Analysis of ionizing radiation‐induced DNA damage and repair in three‐dimensional human skin model system

Please cite this paper as: Analysis of ionizing radiation‐induced DNA damage and repair in three‐dimensional human skin model system. Experimental Dermatology 2010; 19 : e16–e22. Abstract: Knowledge of cellular responses in tissue microenvironment is crucial for the accurate prediction of human health risks following chronic or acute exposure to ionizing radiation (IR). With this objective, we investigated the radio responses for the first time in three‐dimensional (3D) artificial human skin tissue microenvironment after γ‐rays radiation. IR‐induced DNA damage/repair response was assessed by immunological analysis of well‐known DNA double strand break (DSB) repair proteins, i.e. 53BP1 and phosphorylated ataxia telangiectasia mutated^ser1981 (ATM^ser1981). Efficient 53BP1 and phosphorylated ATM foci formation was observed in human EpiDerm tissue constructs after low and high doses of γ‐rays. Interestingly, EpiDerm tissue constructs displayed less 53BP1 and ATM foci number at all radiation doses (0.1, 1, 2.5 and 5 Gy) than that observed for 2D human fibroblasts. DSB repair efficiency judged by the disappearance of 53BP1 foci declined with increasing doses of γ‐rays and tissue constructs irradiated with 2.5 and 5 Gy of γ‐rays displayed 53BP1 foci persisting up to 72 h of analysis. Pretreatment of EpiDerm tissue constructs with LY294002, [an inhibitor of phosphatidylinositol‐3 kinase and PI‐3 kinase like kinases (PIKK)] completely abolished IR‐induced 53BP1 foci formation and increased the apoptotic death. This observation indicates the importance of PIKK signalling pathway for efficient radiation responses in intact tissue constructs. In summary, we have successfully demonstrated the feasibility of monitoring the DNA damage response in human skin tissue microenvironment. In this system, 53BP1 can be used as a useful marker for monitoring the DSB repair efficiency.     

Median cleft lip: A new method of surgical repair

The aim is to discuss a new method of muscle repair in midline cleft lip. Three patients with midline cleft lip were repaired with our technique of muscle repair and the results evaluated. Our new method of muscle repair in the form of ''Z'' helps in forming the philtral dimple.     

A possible mechanism for exonuclease 1-independent eukaryotic mismatch repair

Mismatch repair contributes to genetic stability, and inactivation of the mammalian pathway leads to tumor development. Mismatch correction occurs by an excision-repair mechanism and has been shown to depend on the 5′ to 3′ hydrolytic activity exonuclease 1 (Exo1) in eukaryotic cells. However, genetic and biochemical studies have indicated that one or more Exo1-independent modes of mismatch repair also exist. We have analyzed repair of nicked circular heteroduplex DNA in extracts of Exo1-deficient mouse embryo fibroblast cells. Exo1-independent repair under these conditions is MutLα-dependent and requires functional integrity of the MutLα endonuclease metal-binding motif. In contrast to the Exo1-dependent reaction, we have been unable to detect a gapped excision intermediate in Exo1-deficient extracts when repair DNA synthesis is blocked. A possible explanation for this finding has been provided by analysis of a purified system comprised of MutSα, MutLα, replication factor C, proliferating cell nuclear antigen, replication protein A, and DNA polymerase δ that supports Exo1-independent repair in vitro. Repair in this system depends on MutLα incision of the nicked heteroduplex strand and dNTP-dependent synthesis-driven displacement of a DNA segment spanning the mismatch. Such a mechanism may account, at least in part, for the Exo1-independent repair that occurs in eukaryotic cells, and hence the modest cancer predisposition of Exo1-deficient mammalian cells.     

Using human hair follicle‐derived keratinocytes and melanocytes for constructing pigmented tissue‐engineered skin

Background: Traditional tissue‐engineered skin does not produce a satisfactory long‐term result because it lacks natural skin pigmentation and leads to discolored cosmetically unpleasing skin that only functions to cover the body of patients. Additionally, the cell sources for tissue‐engineered skin are generally derived from normal skin, which is often limited in patients with skin defects. Methods: In this study, hair follicle melanocytes and keratinocytes were isolated from human scalp. The melanocytes were co‐cultured with keratinocytes until the second passage and then purified. Purified melanocytes and keratinocytes were seeded onto the chitosan–gelatin membrane for 1 week to construct pigmented tissue‐engineered skin. The pigmented skin equivalent was used to resurface the skin defect in nude mice. Four weeks after grafting, skin biopsies were harvested to take hematoxylin and eosin staining and immunohistochemistry staining of Melan‐A and HLA‐ABC. Results: Large quantities of purified melanocytes can be obtained with co‐culture method. The hematoxylin and eosin staining of repaired skin biopsy demonstrated that the tissue‐engineered skin can repair skin defects successfully. Engineered skin contained pigmentation and stained positive for Melan‐A and HLA‐ABC, which confirmed the presence of melanocytes and its sources were of human origin. Conclusion: This study demonstrated the possibility of constructing pigmented tissue‐engineered skin with human hair follicle‐derived keratinocytes and melanocytes, which brings a promising method to make up for the deficiency of traditional tissue‐engineered skin and provides an alternative treatment for depigmentation diseases.     

Auditory stream segregation in children with Asperger syndrome

Individuals with Asperger syndrome (AS) often have difficulties in perceiving speech in noisy environments. The present study investigated whether this might be explained by deficient auditory stream segregation ability, that is, by a more basic difficulty in separating simultaneous sound sources from each other. To this end, auditory event-related brain potentials were recorded from a group of school-aged children with AS and a group of age-matched controls using a paradigm specifically developed for studying stream segregation. Differences in the amplitudes of ERP components were found between groups only in the stream segregation conditions and not for simple feature discrimination. The results indicated that children with AS have difficulties in segregating concurrent sound streams, which ultimately may contribute to the difficulties in speech-in-noise perception.     

The Loss of Pleasure,or Why We Are Still Talking about Oedipus

This article brings psychoanalysis to the fore in grappling with the question: Why does patriarchy persist? It highlights the psychological function of patriarchy as a defense against loss by connecting Gilligan's research on development with Bowlby's studies of attachment. Pathological responses to loss parallel the gender codes of patriarchal masculinity and femininity, which are internalized through an initiation that forces ruptures in relationship and subverts the capacity for repair. This parallel suggests that the gender roles, which uphold a patriarchal order, simultaneously defend against the loss of connection inherent in that order. The loss of pleasure and a change in voice signal the psyche's induction into patriarchy and highlight a potential within psychoanalysis to foster a healthy resistance against losses that otherwise appear necessary or natural.     

Dynamics of the bacterial SMC complex and SMC-like proteins involved in DNA repair

Bacteria and archaea possess several different SMC-like proteins, which perform essential functions in a variety of chromosome dynamics, such as chromosome compaction, segregation, and DNA repair. SMC-like proteins localize to distinct sites within the cells at different time points in the cell cycle, or are recruited to sites of DNA breaks and damage. The bacterial SMC (MukB) complex appears to perform a condensin-like function, while SbcC and RecN act early during DNA repair, but apparently at different sites within the cells. Thus, bacterial SMC-like proteins have dynamic functions in chromosome segregation and maintenance of genetic stability.     

Strength of Denture Base Resins Repaired with Auto- and Visible Light-Polymerized Materials

Purpose: Clinicians are still confused about the choice of repair method, which depends on factors such as the length of time required for processing, the mechanical strength of the repaired material, and the effect of stress concentration in the acrylic resins before the repair. The aim was to determine the impact and flexural strength characteristics, such as stress at yield, Young's modulus, and displacement at yield of denture base resins fractured and repaired by three methods using heat‐, auto‐, and visible light‐polymerized acrylic resins. Material and Methods: For impact and flexural strength tests, 18 rectangular specimens measuring 50 × 6 × 4 mm³ and 64 × 10 × 3.3 mm³, respectively, were processed using Impact 2000, Lucitone 550, Impact 1500, and QC‐20 acrylic resins. Fracture tests were performed according to ISO1567:1999. Afterward, all fractured specimens were stored in distilled water at 37°C for 7 days, and then repaired with (1) the same acrylic resin used for specimen fabrication (n = 6), (2) an autopolymerized acrylic resin (TruRepair, n = 6), and (3) a visible light acrylic resin (Versyo.com, n = 6). The repaired specimens were again submitted to the same fracture tests, and the failures were classified as adhesive or cohesive. Data from all mechanical tests after repair by the different methods were submitted to two‐way ANOVA, and mean values were compared by the Tukey test. Results: All acrylic resins showed adhesive fractures after impact and flexural strength tests. Differences (p < 0.05) were found among repair methods for all acrylic resins studied, with the exception of displacement at yield, which showed similar values for repairs with auto‐ and visible light‐polymerized acrylic resins. The highest values for impact strength, stress, and displacement at yield were obtained when the repair was made with the same resin the specimen was made of. Conclusion: Denture base acrylic resins repaired with the same resin they were made of showed greater fracture strength.