ATM phosphorylates PP2A subunit A resulting in nuclear export and spatiotemporal regulation of the DNA damage response.

Ataxia telangiectasia mutated (ATM) is a serine-threonine protein kinase and important regulator of the DNA damage response (DDR). One critical ATM target is the structural subunit A (PR65-S401) of protein phosphatase 2A (PP2A), known to regulate diverse cellular processes such as mitosis and cell growth as well as dephosphorylating many proteins during the recovery from the DDR. We generated mouse embryonic fibroblasts expressing PR65-WT, -S401A (cannot be phosphorylated), and -S401D (phospho-mimetic) transgenes. Significantly, S401 mutants exhibited extensive chromosomal aberrations, impaired DNA double-strand break (DSB) repair and underwent increased mitotic catastrophe after radiation. Both S401A and the S401D cells showed impaired DSB repair (nonhomologous end joining and homologous recombination repair) and exhibited delayed DNA damage recovery, which was reflected in reduced radiation survival. Furthermore, S401D cells displayed increased ERK and AKT signaling resulting in enhanced growth rate further underscoring the multiple roles ATM-PP2A signaling plays in regulating prosurvival responses. Time-lapse video and cellular localization experiments showed that PR65 was exported to the cytoplasm after radiation by CRM1, a nuclear export protein, in line with the very rapid pleiotropic effects observed. A putative nuclear export sequence (NES) close to S401 was identified and when mutated resulted in aberrant PR65 shuttling. Our study demonstrates that the phosphorylation of a single, critical PR65 amino acid (S401) by ATM fundamentally controls the DDR, and balances DSB repair quality, cell survival and growth by spatiotemporal PR65 nuclear-cytoplasmic shuttling mediated by the nuclear export receptor CRM1.

Reaching a Tipping Point: A Qualitative Exploration of Quality of Life and Treatment Decision-Making in People Living With Benign Prostatic Hyperplasia.

Benign prostatic hyperplasia (BPH) is a common condition amongst older men and is associated with lower urinary tract symptoms and erectile dysfunction; these symptoms can be burdensome and negatively affect quality of life. Various surgical and pharmaceutical treatment options exist but there is a paucity of qualitative research exploring men's decision-making when seeking BPH treatment. This study qualitatively explored men's experience of living with BPH and seeking treatment for BPH. Twenty men (aged 52-75) were recruited from outpatient urology clinics at a hospital trust in Southern England. Data were collected using semi-structured interviews (via video or telephone call) and were audio-recorded; transcripts were analysed using thematic analysis. Four themes were generated: 'Impacts are about more than just physical symptoms', 'The path towards treatment', 'The process of information gathering' and 'Navigating hopes, fears and uncertainty'. Results indicate most men appear to seek treatment for BPH following a gradual, and sometimes lengthy, period of deterioration in symptoms; for some men, however, treatment seeking follows an acute episode of sudden or severe symptoms. The decision to proceed with surgical or minimally invasive treatment options appears to be dependent on men reaching a tipping point; they no longer perceive their symptoms as tolerable and feel their ability to cope with symptoms is reduced. Men each bring their own set of concerns and preferences about side effects and risk-benefit profiles of different treatments. Clinicians need to be sensitive to these individual differences and incorporate them into shared decision-making for choosing between treatment options for BPH.

Corrigendum: A longitudinal investigation of quality of life and negative emotions in misophonia.

[This corrects the article DOI: 10.3389/fnins.2022.900474.].

Antibiotic prescription patterns and non-clinical factors influencing antibiotic use by Ecuadorian veterinarians working on cattle and poultry farms: A cross-sectional study.

Understanding antibiotic prescription patterns and non-clinical factors influencing antibiotic use is essential for implementing strategies to promote appropriate antibiotic use. There is, however, limited research exploring these issues with Ecuadorian veterinarians. Therefore, a questionnaire was developed and applied cross-sectionally to veterinarians (n = 173) from two professional organizations to explore the antibiotic prescription patterns and non-clinical factors (e.g., attitudes and perceptions) influencing antibiotic use, and to identify strategies to reduce antibiotic use. The response rate was 78.4%. Responses were compared between veterinarians working mainly on cattle and poultry farms using Mann-Whitney U tests. The most important attitudes, beliefs and perceptions towards antimicrobial resistance (AMR) and antibiotic use were identified with the Relative Importance Index (RII). Veterinarians showed high awareness of AMR and its implications for public health, as well as the necessity of reducing antibiotic use. However, some veterinarians appear to underestimate the potential contribution of veterinary antibiotic use on AMR in humans. Veterinarians self-reported high prescription (> 20%) of antibiotics for cattle and poultry that are critically important for human medicine, such as 3rd and 4th generation cephalosporins, polymyxins and quinolones. Further, antibiotic therapy was not tailored to disease type. Cattle and poultry veterinarians perceived similar barriers to increasing antibiotic stewardship including: poor biosecurity measures, animal confinement, low feed quality, farmers' behaviors (such as stopping antibiotic treatment, storing antibiotics on farms, buying antibiotics in veterinary supply stores), and sales agents' roles as non-professional prescribers of antibiotics. Overall, veterinarians were broadly supportive (>90%) of most strategies to promote appropriate antibiotic use. They saw more merit in improving biosecurity of farms and implementing educational programs for farmers and veterinarians. This study provides insight into the complexity of antibiotic use on Ecuadorian farms and the need for holistic strategies in a One Health context, to achieve antibiotic stewardship.

Work fatigue during COVID-19 lockdown teleworking: the role of psychosocial, environmental, and social working conditions.

Background: During national lockdowns in response to the COVID-19 pandemic, previously office-based workers who transitioned to home-based teleworking faced additional demands (e.g., childcare, inadequate homeworking spaces) likely resulting in poor work privacy fit. Previous office research suggests poor work privacy fit is associated with lower wellbeing and higher work fatigue. Emerging evidence suggests a relationship between childcare duties during pandemic teleworking and work fatigue. In addition to psychosocial working conditions (job demand, job control, and job change management), which are acknowledged predictors of work fatigue, this poses a significant threat to occupational health during pandemic teleworking. However, the relative effects of aspects of the psychosocial environment (job demands and resources), the home office environment (including privacy fit), and the social environment (childcare) on work fatigue as well as their interactions are under-explored. Objective: This study examined the relationships between the psychosocial, environmental, and social working conditions of teleworking during the first COVID-19 lockdown and work fatigue. Specifically, the study examined teleworkers' physical work environment (e.g., if and how home office space is shared, crowding, and noise perceptions) as predictors of privacy fit and the relationship between privacy fit, childcare, psychosocial working conditions (job demand, job control, and job change management), and work fatigue. Work privacy fit was hypothesized to mediate the relationship between childcare and work fatigue. Methods: An online cross-sectional survey was conducted with teleworkers (n = 300) during the first COVID-19 lockdown in April and May 2020; most participants were in Germany, Switzerland, and the United Kingdom. Results: Path analysis was used to examine the hypothesized relationships. Privacy fit was lower for those reporting greater levels of noise in home-working spaces and those feeling crowded at home. Work fatigue was lower amongst those with greater privacy fit and higher amongst those with high levels of job demand. An indirect relationship was observed between childcare and work fatigue with privacy fit mediating this relationship. Conclusion: The influence of privacy fit has so far been largely neglected in research on teleworking, especially during the pandemic. However, its contribution to workers' wellbeing should be acknowledged in occupational health strategies.

A longitudinal investigation of quality of life and negative emotions in misophonia.

Aims: This longitudinal study examined the role of anger, disgust, and anxiety in the experience of misophonia, the quality of life of those with self-reported misophonia in comparison to those without misophonia, and the association of misophonia and quality of life over time. Methods: An online longitudinal survey was conducted, with misophonia, anger, disgust, anxiety, depression, self-esteem, and quality of life measured at two time points (6-months apart) in two groups of people (those with self-reported misophonia and those without misophonia). Results: Anger and disgust emerged as the primary predictors of misophonic responses. Anxiety and depression were not significantly associated with misophonia over time. Differences in quality of life were observed between those with and without self-reported misophonia in the current study, with lower scores across the SF-36 domains of role limitations due to emotional problems, energy/fatigue, emotional wellbeing, social functioning, and general health for those with misophonia compared to those without misophonia. Compared with other studies, scores for those with self-reported misophonia were lower than those with long-term physical conditions, similar to those with tinnitus, but higher than those with obsessive compulsive disorder. Misophonia was predictive of quality of life over time but only on two domains: role limitations due to emotional problems (predictors: avoidance, emotional responses, and impact on participation in life) and pain (predictor: impact on participation in life). Depression remained a strong predictor of quality of life over time. Conclusion: Anger and disgust are more strongly associated with the experience of misophonia than anxiety. Quality of life in people with self-reported misophonia is lower than in the general population and may be similar to those with tinnitus. Depression, avoiding triggers, the extent of the emotional response, and perceived impact on participation in life are associated with perceptions of lower quality of life over time for people with self-reported misophonia.

Assessing Knowledge, Beliefs, and Behaviors around Antibiotic Usage and Antibiotic Resistance among UK Veterinary Students: A Multi-Site, Cross-Sectional Survey.

Antimicrobial resistance (AMR) is a profound threat to human and animal health. Antimicrobial prescribing behaviours are influenced by psychological factors such as knowledge, beliefs, and emotions. As future antimicrobial prescribers, it is important to understand beliefs about AMR and stewardship among veterinary (vet) students. This cross-sectional online survey assessed vet students' self-reported behavior, knowledge, and beliefs in specific relation to antibiotic resistance (ABR) and antibiotic usage. Participants were early years (first- and second-year; n = 460) and later-years (third- and fourth-year; n = 113) undergraduate vet students from three UK universities. Self-reported antibiotic-related behaviors were responsible among most students. Knowledge about ABR and stewardship was moderate among early years students and good among later years students. Vet students typically believed that vets had less responsibility for both causing and preventing ABR than other groups (animal owners, human medics, and the public). This study offers evidence that vet students (along with other groups) tend to lay greater responsibility for ABR/AMR outside of their own profession, which may impact their future prescribing behaviors. It is vital that AMR and antimicrobial stewardship are embedded across veterinary curricula, and that the One Health nature of the challenge posed by AMR is emphasized to encourage shared responsibility across all stakeholder groups, thereby helping to reduce 'other-blaming' for AMR.

The role of emotions and injunctive norms in breastfeeding: a systematic review and meta-analysis.

Breastfeeding has many known benefits, but rates vary globally. We propose two main reasons why psychological theory and interventions have not been successful to date in explaining breastfeeding behaviours. Specifically, prior research underestimates the importance of (1) specific emotions and (2) wider injunctive influences (i.e., societal and moral norms about what women feel they ought to be doing) in the breastfeeding experience. Therefore, we conducted a systematic review of quantitative, qualitative, and mixed-methods studies that explored whether injunctive norms and/ or specific emotions are associated with breastfeeding behaviours (i.e., intentions, initiation and duration). Seventy-two papers were included in this review; data were extracted and quality appraisals conducted for all included studies. A meta-analysis of effect sizes was performed with the quantitative data. A convergent qualitative synthesis of the data was conducted, resulting in the following line of argument: Breastfeeding is a social behaviour and not a personal/individual behaviour. From this line of argument, three themes with associated sub-themes were developed, highlighting the importance of both specific emotions and injunctive norms on breastfeeding behaviours. These influences are discussed in relation to both theoretical and practical implications, as well as future research.

Future Teleworking Inclinations Post-COVID-19: Examining the Role of Teleworking Conditions and Perceived Productivity.

Organisations have implemented intensive home-based teleworking in response to global COVID-19 lockdowns and other pandemic-related restrictions. Financial pressures are driving organisations to continue intensive teleworking after the pandemic. Understanding employees' teleworking inclinations post COVID-19, and how these inclinations are influenced by different factors, is important to ensure any future, more permanent changes to teleworking policies are sustainable for both employees and organisations. This study, therefore, investigated the relationships between the context of home-based teleworking during the pandemic (pandemic-teleworking conditions), productivity perceptions during home-based teleworking, and employees' future teleworking inclinations (FTI) beyond the pandemic. Specifically, the study examined whether pandemic-teleworking conditions related to the job, and the physical and social environments at home, influenced employees' FTI, and if perceptions of improved or reduced productivity mediated these relationships. Data were collected during April and May 2020 with a cross-sectional online survey of teleworkers (n = 184) in Germany, Switzerland, the United Kingdom, and other countries during the first COVID-19 lockdowns. Reported FTI were mixed. Most participants (61%) reported wanting to telework more post-pandemic compared to before the pandemic; however, 18% wanted to telework less. Hierarchical multiple regression analysis revealed that some teleworking conditions (job demands and work privacy fit) were positively associated with FTI. Other teleworking conditions (specifically, job change, job control, home office adequacy, and childcare) were not associated with FTI. Perceived changes in productivity mediated the relationship between work privacy fit and FTI. Findings highlight the role of work privacy fit and job demands in influencing pandemic productivity perceptions and teleworking inclinations post-pandemic. Results raise questions about the suitability and sustainability of home-based teleworking for all staff. As organisations plan to increase the proportion of teleworking post-pandemic, this study suggests there is a need to support employees who perceived their productivity to be poor while home-working during the pandemic.

Interventions to change purchasing behaviour in supermarkets: a systematic review and intervention content analysis.

This systematic review and intervention content analysis used behavioural science frameworks to characterise content and function of interventions targeting supermarket shoppers' purchasing behaviour, and explore if coherence between content and function was linked to intervention effectiveness. Study eligibility: in-store interventions (physical supermarkets) with control conditions, targeting objectively measured food and/or non-alcoholic drink purchases, published in English (no date restrictions). Eleven electronic databases were searched; reference lists of systematic reviews were hand-searched. Methodological quality was assessed using the GATE checklist. A content analysis was performed to characterise intervention content and function, and theoretical coherence between these, using the Behaviour Change Wheel, Behaviour Change Techniques Taxonomy, and Typology of Interventions in Proximal Physical Micro-Environments (TIPPME). Forty-six articles (49 interventions) met inclusion criteria; 26 articles (32 interventions) were included in the content analysis. Twenty behaviour change techniques (BCTs), and four TIPPME intervention types were identified; three BCTs ('Prompts/cues', 'Material incentive', and 'Material reward') were more common in effective interventions. Nineteen interventions solely employed theoretically appropriate BCTs. Theoretical coherence between BCTs and intervention functions was more common in effective interventions. Effective interventions included price promotions and/or in-store merchandising. Future research should explore the effect of specific BCTs using factorial study designs. PROSPERO Registration: CRD42017071065.

Encouraging healthier grocery purchases online: A randomised controlled trial and lessons learned.

Online supermarket platforms present an opportunity for encouraging healthier consumer purchases. A parallel, double-blind randomised controlled trial tested whether promoting healthier products (e.g. lower fat and lower calorie) on the Sainsbury's online supermarket platform would increase purchases of those products. Participants were Nectar loyalty membership scheme cardholders who shopped online with Sainsbury's between 20th September and 10th October 2017. Intervention arm customers saw advertisement banners and recipe ingredient lists containing healthier versions of the products presented in control arm banners and ingredient lists. The primary outcome measure was purchases of healthier products. Additional outcome measures were banner clicks, purchases of standard products, overall purchases and energy (kcal) purchased. Sample sizes were small due to customers navigating the website differently than expected. The intervention encouraged purchases of some promoted healthier products (spaghetti [B = 2.10, p < 0.001], spaghetti sauce [B = 2.06, p < 0.001], spaghetti cheese [B = 2.45, p = 0.001], sour cream [B = 2.52, p < 0.001], fajita wraps [B = 2.10, p < 0.001], fajita cheese [B = 1.19, p < 0.001], bakery aisle products (B = 3.05, p = 0.003) and cola aisle products [B = 0.97, p < 0.002]) but not others (spaghetti mince, or products in the yogurt and ice cream aisles). There was little evidence of effects on banner clicks and energy purchased. Small sample sizes may affect the robustness of these findings. We discuss the benefits of collaborating to share expertise and implement a trial in a live commercial environment, alongside key learnings for future collaborative research in similar contexts.

Erratum: Golding et al. Examining the Effect of Context, Beliefs, and Values on UK Farm Veterinarians' Antimicrobial Prescribing: A Randomized Experimental Vignette and Cross-Sectional Survey. Antibiotics 2021, 10, 445.

The authors wish to make the following corrections to their published paper [...].

Examining the Effect of Context, Beliefs, and Values on UK Farm Veterinarians' Antimicrobial Prescribing: A Randomized Experimental Vignette and Cross-Sectional Survey.

Antimicrobial resistance (AMR) is a pressing threat to public and animal health. There is evidence that antimicrobial prescribing and stewardship behaviors by veterinarians (vets) are influenced by non-clinical factors, such as psychological, social, and environmental factors. This study explored the role of context, beliefs, and values on vets' antimicrobial prescribing decisions. UK-based practicing farm vets (n = 97) were recruited to an online study. Using an experimental vignette methodology, vets were randomly assigned across four conditions, to examine the effects of different contexts (pressure on farm economics, the farmer, or the vet-farmer relationship, compared to a control condition) on vets' likelihood of prescribing antibiotics. Vets' beliefs about different groups' responsibility for causing and preventing AMR and vets' values were also measured. Key findings were that context alone, values, and beliefs about groups' responsibilities for causing AMR were not predictive of vets' likelihood of prescribing antibiotics. However, vets' beliefs about groups' responsibilities for preventing AMR were predictive of an increased likelihood of prescribing antibiotics, when vets were exposed to the experimental condition of the vignette in which the vet-farmer relationship was under pressure. Farm vets also believed that different groups have different levels of responsibility for causing and preventing AMR. Results should be interpreted cautiously, given the smaller than planned for sample size, and the possibility for both false negatives and false positives. Further research is needed to explore how these findings could inform antimicrobial stewardship interventions in veterinary medicine.

#Springwatch #WildMorningswithChris: Engaging With Nature via Social Media and Wellbeing During the COVID-19 Lockdown.

It is widely understood that nature engagement benefits human wellbeing. Such benefits have been found for real as well as virtual engagements. However, little is known about the role of nature-based videos in social media on wellbeing. With COVID-19 restrictions limiting people's direct engagement with natural environments, this study critically examined people's reactions to nature videos posted on Facebook during the first UK COVID-19 lockdown in 2020. Data consisted of comments on videos containing highlights from the British Broadcasting Corporation's (BBC) Springwatch 2020 television series, and from a UK television presenter and naturalist's (Chris Packham) livestream videos, posted on Facebook from March to July, 2020. Looking at the quantitative profile of a range of videos (i.e. views, likes and shares) and a detailed analysis of the 143,265 comments using thematic analysis, three major themes were generated as: (1) engaging with nature via social media is emotionally complicated, (2) cognitive and reflective reactions are generated from social media nature engagement and (3) engagement with nature-based social media as a mechanism for coping with stress during COVID-19. These findings inform understanding of how nature-related social media content and associated commentary have supported wellbeing throughout the ongoing pandemic and their importance as a means of continued support for wellbeing. 'We feel that the injection of wildlife into people's homes, particularly at this point, would be really valuable and uplifting'. - Chris Packham, 2020.

Extracellular signal-related kinase positively regulates ataxia telangiectasia mutated, homologous recombination repair, and the DNA damage response.

The accurate joining of DNA double-strand breaks by homologous recombination repair (HRR) is critical to the long-term survival of the cell. The three major mitogen-activated protein (MAP) kinase (MAPK) signaling pathways, extracellular signal-regulated kinase (ERK), p38, and c-Jun-NH(2)-kinase (JNK), regulate cell growth, survival, and apoptosis. To determine the role of MAPK signaling in HRR, we used a human in vivo I-SceI-based repair system. First, we verified that this repair platform is amenable to pharmacologic manipulation and show that the ataxia telangiectasia mutated (ATM) kinase is critical for HRR. The ATM-specific inhibitor KU-55933 compromised HRR up to 90% in growth-arrested cells, whereas this effect was less pronounced in cycling cells. Then, using well-characterized MAPK small-molecule inhibitors, we show that ERK1/2 and JNK signaling are important positive regulators of HRR in growth-arrested cells. On the other hand, inhibition of the p38 MAPK pathway generated an almost 2-fold stimulation of HRR. When ERK1/2 signaling was stimulated by oncogenic RAF-1, an approximately 2-fold increase in HRR was observed. KU-55933 partly blocked radiation-induced ERK1/2 phosphorylation, suggesting that ATM regulates ERK1/2 signaling. Furthermore, inhibition of MAP/ERK kinase (MEK)/ERK signaling resulted in severely reduced levels of phosphorylated (S1981) ATM foci but not gamma-H2AX foci, and suppressed ATM phosphorylation levels >85% throughout the cell cycle. Collectively, these results show that MAPK signaling positively and negatively regulates HRR in human cells. More specifically, ATM-dependent signaling through the RAF/MEK/ERK pathway is critical for efficient HRR and for radiation-induced ATM activation, suggestive of a regulatory feedback loop between ERK and ATM.

Shared Goals, Different Barriers: A Qualitative Study of UK Veterinarians' and Farmers' Beliefs About Antimicrobial Resistance and Stewardship.

Although much research has investigated the drivers of inappropriate antimicrobial prescribing in human medicine, equivalent research in veterinary medicine is in its infancy. This qualitative study used a critical incident approach to explore farm veterinarians' (vets) and farmers' beliefs about antimicrobial use and antimicrobial stewardship. Semi-structured interviews were conducted with 13 vets and 12 farmers in the UK, who worked mostly with beef cattle, dairy cattle and sheep, but a minority also worked with pigs or poultry. An inductive thematic analysis was conducted to explore how vets and farmers understood their responsibilities toward stewardship and antimicrobial resistance (AMR) and to identify key similarities and differences between the professions. The analysis generated four themes: "A shared conflict between ideals and behaviour," "Barriers to stewardship: the vets' perspective," "Barriers to stewardship: the farmers' perspective," and "A shared ambivalence: ownership vs. other-blaming." Vets and farmers demonstrated good understanding of stewardship but their treatment decisions are not always aligned to stewardship principles. Various barriers to improving antimicrobial stewardship were discussed by vets and farmers, but they placed differing emphasis on specific barriers. Faced with these barriers and an awareness that antimicrobial usage is not always aligned to stewardship principles, vets and farmers expressed frustration and a sense of ambivalence toward stewardship, and also engaged in other-blaming for the problem of AMR. In conclusion, vets and farmers in this study seem motivated to be antimicrobial stewards but feel challenged by the day-to-day reality of their jobs; they experience ambivalence toward their responsibilities for AMR, which may negatively impact their motivation to always act as antimicrobial stewards. Successfully tackling AMR will require change at the individual-, group-, and societal-level. Future interventions to improve antimicrobial usage in livestock farming could be situated within a social ecological framework, where other-blaming between professions is seen as a result of the interplay between psychological and contextual factors. Other-blaming could be reduced using a social identity approach; a common ingroup identity could be created by encouraging vets and farmers to focus on their common goal, namely a shared desire to promote animal welfare through optimal antimicrobial stewardship.

Exploring the psychological health of emergency dispatch centre operatives: a systematic review and narrative synthesis.

BACKGROUND: The study objective was to investigate and synthesize available evidence relating to the psychological health of Emergency Dispatch Centre (EDC) operatives, and to identify key stressors experienced by EDC operatives. METHODS: Eight electronic databases (Embase, PubMed, Medline, CINAHL, PsycInfo, PsycArticles, The Psychology and Behavioural Sciences Collection, and Google Scholar) were searched. All study designs were included, and no date limits were set. Studies were included if they were published in English, and explored the psychological health of any EDC operatives, across fire, police, and emergency medical services. Studies were excluded if they related solely to other emergency workers, such as police officers or paramedics. Methodological quality of included studies was assessed using checklists adapted from the Critical Appraisal Skills Programme. A narrative synthesis was conducted, using thematic analysis. RESULTS: A total of 16 articles were included in the review. Two overarching themes were identified during the narrative synthesis: 'Organisational and Operational Factors' and 'Interactions with Others'. Stressors identified included being exposed to traumatic calls, lacking control over high workload, and working in under-resourced and pressured environments. Lack of support from management and providing an emotionally demanding service were additional sources of stress. Peer support and social support from friends and family were helpful in managing work-related stress. DISCUSSION: EDC operatives experience stress as a result of their work, which appears to be related to negative psychological health outcomes. Future research should explore the long-term effects of this stress, and the potential for workplace interventions to alleviate the negative impacts on psychological health. PROSPERO REGISTRATION NUMBER: CRD42014010806.

Mutation of the BRCA1 SQ-cluster results in aberrant mitosis, reduced homologous recombination, and a compensatory increase in non-homologous end joining.

Mutations in the breast cancer susceptibility 1 (BRCA1) gene are catalysts for breast and ovarian cancers. Most mutations are associated with the BRCA1 N- and C-terminal domains linked to DNA double-strand break (DSB) repair. However, little is known about the role of the intervening serine-glutamine (SQ) - cluster in the DNA damage response beyond its importance in regulating cell cycle checkpoints. We show that serine-to-alanine alterations at critical residues within the SQ-cluster known to be phosphorylated by ATM and ATR result in reduced homologous recombination repair (HRR) and aberrant mitosis. While a S1387A BRCA1 mutant - previously shown to abrogate S-phase arrest in response to radiation - resulted in only a modest decrease in HRR, S1387A together with an additional alteration, S1423A (BRCA12P), reduced HRR to vector control levels and similar to a quadruple mutant also including S1457A and S1524A (BRCA14P). These effects appeared to be independent of PALB2. Furthermore, we found that BRCA14P promoted a prolonged and struggling HRR late in the cell cycle and shifted DSB repair from HRR to non-homologous end joining which, in the face of irreparable chromosomal damage, resulted in mitotic catastrophe. Altogether, SQ-cluster phosphorylation is critical for allowing adequate time for completing normal HRR prior to mitosis and preventing cells from entering G1 prematurely resulting in gross chromosomal aberrations.

Subcutaneous administration of D-luciferin is an effective alternative to intraperitoneal injection in bioluminescence imaging of xenograft tumors in nude mice.

Currently, intraperitoneal (IP) injection of D-luciferin is the preferred method of providing substrate for bioluminescent imaging (BLI); however it has a failure rate of 3-10% due to accidental intestinal injection. The present study evaluates the quality of BLI after subcutaneous (SC) injection of D-luciferin and demonstrates the effectiveness of SC injection in anatomically disparate tumor models. Mice bearing luciferase-expressing tumors underwent BLI after SC or IP injection of D-luciferin. The average time to maximal luminescence was 6 min (range 5-9 min) after SC injection and 8 min (range 5-8 min) after IP injection. Within 7 minutes of injection, SC and IP routes yielded similar luminescence in subcutaneous, intracranial, tongue, and lung xenograft tumor models. In a model of combined subcutaneous and intracranial xenografts, SC injection resulted in proportional luminescence at all sites, confirming that preferential delivery of substrate does not occur. While tumors were occasionally not visualized with IP injection, all tumors were visualized reliably with SC injection. Thus, SC injection of D-luciferin is a convenient and effective alternative to IP injection for BLI in nude mice. It may be a preferable approach, particularly for tumors with weaker signals and/or when greater precision is required.

The Influence of Hypoxia and pH on Bioluminescence Imaging of Luciferase-Transfected Tumor Cells and Xenografts.

Bioluminescence imaging (BLI) is a relatively new noninvasive technology used for quantitative assessment of tumor growth and therapeutic effect in living animal models. BLI involves the generation of light by luciferase-expressing cells following administration of the substrate luciferin in the presence of oxygen and ATP. In the present study, the effects of hypoxia, hypoperfusion, and pH on BLI signal (BLS) intensity were evaluated in vitro using cultured cells and in vivo using a xenograft model in nude mice. The intensity of the BLS was significantly reduced in the presence of acute and chronic hypoxia. Changes in cell density, viability, and pH also affected BLS. Although BLI is a convenient non-invasive tool for tumor assessment, these factors should be considered when interpreting BLS intensity, especially in solid tumors that could be hypoxic due to rapid growth, inadequate blood supply, and/or treatment.