Protocol for mapping physiological DSBs using in-suspension break labeling in situ and sequencing.

Physiological double-stranded breaks (DSBs) are a major source of genomic instability. Here, we present a protocol for mapping physiological DSBs by in-suspension break labeling in situ and sequencing (sBLISS) in a single-nucleotide resolution. We describe steps for cell fixation, labeling of DSBs, DNA isolation followed by in vitro transcription (IVT), reverse transcription, and library preparation. sBLISS provides a map of DSBs over the genome and can be used to study the role of different factors in DSB formation. For complete details on the use and execution of this protocol, please refer to Hidmi et al.1.

Hepatocyte regeneration is driven by embryo-like DNA methylation reprogramming.

As a result of partial hepatectomy, the remaining liver tissue undergoes a process of renewed proliferation that leads to rapid regeneration of the liver. By following the early stages of this process, we observed dramatic programmed changes in the DNA methylation profile, characterized by both de novo and demethylation events, with a subsequent return to the original adult pattern as the liver matures. Strikingly, these transient alterations partially mimic the DNA methylation state of embryonic hepatoblasts (E16.5), indicating that hepatocytes actually undergo epigenetic dedifferentiation. Furthermore, Tet2/Tet3-deletion experiments demonstrated that these changes in methylation are necessary for carrying out basic embryonic functions, such as proliferation, a key step in liver regeneration. This implies that unlike tissue-specific regulatory regions that remain demethylated in the adult, early embryonic genes are programmed to first undergo demethylation, followed by remethylation as development proceeds. The identification of this built-in system may open targeting opportunities for regenerative medicine.

TOP1 and R-loops facilitate transcriptional DSBs at hypertranscribed cancer driver genes.

DNA double-stranded breaks (DSBs) pose a significant threat to genomic integrity, and their generation during essential cellular processes like transcription remains poorly understood. In this study, we employ several techniques to map DSBs, R-loops, and topoisomerase 1 cleavage complex (TOP1cc) to comprehensively investigate the interplay between transcription, DSBs, topoisomerase 1 (TOP1), and R-loops. Our findings reveal the presence of DSBs at highly expressed genes enriched with TOP1 and R-loops. Remarkably, transcription-associated DSBs at these loci are significantly reduced upon depletion of R-loops and TOP1, uncovering the pivotal roles of TOP1 and R-loops in transcriptional DSB formation. By elucidating the intricate interplay between TOP1cc trapping, R-loops, and DSBs, our study provides insights into the mechanisms underlying transcription-associated genomic instability. Moreover, we establish a link between transcriptional DSBs and early molecular changes driving cancer development, highlighting the distinct etiology and molecular characteristics of driver mutations compared to passenger mutations.

Flight safety risk assessment of self-medication among fighter pilots: a cross-sectional study.

BACKGROUND: Fighter pilots are a specific population in which any adverse drug reaction can unpredictably interact with aeronautical constraints and thus compromise flight safety. This issue has not been evaluated in risk assessments. AIM: To provide a semi-quantitative assessment of the risk to flight safety of self-medication in fighter pilots. METHOD: A cross-sectional survey that aimed at identifying the determinants of self-medication in fighter pilots was conducted. All medications consumed within 8 h preceding a flight were listed. A modified Failure Mode and Effects Analysis was performed, and any adverse drug reaction reported in the French marketing authorization document of a drug was considered a failure mode. The frequency of occurrence and severity were evaluated using specific scales to assign each to three risk criticality categories: acceptable, tolerable, and unacceptable. RESULTS: Between March and November 2020, the responses of 170 fighter pilots were analyzed, for an overall return rate of approximately 34%. Among them, 78 reported 140 self-medication events within 8 h preceding a flight. Thirty-nine drug trade names (48 different international nonproprietary names) were listed, from which 694 potential adverse drug reactions were identified. The risk criticality was considered unacceptable, tolerable and acceptable for 37, 325 and 332 adverse drug reactions, respectively. Thus, the risk criticality was considered unacceptable, tolerable and acceptable for 17, 17, and 5 drugs, respectively. CONCLUSION: This analysis suggests that the overall risk to flight safety of the current practice of self-medication in fighter pilots may be considered at least tolerable, or even unacceptable.

Residual Sleepiness Risk in Aircrew Members with -Obstructive Sleep Apnea Syndrome.

BACKGROUND: Obstructive sleep apnea syndrome (OSAS) is a major problem in aviation medicine because it is responsible for sleepiness and high cardiovascular risk, which could jeopardize flight safety. Residual sleepiness after the treatment is not a rare phenomenon and its management is not homogenous in aviation medicine. Thus, we decided to perform a study to describe this management and propose guidelines with the help of the literature.METHODS: This is a retrospective study including all aircrew members with a history of OSAS who visited our aeromedical center between 2011 and 2018. Residual sleepiness assessment was particularly studied.RESULTS: Our population was composed of 138 aircrew members (mean age 50.1 ± 9.6 yr, 76.8% civilians, 80.4% pilots); 65.4% of them had a severe OSAS with a mean Epworth Sleepiness Scale (ESS) at 8.5 ± 4.7 and a mean apnea hypopnea index of 36.2 ± 19.2/h. Of our population, 59.4% performed maintenance of wakefulness tests (MWT) and 10.1% had a residual excessive sleepiness. After the evaluation, 83.1% of our population was fit to fly.DISCUSSION: An evaluation of treatment efficiency is required in aircrew members with OSAS. Furthermore, it is important to have an objective proof of the absence of sleepiness. In this case, ESS is not sufficient and further evaluation is necessary. Many tests exist, but MWT are generally performed and the definition of a normal result in aeronautics is important. This evaluation should not be reserved to solo pilots only.Monin J, Rebiere E, Guiu G, Bisconte S, Perrier E, Manen O. Residual sleepiness risk in aircrew members with obstructive sleep apnea syndrome. Aerosp Med Hum Perform. 2023; 94(2):74-78.

Muscle injury causes long-term changes in stem-cell DNA methylation.

Injury to muscle brings about the activation of stem cells, which then generate new myocytes to replace damaged tissue. We demonstrate that this activation is accompanied by a dramatic change in the stem-cell methylation pattern that prepares them epigenetically for terminal myocyte differentiation. These de- and de novo methylation events occur at regulatory elements associated with genes involved in myogenesis and are necessary for activation and regeneration. Local injury of one muscle elicits an almost identical epigenetic change in satellite cells from other muscles in the body, in a process mediated by circulating factors. Furthermore, this same methylation state is also generated in muscle stem cells (MuSCs) of female animals following pregnancy, even in the absence of any injury. Unlike the activation-induced expression changes, which are transient, the induced methylation profile is stably maintained in resident MuSCs and thus represents a molecular memory of previous physiological events that is probably programmed to provide a mechanism for long-term adaptation.

Prevalence of sleep disorders in a large French cohort of aircrew members and risk of in-flight sleepiness.

BACKGROUND: /objectives: Sleep disorders are a critical issue for flight safety. Previous studies have shown a high prevalence of sleep disorders and excessive sleepiness in the general population and some aircrews. The objectives of this study are to measure the prevalence of excessive daytime sleepiness and sleep disorders in aircrews, and to determine the risk factors of falling asleep during a flight. METHODS: this is a monocentric study based on questionnaires, including all professional civilian and military aircrews examined in an aeromedical center between January and May 2021. The questionnaire, created for this study, included information about socio-demographic characteristics, aeronautical experience, lifestyle, sleep habits, an Epworth sleepiness scale, and screening tests for chronic insomnia, sleep apnea syndrome and restless legs syndrome. RESULTS: 749 aircrew members were included (86.2% male, 58.9% civilian, 74.1% pilot, mean age 43.4 ± 9.6 years), 45.9% of the population had at least one sleep disorder (chronic insomnia 39.5%, sleep apnea syndrome 10.5%, restless legs syndrome 4.1%), 15.5% had an excessive daytime sleepiness, and 24.6% reported in-flight sleep while on duty. Chronic insomnia, screen use before bedtime, use of sleeping pills, inadequate recovery time after a flight, female gender and civilian status were found as risk factors of in-flight sleep in the multivariate analysis. CONCLUSION: this study emphasizes the need to improve the screening and prevention of sleep disorders in this particular population.

Self-Medication Among Military Fighter Aircrews.

BACKGROUND: The practice of self-medication among military fighter aircrew could compromise flight safety because of the adverse effects that can occur in flight. However, data on this subject is scarce. The aim of this study was to identify the determinants of the practice in this population.METHODS: A cross-sectional study was carried out among the French Air Force fighter aircrew based on an anonymous questionnaire distributed electronically. The questions included personal characteristics, opinions, and relations with the healthcare domain as well as the use of self-medication in general and before a flight.RESULTS: Between March and November 2020, 170 questionnaires were reviewed for an overall return rate of approximately 34%. Our data showed an absolute self-medication rate of 97.6%, but the frequency of its use was rare or nonexistent in 53.5% of cases. Factors associated with a more frequent use of self-medication were the function of pilot, age under 35, having a regular prescription, lacking intentionality toward getting enough sleep, having confidence in the medical profession, and some specific clinical situations. The consumption of 97 medications was recorded and 49 before a flight.DISCUSSION: Despite the limitations due to the design of this survey, results suggest that the use of self-medication in fighter aircrews is a reality, but that the frequency of its use is less common. This practice is probably the result of a complex interaction between many personal factors. However, its impact on flight safety remains uncertain.du Baret de Limé M, Monin J, Leschiera J, Duquet J, Manen O, Chiniard T. Self-medication among military fighter aircrews. Aerosp Med Hum Perform. 2022; 93(7):571-580.

The impact of 16-h heat exposure on appetite and food reward in adults.

Heat exposure is thought to reduce energy intake (EI) but studies are sparse and results not always concordant. The aim of this study was to examine whether a 16-h exposure to 32 °C leads to reduced EI compared to a control session (22 °C) and whether modifications in appetite sensations or food reward are implied. Sixteen healthy, lean, and active participants (9 women and 7 men, 25 ± 5 yo, body mass index: 22.0 ± 2.4 kg m-2) were passively exposed to two different thermal temperatures from 4:00 p.m. to 8:00 a.m. under controlled conditions. Hunger and thirst scores were regularly assessed using visual analogue scales. A fixed dinner meal (3670 ± 255 kJ) was consumed at 7:30 p.m. and an ad libitum breakfast buffet (20 foods/drinks varying in temperature, fat, and carbohydrate content) at 7:30 a.m. Components of reward (explicit liking [EL] and implicit wanting [EI]) for fat and sweet properties of food were assessed before each meal using the Leeds Food Preference Questionnaire (LFPQ). Ad libitum EI at breakfast did not differ between sessions (2319 ± 1108 vs 2329 ± 1141 kJ, in 22 and 32 °C sessions, respectively; p = 0.955). While thirst scores were higher in the 32 than the 22 °C session (p < 0.001), hunger scores did not differ (p = 0.580). EL and IW for high fat foods relative to low fat foods were decreased in 32 compared to 22 °C before dinner and breakfast (p < 0.001 for all). Although EI and hunger were not affected by a 16-h exposure to heat, modifications in food reward suggested a reduction in the preference of high-fat foods. Future research should investigate whether reduced EI in response to heat exposure is due to spontaneous selection of low-fat foods rather than altered appetite sensations.

Travel health study in commercial aircrew members.

BACKGROUND: Aircrew members of airlines are exposed to travel risks. The objectives of our study are to assess the experience of aircrews about these risks and their knowledge about prevention means. METHODS: We conducted an observational qualitative study in commercial aircrews at the aeromedical center of Percy Military Hospital between November 2018 and June 2019. RESULTS: 200 aircrews answered the questionnaire, 54% of which were pilots, 91% work on long and/or medium-haul flights, 82.5% of airmen are concerned by their immunization status. Vaccination rate varied according to the vaccine. Two third of airmen usually go to malaria-endemic countries, 12% of respondents use antimalarial treatment in such infected countries, while 93.5% protect themselves against mosquito bites mainly with insect repellent. In case of a fever after a stay in a malaria-endemic country, only 51.5% of respondents immediately think about acute malaria. Aircrews are very motivated by their job but 58% of them feel tired probably linked to quality of sleep and effects of jet-lag, with a statistically significant difference between pilots and cabin crews (43% vs 75% [p < 0.01]). CONCLUSION: Aircrew members know a lot about travel issues. Malaria remains a major concern for aircrews, but it is necessary to maintain information about this topic throughout their career and to provide them with repellents, what many airlines actually do. Fatigue management is also important for airmen, so as they use different technics to accelerate recovery. Some airlines try to help them with a guide for aircrew fatigue management. This particular population involved in flight safety has few risky behaviors; nevertheless, alcohol misuse and drug use are screened during medical examinations and by airlines.

Activation of Oncogenic Super-Enhancers Is Coupled with DNA Repair by RAD51.

DNA double-strand breaks (DSBs) are deleterious and tumorigenic but could also be essential for DNA-based processes. Yet the landscape of physiological DSBs and their role and repair are still elusive. Here, we mapped DSBs at high resolution in cancer and non-tumorigenic cells and found a transcription-coupled repair mechanism at oncogenic super-enhancers. At these super-enhancers the transcription factor TEAD4, together with various transcription factors and co-factors, co-localizes with the repair factor RAD51 of the homologous recombination pathway. Depletion of TEAD4 or RAD51 increases DSBs at RAD51/TEAD4 common binding sites within super-enhancers and decreases expression of related genes, which are mostly oncogenes. Co-localization of RAD51 with transcription factors at super-enhancers occurs in various cell types, suggesting a broad phenomenon. Together, our findings uncover a coupling between transcription and repair mechanisms at oncogenic super-enhancers, to control the hyper-transcription of multiple cancer drivers.

WWOX Inhibits Metastasis of Triple-Negative Breast Cancer Cells via Modulation of miRNAs.

Triple-negative breast cancer (TNBC) is a heterogeneous, highly aggressive, and difficult to treat tumor type. The tumor suppressor WWOX spans FRA16D, a common fragile site that is commonly altered in breast cancer. Despite recent progress, the role of WWOX in TNBC metastasis is unknown. Here we report that WWOX inactivation correlates with advanced stages of TNBC and that its levels are frequently altered in TNBC cells. Ectopic restoration of WWOX in WWOX-negative TNBC cells inhibited metastasis while its depletion in WWOX-positive TNBC cells promoted metastasis. WWOX was a negative regulator of c-MYC, which regulated miR-146a expression and consequently fibronectin levels, contributing to an epithelial status of the cell. Treatment of TNBC cells with anti-miR-146a rescued the WWOX antimetastatic phenotype. Moreover, overexpression of MYC in WWOX-expressing TNBC cells overrode WWOX effects on miR-146a and fibronectin levels. Altogether, our data uncover an essential role for WWOX in antagonizing TNBC progression and highlight its potential use as a biomarker for metastasis. SIGNIFICANCE: These findings highlight the mechanism by which the tumor suppressor WWOX regulates metastasis of triple-negative breast cancer.See related commentary by Sharma, p. 1746.

Prevalence of benign epileptiform variants during initial EEG examination in French military aircrew.

INTRODUCTION: In France, a systematic EEG is performed during initial examination in military aircrew applicants, which may provide an estimation of the prevalence of benign epileptiform variants in healthy adults. METHODS: We analyzed standard EEG (21 scalp electrodes, 20minutes, 400Hz sampling rate) of military aircrew applicants examined in the French Main Aeromedical Center in 2016. EEGs were analyzed using both bipolar and referential montages. The collected data were EEG abnormalities and benign epileptiform variants. The kappa inter-observer index for the detection of benign epileptiform variants was calculated. RESULTS: Our population was composed of 495 subjects (86.7% males, mean age 22.5±4.8 years), wishing to become a pilot in 69.7% of cases. None of the applicants reported any neurological disease and none was taking regular medication. EEG was considered as normal for 96.4% of them. Encountered EEG abnormalities were mainly asymmetric and sharp slow wave bursts. Drowsiness was recorded during 13.9% of these EEG. Benign epileptiform variants were present in 7.7% of our population: anterior theta activities (4%), posterior slow waves (2.8%), alpha variants (0.6%) and wicket spikes (0.2%). Hyperventilation induced EEG slowing in 14.1% of cases. During intermittent photic stimulation, physiological photic driving was observed in 15.2% of subjects. DISCUSSION: Many previous studies have been dedicated to the prevalence of benign epileptiform variants but results are often heterogeneous and based on patients in whom there was an indication for EEG. Our results thus bring data on benign epileptiform variants prevalence in a young adult population characterized by the absence of neurologic disorders. Our study demonstrates that anterior theta activities, posterior slow waves, alpha variants and wicket spikes are the most frequent benign EEG variants in such a young adult population.

Epidemiology of Airmen Treated with Immunosuppressive Drugs and Vaccination Concerns.

BACKGROUND: Immunosuppressive treatments are increasingly prescribed in a variety of diseases. This issue concerns airmen. METHODS: To assess the problem, we conducted an observational retrospective study in the aircrew population examined in 2014 at the Aeromedical Center of Percy Military Hospital. RESULTS: Airmen treated with immunosuppressive drugs accounted for 0.5% of the total population (N = 13,326). Rheumatic and digestive diseases were the main etiologies, respectively 43% and 35% of cases. One-third of airmen took such medications during at least 3 yr and three-quarters of airmen were declared fit to fly, with some limitations. DISCUSSION: Due to their working conditions, airmen are exposed to a real infectious risk, which is, however, difficult to evaluate. The risk is obviously increased by immunosuppressive drugs and may affect flight safety. Aeromedical evaluation should consider this problem. Vaccination plays a central role in the prevention of infectious risk. Based on French recommendations, we propose a vaccination schedule for these particular patients.Guiu G, Monin J, Hamm-Hornez A-P, Manen O, Perrier E. Epidemiology of airmen treated with immunosuppressive drugs and vaccination concerns. Aerosp Med Hum Perform. 2018; 89(4):377-382.

Progression of early repolarization patterns at a four year follow-up in a female flight crew population: Implications for aviation medicine.

BACKGROUND: To assess the prevalence, the appearance, and the distribution, as well as the fluctuation over time of early repolarization patterns after four years in a female population derived from the French aviation sector. METHODS: This was a retrospective longitudinal study from 1998 to 2010 of a population of female employees who received a full clinical examination and an electrocardiogram (ECG) upon their recruitment and after a period of four years. RESULTS: A total of 306 women were included (average of 25.87 ± 3.3 years of age). The prevalence of early repolarization was 9.2%. The most common appearance was J-point slurring for 64.3% (i.e. 20/28 subjects) that occurred in the inferior leads for 28.6% (i.e. 8/28 subjects). After four years, the prevalence was 7.5%, with a regression of this aspect in five of the subjects. There were no changes in the ECG in terms of the distribution and the appearance among the 23 subjects for whom the aspect persisted. Over the course of this four year period all of the subjects remained asymptomatic. CONCLUSIONS: Early repolarization in this largely physically inactive female population was common, and it fluctuated over time. At present, no particular restrictions can be placed on asymptomatic flight crew who exhibit this feature in the absence of a prior medical history for heart disease.

Prevalence of Intraventricular Conduction Disturbances in a Large French Population.

BACKGROUND: Recommendations for the interpretation of electrocardiogram have been published in 2009. The aim of this study was to define the prevalence of intraventricular conduction disturbances (ICoDs) in a large population, using these recommendations. METHODS: From 01/31/1996 to 09/22/2010, an electrocardiogram was performed at each visit for all aircrew members examined for fitness assessment in an aeromedical center. The prevalence of left bundle branch block (LBBB), right bundle branch block (RBBB), incomplete LBBB, incomplete RBBB, nonspecific intraventricular disturbance (NIVCD), left anterior fascicular block (LAFB), and left posterior fascicular block (LPFB) was measured and compared by age and gender. RESULTS: The global prevalence of ICoD was 3.09% in our population of 69,186 patients. The most frequent types of ventricular blocks were IRBBB (1.25%) and LAFB (1.10%), whereas RBBB (0.46%), LBBB (0.08%), ILBBB (0.03%), NIVCD (0.05%), and LPFB (0.13%) were rare findings. ICoDs are more frequent for males and older age groups (P < 0.001). DISCUSSION: Our results are comparable to studies concerning low cardiovascular risks populations. The association between ICoD and cardiovascular diseases needs to be studied in this population.