional one.

XLF/Cernunnos loss impairs mouse brain development by altering symmetric proliferative divisions of neural progenitors.

XLF/Cernunnos is a component of the ligation complex used in classical non-homologous end-joining (cNHEJ), a major DNA double-strand break (DSB) repair pathway. We report neurodevelopmental delays and significant behavioral alterations associated with microcephaly in Xlf-/- mice. This phenotype, reminiscent of clinical and neuropathologic features in humans deficient in cNHEJ, is associated with a low level of apoptosis of neural cells and premature neurogenesis, which consists of an early shift of neural progenitors from proliferative to neurogenic divisions during brain development. We show that premature neurogenesis is related to an increase in chromatid breaks affecting mitotic spindle orientation, highlighting a direct link between asymmetric chromosome segregation and asymmetric neurogenic divisions. This study reveals thus that XLF is required for maintaining symmetric proliferative divisions of neural progenitors during brain development and shows that premature neurogenesis may play a major role in neurodevelopmental pathologies caused by NHEJ deficiency and/or genotoxic stress.

Operator versus material influence on film thickness using adhesive resin cement or pre-heated resin composite.

OBJECTIVE: There is a growing interest in using pre-heated composites instead of dual-cured cements when luting indirect restorations. This study evaluated the film thickness obtained from two pre-heated composites and two resin cements, by two different operators. The influence of the materials and the level of expertise of the operator were analyzed. MATERIALS AND METHODS: Forty specimens of human dentin and composite discs were prepared and divided into four groups depending on the luting process. Each group was randomly equally divided to be handled by two operators with different levels of experience. Two of the initial four groups were luted using dual-cured cements and the two remaining groups using light-cured pre-heated composites. Specimen discs were cut after luting, and film thickness was measured using a Digital microscope. Data were analyzed using a 2-way ANOVA with the Holm-Sidak pairwise multiple comparison procedure (p < 0.05). RESULTS: Mean film thickness ranged from 156.16 ± 4.7 to 33.82 ± 0.7 µm. Significant differences (p < 0.001) were noticed between expert and novice results with pre-heated composites. CONCLUSION: Within the limits of this study, using pre-heated composites as a luting cement requires a better level of expertise to achieve a clinically acceptable film thickness. CLINICAL SIGNIFICANCE: Using pre-heated composites as luting agent for indirect restorations requires an experimented skill level to achieve a clinically recommended film thickness.

Digital evaluation of dental bleaching using a new methodology: an in vivo study.

OBJECTIVE: The objective of the present study was to assess the progress and efficiency of at-home bleaching protocols with 10% carbamide peroxide using a new methodology based on dental photography. MATERIALS AND METHODS: A 4-week overnight at-home bleaching protocol using whitening trays and 10% carbamide peroxide was performed on 52 patients. The tooth color was analyzed using standardized photographs taken every week for 4 weeks and at 4 months posttreatment. The values of the color evolution (ΔE00), L*, a*, and b* were also measured and used to assess the evolution of the chroma, luminosity, and hue using the CIEDE2000 formula. The statistical analyses were conducted at a significance level of P < 0.05 by means of a repeated measures analysis of variance (ANOVA). RESULTS: The tooth color changed the most, and thus the highest ΔE00 was observed, after the first week of treatment. The color continued to change but to a lesser degree during the following weeks. After 4 weeks, the treatment proved to be very effective. Four months after the end of treatment, a color relapse was observed, though it was hardly perceptible to the human eye. The luminosity (L') changed significantly between the beginning and the end of treatment, affecting the maxilla to a greater extent. The chroma evolution showed a high variance and a low relapse for both jaws. The hue was not affected significantly during the treatment and the stabilization. CONCLUSIONS: Within the limitations of the present study, the authors were able to assess the progress and efficiency of at-home bleaching with 10% carbamide peroxide in terms of chroma, luminosity, and hue using a new methodology based on dental photography. CLINICAL SIGNIFICANCE: This new method is effective and enables a reliable analysis of the evolution of a dental bleaching treatment, turning dental photo-graphy into an even more powerful tool for analysis and communication. It can also be used as a proof-of-concept, paving the way for further research on objective monitoring and evaluation of dental treatments using dental photography.

Treatment of Tooth Wear Using Direct or Indirect Restorations: A Systematic Review of Clinical Studies.

Tooth wear is considered a well-developed issue in daily clinical practice; however, there is no standard protocol for treatment. The aim of this manuscript was to systematically review the literature to evaluate the clinical outcomes of direct or indirect restorations for treating tooth wear. A literature search was conducted through the PubMed MedLine, Scopus, ISI Web of Science, Scielo, and EMBASE databases up to 29 April 2022. Clinical studies evaluating the clinical performance of direct or indirect restorations for treating tooth wear for a minimum follow-up of 6 months were included in the review. A total of 2776 records were obtained from the search databases. After full-text reading, 16 studies were included in the qualitative analysis. Considering the high heterogenicity of the studies included, a meta-analysis could not be performed. All studies included the rehabilitation of anterior and posterior teeth with extensive wear, using both indirect and direct restorations for a maximum follow-up of 10 years. Restoration materials included ceramo-metal crowns, full gold crowns, lithium disilicate ceramic, zirconia, polymer infiltrated ceramic networks, and resin composites. Most of the reports assessed the survival rate of the restorations and the clinical features using the United States Public Health Service (USPHS) Evaluation System criteria. Contradictory discoveries were perceived concerning the type of restoration with better clinical performance. Considering the current literature available, there is no evidence in the superiority of any restoration technique to ensure the highest clinical performance for treating tooth wear.

An XRCC4 mutant mouse, a model for human X4 syndrome, reveals interplays with Xlf, PAXX, and ATM in lymphoid development.

We developed an Xrcc4M61R separation of function mouse line to overcome the embryonic lethality of Xrcc4-deficient mice. XRCC4M61R protein does not interact with Xlf, thus obliterating XRCC4-Xlf filament formation while preserving the ability to stabilize DNA ligase IV. X4M61R mice, which are DNA repair deficient, phenocopy the Nhej1-/- (known as Xlf -/-) setting with a minor impact on the development of the adaptive immune system. The core non-homologous end-joining (NHEJ) DNA repair factor XRCC4 is therefore not mandatory for V(D)J recombination aside from its role in stabilizing DNA ligase IV. In contrast, Xrcc4M61R mice crossed on Paxx-/-, Nhej1-/-, or Atm-/- backgrounds are severely immunocompromised, owing to aborted V(D)J recombination as in Xlf-Paxx and Xlf-Atm double Knock Out (DKO) settings. Furthermore, massive apoptosis of post-mitotic neurons causes embryonic lethality of Xrcc4M61R -Nhej1-/- double mutants. These in vivo results reveal new functional interplays between XRCC4 and PAXX, ATM and Xlf in mouse development and provide new insights into the understanding of the clinical manifestations of human XRCC4-deficient condition, in particular its absence of immune deficiency.

Esthetic mock-ups: a clinical study of reproducibility.

The mock-up technique is a widely used clinical method to achieve successful esthetic and functional treatments. Several methods have been described to fabricate the mock-up, either directly in the mouth or indirectly using a silicone index or CAD/CAM options. The mock-up mainly serves as a guide during the preparation stage, but it is also a useful communication and validation tool. Based on the controlled depth preparation concept, the mock-up ensures a specific restoration thickness sufficient for the ceramic and respectful of the enamel tissue. Any error in fabricating the mock-up may cause debonding complications and have a negative impact on the esthetic result. Currently, most mock-ups are created indirectly using a silicone index injected with a temporary resin that mimics the final esthetic project. The present article reports on the protocol involved in a clinical study conducted to validate the precision of reproducibility of this indirect method. Two different options for creating the silicone index were investigated. Twenty mock-ups were realized on the same subject by the same operator. Each mock-up was 3D scanned to compare its reproducibility using the superimposition of 3D meshes. The results show that the reproducibility of indirect mock-ups is clinically acceptable (~ 100 µm), regardless of the fabrication method used. However, a combination of standard silicone and a rigid tray produces better results than hard silicone alone.

Dentine disorders and adhesive treatments: A systematic review.

OBJECTIVES: A better understanding of the microstructure and mechanical properties of enamel and dentine may enable practitioners to apply the current adhesive dentistry protocols to clinical cases involving dentine disorders (dentinogenesis imperfecta or dentine dysplasia). DATA/SOURCES: Publications (up to June 2020) investigating the microstructure of dentine disorders were browsed in a systematic search using the PubMed/Medline, Embase and Cochrane Library electronic databases. Two authors independently selected the studies, extracted the data in accordance with the PRISMA statement, and assessed the risk of bias with the Critical Appraisal Checklist. A Mann-Whitney U test was computed to compare tissues damage related to the two dentine disorders of interest. STUDY SELECTION: From an initial total of 642 studies, only 37 (n = 164 teeth) were included in the present analysis, among which 18 investigating enamel (n = 70 teeth), 15 the dentine-enamel junction (n = 62 teeth), and 35 dentine (n = 156 teeth). Dentine is damaged in cases of dentinogenesis imperfecta and osteogenesis imperfecta (p = 2.55E-21 and p = 3.99E-21, respectively). These studies highlight a reduction in mineral density, hardness, modulus of elasticity and abnormal microstructure in dentine disorders. The majority of studies report an altered dentine-enamel junction in dentinogenesis imperfecta and in osteogenesis imperfecta (p = 6.26E-09 and p = 0.001, respectively). Interestingly, enamel is also affected in cases of dentinogenesis imperfecta (p = 0.0013), unlike to osteogenesis imperfecta (p = 0.056). CONCLUSIONS: Taking into account all these observations, only a few clinical principles may be favoured in the case of adhesive cementation: (i) to preserve the residual enamel to enhance bonding, (ii) to sandblast the tooth surfaces to increase roughness, (iii) to choose a universal adhesive and reinforce enamel and dentine by means of infiltrant resins. As these recommendations are mostly based on in vitro studies, future in vivo studies should be conducted to confirm these hypotheses.

Microguided endodontics: Accuracy evaluation for access through intraroot fibre-post.

In this work, we intended to assess the reliability of guided endodontic technique to remove a bonded fibre-post when there are artefacts in the cone-beam computed tomography (CBCT) images caused by composite dental materials. We mounted natural posterior teeth on ten simulated models. Forty fibre-post and composite-core restorations were inserted in the teeth. We merged a pre-operative CBCT and optical surface scan on the BlueskyplanTM software to digitally design and subsequently 3D-printed the guides. Two operators initiated endodontic access into the fibre-post restorations using the template to guide the drill. Post-operative CBCT was taken and merged onto the pre-operative plan to measure the deviations at the coronal and apical segments. The mean deviation between the planned and actual drill paths were, respectively, of 0.39 ± 0.14 mm coronally and 0.40 ± 0.19 mm apically. Microguided endodontics is a predictable and accurate method to remove fibre-post restorations efficiently.

In vitro comparison of the surface roughness of polymethyl methacrylate and bis-acrylic resins for interim restorations before and after polishing.

STATEMENT OF PROBLEM: Polymethyl methacrylate and bis-acrylic based resins are widely used for interim restorations. Their initial surface roughness is important because it determines their aesthetic properties and the potential for biofilm adhesion. PURPOSE: The purpose of this in vitro study was to assess the surface roughness and morphology of 6 bis-acrylic and 2 polymethyl methacrylate resins widely used for interim dental restorations, both before and after polishing. MATERIAL AND METHODS: Specimens made of different bis-acrylic resins (Protemp 4, Luxatemp Star, Systemp, Telio, Structur Premium, Structur 3) or of polymethyl methacrylate (Unifast Trad, Unifast 3) were polished using a 2-step polishing system (Diatech). The average surface roughness before and after polishing (10 seconds at low speed in dry conditions) was measured by optical profilometry. Atomic force microscopy and scanning electron microscopy were used to analyze surface morphology. The Mann-Whitney and Kruskal-Wallis tests were used to evaluate the differences in roughness among specimens (α=.05), and the Pearson r correlation was computed to assess the relationship between fillers and average surface roughness. RESULTS: In the 8 groups evaluated, the roughness significantly increased (P<.001) for Protemp 4 (from 0.12 to 0.50 µm), Luxatemp Star (0.17 to 1.19 µm), Unifast 3 (0.40 to 1.00 µm), Systemp (0.46 to 1.51 µm), Structur 3 (0.85 to 1.06 µm), Structur Premium (1.00 to 1.74 µm), and Telio (1.13 to 1.21 µm), except for Unifast Trad (9.20 to 2.59 µm). Pairwise multiple comparisons identified Protemp 4 as having the smoothest surface before and after polishing, while Unifast Trad was the roughest in both. Atomic force microscopy and scanning electron microscopy observations showed that the surface roughness of bis-acrylic resins was related to their surface morphology and average filler sizes. A positive relation between fillers and roughness was assessed (r=0.345, P<.001). CONCLUSIONS: For the bis-acrylic interim resins, the surface roughness after polishing was correlated to the material used and its filler sizes. Nanofiller-based resins showed the smoothest surfaces. For the polymethyl methacrylate-based resins, the recently marketed Unifast 3 had the lowest overall roughness values.

Higher chromosome stability in embryonic neural stem and progenitor cells than in fibroblasts in response to acute or chronic genotoxic stress.

High fidelity of genetic transmission in neural stem and progenitor cells (NSPCs) has been long time considered to be crucial for brain development and homeostasis. However, recent studies have identified recurrent DSB clusters in dividing NSPCs, which may underlie the diversity of neuronal cell types. This raised the interest in understanding how NSPCs sense and repair DSBs and how this mechanism could be altered by environmental genotoxic stress caused by pollutants or ionizing radiation. Here, we show that embryonic mouse neural stem and progenitor cells (NSPCs) have significantly higher capacity than mouse embryonic fibroblasts (MEFs) to maintain their chromosome stability in response to acute (γ-radiation) and chronic (tritiated thymidine -3H-T- incorporation into DNA) genotoxic stress. Cells deficient for XLF/Cernunnos, which is involved in non-homologous end joining DNA (NHEJ) repair, highlighted important variations in fidelity of DNA repair pathways between the two cell types. Strikingly, a progressive and generalized chromosome instability was observed in MEFs cultured with 3H-T at long-term, whereas NSPCs cultured in the same conditions, preserved their chromosome stability thanks to higher DNA repair activity further enhanced by an adaptive response and also to the elimination of damaged cells by apoptosis. This specific DNA damage response of NSPCs may rely on the necessity for preservation of their genome stability together with their possible function in creating neuronal genetic diversity.

A comparative in vivo study of new shade matching procedures.

AIM: The aim of the present study was to compare six different methods of in vivo color matching: visual shade matching (3D-Master Linearguide shade guide) performed by 1) a novice practitioner, 2) an expert practitioner, 3) the new Rayplicker spectrometer, 4) the Trios III intraoral scanner, and 5) the Omnicam intraoral scanner compared with 6) the Easyshade V spectrophotometer, which was considered as the reference. MATERIALS AND METHODS: Color matching was performed using the 3D-Master references on the sound maxillary right central incisors of 40 subjects. The study first compared the number of colors found using each of the six methods. The references were then converted to the Commission Internationale de l'Eclairage (CIE) L*a*b* values, from which the difference ?E between either two methods ?was derived. Finally, the L* value was used to compare the luminosity measured by each of the six methods. RESULTS: The Rayplicker showed the smallest ?E compared with the Easyshade V. The expert found a closer color to the Easyshade V than did the novice, and both were closer to the Easyshade V than the two intraoral scanners. The intraoral scanners showed notable differences compared with the Easyshade V. The intraoral scanners also offered a reduced choice of colors and recorded the highest luminosities compared with the other methods. CONCLUSION: Within the limitations of this study, the color matching by the Rayplicker was closest to that of the Easyshade V. The good performance of this new device means that it is a challenging competitor for the Easyshade V. Finally, the new methods based on intraoral scanners were less reliable than the spectrophotometers and the visual shade matching.

Adult T-type lymphoblastic lymphoma presenting as hypercalcemic crisis and aplastic anemia: a case report.

BACKGROUND: Hypercalcemia and aplastic anemia are two uncommon presentations of non-Hodgkin lymphoma that potentially worsen the disease prognosis. Although hypercalcemia has been reported in the B-cell subtypes and some T-cell subtypes of non-Hodgkin lymphoma, it has not been described in T-cell lymphoblastic lymphoma. The same applies to aplastic anemia, which is also not described in T-type lymphomas. CASE PRESENTATION: We report a case of a 52-year-old Cameroonian man with acute kidney injury who presented with confusion, abdominal pain, constipation, polyuria, polydipsia, calciphylaxis, enlarged lymph nodes, tachycardia, and a blood pressure of 170/88 mmHg. Laboratory investigations revealed hypercalcemia (total/ionized 199.5/101.75 mg/L), normal serum phosphorus (40.20 mg/L), and a low intact parathyroid hormone (9.70 pg/ml). Complete blood count revealed pancytopenia. Peripheral blood smear confirmed thrombocytopenia but showed neither blasts nor flower cells. Bone marrow aspirate revealed hypocellularity with no blasts or fibrosis. Lymph node biopsy was suggestive of T-cell precursor lymphoma. T-lymphoblastic lymphoma presenting with hypercalcemic crisis and aplastic anemia was diagnosed, and the patient received the cyclophosphamide-doxorubicin-vincristine-prednisone protocol of chemotherapy together with filgrastim and whole-blood transfusion for aplastic anemia. The short-term outcome was fatal, however. CONCLUSIONS: Severe hypercalcemia and aplastic anemia are potential paraneoplastic syndromes of adult T-type lymphoblastic lymphoma, with fatal short-term outcome.

Longitudinal Study of Irradiation-Induced Brain Microstructural Alterations With S-Index, a Diffusion MRI Biomarker, and MR Spectroscopy.

PURPOSE: Radiation therapy is widely used for the treatment of brain tumors, but it may lead to severe cognitive impairments. Previous studies have shown that ionizing irradiation induces demyelination, blood-brain barrier alterations, and impaired neurogenesis in animal models. Hence, noninvasive and sensitive biomarkers of irradiation injury are needed to investigate these effects in patients and improve radiation therapy protocols. METHODS AND MATERIALS: The heads of 3-month-old male C57BL/6RJ mice (15 control mice and 15 irradiated mice) were exposed to radiation doses of 3 fractions of 5 Gy from a 60Co source with a medical irradiator. A longitudinal study was performed to investigate cranial irradiation-induced (3 fractions of 5 Gy) microstructural tissue alterations using water diffusion magnetic resonance imaging and magnetic resonance spectroscopy in different areas of the mouse brain (cortex, thalamus, striatum, olfactory bulbs [OBs], hippocampus, and subventricular zone [SVZ]). In addition to the quantification of standard non-Gaussian diffusion parameters, apparent diffusion coefficient (ADC0) and kurtosis (K), we evaluated a new composite diffusion metric, designated the S-index (ie, "signature index"). RESULTS: We observed a significant decrease in the S-index in the SVZ from 1 month to 8 months after brain irradiation (P < .05). An interesting finding was that, along with a decrease in taurine levels (up to -15% at 2 months, P < .01), a delayed S-index drop was observed in the OBs from 4 months after irradiation and maintained until the end of our experiment (P < .0001). These observations suggest that S-index variations revealed the irradiation-induced decline of neurogenesis that was further confirmed by a decrease in neural stem cells in the SVZ and in newborn neurons in the OBs of irradiated animals. CONCLUSIONS: This study demonstrates that diffusion magnetic resonance imaging, especially through the S-index approach, is a relevant imaging modality to monitor brain irradiation injury and probe microstructural changes underlying irradiation-induced cognitive deficits.

PAXX and Xlf interplay revealed by impaired CNS development and immunodeficiency of double KO mice.

The repair of DNA double-stranded breaks (DNAdsb) through non-homologous end joining (NHEJ) is a prerequisite for the proper development of the central nervous system and the adaptive immune system. Yet, mice with Xlf or PAXX loss of function are viable and present with very mild immune phenotypes, although their lymphoid cells are sensitive to ionizing radiation attesting for the role of these factors in NHEJ. In contrast, we show here that mice defective for both Xlf and PAXX are embryonically lethal owing to a massive apoptosis of post-mitotic neurons, a situation reminiscent to XRCC4 or DNA Ligase IV KO conditions. The development of the adaptive immune system in Xlf-/-PAXX-/- E18.5 embryos is severely affected with the block of B- and T-cell maturation at the stage of IgH and TCRß gene rearrangements, respectively. This damaging phenotype highlights the functional nexus between Xlf and PAXX, which is critical for the completion of NHEJ-dependent mechanisms during mouse development.

Antibacterial Peptide-Based Gel for Prevention of Medical Implanted-Device Infection.

Implanted medical devices are prone to infection. Designing new strategies to reduce infection and implant rejection are an important challenge for modern medicine. To this end, in the last few years many hydrogels have been designed as matrices for antimicrobial molecules destined to fight frequent infection found in moist environments like the oral cavity. In this study, two types of original hydrogels containing the antimicrobial peptide Cateslytin have been designed. The first hydrogel is based on alginate modified with catechol moieties (AC gel). The choice of these catechol functional groups which derive from mussel's catechol originates from their strong adhesion properties on various surfaces. The second type of gel we tested is a mixture of alginate catechol and thiol-terminated Pluronic (AC/PlubisSH), a polymer derived from Pluronic, a well-known biocompatible polymer. This PlubisSH polymer has been chosen for its capacity to enhance the cohesion of the composition. These two gels offer new clinical uses, as they can be injected and jellify in a few minutes. Moreover, we show these gels strongly adhere to implant surfaces and gingiva. Once gelled, they demonstrate a high level of rheological properties and stability. In particular, the dissipative energy of the (AC/PlubisSH) gel detachment reaches a high value on gingiva (10 J.m-2) and on titanium alloys (4 J.m-2), conferring a strong mechanical barrier. Moreover, the Cateslytin peptide in hydrogels exhibited potent antimicrobial activities against P. gingivalis, where a strong inhibition of bacterial metabolic activity and viability was observed, indicating reduced virulence. Gel biocompatibility tests indicate no signs of toxicity. In conclusion, these new hydrogels could be ideal candidates in the prevention and/or management of periimplant diseases.

Brain development is impaired in c-fos -/- mice.

c-Fos is a proto-oncogene involved in diverse cellular functions. Its deregulation has been associated to abnormal development and oncogenic progression. c-fos-/- mice are viable but present a reduction in their body weight and brain size. We examined the importance of c-Fos during neocortex development at 13.5, 14.5 and 16.5 days of gestation. At E14.5, neocortex thickness, apoptosis, mitosis and expression of markers along the different stages of Neural Stem Progenitor Cells (NSPCs) differentiation in c-fos-/- and wild-type mice were analyzed. A ~15% reduction in the neocortex thickness of c-fos-/- embryos was observed which correlates with a decrease in the number of differentiated cells and an increase in apoptosis at the ventricular zone. No difference in mitosis rate was observed, although the mitotic angle was predominantly vertical in c-fos-/- embryos, suggesting a reduced trend of NSPCs to differentiate. At E13.5, changes in differentiation markers start to be apparent and are still clearly observed at E16.5. A tendency of more AP-1/DNA complexes present in nuclear extracts of cerebral cortex from c-fos-/- embryos with no differences in the lipid synthesis activity was found. These results suggest that c-Fos is involved in the normal development of NSPCs by means of its AP-1 activity.

The prion protein is critical for DNA repair and cell survival after genotoxic stress.

The prion protein (PrP) is highly conserved and ubiquitously expressed, suggesting that it plays an important physiological function. However, despite decades of investigation, this role remains elusive. Here, by using animal and cellular models, we unveil a key role of PrP in the DNA damage response. Exposure of neurons to a genotoxic stress activates PRNP transcription leading to an increased amount of PrP in the nucleus where it interacts with APE1, the major mammalian endonuclease essential for base excision repair, and stimulates its activity. Preventing the induction of PRNP results in accumulation of abasic sites in DNA and impairs cell survival after genotoxic treatment. Brains from Prnp(-/-) mice display a reduced APE1 activity and a defect in the repair of induced DNA damage in vivo. Thus, PrP is required to maintain genomic stability in response to genotoxic stresses.

Assessing cell cycle progression of neural stem and progenitor cells in the mouse developing brain after genotoxic stress.

Neurons of the cerebral cortex are generated during brain development from different types of neural stem and progenitor cells (NSPC), which form a pseudostratified epithelium lining the lateral ventricles of the embryonic brain. Genotoxic stresses, such as ionizing radiation, have highly deleterious effects on the developing brain related to the high sensitivity of NSPC. Elucidation of the cellular and molecular mechanisms involved depends on the characterization of the DNA damage response of these particular types of cells, which requires an accurate method to determine NSPC progression through the cell cycle in the damaged tissue. Here is shown a method based on successive intraperitoneal injections of EdU and BrdU in pregnant mice and further detection of these two thymidine analogues in coronal sections of the embryonic brain. EdU and BrdU are both incorporated in DNA of replicating cells during S phase and are detected by two different techniques (azide or a specific antibody, respectively), which facilitate their simultaneous detection. EdU and BrdU staining are then determined for each NSPC nucleus in function of its distance from the ventricular margin in a standard region of the dorsal telencephalon. Thus this dual labeling technique allows distinguishing cells that progressed through the cell cycle from those that have activated a cell cycle checkpoint leading to cell cycle arrest in response to DNA damage. An example of experiment is presented, in which EdU was injected before irradiation and BrdU immediately after and analyzes performed within the 4 hr following irradiation. This protocol provides an accurate analysis of the acute DNA damage response of NSPC in function of the phase of the cell cycle at which they have been irradiated. This method is easily transposable to many other systems in order to determine the impact of a particular treatment on cell cycle progression in living tissues.