Introducing the NEMO-Lowlands iconic gesture dataset, collected through a gameful human-robot interaction.

This paper describes a novel dataset of iconic gestures, together with a publicly available robot-based elicitation method to record these gestures, which consists of playing a game of charades with a humanoid robot. The game was deployed at a science museum (NEMO) and a large popular music festival (Lowlands) in the Netherlands. This resulted in recordings of 428 participants, both adults and children, performing 3715 silent iconic gestures for 35 different objects in a naturalistic setting. Our dataset adds to existing collections of iconic gesture recordings in two important ways. First, participants were free to choose how they represented the broad concepts using gestures, and they were asked to perform a second attempt if the robot did not recognize their gesture the first time. This provides insight into potential repair strategies that might be used. Second, by making the interactive game available we enable other researchers to collect additional recordings, for different concepts, and in diverse cultures or contexts. This can be done in a consistent manner because a robot is used as a confederate in the elicitation procedure, which ensures that every data collection session plays out in the same way. The current dataset can be used for research into human gesturing behavior, and as input for the gesture recognition and production capabilities of robots and virtual agents.

Disruption of TTDA results in complete nucleotide excision repair deficiency and embryonic lethality.

The ten-subunit transcription factor IIH (TFIIH) plays a crucial role in transcription and nucleotide excision repair (NER). Inactivating mutations in the smallest 8-kDa TFB5/TTDA subunit cause the neurodevelopmental progeroid repair syndrome trichothiodystrophy A (TTD-A). Previous studies have shown that TTDA is the only TFIIH subunit that appears not to be essential for NER, transcription, or viability. We studied the consequences of TTDA inactivation by generating a Ttda knock-out (Ttda(-/-) ) mouse-model resembling TTD-A patients. Unexpectedly, Ttda(-/-) mice were embryonic lethal. However, in contrast to full disruption of all other TFIIH subunits, viability of Ttda(-/-) cells was not affected. Surprisingly, Ttda(-/-) cells were completely NER deficient, contrary to the incomplete NER deficiency of TTD-A patient-derived cells. We further showed that TTD-A patient mutations only partially inactivate TTDA function, explaining the relatively mild repair phenotype of TTD-A cells. Moreover, Ttda(-/-) cells were also highly sensitive to oxidizing agents. These findings reveal an essential role of TTDA for life, nucleotide excision repair, and oxidative DNA damage repair and identify Ttda(-/-) cells as a unique class of TFIIH mutants.

An Xpd mouse model for the combined xeroderma pigmentosum/Cockayne syndrome exhibiting both cancer predisposition and segmental progeria.

Inborn defects in nucleotide excision DNA repair (NER) can paradoxically result in elevated cancer incidence (xeroderma pigmentosum [XP]) or segmental progeria without cancer predisposition (Cockayne syndrome [CS] and trichothiodystrophy [TTD]). We report generation of a knockin mouse model for the combined disorder XPCS with a G602D-encoding mutation in the Xpd helicase gene. XPCS mice are the most skin cancer-prone NER model to date, and we postulate an unusual NER dysfunction that is likely responsible for this susceptibility. XPCS mice also displayed symptoms of segmental progeria, including cachexia and progressive loss of germinal epithelium. Like CS fibroblasts, XPCS and TTD fibroblasts from human and mouse showed evidence of defective repair of oxidative DNA lesions that may underlie these segmental progeroid symptoms.

Differentiation driven changes in the dynamic organization of Basal transcription initiation.

Studies based on cell-free systems and on in vitro-cultured living cells support the concept that many cellular processes, such as transcription initiation, are highly dynamic: individual proteins stochastically bind to their substrates and disassemble after reaction completion. This dynamic nature allows quick adaptation of transcription to changing conditions. However, it is unknown to what extent this dynamic transcription organization holds for postmitotic cells embedded in mammalian tissue. To allow analysis of transcription initiation dynamics directly into living mammalian tissues, we created a knock-in mouse model expressing fluorescently tagged TFIIH. Surprisingly and in contrast to what has been observed in cultured and proliferating cells, postmitotic murine cells embedded in their tissue exhibit a strong and long-lasting transcription-dependent immobilization of TFIIH. This immobilization is both differentiation driven and development dependent. Furthermore, although very statically bound, TFIIH can be remobilized to respond to new transcriptional needs. This divergent spatiotemporal transcriptional organization in different cells of the soma revisits the generally accepted highly dynamic concept of the kinetic framework of transcription and shows how basic processes, such as transcription, can be organized in a fundamentally different fashion in intact organisms as previously deduced from in vitro studies.

Electronic Brainstorming With a Chatbot Partner: A Good Idea Due to Increased Productivity and Idea Diversity.

Brainstorming is a creative technique that fosters collaboration to enhance idea generation. The occurrence of evaluation apprehension, a fear of being evaluated negatively by others, however, can stymy brainstorming. How the advantages of collaboration can be leveraged while evaluation apprehension is prevented is an open scientific and practical problem. In this brief research report, it is proposed that chatbots could provide a solution. Chatbots can be designed to share ideas with their users, facilitating inspiration. Compared to human beings, chatbots are also perceived as possessing limited agency and evaluative capacity. This could reduce evaluation apprehension. Given that chatbots are often embedded in a textual chat interface, social cues (picture, name, and description) can reinforce the perceived chatbot identity, enhancing its alleged effects on evaluation apprehension and subsequently on brainstorming performance. These conjectures were tested in an online 2 × 2 between-subjects experiment (n = 120) where people were instructed to brainstorm with a partner that was framed as either a chatbot or human being (but followed the same automated script), with or without the presence of social cues. The results showed that brainstorming with a chatbot led participants to produce more ideas, with more diversity than brainstorming with an alleged human being. Social cues enhanced the effect on idea diversity, but only with the chatbot. No significant effects on evaluation apprehension were found. The contribution of this study is therefore that chatbots can be used for effective human-machine teaming during brainstorming, but this enhancement is not explained by its effects on evaluation apprehension.

Brainstorming With a Social Robot Facilitator: Better Than Human Facilitation Due to Reduced Evaluation Apprehension?

Brainstorming is a creative technique used to support productivity and creativity during the idea generation phase of an innovation process. In professional practice, a facilitator structures, regulates, and motivates those behaviors of participants that help maintain productivity and creativity during a brainstorm. Emerging technologies, such as social robots, are being developed to support or even automate the facilitator's role. However, little is known about whether and how brainstorming with a social robot influences productivity. To take a first look, we conducted a between-subjects experiment (N = 54) that explored 1) whether brainstorming with a Wizard-of-Oz operated robot facilitator, compared to with a human facilitator, influences productivity; and 2) whether any effects on productivity might be explained by the robot's negative effects on social anxiety and evaluation apprehension. The results showed no evidence for an effect of brainstorming with a teleoperated robot facilitator, compared to brainstorming directly with a human facilitator, on productivity. Although the results did suggest that overall, social anxiety caused evaluation apprehension, and evaluation apprehension negatively affected productivity, there was no effect of brainstorming with a robot facilitator on this relationship. Herewith, the present study contributes to an emerging body of work on the efficacy and mechanisms of the facilitation of creative work by social robots.

The Critical Need to Build a European Governance Model for Online Access to Medical Information Services: A Position Paper.

European pharmaceutical companies have a legal requirement to provide non-promotional Medical Information (MI) services to support healthcare professionals (HCPs) who are using their medicinal products. While the industry has self-regulating bodies with established Codes of Practice, these mainly focus on promotional messaging and commercial activities. In the absence of similar frameworks for MI, such services struggle to understand how to meet HCP digital expectations, often in fear of breaching the promotional codes. This is limiting access to the wealth of non-promotional patient-focussed information held within the industry. Meanwhile, a large volume of unregulated, low-quality information can be readily found on the internet. To understand the current status, the Medical Information Leaders in Europe (MILE) industry association performed a benchmarking survey which explored the online MI service provision of 13 mid-large pharmaceutical companies across Europe. This highlighted a great diversity in approach in terms of geographical coverage and content. Visibility and access for HCPs is complex, compromising online engagement and website utilisation. This MILE position paper highlights the critical need to establish a clear governance model, which empowers pharmaceutical company MI functions to provide unbranded, non-promotional, medicinal product information sources to support HCP online information needs. It is essential to build confidence, transparency and trust by establishing a practical quality framework with principles and standards for online MI services for HCPs.

Individual Differences in Children's (Language) Learning Skills Moderate Effects of Robot-Assisted Second Language Learning.

The current study investigated how individual differences among children affect the added value of social robots for teaching second language (L2) vocabulary to young children. Specifically, we investigated the moderating role of three individual child characteristics deemed relevant for language learning: first language (L1) vocabulary knowledge, phonological memory, and selective attention. We expected children low in these abilities to particularly benefit from being assisted by a robot in a vocabulary training. An L2 English vocabulary training intervention consisting of seven sessions was administered to 193 monolingual Dutch five-year-old children over a three- to four-week period. Children were randomly assigned to one of three experimental conditions: 1) a tablet only, 2) a tablet and a robot that used deictic (pointing) gestures (the no-iconic-gestures condition), or 3) a tablet and a robot that used both deictic and iconic gestures (i.e., gestures depicting the target word; the iconic-gestures condition). There also was a control condition in which children did not receive a vocabulary training, but played dancing games with the robot. L2 word knowledge was measured directly after the training and two to four weeks later. In these post-tests, children in the experimental conditions outperformed children in the control condition on word knowledge, but there were no differences between the three experimental conditions. Several moderation effects were found. The robot's presence particularly benefited children with larger L1 vocabularies or poorer phonological memory, while children with smaller L1 vocabularies or better phonological memory performed better in the tablet-only condition. Children with larger L1 vocabularies and better phonological memory performed better in the no-iconic-gestures condition than in the iconic-gestures condition, while children with better selective attention performed better in the iconic-gestures condition than the no-iconic-gestures condition. Together, the results showed that the effects of the robot and its gestures differ across children, which should be taken into account when designing and evaluating robot-assisted L2 teaching interventions.

A link between the accumulation of DNA damage and loss of multi-potency of human mesenchymal stromal cells.

Human mesenchymal stromal cells (hMSCs) represent an attractive cell source for clinic applications. Besides being multi-potent, recent clinical trials suggest that they secrete both trophic and immunomodulatory factors, allowing allogenic MSCs to be used in a wider variety of clinical situations. The yield of prospective isolation is however very low, making expansion a required step toward clinical applications. Unfortunately, this leads to a significant decrease in their stemness. To identify the mechanism behind loss of multi-potency, hMSCs were expanded until replicative senescence and the concomitant molecular changes were characterized at regular intervals. We observed that, with time of culture, loss of multi-potency was associated with both the accumulation of DNA damage and the respective activation of the DNA damage response pathway, suggesting a correlation between both phenomena. Indeed, exposing hMSCs to DNA damage agents led to a significant decrease in the differentiation potential. We also showed that hMSCs are susceptible to accumulate DNA damage upon in vitro expansion, and that although hMSCs maintained an effective nucleotide excision repair activity, there was a progressive accumulation of DNA damage. We propose a model in which DNA damage accumulation contributes to the loss of differentiation potential of hMSCs, which might not only compromise their potential for clinical applications but also contribute to the characteristics of tissue ageing.

Impaired genome maintenance suppresses the growth hormone--insulin-like growth factor 1 axis in mice with Cockayne syndrome.

Cockayne syndrome (CS) is a photosensitive, DNA repair disorder associated with progeria that is caused by a defect in the transcription-coupled repair subpathway of nucleotide excision repair (NER). Here, complete inactivation of NER in Csb(m/m)/Xpa(-/-) mutants causes a phenotype that reliably mimics the human progeroid CS syndrome. Newborn Csb(m/m)/Xpa(-/-) mice display attenuated growth, progressive neurological dysfunction, retinal degeneration, cachexia, kyphosis, and die before weaning. Mouse liver transcriptome analysis and several physiological endpoints revealed systemic suppression of the growth hormone/insulin-like growth factor 1 (GH/IGF1) somatotroph axis and oxidative metabolism, increased antioxidant responses, and hypoglycemia together with hepatic glycogen and fat accumulation. Broad genome-wide parallels between Csb(m/m)/Xpa(-/-) and naturally aged mouse liver transcriptomes suggested that these changes are intrinsic to natural ageing and the DNA repair-deficient mice. Importantly, wild-type mice exposed to a low dose of chronic genotoxic stress recapitulated this response, thereby pointing to a novel link between genome instability and the age-related decline of the somatotroph axis.

Live Streams on Twitch Help Viewers Cope With Difficult Periods in Life.

Live streaming platforms such as Twitch that facilitate participatory online communities have become an integral part of game culture. Users of these platforms are predominantly teenagers and young adults, who increasingly spend time socializing online rather than offline. This shift to online behavior can be a double-edged sword when coping with difficult periods in life such as relationship issues, the death of a loved one, or job loss. On the one hand, platforms such as Twitch offer pleasure, distraction, and relatedness with others to help with coping, and the increased sense of anonymity and control could stimulate self-disclosure. However, the prevalence of trolling and memes may also discourage people from opening up, and relationships that are built online-especially those with microcelebrity streamers-could be perceived as more meaningful than they actually are. To create a deeper understanding of Twitch as a new media platform embedded in game culture, and how users perceive its potential as a coping mechanism, we have conducted a first exploration by means of a survey. The questions focused on general Twitch behavior, the difficult period in life, and the role of Twitch during this period. It was distributed online among people who considered themselves active Twitch users, and who had gone through a difficult period. Eighty-four participants completed the entire survey. The majority of participants indicated that Twitch helped them cope, and that it became a larger part of their lives during the difficult period compared to regular viewing. Recurring themes were the entertainment, distraction, and sense of community Twitch offers. Viewing behavior during difficult periods appears to remain largely the same in terms of the streamers that are watched, although time spent viewing increases, and there is a change toward more time spent actively watching rather than having the stream on in the background. With this work, we aim to create a deeper understanding of Twitch as a platform, and its importance for gamers that are going through difficult periods in life.

Principles and Considerations for Responsible Sharing of Safety Information Via the Medical Information Channel.

The approach used by medical information services in answering unsolicited safety-related questions from health care professionals regarding prescription medicines varies widely across the pharmaceutical industry. A significant amount of information is available in the public domain, but this can be difficult to filter and determine what is most appropriate for a given situation. A team representing the medical information group MILE (Medical Information Leaders Europe) and European Federation of Pharmaceutical Industries and Associations Pharmacovigilance Expert Group have partnered to develop principles and considerations on how to answer unsolicited safety questions. Essentially two key principles are important in ensuring success: (1) Effective collaboration between medical information and patient safety teams is important for an optimal outcome providing accurate, useful, and timely information. This article discusses considerations for an effective, efficient collaboration between medical information and patient safety and suggests a way of working. (2) Collaborating teams will need to evaluate and select the most appropriate sources of information to answer the question. Sources of information that may or may not be in the public domain are discussed. Adoption of principles and considerations discussed in this article may be expected to improve current safety information-sharing practices that tend to be conservative and risk averse. In addition, this presents the opportunity to initiate discussions with regulatory authorities to realize the benefits that will come through greater transparency and communication to support safe and effective use of medicines.

A Mobile App for Longterm Monitoring of Narcolepsy Symptoms: Design, Development, and Evaluation.

BACKGROUND: Narcolepsy is a chronic sleep disorder with a broad variety of symptoms. Although narcolepsy is primarily characterized by excessive daytime sleepiness and cataplexy (loss of muscle control triggered by emotions), patients may suffer from hypnagogic hallucinations, sleep paralysis, and fragmented night sleep. However, the spectrum of narcolepsy also includes symptoms not related to sleep, such as cognitive or psychiatric problems. Symptoms vary greatly among patients and day-to-day variance can be considerable. Available narcolepsy questionnaires do not cover the whole symptom spectrum and may not capture symptom variability. Therefore, there is a clinical need for tools to monitor narcolepsy symptoms over time to evaluate their burden and the effect of treatment. OBJECTIVE: This study aimed to describe the design, development, implementation, and evaluation of the Narcolepsy Monitor, a companion app for long-term symptom monitoring in narcolepsy patients. METHODS: After several iterations during which content, interaction design, data management, and security were critically evaluated, a complete version of the app was built. The Narcolepsy Monitor allows patients to report a broad spectrum of experienced symptoms and rate their severity based on the level of burden that each symptom imposes. The app emphasizes the reporting of changes in relative severity of the symptoms. A total of 7 patients with narcolepsy were recruited and asked to use the app for 30 days. Evaluation was done by using in-depth interviews and user experience questionnaire. RESULTS: We designed and developed a final version of the Narcolepsy Monitor after which user evaluation took place. Patients used the app on an average of 45.3 (SD 19.2) days. The app was opened on 35% of those days. Daytime sleepiness was the most dynamic symptom, with a mean number of changes of 5.5 (SD 3.7) per month, in contrast to feelings of anxiety or panic, which was only moved 0.3 (SD 0.7) times per month. Mean symptom scores were highest for daytime sleepiness (1.8 [SD 1.0]), followed by lack of energy (1.6 [SD 1.4]) and often awake at night (1.5 [SD 1.0]). The personal in-depth interviews revealed 3 major themes: (1) reasons to use, (2) usability, and (3) features. Overall, patients appreciated the concept of ranking symptoms on subjective burden and found the app easy to use. CONCLUSIONS: The Narcolepsy Monitor appears to be a helpful tool to gain more insight into the individual burden of narcolepsy symptoms over time and may serve as a patient-reported outcome measure for this debilitating disorder.

Cell-type-specific consequences of nucleotide excision repair deficiencies: Embryonic stem cells versus fibroblasts.

Pluripotent embryonic stem cells (ES cells) are the precursors of all different cell types comprising the organism. Since persistent DNA damage in this cell type might lead to mutations that cause huge malformations in the developing organism, genome caretaking is of prime importance. We first compared the sensitivity of wild type mouse embryonic fibroblasts (MEFs) and ES cells for various genotoxic agents and show that ES cells are more sensitive to treatment with UV-light, gamma-rays and mitomycin C than MEFs. We next investigated the contribution of the transcription-coupled (TC-NER) and global genome (GG-NER) sub-pathways of nucleotide excision repair (NER) in protection of ES cells, using cells from mouse models for the NER disorders xeroderma pigmentosum (XP) and Cockayne syndrome (CS). TC-NER-deficient Csb(-/-) and GG-NER/TC-NER-defective Xpa(-/-) MEFs are hypersensitive to UV, whereas GG-NER-deficient Xpc(-/-) MEFs attribute intermediate UV sensitivity. The observed UV-hypersensitivity in Csb(-/-) and Xpa(-/-) MEFs correlates with increased apoptosis. In contrast, Xpa(-/-) and Xpc(-/-) ES cells are highly UV-sensitive, while a Csb deficiency only causes a mild increase in UV-sensitivity. Surprisingly, a UV-induced hyperapoptotic response is mainly observed in Xpa(-/-) ES cells, suggesting a different mechanism of apoptosis induction in ES cells, mainly triggered by damage in the global genome rather than in transcribed genes (as in MEFs). Moreover, we show a pronounced S-phase delay in Xpa(-/-) and Xpc(-/-) ES cells, which might well function as a safeguard mechanism for heavily damaged ES cells in case the apoptotic response fails. Although Xpa(-/-) and Xpc(-/-) ES cells are totally NER-defective or GG-NER-deficient respectively, mutation induction upon UV is similar compared to wild type ES cells indicating that the observed apoptotic and cell cycle responses are indeed sufficient to protect against proliferation of damaged cells. In conclusion, we show a double safeguard mechanism in ES cells against NER-type of damages, which mainly relies on damage detection in the global genome.

Rescue of progeria in trichothiodystrophy by homozygous lethal Xpd alleles.

Although compound heterozygosity, or the presence of two different mutant alleles of the same gene, is common in human recessive disease, its potential to impact disease outcome has not been well documented. This is most likely because of the inherent difficulty in distinguishing specific biallelic effects from differences in environment or genetic background. We addressed the potential of different recessive alleles to contribute to the enigmatic pleiotropy associated with XPD recessive disorders in compound heterozygous mouse models. Alterations in this essential helicase, with functions in both DNA repair and basal transcription, result in diverse pathologies ranging from elevated UV sensitivity and cancer predisposition to accelerated segmental progeria. We report a variety of biallelic effects on organismal phenotype attributable to combinations of recessive Xpd alleles, including the following: (i) the ability of homozygous lethal Xpd alleles to ameliorate a variety of disease symptoms when their essential basal transcription function is supplied by a different disease-causing allele, (ii) differential developmental and tissue-specific functions of distinct Xpd allele products, and (iii) interallelic complementation, a phenomenon rarely reported at clinically relevant loci in mammals. Our data suggest a re-evaluation of the contribution of "null" alleles to XPD disorders and highlight the potential of combinations of recessive alleles to affect both normal and pathological phenotypic plasticity in mammals.

Principles and Considerations for Responsible Sharing of Safety Information Via the Medical Information Channel.

The approach used by medical information services in answering unsolicited safety-related questions from health care professionals regarding prescription medicines varies widely across the pharmaceutical industry. A significant amount of information is available in the public domain, but this can be difficult to filter and determine what is most appropriate for a given situation. A team representing the medical information group MILE (Medical Information Leaders Europe) and European Federation of Pharmaceutical Industries and Associations Pharmacovigilance Expert Group have partnered to develop principles and considerations on how to answer unsolicited safety questions. Essentially two key principles are important in ensuring success: (1) Effective collaboration between medical information and patient safety teams is important for an optimal outcome providing accurate, useful, and timely information. This article discusses considerations for an effective, efficient collaboration between medical information and patient safety and suggests a way of working. (2) Collaborating teams will need to evaluate and select the most appropriate sources of information to answer the question. Sources of information that may or may not be in the public domain are discussed. Adoption of principles and considerations discussed in this article may be expected to improve current safety information-sharing practices that tend to be conservative and risk averse. In addition, this presents the opportunity to initiate discussions with regulatory authorities to realize the benefits that will come through greater transparency and communication to support safe and effective use of medicines.

Guidelines for Designing Social Robots as Second Language Tutors.

In recent years, it has been suggested that social robots have potential as tutors and educators for both children and adults. While robots have been shown to be effective in teaching knowledge and skill-based topics, we wish to explore how social robots can be used to tutor a second language to young children. As language learning relies on situated, grounded and social learning, in which interaction and repeated practice are central, social robots hold promise as educational tools for supporting second language learning. This paper surveys the developmental psychology of second language learning and suggests an agenda to study how core concepts of second language learning can be taught by a social robot. It suggests guidelines for designing robot tutors based on observations of second language learning in human-human scenarios, various technical aspects and early studies regarding the effectiveness of social robots as second language tutors.

The ubiquitin-conjugating DNA repair enzyme HR6A is a maternal factor essential for early embryonic development in mice.

The Saccharomyces cerevisiae RAD6 protein is required for a surprising diversity of cellular processes, including sporulation and replicational damage bypass of DNA lesions. In mammals, two RAD6-related genes, HR6A and HR6B, encode highly homologous proteins. Here, we describe the phenotype of cells and mice deficient for the mHR6A gene. Just like mHR6B knockout mouse embryonic fibroblasts, mHR6A-deficient cells appear to have normal DNA damage resistance properties, but mHR6A knockout male and female mice display a small decrease in body weight. The necessity for at least one functional mHR6A (X-chromosomal) or mHR6B (autosomal) allele in all somatic cell types is supported by the fact that neither animals lacking both proteins nor females with only one intact mHR6A allele are viable. In striking contrast to mHR6B knockout males, which show a severe spermatogenic defect, mHR6A knockout males are normally fertile. However, mHR6A knockout females fail to produce offspring despite a normal ovarian histology and ovulation. The absence of mHR6A in oocytes prevents development beyond the embryonic two-cell stage but does not result in an aberrant methylation pattern of histone H3 at this early stage of mouse embryonic development. These observations support redundant but dose-dependent roles for HR6A and HR6B in somatic cell types and germ line cells in mammals.

Different effects of CSA and CSB deficiency on sensitivity to oxidative DNA damage.

Mutations in the CSA and CSB genes cause Cockayne syndrome, a rare inherited disorder characterized by UV sensitivity, severe neurological abnormalities, and progeriod symptoms. Both gene products function in the transcription-coupled repair (TCR) subpathway of nucleotide excision repair (NER), providing the cell with a mechanism to remove transcription-blocking lesions from the transcribed strands of actively transcribed genes. Besides a function in TCR of NER lesions, a role of CSB in (transcription-coupled) repair of oxidative DNA damage has been suggested. In this study we used mouse models to compare the effect of a CSA or a CSB defect on oxidative DNA damage sensitivity at the levels of the cell and the intact organism. In contrast to CSB(-/-) mouse embryonic fibroblasts (MEFs), CSA(-/-) MEFs are not hypersensitive to gamma-ray or paraquat treatment. Similar results were obtained for keratinocytes. In contrast, both CSB(-/-) and CSA(-/-) embryonic stem cells show slight gamma-ray sensitivity. Finally, CSB(-/-) but not CSA(-/-) mice fed with food containing di(2-ethylhexyl)phthalate (causing elevated levels of oxidative DNA damage in the liver) show weight reduction. These findings not only uncover a clear difference in oxidative DNA damage sensitivity between CSA- and CSB-deficient cell lines and mice but also show that sensitivity to oxidative DNA damage is not a uniform characteristic of Cockayne syndrome. This difference in the DNA damage response between CSA- and CSB-deficient cells is unexpected, since until now no consistent differences between CSA and CSB patients have been reported. We suggest that the CSA and CSB proteins in part perform separate roles in different DNA damage response pathways.

The structure-specific endonuclease Ercc1-Xpf is required to resolve DNA interstrand cross-link-induced double-strand breaks.

Interstrand cross-links (ICLs) are an extremely toxic class of DNA damage incurred during normal metabolism or cancer chemotherapy. ICLs covalently tether both strands of duplex DNA, preventing the strand unwinding that is essential for polymerase access. The mechanism of ICL repair in mammalian cells is poorly understood. However, genetic data implicate the Ercc1-Xpf endonuclease and proteins required for homologous recombination-mediated double-strand break (DSB) repair. To examine the role of Ercc1-Xpf in ICL repair, we monitored the phosphorylation of histone variant H2AX (gamma-H2AX). The phosphoprotein accumulates at DSBs, forming foci that can be detected by immunostaining. Treatment of wild-type cells with mitomycin C (MMC) induced gamma-H2AX foci and increased the amount of DSBs detected by pulsed-field gel electrophoresis. Surprisingly, gamma-H2AX foci were also induced in Ercc1(-/-) cells by MMC treatment. Thus, DSBs occur after cross-link damage via an Ercc1-independent mechanism. Instead, ICL-induced DSB formation required cell cycle progression into S phase, suggesting that DSBs are an intermediate of ICL repair that form during DNA replication. In Ercc1(-/-) cells, MMC-induced gamma-H2AX foci persisted at least 48 h longer than in wild-type cells, demonstrating that Ercc1 is required for the resolution of cross-link-induced DSBs. MMC triggered sister chromatid exchanges in wild-type cells but chromatid fusions in Ercc1(-/-) and Xpf mutant cells, indicating that in their absence, repair of DSBs is prevented. Collectively, these data support a role for Ercc1-Xpf in processing ICL-induced DSBs so that these cytotoxic intermediates can be repaired by homologous recombination.